Expanding the payload scope in antibody-drug conjugates: Unprecedented delivery of hydroxy-containing drugs through self-immolative phosphoramidates

Expanding the payload scope in antibody-drug conjugates: Unprecedented delivery of hydroxy-containing drugs through self-immolative phosphoramidates | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Expanding the payload scope in antibody-drug conjugates: Unprecedented delivery of hydroxy-containing drugs through self-immolative phosphoramidates Marc-André Kasper, Philipp Ochtrop, Anil Jagtap, Jan Felber, Simon Vogt, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5454963/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Jan, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Despite recent advances in targeted drug delivery, approved Antibody-Drug-Conjugates (ADCs) are still limited by the delivery of a restricted set of payloads with limited modes of action (MOA). Versatile linkers, applicable to functional groups prevalent across diverse pharmacophores are needed to expand this space. We present phosphoramidate-based self-immolative linker-units that facilitate stable attachment in serum and traceless drug release in the target cell from aliphatic and aromatic alcohols. Studies with camptothecins show that stability and release are tunable and that various intracellular trigger events can be exploited to ensure traceless drug delivery. Superior stability, in vivo efficacy, and pharmacokinetics (PK) compared to approved ADCs are demonstrated. Moreover, we report targeted delivery of 10 different hydroxy-containing cytotoxins with different intracellular MOAs. In vivo studies with gemcitabine show excellent PK and efficacy, unlocking gemcitabine’s full potential and illustrating the ability of the phosphoramidate-based linker system to expand the payload space for ADCs. Biological sciences/Drug discovery/Drug delivery Biological sciences/Cancer/Cancer therapy/Targeted therapies Physical sciences/Chemistry/Chemical biology/Drug delivery Physical sciences/Chemistry/Biochemistry/Bioconjugate chemistry Biological sciences/Chemical biology/Chemical tools Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Main Targeted drug delivery for the treatment of malignant diseases holds great promise in reducing undesired side effects while increasing efficacy by precisely hitting cancer cells. 1 Most prominently, antibodies conjugated to highly potent cytotoxic drugs, a modality known as antibody-drug conjugates (ADCs), have been utilized as targeting vehicles. 2 , 3 The pace of more new ADCs entering clinical trials and receiving FDA approval has accelerated over the past decade, underlining the benefits of targeted drug delivery for patients. 4 Still, challenges remain to be solved, such as chemotherapy-like toxicities, lack of efficacy when applied as monotherapy and, eventually, acquired resistance. 5 The potency and mode of action (MOA) of the cytotoxic payload that is being delivered is considered as one of the major drivers of these shortcomings. 6 With a small repertoire of only three MOAs in approved ADCs, namely tubulin-inhibition, topoisomerase-I (TOP1)-inhibition and DNAdamaging, it is anticipated that MOA diversification will be a key factor in the development of next generation targeted cancer therapeutics. 7 Another Achilles’ heel that has been identified in the current generation of ADCs is the linker between antibody and drug. Ongoing research efforts aim to improve linker associated limitations such as premature drug loss, 8 unspecific uptake 9, 1 0 and undesired aggregation. 1 1 On top of that, linker design determines the cleavability and drug release at the targeted location. In particular, efficient, traceless drug release after receptor-mediated uptake can be crucial for the intracellular function of the payload 1 2 and for its permeability, which allows effective eradication of tumors with heterogenous expression of the antibody’s target via the bystander effect. 1 3, 1 4 Hence, cleavable linker systems are required that provide a stable attachment of a payload via one of its functional groups during circulation and, importantly, simultaneously allow for traceless release of this functional group upon uptake into the targeted cell. Cleavable linker systems usually consist of a release unit that is activated by an intracellular trigger event, and a self-immolative moiety that subsequently liberates the unmodified functional group. 1, 1 5 Commonly exploited trigger events include a variety of intracellular or intra-lysosomal conditions such as acidic and reductive environment or specific enzymatic activities from esterases, 1 6 proteases, 1 7 glycosidases, 1 8 phosphatases, 1 9 reductases, 2 0 or sulfatases 2 1 . Such cleavable linkers have been designed for the attachment of different functional groups on payloads including primary-, 22 secondary-, 23 tertiary- and heteroaryl-amines, 24 thiols, amides, 25 ortho-quinones 26 and alcohols. 27 In particular, hydroxyl linkages are attractive since this functionality is frequently present in synthetic small molecule drugs and natural product derivatives. 28 However, based on their chemical environment, hydroxyl groups can differ tremendously in steric accessibility and pKa, ranging from 7 (phenols) to 16 (aliphatic alcohols). This broad scope poses a challenge for the design of broadly applicable linker systems that allow for both stable attachment and traceless release of chemically distinct alcohols. Available linker systems for alcohol attachment rely on carbamates, 29 carbonates, 30 esters, 31 phosphates, pyrophosphates, 32 1,6-benzylamines, 33 hemiaminals, 34 disulfides, 20 , 35 or methylene carbamates. 27 However, none of these functional groups combine broad applicability, stability in serum and efficient traceless release. Here, we report a new self-immolative moiety providing stable attachment and traceless release of alcohols for targeted drug delivery. In our design, we make use of P(V)-based motifs of the clinically validated ProTide technology, 36 a prodrug system that has been developed to mask hydrophilicity of nucleoside-like drug molecules to enhance cell permeability. 37 After passive membrane diffusion, these prodrugs are activated via esterase cleavage of an amino acid ester followed by self-immolation via a 5-membered cyclization, release of a sacrificial phenol and finally, enzymatical release of the (phosphorylated) nucleoside analog (Fig. 1 a). In our work we have redesigned the central phosphorous core by introducing a conjugation handle to one of the three P -substituents that can form a covalent bond with a targeting moiety like an antibody. This allows to transform a prodrug- into a targeted drug delivery technology. Exploiting the fact that Protides liberate an aromatic- and an aliphatic alcohol intracellularly, we transferred and developed this concept even further to release not only nucleoside analogs but instead designed a linker-technology to deliver a broad spectrum of aliphatic- and aromatic alcohols (Fig. 1 b). In contrast to the earlier described nucleoside prodrugs which are restricted to esterase cleavage after passive uptake into the cytosol, the release from our phosphoramidate-based targeted drug delivery system can be initiated by various intracellular and endosomal triggers including esterases, proteases, glucoronidases and reductive conditions due to the uptake mechanism via receptor-mediated endocytosis. Results and discussion Establishment of phosphorus-based self-immolative handles for the targeted delivery of aromatic alcohols exemplified with SN38 To start our endeavors, we chose the TOP1-inhibitor SN38, the active pharmaceutical ingredient (API) of the clinically approved ADC sacituzumab govitecan (SG, Trodelvy), as our first model drug. 38 Guided by previous experience with unsaturated ethynyl- 39 and vinyl-phosphonamidate 40 -based linker technologies, we designed linker structures 1 and 2 for cysteine-selective antibody conjugation and attached SN38 via its aromatic 10-hydroxyl group as O -substituent of the phosphonamidates. In this design, the alaninyl tert butyl ester at the phosphorus N -substituent is supposed to act as release trigger. Upon intracellular tert butyl ester hydrolysis, cyclization via a 5-membered intermediate could lead to a traceless release of unmodified SN38 via a similar mechanism as described for the phenol in Fig. 1 a. Synthesis of 1 and 2 was performed as outlined in Fig. 2 a (See SI for detailed information). Conjugation of 1 -SN38 to sacituzumab and an isotype antibody delivered homogenous ADCs with a drug-to-antibody ratio (DAR) of 8, while a conjugation with 2 -SN38 was not suitable to produce ADCs with DAR higher than 0.5. These observations reflect the previously reported reactivity differences between ethynyl and vinyl-phosphonamidates with cysteines and consequently disqualified 2 -SN38 as an efficient drug-linker for SN38. 40 Next, we wanted to explore the properties of the linker systems 1 and 2 and compare them with the established linker system of SG (CL2A-SN38). In the linker (CL2A-SN38) of SG, SN38 is attached at its tertiary 20hydroxyl group via a carbonate moiety (Fig. 3 a). This linker has been designed to rapidly hydrolyze under physiological conditions. As previously reported 4 1 and confirmed by our in vitro cell culture assays, this lability leads to extracellular release of SN38, causing cell killing also of non-targeted cells with isotype ADCs that are not actively internalized. Consequently, the IC 50 of targeted vs. isotype ADC in cell viability experiments with multiple days of incubation are highly similar (0.2 nM versus 0.3 nM, Fig. 2 b). Previously reported efforts to generate a serum stable linker, in which SN38 is attached via a carbamate to the aromatic 10-hydroxyl group, failed to show efficacy due to insufficient intracellular release of SN38 as indicated by higher IC 50 than for SN38 as free drug. 3 0 These two scenarios observed for SN38-containing linker systems inspired us to employ in vitro proliferation assays with isotype versus targeted ADCs as a reliable, close-to-application readout to optimize our linker system for stability and release efficiency. For this reason, we analyzed the ratio of IC 50 from isotype- over targeted ADC as measure for linkage stability, and absolute IC 50 of the targeted ADC as measure for effective release. We subjected sacituzumab- 1 -SN38 to in vitro cytotoxicity assays with the Trop2-positive MDA-MB-468 cancer cell line and observed almost identical IC 50 values for targeted and isotype ADCs of 0.4 and 0.6 nM, respectively, indicating insufficient stability under cell culture conditions, similar as observed for SG (Fig. 2 b). This instability stands in strong contrast to previously reported O -alkyl ethynylphosphonamidate-based ADCs that were shown to be highly stable under physiological conditions, in vitro and even in vivo. We attribute the lability of 1 -SN38 to the strong leaving group character of the aromatic SN38, which leads to fast hydrolysis of the phosphonamidate under assay conditions. To stabilize the linker system against hydrolysis, we envisioned exchanging the carbon at the phosphorous core to oxygen and introducing a short amino pentyl linker as second O -substituent while keeping the N -alanine tert -butyl ester as release handle. By attaching a suitable antibody conjugation handle such as ethynylphosphonamidates (P5-conjugation) 12 , 42 , this resulted in phosphoramidate-based linker-payloads 3 and its variations 4 and 5 . In linker 4 O - and N - substituted linker and release handle are swapped, while in 5 the release handle is exchanged to a non-cleavable 4-methylbenzyl group. Linkers 3 – 5 were synthesized as outlined in Fig. 2 c (See SI for detailed information) and proved to be superior to 1 SN38, since 3-4 -SN38, conjugated to sacituzumab, exhibited excellent anti-proliferative activity with a favorable potency window between targeted and isotype ADC (Fig. 2 d). An IC 50 of 0.4 nM and a 13-fold difference for the Trop2-targeted ADC saci- 3 -SN38 vs. the isotype ADC indicates a more stable SN38 conjugation to the mAb compared to SG or 1 -SN38, while allowing an efficient release of the drug after receptor-mediated cellular uptake. A similar behavior was observed for saci- 4 SN38. However, since the selectivity window was slightly lower (5-fold) we decided to continue our studies using linker 3 . Interestingly, targeted ADCs based on the non-cleavable control 5 SN38, lacking an intracellular release trigger, only showed an anti-proliferative effect at the highest concentration in vitro . These results indicate that phosphoramidate based linker systems 3 and 4 provide a stable connection of SN38 via its aromatic alcohol which can be cleaved intracellularly, involving enzyme-mediated hydrolysis of the ester in the release handle. Next, we set out to investigate the bystander effect of saci- 3 -SN38. 14 As shown in Fig. 2 e, saci- 3 -SN38 exhibits strong bystander killing of Trop2- cells after supernatant transfer from Trop2 + cells preincubated with ADC, but only very low direct cell killing of the target-negative cells. Since traceless payload release from the linker has been described as a key requirement for bystander activity, this data further indicates efficient, traceless SN38 release after receptor-mediated uptake from 3 -SN38,. 