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

Domain: chemistry
Published: 2026-01-20
Editorial outcome: major_revision

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 of 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 camptothecin 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.

Introduction
Targeted drug delivery for the treatment of malignant diseases holds great promise in reducing undesired side effects while increasing efficacy by precisely hitting cancer cells1. Most prominently, antibodies conjugated to highly potent cytotoxic drugs, a modality known as antibody-drug conjugates (ADCs), have been utilized as targeting vehicles2,3. 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 patients4. Still, challenges remain to be solved, such as chemotherapy-like toxicities, lack of efficacy when applied as monotherapy and, eventually, acquired resistance5. 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 shortcomings6. With a small repertoire of only three MOAs in approved ADCs, namely tubulin-inhibition, topoisomerase-I (TOP1)-inhibition and DNA-damaging, it is anticipated that MOA diversification will be a key factor in the development of next generation targeted cancer therapeutics7. 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 loss8, unspecific uptake9,10 and undesired aggregation11.

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 payload12 and for its permeability, which allows effective eradication of tumors with heterogeneous expression of the antibody’s target via the bystander effect13,14. 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 group1,15. Commonly exploited trigger events include a variety of intracellular or intra-lysosomal conditions such as acidic and reductive environment or specific enzymatic activities from esterases16, proteases17, glycosidases18, phosphatases19, reductases20, or sulfatases21. Recent developments in the field focused on enhancing the stability and efficacy of widely used cathepsin B cleavable linkers by tackling the premature payload release through unspecific cleavage of the VC peptide. Especially the introduction of exo-cleavable22, tandem-cleavable23 or carboxylesterase resistant linker24 showcase the impact of linker design on ADC stability and efficacy.

Such cleavable linkers have been designed for the attachment of different functional groups on payloads including primary-25, secondary-26, tertiary- and heteroaryl-amines27, thiols, amides28, ortho-quinones29 and alcohols30. In particular, hydroxyl linkages are attractive since this functionality is frequently present in synthetic small molecule drugs and natural product derivatives31. 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 carbamates32, carbonates33, esters34, phosphates, pyrophosphates35, 1,6-benzylamines36, hemiaminals37,38, disulfides20,39, or methylene carbamates30. DXd represents one of the clinically successful examples in which a primary alcohol has been stably linked and its sufficient release can be ensured via a cleavable hemiaminal structure40,41. However, the broad applicability of hemiaminals to hydroxy functions of different pKa remains to be shown. Therefore, none of the functional groups that have been used so far for hydroxyl group attachment combine broad applicability, stability in serum and efficient traceless release.

To address these limitations, we took inspiration from the clinically validated ProTide technology42, a prodrug system that has been developed to mask hydrophilicity of nucleoside-like drug molecules to enhance cell permeability43. This approach has yielded three FDA-approved antiviral drugs, sofosbuvir44, tenofovir alafenamide45, and remdesivir46, that have become blockbuster therapeutics, underscoring the ProTide strategy’s success in antiviral therapy and its growing promise in oncology47–49. Continued advancements in ProTide chemistry, including applications beyond traditional nucleoside analogs, further highlight its versatility50. 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. 1a).

In this work, we redesign the central phosphorous core of the phosphoramidate moiety 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. Therby we transform a prodrug system into a targeted drug delivery technology. Exploiting the fact that ProTides liberate an aromatic- and an aliphatic alcohol intracellularly, we extend this concept to create a linker-technology capable of releasing not only nucleoside analogs but delivering a broad spectrum of aliphatic and aromatic alcohols (Fig. 1b). In contrast to the earlier described nucleoside prodrugs which are restricted to esterase cleavage after passive uptake into the cytosol42, 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
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 ADCs sacituzumab govitecan (SG, Trodelvy), as our first model drug51. Guided by previous experience with unsaturated ethynyl-52 and vinyl-phosphonamidate53-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. 1a. Synthesis of 1 and 2 was performed as outlined in Fig. 2a (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 SN3853.

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 20-hydroxyl group via a carbonate moiety (Fig. 3a). This linker has been designed to rapidly hydrolyze under physiological conditions. As previously reported54 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 IC50 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. 2b). 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 IC50 than for SN38 as free drug33. 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 IC50 from isotype- over targeted ADC as measure for linkage stability, and absolute IC50 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 IC50 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. 2b). 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 aminopentyl 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)12,55, 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. 2c (See SI for detailed information) and proved to be superior to 1-SN38, since 3-4-SN38, conjugated to sacituzumab, exhibited strong anti-proliferative activity with a favorable potency window between targeted and isotype ADC (Fig. 2d). An IC50 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-SN3814. As shown in Fig. 2e, 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-SN3856.

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, (Fig. S1) and observed improved rat serum stability of saci-3-SN38 with more than 50% intact ADC after 7 days of incubation (Fig. 2f). In contrast, SG lost almost 100% of conjugated drug already after 24 hours, mainly caused by carbonate hydrolysis. Since tumor-target specificity of SG has been demonstrated in vivo, contrary to the in vitro observations54, we compared both linker-payloads head-to-head in an in vivo efficacy experiment (Fig. 2g). 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 systems55,57, ensuring a higher tumor exposure to SN38. In addition, we tested saci-3-SN38 and SG on a panel of healthy human primary cells to assess differences of the linker structures on the undesired effect on normal tissue (Fig. S1c). On all of the tested healthy cells, saci-3-SN38 had significantly less effect compared to SG, suggesting not only a higher anti-tumor efficacy of the linker systems described herein but also reduced effect on healthy tissue and thereby a broader therapeutic window.

Linker stability tuning and combination with different release triggers
Encouraged by reports on structural optimization of ProTides42, 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 (IC50 targeted ADC) and selectivity (ratio IC50 isotype/targeted ADC) on two target-positive cell lines for each ADC (Fig. 3a and Fig. S2). 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. 3b). 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 phosphorus, showed a decreased window (Fig. 3c). 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 esterases58. This trend could be confirmed with esterase cleavage assays on N-Acetyl-Cysteine adducts of the isolated linker payloads 3-, 9- and 10-SN38, showing a faster release of the payload from the isopropyl esters 9 and 10 compared to the tert-butyl ester in 3 (Fig. S3). 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-delivery2. 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 cancers59. 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. Although such structures have not previously been explored in the context of ProTide prodrugs, they were recently described in phosphonamidite- and phosphoramidate-based fluorescent probes designed to study carboxypeptidases, representing the first report to implicate carboxypeptidases in ProTide activation60. 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. 3c).

Other known release triggers exploit the reductive environment or beta-glucuronidase activity in endo-/lysosomes49,50. 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. 3c). 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 properties61. The free carboxylic acid in dialanine linker 11 comprised the most hydrophilic DAR8 SN38-ADC tested herein (Fig. 3d). 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. Of note, changing the tert-butyl- to the isopropyl-ester did not change the effect on normal human primary cells (Fig. S1c).

Taken together, the results summarized 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 stability tuning could be useful in the future to identify the optimal linker for a certain target that require 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)62. 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. 4a and b). Compared to what we observed for SN38, the phosphonamidate-based linker 1 delivered DXd into the targeted cell line with a 3-fold better