43 We then set out to compare 3 -SN38 head-to-head with SG to evaluate whether phosphoramidates could improve the established Cl2A linker in SG. We confirmed target selectivity for saci- 3 -SN38 in vitro on a larger panel of cell lines, in contrast to CL2A-SN38 that proved to be unselective, (Supp. Figure 1 ) and observed improved serum stability of saci- 3 -SN38 with more than 50% intact ADC after 7 days of incubation (Fig. 2 f). In contrast, SG lost almost 100% of conjugated drug already after 24h, mainly caused by carbonate hydrolysis. Since tumor-target specificity of SG has been demonstrated in vivo , contrary to the in vitro observations, 41 we compared both linker-payloads head-to-head in an in vivo efficacy experiment (Fig. 2 g). Here, Trop2+-tumor bearing mice, were treated twice (day 0 and day 4) with 10 mg/kg of saci- 3 -SN38, iso- 3 -SN38, SG or vehicle. We observed a superior efficacy with complete responses for saci- 3 -SN38 in all mice while SG only led to delayed tumor growth and iso- 3 -SN38 showed no clear responses. We attribute this enhanced anti-cancer activity to improved linker stability, as previously reported in other systems, 42 , 44 ensuring a higher tumor exposure to SN38. Linker stability tuning and combination with different release triggers Encouraged by reports on structural optimization of ProTides, 36 we set out to tune potency and stability of our phosphoramidate-based linker system by synthesizing derivatives of linker 3 attached to SN38. For evaluation, we conjugated the resulting linker-payloads to two different antibodies, targeting the cell-surface cancer antigens Trop2 and CD30, which were characterized for potency (IC 50 targeted ADC) and selectivity (ratio IC 50 isotype/targeted ADC) on two target-positive cell lines for each ADC (Fig. 3 a). To increase electron density at P, we synthesized linker 6 , in which the primary pentyl O -substituent of linker 3 is exchanged to a secondary cyclohexyl O -substituent, and linker 7 , in which we introduced an additional methyl group at the amino acid alpha carbon. We also aimed to reduce electron density at the phosphorus by exchanging the alanine in linker 3 to a glycine in linker 8 (Fig. 3 b). We observed that an increase in electron density in linkers 6 and 7 lead to a larger difference between targeted and isotype ADCs, whereas linker 8 , possessing less electron density at the phosphorous, showed a decreased window (Fig. 3 c). At the same time, we observed slightly reduced median potencies for the ADCs based on 6 -SN38 and 7 -SN38 compared to 3 -SN38 and 8 -SN38, which might be attributed to steric hindrance leading to either reduced esterase activity or reduced cyclization rates between the liberated carboxylic acid and the central phosphorus atom. To further study this, we exchanged the esterase cleavable tert -butyl ester in the derivatives 3 and 6 to isopropyl esters in linkers 9 and 10 , following previous reports on ProTides that tert -butyl esters are less efficiently cleaved by esterases. 45 Interestingly, this change did not have an effect on the median potency on the targeted cell line but the selectivity slightly increased from tert -butyl- to isopropyl esters in both cases. The cleavable phosphoramidate-based linkers described to this point are all designed to be activated analogously by esterase-mediated cleavage of the respective amino-acid ester. Next, we wanted to explore if the cleavage can also be initiated via alternative release triggers that are commonly applied in antibody-mediated drug-delivery. 2 In contrast to prodrugs, that passively diffuse into the cytosol, ADCs are actively taken up by targeted cells via the endosomal and lysosomal pathway, which exposes them to high levels of various proteases that are often overexpressed in human cancers. 46 Hence, we exchanged the amino-acid ester release handle with an alanine-alanine dipeptide in linker 11- SN38, with the expectation that 11- SN38 forms the same carboxylic acid intermediate after protease mediated cleavage that 6- and 10 -SN38 form upon esterase cleavage. 11 -SN38 exhibited similar potencies on the targeted cell lines, but reduced selectivity (targeted vs. isotype-ADC) compared to the esters 3 , 8 , and 9 (Fig. 3 c). Other known release triggers exploit the reductive environment or beta-glucuronidase activity in endo-/lysosomes. 49,50 Hence, we also synthesized reductively cleavable disulfide 12 and GlcA protected alcohol 13 and explored if liberated sulfhydryl or hydroxyl groups would perform a 5-membered cyclization at the phosphorus and facilitate release of the attached drug. The selectivity of ADCs 12 -SN38 and 13 -SN38 for the targeted cell lines was comparable to the glycine derivative 8 (Fig. 3 c). Even though slightly reduced, both targeted constructs showed decent activities, supporting a release mechanism that involves the liberation of a nucleophile (COOH, OH and SH) in delta position of the central P-atom which can release the attached drug by five-membered cyclization. Another property of ADCs that is discussed to have a crucial impact on their performance is their hydrophobicity. To determine the hydrophobicity of the synthesized ADCs and how the different linkers 3 and 6–13 influence these, we characterized the conjugates by hydrophobic interaction chromatography (HIC). The HIC-retention time can be used as a measure for overall ADC hydrophobicity and previous reports conclude that earlier eluting, more hydrophilic ADCs have beneficial PK properties. 47 The free carboxylic acid in dialanine linker 12 comprised the most hydrophilic DAR8 SN38-ADC tested herein (Fig. 3 d). From the ester series linker 6 and 7 delivered more hydrophobic ADCs, whereas the glycine derivative in linker 8 yielded a slightly more hydrophilic ADC compared to linker 3 . At the same time, an increased hydrophilicity of the ADCs was observed with the isopropyl substituent in 9 and 10 compared to all the tert -butyl derivatives. Taken together, the results summerized in Fig. 3 illustrate that stability, release efficiency and hydrophilicity of phosphoramidate-based drug-linker can be tuned by altering the substitution pattern around the phosphorous atom, covering a range from SG-like instability to a more targeted delivery of SN38. This unprecedented tuning could be useful in the future to identify the optimal linker for a certain target that requires either stable delivery or a certain level of instability to match different levels of healthy tissue expression. Establishment of phosphorus-based self-immolative handles for the targeted delivery of aliphatic alcohols exemplified with DXd Next, we chose the TOP1-Inhibitor DXd as model payload, carrying a primary alcohol. DXd is the payload of the marketed ADC trastuzumab deruxtecan (TD/Enhertu). 48 We synthetically introduced DXd into the phosphorus(V)-based linker systems 1 and 3 as already described for SN38 and conjugated the resulting linker-payloads to trastuzumab and an isotype antibody to test for targeted delivery via the HER2 antigen (Fig. 4 a and 4 b). Compared to what we observed for SN38, the phosphonamidatebased linker 1 delivered DXd into the targeted cell line with a 3-fold better selectivity compared to the isotype ADC (Fig. 4 d). Like for SN38, a phosphoramidate release handle in 3 -DXd was superior to phosphonamidates in 1 -DXd (Fig. 4 d), displaying a higher potency (IC 50 = 0.2 nM versus 0.8 nM) and a larger selectivity (19x versus 3.4x) for the targeted cell line. Next, we exchanged the esterase cleavable handle to a protease responsive dialaninyl-cleavage site in linker 14 -DXd (Fig. 4 c). In stark contrast to the protease cleavable 11 -SN38, we observed an improved linker stability that resulted in a more selective (50×) delivery of the payload. We assign this enhanced stability to decreased leaving group properties (= higher pKa) of the aliphatic hydroxyl group in DXd. The selectivity of 14 -DXd was even more increased using linker 15 (55×) that possesses a terminal carboxy group instead of the tert-butyl ester as shown in Fig. 5 c. The terminal carboxy group is also desired, since it adds additional hydrophilicity to the linker system, as shown for SN38 in Fig. 4 d. To better understand whether Dxd release involves an attack of the alanine carboxy group on the central phosphorous via a 5-membered intermediate, we synthesized linker 16 - and 17 -DXd, requiring formation of less favored 6- or 7-membered rings for a similar drug release after protease cleavage. ADCs of both linkers were almost inactive on the targeted cell lines (Fig. 4 d), underlining the mechanism that is shown in Fig. 1 b, requiring a 5-membered cyclization from a free carboxylate to the central phosphorus atom for efficient release. Comparing the high efficiency of linker 15 with the inactive 16 and 17 further allows the conclusion that release of non-derivatized DXd-OH is required to exhibit in vitro potency and that linker 15 is the most suited for delivery of aliphatic alcohols. Next, we compared 15 -Dxd head to head with the state-of-the-art protease-cleavable linker-payload GGFG-DXd in a set of in vitro and in vivo experiments. In Fig. 4 e, we assessed ADC-stability in rat serum and demonstrate increased stability of tras- 15 -DXd compared to TD. It should be noted that the serum stability of 15 -DXd is even higher than that of linker 3 -SN38 (Fig. 2 f). We attribute this to the higher pKa of the aliphatic alcohol in Dxd making it a worse leaving group compared to the aromatic alcohol in SN38, leading to less drug hydrolysis in serum. We confirmed target selectivity for tras- 15 -DXd and TD in vitro on a larger panel of cell lines without activity on non-targeted cell lines (Supp. Figure 3 ). Finally, we showed that tras- 15 -DXd effectively eradicates HER2 + tumors in vivo , whereas an iso- 15 -DXd did not show any anti-tumor effect (Fig. 4 f). Only the linker system described herein showed complete responses in three out of five animals at a dose of 1 mg/kg, whereas no complete responses were observed for the group that has been treated with TD at the same dose. Pharmacokinetic analyses by ELISA of blood samples drawn from this study revealed an excellent PK profile with slow clearance of tras- 15 -DXd and superimposable assay results for total antibody and intact ADC, highlighting outstanding linker stability. In contrast, TD was more rapidly cleared from circulation (Fig. 4 f). Collectively, these results underline that the phosphoramidate linker systems described herein enable an efficient and selective delivery of DXd into the targeted cell with a superior in vivo efficacy compared to an approved ADC based on the same antibody and payload. Application of phosphoramidate linkers to the antibody-mediated delivery of structurally diverse hydroxy-containing cytotoxins exhibiting diverse MOAs Finally, with the overall goal to identify novel ADC payloads, we aimed to apply our linker systems to a wider range of hydroxycontaining antiproliferative agents. The chosen drugs cover a broad spectrum of MOA and have been developed for chemotherapeutic treatment in oncology but were rarely used in an ADC context before. For all drugs with aromatic alcohols, we employed linker 9 and for all drugs with aliphatic alcohols linker 15 , both identified as optimal linker in terms of potency, selectivity and ADC hydrophilicity. We employed the HSP90-inhibitor ganetespib 4 9 and the elongation factor inhibitor ON013100 50 as examples for aromatic payloads; the nucleoside analogue gemcitabine 5 1 and the dihydroorotate dehydrogenase (DHODH) inhibitors BAY 2402234 52 and DHODH-IN-16, 5 3 as primary alcohols; the HSP90-inhibitor SNX2112 54 and the tubulin inhibitor paclitaxel 5 5 as secondary alcohols and the nicotinamid phosphoribosyltransferase (NAMPT) inhibitor (LSN3154567) 5 6 as example for a tertiary alcohol (Fig. 5 b). All linker-drugs were conjugated to the three different antibodies trastuzumab (HER2-targeting), datopotamab (Trop2-targeting) and brentuximab (CD30-targeting) (Fig. 5 a), generating homogenous DAR8 constructs (Fig. 5 c). Next, to investigate the applicability of those payloads, the ADCs were tested in eight different cell lines. Trastuzumab-based ADCs were investigated in cell killing assays on the HER2-positive solid cancer cell lines N87, SKBR-3 and HCC1569; datopotamab-based ADCs on the Trop2-positive solid cancer cell lines H441, HCC-78, and MDAMB-468, and brentuximab-based ADCs on the CD30-positive lymphoma cancer cell lines L-540 and Karpas-299. This in vitro evaluation revealed successful target-dependent delivery of all chosen payloads as can be seen by the IC50s and selectivity factors in Fig. 7e. This is highly remarkable, since it firstly confirms broad applicability to very different chemical hydroxyl groups and secondly shows the ability to unlock novel payloads using phosphoramidate based self-immolative handles. Even though single examples of ADCs carrying HSP90- 57 , NAMPT-inhibitors 56 and paclitaxel 58 have been described in the literature, this is the first time that ADCs carrying nucleoside analogues, DHODH- or an elongation factor- inhibitors are described and documented with in vitro potency. 7 Potency generally ranged between IC50s of 0.1 nM and 60 nM. Whereas most of the payloads showed a beneficial ratio between targeted and non-targeted ADCs with a targeting factor of 10x, paclitaxel and LSN315456 demonstrated only modest selectivity for the targeted cell line (Fig. 5 d). It should be noted that universal linker 9 and 15 for aromatic or aliphatic alcohols were used in this screen and that stability tuning by phosphoramidate modifications, as reported for SN38, might further optimize the stability and release for each payload described here. To better understand the potency differences of the different payloads across cell lines, we plotted the antiproliferative activity of all the unconjugated payloads against the effectivity of the targeted ADCs (Fig. 5 e). As expected, the more effective an antiproliferative drug is in its unconjugated form on a certain cell line, the more active is also its respective ADC. Remarkably, all payloads with activity below 1 nM also showed activity when delivered by an ADC, clearly demonstrating the broad applicability of the described linker system to efficiently deliver chemically diverse hydroxyl groups. Payloads with a lower activity than 1 nM led to active ADCs in some cases but were more likely to be inactive. The observation that subnanomolar potency of a cytotoxin is a requirement for an ADC payload is in line with previous predictions. 4 , 59 However, best to our knowledge, this is the first time that this is investigated more systematically in one comparable experiment with a broad range of different drugs conjugated via the same linker system. Finally, we chose gemcitabine as a showcase payload to further highlight the potential of the phosphoramidate-based linker system described herein to unlock unusual and scarcely described payloads for targeted drug delivery with ADCs. Gemcitabine attracted our interest as potential ADC-payload since it is a clinically-established chemotherapeutic with a known safety profile but significant limitations in rapid clearance from the body, requiring high gram doses over time. 60 Antibody-mediated drug delivery could overcome those pharmacokinetic limitations, unlocking gemcitabines’ full therapeutic potential. ADCs with both regioisomers of 15 -Gemcitabine, primary 5’-hydroxy and secondary 3’-hydroxy were synthesized from trastuzumab (Fig. 6 a) and evaluated for target-mediated antiproliferative activity in vitro . Confirming the broad applicability of the linker systems described herein, ADCs attached via the primary or secondary alcohol of gemcitabine proved to be equally active on the targeted cell line (Fig. 6 b). As depicted in Fig. 8a, gemcitabine requires 5’-phosphorylation via deoxycytidine kinase followed by pyrophosphorylation to exhibit its cytotoxic activity. Co-incubation of ADC-treated cells with DI-87, an inhibitor for the initial phosphorylation, depleted activity for both ADCs to the level of unmodified trastuzumab only. Thus, we conclude that the ADCs tracelessly release gemcitabine and not any related phosphorylated species originating from the phosphoramidate linker system described herein. Finally, tras- 15 -5’-gemcitabine was evaluated in vivo in the N87 tumor model. Encouragingly, we observed a strong anti-tumor effect after a single dose of 20 mg/kg (Fig. 6 c). This effect proved to be selective, since the isotype ADC was completely inactive at the same dose. In parallel, the anti-tumor effect of trastuzumab (20 mg/kg) and unconjugated gemcitabine, dosed at 0.28 mg/kg corresponding to the dose administered in the DAR8 ADC format, was only slightly higher than for the vehicle control. This effect most likely reflects the tumor growth inhibition of trastuzumab alone, already observed in vitro (Fig. 6 b), while unconjugated gemcitabine can be considered inactive at this low dose due to rapid clearance from circulation, as previously reported. Considering that the dose of gemcitabine administered in the ADC format is almost 1000-times lower than that typically administered over multiple injections to achieve tumor regression, 61 the in vivo effect observed here is particularly remarkable. We attribute this enhanced activity to improved pharmacokinetics. In fact, clearance of the DAR8 tras- 15 -5’-gemcitabine is highly similar to unconjugated trastuzumab, as shown in Fig. 8c. Hence, the linker system described here widens the therapeutic window for gemcitabine by stably delivering the active payload to the tumor over a long period of time after single administration, whereas unconjugated gemcitabine is rapidly metabolized and excreted in vivo . 62 Conclusion In the current work, we identified phosphoramidates, substituted with a release handle at the nitrogen atom, a linker for antibody modification and the to-be-delivered drug at the oxygen substituents as broadly applicable functional motifs for the delivery of aromatic- as well as primary-, secondary- and tertiary-alcohol-containing payloads. The stability and release of the payload can be tuned via simple modifications of the phosphoramidate substituents, allowing adaptation to various alcohols of different pka or matching the linkage stability to the requirements of the antibodies’ target. Moreover, as long as the trigger releases a nucleophile that can form a five-membered ring with the central phosphorus atom, the release can be initiated via various intracellular stimuli including esterases, proteases and glucuronidase, as well as reductive conditions. The linker system outperformed the ones of the approved ADCs SG and TD in delivery of the respective hydroxy-containing TOP1-Inhibitors SN38 and DXd. Here, phosphoramidate based linker were superior in serum stability, in vivo efficacy and PK. More importantly, the simple drug-linker synthesis and conjugation of highly loaded homogenous DAR8 ADCs enabled us to repurpose and screen existing small molecule cytotoxins with various MOAs as novel ADC payloads. Highly efficacious ADCs with IC50s in the nanomolar to sub-nanomolar range were synthesized. Many of those cytotoxins and MOAs were without literature precedence in drug delivery. The broad dataset with ten cytotoxins conjugated to a variety of antibodies allowed us to support the general hypothesis in targeted drug delivery that sub-nanomolar potency of a small molecule cytotoxin is needed to generate effective ADCs. Payloads with lower potency on a certain cell line on the other hand can completely lack activity in vitro . Finally, for the first time, we demonstrate in vivo efficacy of anADC based on gemcitabine. The excellent PK profile of this DAR8 ADC, enabled by the phosphoramidate linker system, leads to in vivo activity at doses 1000 times lower than those typically administered for unconjugated gemcitabine. We are convinced that the broad compatibility of the linker system described herein with structurally diverse alcohols, combined with the efficient production of homogenous DAR8 ADCs from readily available pharmacologically active small molecules will allow for faster identification of suitable payloads to expand the currently limited panel of only three MOAs in approved ADCs. This will broaden the scope of ADC modalities with great potential in addressing drug resistance mechanisms in patients. Materials and methods General method for the conjugation of linker payloads 50 µl of the antibody solution of 10.0 mg/ml in P5-conjugation buffer (50 mM Tris, 1 mM EDTA, 100 mM NaCl, pH 8.3 at RT) were mixed with 3.33 µl of a 10 mM TCEP solution in P5-conjugation buffer. Directly afterwards, 1.67 µl of a 40 mM solution of the linker-payload constructs dissolved in DMSO were added. The mixture was shaken at 350 rpm and 25°C for 16 hours. The reaction mixtures were purified by preparative size-exclusion chromatography with a 25 ml Superdex™ 200 Increase 10/300GL (Cytiva, Sweden) and a flow of 0.8 ml/min eluting with sterile PBS (Merck, Germany). The antibody containing fractions were pooled and concentrated by spin-filtration (Amicon® Ultra- 2mL MWCO: 30 kDa, Merck, Germany). In vitro cytotoxicity on cancer cells To investigate the cytotoxicity of ADCs and unconjugated cytotoxins, 5.000 cells per well were incubated for 7 days or 4 days, respectively with increasing concentrations of the ADCs (0.18–12000 ng/ml = 0.0012-80 nM) or small molecules (0.015 nM – 1000 nM) to generate a dose–response curve. Before the analysis of cell viability, the spent medium containing dead cells was removed and fresh medium was added. Killing was analysed using resazurin cell viability dye at a final concentration of 55 µmol/l (Merck) by dividing the fluorescence from control cells in medium by the fluorescence of ADC-treated cells. Fluorescence emission at 590 nmol/l was measured on a microplate reader Infinite 200 PRO (Tecan Group Ltd.). In vitro bystander activity For the supernatant-based bystander experiment, 20,000 Trop2-positive MDA-MB-468 cells were seeded in 100 µl medium and treated with saci- 3 -SN38 at concentrations ranging from 0.18 to 12,000 ng/ml. After 5 days, 50 µl of the supernatant of the treated cells was transferred to 50 µl of Trop-negative SW-620 (5,000 cells/well) and incubated for 5 days. Resazurin readout was performed as described for the in vitro toxicity evaluation. In vivo efficacy All animal experiments were conducted in accordance with German animal welfare law and approved by local authorities. For the SN38 study, 1x10 7 MDA-MB-468 cells (50µl + 50µl Matrigel) were subcutaneously injected to CB17-Scid mice. Treatment was initiated when tumours reached a tumour volume of about 0.1 cm 3 9 days after implantation. 5 animals per group were treated twice at day 9 and day 13 with 10 mg/kg each treatment day of either saci- 3 -SN38, iso- 3 -SN38 or Trodelvy and compared to a vehicle treated group. For the DXd study, 2x10 6 N87 cells were subcutaneously injected to CB17-Scid mice. Treatment was initiated when tumours reached a tumour volume of about 0.1 cm 3 7 days after implantation. 5 animals per group were treated at day 7 with 1 mg/kg of either tras- 15 -DXd, iso- 15 -DXd or Enhertu and compared to a vehicle treated group. For the gemcitabine study, 2x10 6 N87 cells were subcutaneously injected to CB17-Scid mice. Treatment was initiated when tumours reached a tumour volume of about 0.1 cm 3 4 days after implantation. 5 animals per group were treated at day 4 with either 20 mg/kg of tras- 15 -gemcitabine, 20 mg/kg of iso- 15 -gemcitabine or 20mg/kg tras and 0.28 mg/kg gemcitabine and compared to a vehicle treated group. Mice were treated via intravenous injection after randomisation into treatment and control groups. Tumour volumes, body weights and general health conditions were recorded throughout the whole study. Declarations Conflict of interest: The authors declare competing financial interests: All authors, except from C.P.R.H are full-time employees of Tubulis. The presented work is part of a pending patent application by J.H., D.S., P.O., A.P.J., S.V, S.W. and M.A.K. Author Contributions: Philipp Ochtrop: Data curation, Formal Analysis, Investigation, Methodology, Supervision, Writing – review & editing. Anil P. Jagtap: Data curation, Investigation, Writing – review & editing. Jan G. Felber: Data curation, Formal Analysis, Visualization, Writing – review & editing. Simon Vogt: Investigation. Isabelle Mai: Data curation, Investigation. Philipp Cyprys: Data curation. Saskia Schmitt: Data curation, Formal Analysis, Formal Analysis. Sarah Payer: Data curation. Annabel Kitowski: Data curation. Swetlana Wunder: Investigation. Paul Machui: Data curation. Sarah Herterich: Data curation, Natascia Leonardi: Data curation, Elizaveta Poliak: Data curation, Christian P.R. Hackenberger: Writing – review & editing. Olivier Marcq: Supervision. Dominik Schumacher: Funding acquisition, Project administration, Supervision. Jonas Helma: Methodology, Funding acquisition, Project administration, Supervision. Annette M. Vogl: Methodology, Supervision. Marc-André Kasper: Conceptualization, Data curation, Investigation, Methodology, Supervision, Visualization, Writing – original draft. 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15:10:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5454963/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5454963/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-026-68605-y","type":"published","date":"2026-01-20T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82850877,"identity":"223b064b-25a4-4846-a396-bc3d47d3dd1f","added_by":"auto","created_at":"2025-05-16 03:19:41","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":218763,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural comparison between ProTide Prodrugs and development of the phosphoramidate linker moiety for traceless release of aromatic- and aliphatic alcohols in targeted drug delivery (a) \u003c/strong\u003eMechanism of ProTide release after passive diffusion into the cytosol.\u003ca href=\"#_ENREF_36\" title=\"Mehellou, 2018 #143\"\u003e\u003csup\u003e36\u003c/sup\u003e\u003c/a\u003e AlaOiPr release handle is highlighted. \u003cstrong\u003e(b)\u003c/strong\u003e Re-design of phosphoramidates as linkers for targeted drug delivery. Phosphoramidate-based structures and proposed mechanism for intracellular release of aromatic and aliphatic drugs via the linker systems described herein. AlaOiPr-(esterase trigger) and AlaAlaOH- (protease trigger) release handles are highlighted. Representation of the aromatic and aliphatic alcohols with MOAs that have been used as ADC payloads in the current work.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5454963/v1/1693bec34f07ad2d3bee31d0.jpeg"},{"id":82850753,"identity":"ea044ee0-4927-4cbb-954b-4151bdfc6f21","added_by":"auto","created_at":"2025-05-16 03:11:41","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":475408,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of ADCs with phosphorus-based sefl-immolative linkers and aromatic alcohols, exemplified with SN38.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Synthesis of phosphonamidate-based linker-payloads \u003cstrong\u003e1/2\u003c/strong\u003e-SN38: i) ethynyl or vinyl magnesium bromide, THF; ii) tetrazole, \u003cem\u003ethen\u003c/em\u003e SN38, tetrazole, MeCN/THF,\u003cem\u003e then\u003c/em\u003e I\u003csub\u003e2\u003c/sub\u003e, pyridine; (iii) Sacituzumab (saci) or Isotype (iso) mAb, TCEP, \u003cstrong\u003e1/2\u003c/strong\u003e-SN38, 15\u0026nbsp;h at pH\u0026nbsp;8.3. (\u003cstrong\u003eb\u003c/strong\u003e) Evaluation of SG (Trodelvy) and sacituzumab (saci/anti-Trop2)-\u003cstrong\u003e1/2\u003c/strong\u003e‑SN38 in comparison to isotype controls. Anti-proliferative dose response curves for target-positive saci-\u003cstrong\u003e1/2\u003c/strong\u003e‑SN38-ADCs (solid) and isotype ADCs (dashed) on MDA-MB-468 cells (Trop2+). (\u003cstrong\u003ec\u003c/strong\u003e) Synthesis of phosphoramidate-based linker-payloads \u003cstrong\u003e3\u003c/strong\u003e/\u003cstrong\u003e4\u003c/strong\u003e-SN38. iv) alanine \u003cem\u003etert\u003c/em\u003e-butyl ester or isopropyl lactate, \u003cem\u003ethen\u003c/em\u003e \u003cem\u003eN\u003c/em\u003e‑Boc 1,5-diaminopentane or \u003cem\u003eN\u003c/em\u003e‑Boc-aminopentanol, Et\u003csub\u003e3\u003c/sub\u003eN, THF; v) SN38, DBU, DMSO; vi) TFA, CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e; vii) P5(PEG24)-COOH, PyBOP, DIPEA, DMSO. viii) Sacituzumab (saci/anti-Trop2), brentuximab (bren/anti-CD30), TCEP, \u003cstrong\u003e3\u003c/strong\u003e/\u003cstrong\u003e4\u003c/strong\u003e-SN38, pH 8.3, 15\u0026nbsp;h. (\u003cstrong\u003ed\u003c/strong\u003e) Cytotoxicity of saci-\u003cstrong\u003e3/4\u003c/strong\u003e-SN38 and the non-cleavable saci-\u003cstrong\u003e5\u003c/strong\u003e-SN38 against their respective isotype ADCs on MDA-MB-468- (Trop2+). Graphs show mean, \u003cem\u003en\u003c/em\u003e = 2. (\u003cstrong\u003ee\u003c/strong\u003e) Bystander killing: SW-620 cells (Trop2-) treated with increasing concentrations of Saci-\u003cstrong\u003e3\u003c/strong\u003e-SN38 or with the supernatant of MDA-MB-468 cells (Trop2+) that have been pre-treated with increasing concentrations of saci-\u003cstrong\u003e3\u003c/strong\u003e-SN38. Dose-response curves show the direct antiproliferative effect or the indirect effect (bystander) of released payload in the supernatant on SW-620. Graphs show mean ± SEM (or SD), \u003cem\u003en\u003c/em\u003e = 2. (\u003cstrong\u003ef\u003c/strong\u003e) Rat serum stability: DAR measured by mass spectrometry of SG or saci-\u003cstrong\u003e3\u003c/strong\u003e-SN38 after incubation for up to 7 days in rat serum at 37°C. (\u003cstrong\u003eg\u003c/strong\u003e) \u003cem\u003eIn vivo\u003c/em\u003e efficacy evaluation of saci-\u003cstrong\u003e3\u003c/strong\u003e-SN38, iso-\u003cstrong\u003e3\u003c/strong\u003e-SN38, SG and vehicle (black). Female CB17 SCID mice have been implanted with cell-culture-derived xenograft model based on MDA-MB-468 cells in matrigel (corresponding \u003cem\u003ein vitro\u003c/em\u003e effect for this cell line is shown in Fig. 2b and Fig 2d). Treatment started once tumor volumes reached 0.1-0.15 cm\u003csup\u003e3\u003c/sup\u003e. Mice (n=5 per group) were treated at day 0 and day 4 with 10 mg/kg of the ADCs. Shown is Mean - SEM. n = 5 animals.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5454963/v1/8a1b1ce9b57b05d03bb2346c.jpeg"},{"id":82850754,"identity":"03760c48-9eb0-4792-9a30-ea41f8081d6b","added_by":"auto","created_at":"2025-05-16 03:11:41","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":292656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVariation of the phosphoramidate-based release handles.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) DAR8 ADCs from brentuximab (bren/anti-CD30) and datopotamab (dato/anti-Trop2) using SN38 and various phosphoramidate-based self-immolative release handles, amenable for esterase-, protease-, reductase-, or glucoronidase-initiated release by 5‑membered cyclization. (\u003cstrong\u003eb\u003c/strong\u003e) Chemical structures for various phosphoramidate-based side chains applied in linker \u003cstrong\u003e3\u003c/strong\u003e and \u003cstrong\u003e6\u003c/strong\u003e-\u003cstrong\u003e13\u003c/strong\u003e. (\u003cstrong\u003ec\u003c/strong\u003e) Potency and selectivity of the different ADC constructs for the targeted cell line. Bren-ADCs were tested on L-540 and SR-786 cells (CD30+), dato-ADCs were tested on HCC-78 and MDA-MB-468 cells (Trop2+), while they served as isotypes \u003cem\u003evice versa\u003c/em\u003e. The ADC selectivity is plotted as the ratio of IC\u003csub\u003e50\u003c/sub\u003e isotype/IC\u003csub\u003e50\u003c/sub\u003e targeted ADC. The absolute potencies of the bren-/dato-ADCs are plotted for their target-positive cell lines. The black bars show the mean values each for the four cell lines (targeted via two distinct receptor-antibody-pairs) and serve as orientation to compare the different linker structures. Doses-response curves are shown in Supp. Fig.2. (\u003cstrong\u003ed\u003c/strong\u003e) Hydrophilicity of the DAR8 ADCs for the dato‑conjugates, derived from the retention time in hydrophobic interaction chromatography (HIC).\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5454963/v1/18ca1bf3b74280a205ab149a.jpeg"},{"id":82850763,"identity":"31b20e6b-a828-4e35-8085-165ce568852c","added_by":"auto","created_at":"2025-05-16 03:11:41","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":383488,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of ADCs with phosphorus based self-immolative linkers and aliphatic alcohols, exemplified with DXd. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) DAR8 ADCs from trastuzumab (tras/anti-HER2) using DXd and protease-initiated release by 5‑membered cyclization. (\u003cstrong\u003eb\u003c/strong\u003e) Linker-payload structures \u003cstrong\u003e1\u003c/strong\u003e-DXd and \u003cstrong\u003e3\u003c/strong\u003e-DXd after cysteine conjugation. (\u003cstrong\u003ec\u003c/strong\u003e) Linker-payload structures \u003cstrong\u003e14-17\u003c/strong\u003e-DXd after cysteine conjugation. (\u003cstrong\u003ed\u003c/strong\u003e) Evaluation of trastuzumab \u003cstrong\u003e1- \u003c/strong\u003eand\u003cstrong\u003e 3\u003c/strong\u003e‑DXd in comparison to isotype controls (left). Comparison of esterase cleavable Trastuzumab \u003cstrong\u003e3\u003c/strong\u003e‑DXd to protease cleavable \u003cstrong\u003e14\u003c/strong\u003e-DXd and \u003cstrong\u003e15\u003c/strong\u003e-DXd in comparison to isotype controls (middle). Comparison of trastuzumab-\u003cstrong\u003e15\u003c/strong\u003e-DXd, \u003cstrong\u003e16\u003c/strong\u003e-DXd and \u003cstrong\u003e17\u003c/strong\u003e-DXd that can form a 5-, 6-, or 7-membered ring after protease cleavage in comparison to isotype controls (right). Anti-proliferative dose response curves for targeting ADCs (solid) and isotype-ADCs (dashed) on SKBR-3 cells (HER2+). Mean, n = 2. (\u003cstrong\u003ee\u003c/strong\u003e) Rat serum stability: DAR measured by mass spectrometry of tras-\u003cstrong\u003e15\u003c/strong\u003e-DXd or TD after incubation for up to 5 days in rat serum at 37°C. (\u003cstrong\u003ef\u003c/strong\u003e) \u003cem\u003eIn vivo\u003c/em\u003e efficacy evaluation of tras-\u003cstrong\u003e15\u003c/strong\u003e-DXd, iso-\u003cstrong\u003e15\u003c/strong\u003e-DXd, TD or vehicle (black). Female CB17 SCID mice have been implanted with cell-culture-derived xenograft model based on N87 cells in Matrigel. Treatment started once when tumor volumes reached 0.1-0.15 cm\u003csup\u003e3\u003c/sup\u003e. Mice were treated once at day 0 with 1 mg/kg of the ADCs. Each group consisted of n=5 females each. Shown is mean ± SEM. CR = complete responses of total mice treated. Pharmacokinetic analysis of CB17 SCID mice treated with 1 mg/kg of either tras-\u003cstrong\u003e15\u003c/strong\u003e-DXd or TD. Samples were drawn after several timepoints throughout the 3-week study period. The serum samples were analyzed by ELISA for total mAb with anti-HER2 antibodies (straight line) and intact ADC with anti-payload antibodies (dotted line).\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5454963/v1/e2cdcd8d62687eda525c30a3.jpeg"},{"id":82850769,"identity":"8e758e40-a1b7-49b6-8239-5e6a22078ace","added_by":"auto","created_at":"2025-05-16 03:11:41","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":425877,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombination of the phosphoramidate-based linker systems with structurally diverse hydroxy-containing cytotoxins exhibiting diverse MOAs. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) DAR8 ADCs from trastuzumab (tras/anti-HER2), datopotamab (Dato/anti-Trop2) or brentuximab (bren/anti-CD30) in combination with linker\u003cstrong\u003e 9\u003c/strong\u003e for aromatic and linker \u003cstrong\u003e15\u003c/strong\u003e for aliphatic payloads. (\u003cstrong\u003eb\u003c/strong\u003e) Ganetespib and ON01300 have been conjugated via linker \u003cstrong\u003e9\u003c/strong\u003e; BAY-2402234, DHODH-IN-16, SNX2112, paclitaxel and LSN3154567 have been conjugated via linker \u003cstrong\u003e15\u003c/strong\u003e. (\u003cstrong\u003ec\u003c/strong\u003e) HIC chromatograms to analyze ADC homogeneity from every payload. A DAR of 8 has been confirmed by MS. (\u003cstrong\u003ed\u003c/strong\u003e) Potency and selectivity of the different ADCs for the targeted cell line. Tras-ADCs were tested on N87, SKBR-3 and HCC1569 (HER2+), bren-ADCs were tested on L-540 and Karpas-299 cells (CD30+), dato-ADCs were tested on H441, MDA-MB-468 and HCC-78-cells (Trop2+), while the bren conjugates served as isotypes for the HER2+ and Trop2+-cell lines and \u003cem\u003evice versa.\u003c/em\u003e The ADC selectivity is plotted as the ratio of IC\u003csub\u003e50\u003c/sub\u003e isotype-/IC\u003csub\u003e50\u003c/sub\u003e targeted ADC. The absolute potencies of the ADCs are plotted for their target-positive cell lines. The black bars show the mean values for the eight cell lines (targeted via three distinct receptor-antibody-pairs) and serve as orientation to compare the different payloads. (\u003cstrong\u003ee\u003c/strong\u003e) Correlation between the proliferation inhibition IC\u003csub\u003e50\u003c/sub\u003e of unconjugated payloads (x-axis) over the ADCs on the same cell line (y-axis). ADCs above the dotted line were inactive at the highest tested concentration of 80 nM and excluded from the correlation. Dose-response curves for ADCs and payloads are shown in Supp. Fig.2.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5454963/v1/be8764511f2ee9a3613edd51.jpeg"},{"id":82851786,"identity":"2e561fe8-e5b2-4169-9156-9b454bca7f65","added_by":"auto","created_at":"2025-05-16 03:27:42","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":243295,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of gemcitabine ADCs attached via linker 15 with its’ primary 5’ or its secondary 3’ hydroxyl groups. (a) \u003c/strong\u003eStructure of the two different ADCs and schematic representation of the intracellular MOA. \u003cstrong\u003e(b) \u003c/strong\u003e\u003cem\u003eIn vitro\u003c/em\u003e evaluation of trastuzumab (tras/anti-HER2) ADCs attached via 5’ and 3’ on N87-cells and inhibition of antiproliferative activity via DI-87. Mean, n = 2. \u003cstrong\u003e(c)\u003c/strong\u003e\u003cem\u003e In vivo\u003c/em\u003e PK and efficacy evaluation of tras-\u003cstrong\u003e15\u003c/strong\u003e-gemcitabine. \u003cem\u003eIn vivo\u003c/em\u003e efficacy was evaluated in female CB17 SCID mice have been implanted with cell-culture-derived xenograft model based on N87 cells in Matrigel. Treatment started once tumor volumes reached 0.1-0.15 cm\u003csup\u003e3\u003c/sup\u003e with 20 mg/kg on day 0 of either tras-\u003cstrong\u003e15\u003c/strong\u003e-5’-gemcitabine, iso-\u003cstrong\u003e15\u003c/strong\u003e-gemcitabine or 20 mg/kg tras + 0.28 mg/kg unconjugated gemcitabine or vehicle. Each group consisted of n=5 females each. Shown is mean ± SEM.PK has been evaluated in CB17 SCID mice treated with 20 mg/kg at T=0 of trastuzumab only (grey) or tras-\u003cstrong\u003e15\u003c/strong\u003e-5’-gemcitabine. Samples were drawn after several timepoints throughout the 3-week study period and analyzed by ELISA for total mAb.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5454963/v1/29013705561182f4da62c803.jpeg"},{"id":100765517,"identity":"c11440f3-2195-4f81-af42-b9052c3cc81f","added_by":"auto","created_at":"2026-01-21 08:45:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3449693,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5454963/v1/d67119ed-7309-4dea-8d79-9c7ffc58f3af.pdf"},{"id":82850878,"identity":"7de66410-0f6b-4a38-847a-a0b262108d04","added_by":"auto","created_at":"2025-05-16 03:19:41","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12103622,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"20241114SIselfimmolativephosphoramidates.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5454963/v1/5dcce93c346f4b1808d5170a.pdf"},{"id":82850757,"identity":"7db273f5-6864-473e-806d-751cf6bf8ee6","added_by":"auto","created_at":"2025-05-16 03:11:41","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1667245,"visible":true,"origin":"","legend":"Article File - Reporting summary","description":"","filename":"nrreportingsummaryMAK1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5454963/v1/d7dc0478193e764aa6f22a0c.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nAll authors, except from C.P.R.H are full-time employees of Tubulis. The presented work is part of a pending patent application by J.H., D.S., P.O., A.P.J., S.V, S.W. and M.A.K.","formattedTitle":"Expanding the payload scope in antibody-drug conjugates: Unprecedented delivery of hydroxy-containing drugs through self-immolative phosphoramidates","fulltext":[{"header":"Main","content":"\u003cp\u003eTargeted drug delivery for the treatment of malignant diseases holds great promise in reducing undesired side effects while increasing efficacy by precisely hitting cancer cells.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Most prominently, antibodies conjugated to highly potent cytotoxic drugs, a modality known as antibody-drug conjugates (ADCs), have been utilized as targeting vehicles.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e The pace of more new ADCs entering clinical trials and receiving FDA approval has accelerated over the past decade, underlining the benefits of targeted drug delivery for patients.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Still, challenges remain to be solved, such as chemotherapy-like toxicities, lack of efficacy when applied as monotherapy and, eventually, acquired resistance.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe potency and mode of action (MOA) of the cytotoxic payload that is being delivered is considered as one of the major drivers of these shortcomings.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e With a small repertoire of only three MOAs in approved ADCs, namely tubulin-inhibition, topoisomerase-I (TOP1)-inhibition and DNAdamaging, it is anticipated that MOA diversification will be a key factor in the development of next generation targeted cancer therapeutics.\u003csup\u003e7\u003c/sup\u003e Another Achilles\u0026rsquo; heel that has been identified in the current generation of ADCs is the linker between antibody and drug. Ongoing research efforts aim to improve linker associated limitations such as premature drug loss,\u003csup\u003e8\u003c/sup\u003e unspecific uptake\u003csup\u003e9, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e0\u003c/sup\u003e and undesired aggregation.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e1\u003c/sup\u003e On top of that, linker design determines the cleavability and drug release at the targeted location. In particular, efficient, traceless drug release after receptor-mediated uptake can be crucial for the intracellular function of the payload\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e2\u003c/sup\u003e and for its permeability, which allows effective eradication of tumors with heterogenous expression of the antibody\u0026rsquo;s target via the bystander effect.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e3, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e4\u003c/sup\u003e Hence, cleavable linker systems are required that provide a stable attachment of a payload via one of its functional groups during circulation and, importantly, simultaneously allow for traceless release of this functional group upon uptake into the targeted cell. Cleavable linker systems usually consist of a release unit that is activated by an intracellular trigger event, and a self-immolative moiety that subsequently liberates the unmodified functional group.\u003csup\u003e1, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e5\u003c/sup\u003e Commonly exploited trigger events include a variety of intracellular or intra-lysosomal conditions such as acidic and reductive environment or specific enzymatic activities from esterases,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e6\u003c/sup\u003e proteases,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e7\u003c/sup\u003e glycosidases,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e8\u003c/sup\u003e phosphatases,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e9\u003c/sup\u003e reductases,\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e0\u003c/sup\u003e or sulfatases\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSuch cleavable linkers have been designed for the attachment of different functional groups on payloads including primary-,\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e secondary-,\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e tertiary- and heteroaryl-amines,\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e thiols, amides,\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e ortho-quinones\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and alcohols.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e In particular, hydroxyl linkages are attractive since this functionality is frequently present in synthetic small molecule drugs and natural product derivatives.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e However, based on their chemical environment, hydroxyl groups can differ tremendously in steric accessibility and pKa, ranging from 7 (phenols) to 16 (aliphatic alcohols). This broad scope poses a challenge for the design of broadly applicable linker systems that allow for both stable attachment and traceless release of chemically distinct alcohols. Available linker systems for alcohol attachment rely on carbamates,\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e carbonates,\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e esters,\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e phosphates, pyrophosphates,\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e 1,6-benzylamines,\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e hemiaminals,\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e disulfides,\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e,\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e or methylene carbamates.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e However, none of these functional groups combine broad applicability, stability in serum and efficient traceless release.\u003c/p\u003e \u003cp\u003eHere, we report a new self-immolative moiety providing stable attachment and traceless release of alcohols for targeted drug delivery. In our design, we make use of P(V)-based motifs of the clinically validated ProTide technology,\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e a prodrug system that has been developed to mask hydrophilicity of nucleoside-like drug molecules to enhance cell permeability.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e After passive membrane diffusion, these prodrugs are activated via esterase cleavage of an amino acid ester followed by self-immolation via a 5-membered cyclization, release of a sacrificial phenol and finally, enzymatical release of the (phosphorylated) nucleoside analog (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In our work we have redesigned the central phosphorous core by introducing a conjugation handle to one of the three \u003cem\u003eP\u003c/em\u003e-substituents that can form a covalent bond with a targeting moiety like an antibody. This allows to transform a prodrug- into a targeted drug delivery technology. Exploiting the fact that Protides liberate an aromatic- and an aliphatic alcohol intracellularly, we transferred and developed this concept even further to release not only nucleoside analogs but instead designed a linker-technology to deliver a broad spectrum of aliphatic- and aromatic alcohols (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eIn contrast to the earlier described nucleoside prodrugs which are restricted to esterase cleavage after passive uptake into the cytosol, the release from our phosphoramidate-based targeted drug delivery system can be initiated by various intracellular and endosomal triggers including esterases, proteases, glucoronidases and reductive conditions due to the uptake mechanism via receptor-mediated endocytosis.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of phosphorus-based self-immolative handles for the targeted delivery of aromatic alcohols exemplified with SN38\u003c/h2\u003e \u003cp\u003eTo start our endeavors, we chose the TOP1-inhibitor SN38, the active pharmaceutical ingredient (API) of the clinically approved ADC sacituzumab govitecan (SG, Trodelvy), as our first model drug.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e Guided by previous experience with unsaturated ethynyl-\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e and vinyl-phosphonamidate\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e-based linker technologies, we designed linker structures 1 and 2 for cysteine-selective antibody conjugation and attached SN38 via its aromatic 10-hydroxyl group as \u003cem\u003eO\u003c/em\u003e-substituent of the phosphonamidates. In this design, the alaninyl \u003cem\u003etert\u003c/em\u003ebutyl ester at the phosphorus \u003cem\u003eN\u003c/em\u003e-substituent is supposed to act as release trigger. Upon intracellular \u003cem\u003etert\u003c/em\u003ebutyl ester hydrolysis, cyclization via a 5-membered intermediate could lead to a traceless release of unmodified SN38 via a similar mechanism as described for the phenol in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. Synthesis of \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e was performed as outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea (See SI for detailed information). Conjugation of \u003cb\u003e1\u003c/b\u003e-SN38 to sacituzumab and an isotype antibody delivered homogenous ADCs with a drug-to-antibody ratio (DAR) of 8, while a conjugation with \u003cb\u003e2\u003c/b\u003e-SN38 was not suitable to produce ADCs with DAR higher than 0.5. These observations reflect the previously reported reactivity differences between ethynyl and vinyl-phosphonamidates with cysteines and consequently disqualified \u003cb\u003e2\u003c/b\u003e-SN38 as an efficient drug-linker for SN38.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eNext, we wanted to explore the properties of the linker systems \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e and compare them with the established linker system of SG (CL2A-SN38). In the linker (CL2A-SN38) of SG, SN38 is attached at its tertiary 20hydroxyl group via a carbonate moiety (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). This linker has been designed to rapidly hydrolyze under physiological conditions. As previously reported\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e1\u003c/sup\u003e and confirmed by our \u003cem\u003ein vitro\u003c/em\u003e cell culture assays, this lability leads to extracellular release of SN38, causing cell killing also of non-targeted cells with isotype ADCs that are not actively internalized. Consequently, the IC\u003csub\u003e50\u003c/sub\u003e of targeted vs. isotype ADC in cell viability experiments with multiple days of incubation are highly similar (0.2 nM versus 0.3 nM, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Previously reported efforts to generate a serum stable linker, in which SN38 is attached via a carbamate to the aromatic 10-hydroxyl group, failed to show efficacy due to insufficient intracellular release of SN38 as indicated by higher IC\u003csub\u003e50\u003c/sub\u003e than for SN38 as free drug.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e0\u003c/sup\u003e These two scenarios observed for SN38-containing linker systems inspired us to employ \u003cem\u003ein vitro\u003c/em\u003e proliferation assays with isotype versus targeted ADCs as a reliable, close-to-application readout to optimize our linker system for stability and release efficiency. For this reason, we analyzed the ratio of IC\u003csub\u003e50\u003c/sub\u003e from isotype- over targeted ADC as measure for linkage stability, and absolute IC\u003csub\u003e50\u003c/sub\u003e of the targeted ADC as measure for effective release. We subjected sacituzumab-\u003cb\u003e1\u003c/b\u003e-SN38 to \u003cem\u003ein vitro\u003c/em\u003e cytotoxicity assays with the Trop2-positive MDA-MB-468 cancer cell line and observed almost identical IC\u003csub\u003e50\u003c/sub\u003e values for targeted and isotype ADCs of 0.4 and 0.6 nM, respectively, indicating insufficient stability under cell culture conditions, similar as observed for SG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). This instability stands in strong contrast to previously reported \u003cem\u003eO\u003c/em\u003e-alkyl ethynylphosphonamidate-based ADCs that were shown to be highly stable under physiological conditions, \u003cem\u003ein vitro\u003c/em\u003e and even \u003cem\u003ein vivo.\u003c/em\u003e We attribute the lability of \u003cb\u003e1\u003c/b\u003e-SN38 to the strong leaving group character of the aromatic SN38, which leads to fast hydrolysis of the phosphonamidate under assay conditions.\u003c/p\u003e \u003cp\u003eTo stabilize the linker system against hydrolysis, we envisioned exchanging the carbon at the phosphorous core to oxygen and introducing a short amino pentyl linker as second \u003cem\u003eO\u003c/em\u003e-substituent while keeping the \u003cem\u003eN\u003c/em\u003e-alanine \u003cem\u003etert\u003c/em\u003e-butyl ester as release handle. By attaching a suitable antibody conjugation handle such as ethynylphosphonamidates (P5-conjugation)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, this resulted in phosphoramidate-based linker-payloads \u003cb\u003e3\u003c/b\u003e and its variations \u003cb\u003e4\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e. In linker \u003cb\u003e4\u003c/b\u003e \u003cem\u003eO\u003c/em\u003e- and \u003cem\u003eN\u003c/em\u003e- substituted linker and release handle are swapped, while in \u003cb\u003e5\u003c/b\u003e the release handle is exchanged to a non-cleavable 4-methylbenzyl group. Linkers \u003cb\u003e3\u003c/b\u003e\u0026ndash;\u003cb\u003e5\u003c/b\u003e were synthesized as outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec (See SI for detailed information) and proved to be superior to \u003cb\u003e1\u003c/b\u003eSN38, since \u003cb\u003e3-4\u003c/b\u003e-SN38, conjugated to sacituzumab, exhibited excellent anti-proliferative activity with a favorable potency window between targeted and isotype ADC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). An IC\u003csub\u003e50\u003c/sub\u003e of 0.4 nM and a 13-fold difference for the Trop2-targeted ADC saci-\u003cb\u003e3\u003c/b\u003e-SN38 vs. the isotype ADC indicates a more stable SN38 conjugation to the mAb compared to SG or \u003cb\u003e1\u003c/b\u003e-SN38, while allowing an efficient release of the drug after receptor-mediated cellular uptake. A similar behavior was observed for saci-\u003cb\u003e4\u003c/b\u003eSN38. However, since the selectivity window was slightly lower (5-fold) we decided to continue our studies using linker \u003cb\u003e3\u003c/b\u003e. Interestingly, targeted ADCs based on the non-cleavable control \u003cb\u003e5\u003c/b\u003eSN38, lacking an intracellular release trigger, only showed an anti-proliferative effect at the highest concentration \u003cem\u003ein vitro\u003c/em\u003e. These results indicate that phosphoramidate based linker systems \u003cb\u003e3\u003c/b\u003e and \u003cb\u003e4\u003c/b\u003e provide a stable connection of SN38 via its aromatic alcohol which can be cleaved intracellularly, involving enzyme-mediated hydrolysis of the ester in the release handle. Next, we set out to investigate the bystander effect of saci-\u003cb\u003e3\u003c/b\u003e-SN38.\u003csup\u003e14\u003c/sup\u003e As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, saci-\u003cb\u003e3\u003c/b\u003e-SN38 exhibits strong bystander killing of Trop2- cells after supernatant transfer from Trop2\u0026thinsp;+\u0026thinsp;cells preincubated with ADC, but only very low direct cell killing of the target-negative cells. Since traceless payload release from the linker has been described as a key requirement for bystander activity, this data further indicates efficient, traceless SN38 release after receptor-mediated uptake from \u003cb\u003e3\u003c/b\u003e-SN38,.\u003csup\u003e43\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then set out to compare \u003cb\u003e3\u003c/b\u003e-SN38 head-to-head with SG to evaluate whether phosphoramidates could improve the established Cl2A linker in SG. We confirmed target selectivity for saci-\u003cb\u003e3\u003c/b\u003e-SN38 \u003cem\u003ein vitro\u003c/em\u003e on a larger panel of cell lines, in contrast to CL2A-SN38 that proved to be unselective, (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and observed improved serum stability of saci-\u003cb\u003e3\u003c/b\u003e-SN38 with more than 50% intact ADC after 7 days of incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). In contrast, SG lost almost 100% of conjugated drug already after 24h, mainly caused by carbonate hydrolysis. Since tumor-target specificity of SG has been demonstrated \u003cem\u003ein vivo\u003c/em\u003e, contrary to the \u003cem\u003ein vitro\u003c/em\u003e observations,\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e we compared both linker-payloads head-to-head in an \u003cem\u003ein vivo\u003c/em\u003e efficacy experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Here, Trop2+-tumor bearing mice, were treated twice (day 0 and day 4) with 10 mg/kg of saci-\u003cb\u003e3\u003c/b\u003e-SN38, iso-\u003cb\u003e3\u003c/b\u003e-SN38, SG or vehicle. We observed a superior efficacy with complete responses for saci-\u003cb\u003e3\u003c/b\u003e-SN38 in all mice while SG only led to delayed tumor growth and iso-\u003cb\u003e3\u003c/b\u003e-SN38 showed no clear responses. We attribute this enhanced anti-cancer activity to improved linker stability, as previously reported in other systems,\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e ensuring a higher tumor exposure to SN38.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLinker stability tuning and combination with different release triggers\u003c/h3\u003e\n\u003cp\u003eEncouraged by reports on structural optimization of ProTides,\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e we set out to tune potency and stability of our phosphoramidate-based linker system by synthesizing derivatives of linker \u003cb\u003e3\u003c/b\u003e attached to SN38. For evaluation, we conjugated the resulting linker-payloads to two different antibodies, targeting the cell-surface cancer antigens Trop2 and CD30, which were characterized for potency (IC\u003csub\u003e50\u003c/sub\u003e targeted ADC) and selectivity (ratio IC\u003csub\u003e50\u003c/sub\u003e isotype/targeted ADC) on two target-positive cell lines for each ADC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). To increase electron density at P, we synthesized linker \u003cb\u003e6\u003c/b\u003e, in which the primary pentyl \u003cem\u003eO\u003c/em\u003e-substituent of linker \u003cb\u003e3\u003c/b\u003e is exchanged to a secondary cyclohexyl \u003cem\u003eO\u003c/em\u003e-substituent, and linker \u003cb\u003e7\u003c/b\u003e, in which we introduced an additional methyl group at the amino acid alpha carbon. We also aimed to reduce electron density at the phosphorus by exchanging the alanine in linker \u003cb\u003e3\u003c/b\u003e to a glycine in linker \u003cb\u003e8\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). We observed that an increase in electron density in linkers \u003cb\u003e6\u003c/b\u003e and \u003cb\u003e7\u003c/b\u003e lead to a larger difference between targeted and isotype ADCs, whereas linker \u003cb\u003e8\u003c/b\u003e, possessing less electron density at the phosphorous, showed a decreased window (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). At the same time, we observed slightly reduced median potencies for the ADCs based on \u003cb\u003e6\u003c/b\u003e-SN38 and \u003cb\u003e7\u003c/b\u003e-SN38 compared to \u003cb\u003e3\u003c/b\u003e-SN38 and \u003cb\u003e8\u003c/b\u003e-SN38, which might be attributed to steric hindrance leading to either reduced esterase activity or reduced cyclization rates between the liberated carboxylic acid and the central phosphorus atom. To further study this, we exchanged the esterase cleavable \u003cem\u003etert\u003c/em\u003e-butyl ester in the derivatives \u003cb\u003e3\u003c/b\u003e and \u003cb\u003e6\u003c/b\u003e to isopropyl esters in linkers \u003cb\u003e9\u003c/b\u003e and \u003cb\u003e10\u003c/b\u003e, following previous reports on ProTides that \u003cem\u003etert\u003c/em\u003e-butyl esters are less efficiently cleaved by esterases.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e Interestingly, this change did not have an effect on the median potency on the targeted cell line but the selectivity slightly increased from \u003cem\u003etert\u003c/em\u003e-butyl- to isopropyl esters in both cases.\u003c/p\u003e \u003cp\u003eThe cleavable phosphoramidate-based linkers described to this point are all designed to be activated analogously by esterase-mediated cleavage of the respective amino-acid ester. Next, we wanted to explore if the cleavage can also be initiated via alternative release triggers that are commonly applied in antibody-mediated drug-delivery.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e In contrast to prodrugs, that passively diffuse into the cytosol, ADCs are actively taken up by targeted cells via the endosomal and lysosomal pathway, which exposes them to high levels of various proteases that are often overexpressed in human cancers.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e Hence, we exchanged the amino-acid ester release handle with an alanine-alanine dipeptide in linker \u003cb\u003e11-\u003c/b\u003eSN38, with the expectation that \u003cb\u003e11-\u003c/b\u003eSN38 forms the same carboxylic acid intermediate after protease mediated cleavage that \u003cb\u003e6-\u003c/b\u003e and \u003cb\u003e10\u003c/b\u003e-SN38 form upon esterase cleavage. \u003cb\u003e11\u003c/b\u003e-SN38 exhibited similar potencies on the targeted cell lines, but reduced selectivity (targeted vs. isotype-ADC) compared to the esters \u003cb\u003e3\u003c/b\u003e,\u003cb\u003e8\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOther known release triggers exploit the reductive environment or beta-glucuronidase activity in endo-/lysosomes.\u003csup\u003e49,50\u003c/sup\u003e Hence, we also synthesized reductively cleavable disulfide \u003cb\u003e12\u003c/b\u003e and GlcA protected alcohol \u003cb\u003e13\u003c/b\u003e and explored if liberated sulfhydryl or hydroxyl groups would perform a 5-membered cyclization at the phosphorus and facilitate release of the attached drug. The selectivity of ADCs \u003cb\u003e12\u003c/b\u003e-SN38 and \u003cb\u003e13\u003c/b\u003e-SN38 for the targeted cell lines was comparable to the glycine derivative \u003cb\u003e8\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Even though slightly reduced, both targeted constructs showed decent activities, supporting a release mechanism that involves the liberation of a nucleophile (COOH, OH and SH) in delta position of the central P-atom which can release the attached drug by five-membered cyclization.\u003c/p\u003e \u003cp\u003eAnother property of ADCs that is discussed to have a crucial impact on their performance is their hydrophobicity. To determine the hydrophobicity of the synthesized ADCs and how the different linkers \u003cb\u003e3\u003c/b\u003e and \u003cb\u003e6\u0026ndash;13\u003c/b\u003e influence these, we characterized the conjugates by hydrophobic interaction chromatography (HIC). The HIC-retention time can be used as a measure for overall ADC hydrophobicity and previous reports conclude that earlier eluting, more hydrophilic ADCs have beneficial PK properties.\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e The free carboxylic acid in dialanine linker \u003cb\u003e12\u003c/b\u003e comprised the most hydrophilic DAR8 SN38-ADC tested herein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). From the ester series linker \u003cb\u003e6\u003c/b\u003e and \u003cb\u003e7\u003c/b\u003e delivered more hydrophobic ADCs, whereas the glycine derivative in linker \u003cb\u003e8\u003c/b\u003e yielded a slightly more hydrophilic ADC compared to linker \u003cb\u003e3\u003c/b\u003e. At the same time, an increased hydrophilicity of the ADCs was observed with the isopropyl substituent in \u003cb\u003e9\u003c/b\u003e and \u003cb\u003e10\u003c/b\u003e compared to all the \u003cem\u003etert\u003c/em\u003e-butyl derivatives.\u003c/p\u003e \u003cp\u003eTaken together, the results summerized in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrate that stability, release efficiency and hydrophilicity of phosphoramidate-based drug-linker can be tuned by altering the substitution pattern around the phosphorous atom, covering a range from SG-like instability to a more targeted delivery of SN38. This unprecedented tuning could be useful in the future to identify the optimal linker for a certain target that requires either stable delivery or a certain level of instability to match different levels of healthy tissue expression.\u003c/p\u003e\n\u003ch3\u003eEstablishment of phosphorus-based self-immolative handles for the targeted delivery of aliphatic alcohols exemplified with DXd\u003c/h3\u003e\n\u003cp\u003eNext, we chose the TOP1-Inhibitor DXd as model payload, carrying a primary alcohol. DXd is the payload of the marketed ADC trastuzumab deruxtecan (TD/Enhertu).\u003csup\u003e48\u003c/sup\u003e We synthetically introduced DXd into the phosphorus(V)-based linker systems \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e3\u003c/b\u003e as already described for SN38 and conjugated the resulting linker-payloads to trastuzumab and an isotype antibody to test for targeted delivery via the HER2 antigen (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Compared to what we observed for SN38, the phosphonamidatebased linker \u003cb\u003e1\u003c/b\u003e delivered DXd into the targeted cell line with a 3-fold better selectivity compared to the isotype ADC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Like for SN38, a phosphoramidate release handle in \u003cb\u003e3\u003c/b\u003e-DXd was superior to phosphonamidates in \u003cb\u003e1\u003c/b\u003e-DXd (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), displaying a higher potency (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.2 nM versus 0.8 nM) and a larger selectivity (19x versus 3.4x) for the targeted cell line. Next, we exchanged the esterase cleavable handle to a protease responsive dialaninyl-cleavage site in linker \u003cb\u003e14\u003c/b\u003e-DXd (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). In stark contrast to the protease cleavable \u003cb\u003e11\u003c/b\u003e-SN38, we observed an improved linker stability that resulted in a more selective (50\u0026times;) delivery of the payload. We assign this enhanced stability to decreased leaving group properties (=\u0026thinsp;higher pKa) of the aliphatic hydroxyl group in DXd. The selectivity of \u003cb\u003e14\u003c/b\u003e-DXd was even more increased using linker \u003cb\u003e15\u003c/b\u003e (55\u0026times;) that possesses a terminal carboxy group instead of the tert-butyl ester as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec. The terminal carboxy group is also desired, since it adds additional hydrophilicity to the linker system, as shown for SN38 in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed.\u003c/p\u003e \u003cp\u003eTo better understand whether Dxd release involves an attack of the alanine carboxy group on the central phosphorous via a 5-membered intermediate, we synthesized linker \u003cb\u003e16\u003c/b\u003e- and \u003cb\u003e17\u003c/b\u003e-DXd, requiring formation of less favored 6- or 7-membered rings for a similar drug release after protease cleavage. ADCs of both linkers were almost inactive on the targeted cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), underlining the mechanism that is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, requiring a 5-membered cyclization from a free carboxylate to the central phosphorus atom for efficient release. Comparing the high efficiency of linker \u003cb\u003e15\u003c/b\u003e with the inactive \u003cb\u003e16\u003c/b\u003e and \u003cb\u003e17\u003c/b\u003e further allows the conclusion that release of non-derivatized DXd-OH is required to exhibit \u003cem\u003ein vitro\u003c/em\u003e potency and that linker \u003cb\u003e15\u003c/b\u003e is the most suited for delivery of aliphatic alcohols.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we compared \u003cb\u003e15\u003c/b\u003e-Dxd head to head with the state-of-the-art protease-cleavable linker-payload GGFG-DXd in a set of \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments. In Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, we assessed ADC-stability in rat serum and demonstrate increased stability of tras-\u003cb\u003e15\u003c/b\u003e-DXd compared to TD. It should be noted that the serum stability of \u003cb\u003e15\u003c/b\u003e-DXd is even higher than that of linker \u003cb\u003e3\u003c/b\u003e-SN38 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). We attribute this to the higher pKa of the aliphatic alcohol in Dxd making it a worse leaving group compared to the aromatic alcohol in SN38, leading to less drug hydrolysis in serum. We confirmed target selectivity for tras-\u003cb\u003e15\u003c/b\u003e-DXd and TD \u003cem\u003ein vitro\u003c/em\u003e on a larger panel of cell lines without activity on non-targeted cell lines (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Finally, we showed that tras-\u003cb\u003e15\u003c/b\u003e-DXd effectively eradicates HER2\u0026thinsp;+\u0026thinsp;tumors \u003cem\u003ein vivo\u003c/em\u003e, whereas an iso-\u003cb\u003e15\u003c/b\u003e-DXd did not show any anti-tumor effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Only the linker system described herein showed complete responses in three out of five animals at a dose of 1 mg/kg, whereas no complete responses were observed for the group that has been treated with TD at the same dose. Pharmacokinetic analyses by ELISA of blood samples drawn from this study revealed an excellent PK profile with slow clearance of tras-\u003cb\u003e15\u003c/b\u003e-DXd and superimposable assay results for total antibody and intact ADC, highlighting outstanding linker stability. In contrast, TD was more rapidly cleared from circulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Collectively, these results underline that the phosphoramidate linker systems described herein enable an efficient and selective delivery of DXd into the targeted cell with a superior \u003cem\u003ein vivo\u003c/em\u003e efficacy compared to an approved ADC based on the same antibody and payload.\u003c/p\u003e \u003cp\u003e \u003cb\u003eApplication of phosphoramidate linkers to the antibody-mediated delivery of structurally diverse hydroxy-containing cytotoxins exhibiting diverse MOAs\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFinally, with the overall goal to identify novel ADC payloads, we aimed to apply our linker systems to a wider range of hydroxycontaining antiproliferative agents. The chosen drugs cover a broad spectrum of MOA and have been developed for chemotherapeutic treatment in oncology but were rarely used in an ADC context before. For all drugs with aromatic alcohols, we employed linker \u003cb\u003e9\u003c/b\u003e and for all drugs with aliphatic alcohols linker \u003cb\u003e15\u003c/b\u003e, both identified as optimal linker in terms of potency, selectivity and ADC hydrophilicity. We employed the HSP90-inhibitor ganetespib\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e9\u003c/sup\u003e and the elongation factor inhibitor ON013100\u003csup\u003e50\u003c/sup\u003e as examples for aromatic payloads; the nucleoside analogue gemcitabine\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e1\u003c/sup\u003e and the dihydroorotate dehydrogenase (DHODH) inhibitors BAY 2402234\u003csup\u003e52\u003c/sup\u003e and DHODH-IN-16,\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e3\u003c/sup\u003e as primary alcohols; the HSP90-inhibitor SNX2112\u003csup\u003e54\u003c/sup\u003e and the tubulin inhibitor paclitaxel\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e5\u003c/sup\u003e as secondary alcohols and the nicotinamid phosphoribosyltransferase (NAMPT) inhibitor (LSN3154567)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e6\u003c/sup\u003e as example for a tertiary alcohol (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). All linker-drugs were conjugated to the three different antibodies trastuzumab (HER2-targeting), datopotamab (Trop2-targeting) and brentuximab (CD30-targeting) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), generating homogenous DAR8 constructs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Next, to investigate the applicability of those payloads, the ADCs were tested in eight different cell lines. Trastuzumab-based ADCs were investigated in cell killing assays on the HER2-positive solid cancer cell lines N87, SKBR-3 and HCC1569; datopotamab-based ADCs on the Trop2-positive solid cancer cell lines H441, HCC-78, and MDAMB-468, and brentuximab-based ADCs on the CD30-positive lymphoma cancer cell lines L-540 and Karpas-299. This \u003cem\u003ein vitro\u003c/em\u003e evaluation revealed successful target-dependent delivery of all chosen payloads as can be seen by the IC50s and selectivity factors in Fig.\u0026nbsp;7e. This is highly remarkable, since it firstly confirms broad applicability to very different chemical hydroxyl groups and secondly shows the ability to unlock novel payloads using phosphoramidate based self-immolative handles. Even though single examples of ADCs carrying HSP90-\u003csup\u003e57\u003c/sup\u003e, NAMPT-inhibitors\u003csup\u003e56\u003c/sup\u003e and paclitaxel\u003csup\u003e58\u003c/sup\u003e have been described in the literature, this is the first time that ADCs carrying nucleoside analogues, DHODH- or an elongation factor- inhibitors are described and documented with \u003cem\u003ein vitro\u003c/em\u003e potency.\u003csup\u003e7\u003c/sup\u003e Potency generally ranged between IC50s of 0.1 nM and 60 nM. Whereas most of the payloads showed a beneficial ratio between targeted and non-targeted ADCs with a targeting factor of 10x, paclitaxel and LSN315456 demonstrated only modest selectivity for the targeted cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). It should be noted that universal linker \u003cb\u003e9\u003c/b\u003e and \u003cb\u003e15\u003c/b\u003e for aromatic or aliphatic alcohols were used in this screen and that stability tuning by phosphoramidate modifications, as reported for SN38, might further optimize the stability and release for each payload described here.\u003c/p\u003e \u003cp\u003eTo better understand the potency differences of the different payloads across cell lines, we plotted the antiproliferative activity of all the unconjugated payloads against the effectivity of the targeted ADCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). As expected, the more effective an antiproliferative drug is in its unconjugated form on a certain cell line, the more active is also its respective ADC. Remarkably, all payloads with activity below 1 nM also showed activity when delivered by an ADC, clearly demonstrating the broad applicability of the described linker system to efficiently deliver chemically diverse hydroxyl groups. Payloads with a lower activity than 1 nM led to active ADCs in some cases but were more likely to be inactive. The observation that subnanomolar potency of a cytotoxin is a requirement for an ADC payload is in line with previous predictions.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e However, best to our knowledge, this is the first time that this is investigated more systematically in one comparable experiment with a broad range of different drugs conjugated via the same linker system.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, we chose gemcitabine as a showcase payload to further highlight the potential of the phosphoramidate-based linker system described herein to unlock unusual and scarcely described payloads for targeted drug delivery with ADCs. Gemcitabine attracted our interest as potential ADC-payload since it is a clinically-established chemotherapeutic with a known safety profile but significant limitations in rapid clearance from the body, requiring high gram doses over time.\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e Antibody-mediated drug delivery could overcome those pharmacokinetic limitations, unlocking gemcitabines\u0026rsquo; full therapeutic potential.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eADCs with both regioisomers of \u003cb\u003e15\u003c/b\u003e-Gemcitabine, primary 5\u0026rsquo;-hydroxy and secondary 3\u0026rsquo;-hydroxy were synthesized from trastuzumab (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) and evaluated for target-mediated antiproliferative activity \u003cem\u003ein vitro\u003c/em\u003e. Confirming the broad applicability of the linker systems described herein, ADCs attached via the primary or secondary alcohol of gemcitabine proved to be equally active on the targeted cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). As depicted in Fig.\u0026nbsp;8a, gemcitabine requires 5\u0026rsquo;-phosphorylation via deoxycytidine kinase followed by pyrophosphorylation to exhibit its cytotoxic activity. Co-incubation of ADC-treated cells with DI-87, an inhibitor for the initial phosphorylation, depleted activity for both ADCs to the level of unmodified trastuzumab only. Thus, we conclude that the ADCs tracelessly release gemcitabine and not any related phosphorylated species originating from the phosphoramidate linker system described herein.\u003c/p\u003e \u003cp\u003eFinally, tras-\u003cb\u003e15\u003c/b\u003e-5\u0026rsquo;-gemcitabine was evaluated \u003cem\u003ein vivo\u003c/em\u003e in the N87 tumor model. Encouragingly, we observed a strong anti-tumor effect after a single dose of 20 mg/kg (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). This effect proved to be selective, since the isotype ADC was completely inactive at the same dose. In parallel, the anti-tumor effect of trastuzumab (20 mg/kg) and unconjugated gemcitabine, dosed at 0.28 mg/kg corresponding to the dose administered in the DAR8 ADC format, was only slightly higher than for the vehicle control. This effect most likely reflects the tumor growth inhibition of trastuzumab alone, already observed \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), while unconjugated gemcitabine can be considered inactive at this low dose due to rapid clearance from circulation, as previously reported. Considering that the dose of gemcitabine administered in the ADC format is almost 1000-times lower than that typically administered over multiple injections to achieve tumor regression,\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e the \u003cem\u003ein vivo\u003c/em\u003e effect observed here is particularly remarkable. We attribute this enhanced activity to improved pharmacokinetics. In fact, clearance of the DAR8 tras-\u003cb\u003e15\u003c/b\u003e-5\u0026rsquo;-gemcitabine is highly similar to unconjugated trastuzumab, as shown in Fig.\u0026nbsp;8c. Hence, the linker system described here widens the therapeutic window for gemcitabine by stably delivering the active payload to the tumor over a long period of time after single administration, whereas unconjugated gemcitabine is rapidly metabolized and excreted \u003cem\u003ein vivo\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn the current work, we identified phosphoramidates, substituted with a release handle at the nitrogen atom, a linker for antibody modification and the to-be-delivered drug at the oxygen substituents as broadly applicable functional motifs for the delivery of aromatic- as well as primary-, secondary- and tertiary-alcohol-containing payloads. The stability and release of the payload can be tuned via simple modifications of the phosphoramidate substituents, allowing adaptation to various alcohols of different pka or matching the linkage stability to the requirements of the antibodies\u0026rsquo; target. Moreover, as long as the trigger releases a nucleophile that can form a five-membered ring with the central phosphorus atom, the release can be initiated via various intracellular stimuli including esterases, proteases and glucuronidase, as well as reductive conditions. The linker system outperformed the ones of the approved ADCs SG and TD in delivery of the respective hydroxy-containing TOP1-Inhibitors SN38 and DXd. Here, phosphoramidate based linker were superior in serum stability, \u003cem\u003ein vivo\u003c/em\u003e efficacy and PK. More importantly, the simple drug-linker synthesis and conjugation of highly loaded homogenous DAR8 ADCs enabled us to repurpose and screen existing small molecule cytotoxins with various MOAs as novel ADC payloads. Highly efficacious ADCs with IC50s in the nanomolar to sub-nanomolar range were synthesized. Many of those cytotoxins and MOAs were without literature precedence in drug delivery. The broad dataset with ten cytotoxins conjugated to a variety of antibodies allowed us to support the general hypothesis in targeted drug delivery that sub-nanomolar potency of a small molecule cytotoxin is needed to generate effective ADCs. Payloads with lower potency on a certain cell line on the other hand can completely lack activity \u003cem\u003ein vitro\u003c/em\u003e. Finally, for the first time, we demonstrate \u003cem\u003ein vivo\u003c/em\u003e efficacy of anADC based on gemcitabine. The excellent PK profile of this DAR8 ADC, enabled by the phosphoramidate linker system, leads to \u003cem\u003ein vivo\u003c/em\u003e activity at doses 1000 times lower than those typically administered for unconjugated gemcitabine. We are convinced that the broad compatibility of the linker system described herein with structurally diverse alcohols, combined with the efficient production of homogenous DAR8 ADCs from readily available pharmacologically active small molecules will allow for faster identification of suitable payloads to expand the currently limited panel of only three MOAs in approved ADCs. This will broaden the scope of ADC modalities with great potential in addressing drug resistance mechanisms in patients.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGeneral method for the conjugation of linker payloads\u003c/h2\u003e \u003cp\u003e50 \u0026micro;l of the antibody solution of 10.0 mg/ml in P5-conjugation buffer (50 mM Tris, 1 mM EDTA, 100 mM NaCl, pH 8.3 at RT) were mixed with 3.33 \u0026micro;l of a 10 mM TCEP solution in P5-conjugation buffer. Directly afterwards, 1.67 \u0026micro;l of a 40 mM solution of the linker-payload constructs dissolved in DMSO were added. The mixture was shaken at 350 rpm and 25\u0026deg;C for 16 hours. The reaction mixtures were purified by preparative size-exclusion chromatography with a 25 ml Superdex\u0026trade; 200 Increase 10/300GL (Cytiva, Sweden) and a flow of 0.8 ml/min eluting with sterile PBS (Merck, Germany). The antibody containing fractions were pooled and concentrated by spin-filtration (Amicon\u0026reg; Ultra- 2mL MWCO: 30 kDa, Merck, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ecytotoxicity on cancer cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the cytotoxicity of ADCs and unconjugated cytotoxins, 5.000 cells per well were incubated for 7 days or 4 days, respectively with increasing concentrations of the ADCs (0.18\u0026ndash;12000 ng/ml\u0026thinsp;=\u0026thinsp;0.0012-80 nM) or small molecules (0.015 nM \u0026ndash; 1000 nM) to generate a dose\u0026ndash;response curve. Before the analysis of cell viability, the spent medium containing dead cells was removed and fresh medium was added. Killing was analysed using resazurin cell viability dye at a final concentration of 55 \u0026micro;mol/l (Merck) by dividing the fluorescence from control cells in medium by the fluorescence of ADC-treated cells. Fluorescence emission at 590 nmol/l was measured on a microplate reader Infinite 200 PRO (Tecan Group Ltd.).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ebystander activity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor the supernatant-based bystander experiment, 20,000 Trop2-positive MDA-MB-468 cells were seeded in 100 \u0026micro;l medium and treated with saci-\u003cb\u003e3\u003c/b\u003e-SN38 at concentrations ranging from 0.18 to 12,000 ng/ml. After 5 days, 50 \u0026micro;l of the supernatant of the treated cells was transferred to 50 \u0026micro;l of Trop-negative SW-620 (5,000 cells/well) and incubated for 5 days. Resazurin readout was performed as described for the in vitro toxicity evaluation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eefficacy\u003c/b\u003e\u003c/p\u003e \u003cp\u003e All animal experiments were conducted in accordance with German animal welfare law and approved by local authorities.\u003c/p\u003e \u003cp\u003eFor the SN38 study, 1x10\u003csup\u003e7\u003c/sup\u003e MDA-MB-468 cells (50\u0026micro;l\u0026thinsp;+\u0026thinsp;50\u0026micro;l Matrigel) were subcutaneously injected to CB17-Scid mice. Treatment was initiated when tumours reached a tumour volume of about 0.1 cm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e 9 days after implantation. 5 animals per group were treated twice at day 9 and day 13 with 10 mg/kg each treatment day of either saci-\u003cb\u003e3\u003c/b\u003e-SN38, iso-\u003cb\u003e3\u003c/b\u003e-SN38 or Trodelvy and compared to a vehicle treated group.\u003c/p\u003e \u003cp\u003eFor the DXd study, 2x10\u003csup\u003e6\u003c/sup\u003e N87 cells were subcutaneously injected to CB17-Scid mice. Treatment was initiated when tumours reached a tumour volume of about 0.1 cm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e 7 days after implantation. 5 animals per group were treated at day 7 with 1 mg/kg of either tras-\u003cb\u003e15\u003c/b\u003e-DXd, iso-\u003cb\u003e15\u003c/b\u003e-DXd or Enhertu and compared to a vehicle treated group.\u003c/p\u003e \u003cp\u003eFor the gemcitabine study, 2x10\u003csup\u003e6\u003c/sup\u003e N87 cells were subcutaneously injected to CB17-Scid mice. Treatment was initiated when tumours reached a tumour volume of about 0.1 cm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e 4 days after implantation. 5 animals per group were treated at day 4 with either 20 mg/kg of tras-\u003cb\u003e15\u003c/b\u003e-gemcitabine, 20 mg/kg of iso-\u003cb\u003e15\u003c/b\u003e-gemcitabine or 20mg/kg tras and 0.28 mg/kg gemcitabine and compared to a vehicle treated group.\u003c/p\u003e \u003cp\u003eMice were treated via intravenous injection after randomisation into treatment and control groups. Tumour volumes, body weights and general health conditions were recorded throughout the whole study.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest:\u003c/h2\u003e \u003cp\u003eThe authors declare competing financial interests: All authors, except from C.P.R.H are full-time employees of Tubulis. The presented work is part of a pending patent application by J.H., D.S., P.O., A.P.J., S.V, S.W. and M.A.K.\u003c/p\u003e\u003ch2\u003eAuthor Contributions:\u003c/h2\u003e \u003cp\u003ePhilipp Ochtrop: Data curation, Formal Analysis, Investigation, Methodology, Supervision, Writing \u0026ndash; review \u0026amp; editing. Anil P. Jagtap: Data curation, Investigation, Writing \u0026ndash; review \u0026amp; editing. Jan G. Felber: Data curation, Formal Analysis, Visualization, Writing \u0026ndash; review \u0026amp; editing. Simon Vogt: Investigation. Isabelle Mai: Data curation, Investigation. Philipp Cyprys: Data curation. Saskia Schmitt: Data curation, Formal Analysis, Formal Analysis. Sarah Payer: Data curation. Annabel Kitowski: Data curation. Swetlana Wunder: Investigation. Paul Machui: Data curation. Sarah Herterich: Data curation, Natascia Leonardi: Data curation, Elizaveta Poliak: Data curation, Christian P.R. Hackenberger: Writing \u0026ndash; review \u0026amp; editing. Olivier Marcq: Supervision. Dominik Schumacher: Funding acquisition, Project administration, Supervision. Jonas Helma: Methodology, Funding acquisition, Project administration, Supervision. Annette M. Vogl: Methodology, Supervision. Marc-Andr\u0026eacute; Kasper: Conceptualization, Data curation, Investigation, Methodology, Supervision, Visualization, Writing \u0026ndash; original draft.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eThe authors thank Danila Hauswald for excellent technical assistance in antibody purification.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSrinivasarao M, Low PS (2017) Ligand-Targeted Drug Delivery. 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J Experimental Clin Cancer Res 37:291. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1186/s13046-018-0972-3\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1186/s13046-018-0972-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCiccolini J, Serdjebi C, Peters GJ, Giovannetti E (2016) Pharmacokinetics and pharmacogenetics of Gemcitabine as a mainstay in adult and pediatric oncology: an EORTC-PAMM perspective. Cancer Chemother Pharmacol 78:1\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1007/s00280-016-3003-0\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1007/s00280-016-3003-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5454963/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5454963/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDespite recent advances in targeted drug delivery, approved Antibody-Drug-Conjugates (ADCs) are still limited by the delivery of a restricted set of payloads with limited modes of action (MOA). Versatile linkers, applicable to functional groups prevalent across diverse pharmacophores are needed to expand this space. We present phosphoramidate-based self-immolative linker-units that facilitate stable attachment in serum and traceless drug release in the target cell from aliphatic and aromatic alcohols. Studies with camptothecins show that stability and release are tunable and that various intracellular trigger events can be exploited to ensure traceless drug delivery. Superior stability, \u003cem\u003ein vivo\u003c/em\u003e efficacy, and pharmacokinetics (PK) compared to approved ADCs are demonstrated. Moreover, we report targeted delivery of 10 different hydroxy-containing cytotoxins with different intracellular MOAs. \u003cem\u003eIn vivo\u003c/em\u003e studies with gemcitabine show excellent PK and efficacy, unlocking gemcitabine\u0026rsquo;s full potential and illustrating the ability of the phosphoramidate-based linker system to expand the payload space for ADCs.\u003c/p\u003e","manuscriptTitle":"Expanding the payload scope in antibody-drug conjugates: Unprecedented delivery of hydroxy-containing drugs through self-immolative phosphoramidates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-16 03:11:36","doi":"10.21203/rs.3.rs-5454963/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b82f7648-f1e7-43af-9ba1-3ffc24c88c58","owner":[],"postedDate":"May 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":42537334,"name":"Biological sciences/Drug discovery/Drug delivery"},{"id":42537335,"name":"Biological sciences/Cancer/Cancer therapy/Targeted therapies"},{"id":42537336,"name":"Physical sciences/Chemistry/Chemical biology/Drug delivery"},{"id":42537337,"name":"Physical sciences/Chemistry/Biochemistry/Bioconjugate chemistry"},{"id":42537338,"name":"Biological sciences/Chemical biology/Chemical tools"}],"tags":[],"updatedAt":"2026-01-21T08:26:58+00:00","versionOfRecord":{"articleIdentity":"rs-5454963","link":"https://doi.org/10.1038/s41467-026-68605-y","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-01-20 05:00:00","publishedOnDateReadable":"January 20th, 2026"},"versionCreatedAt":"2025-05-16 03:11:36","video":"","vorDoi":"10.1038/s41467-026-68605-y","vorDoiUrl":"https://doi.org/10.1038/s41467-026-68605-y","workflowStages":[]},"version":"v1","identity":"rs-5454963","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5454963","identity":"rs-5454963","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}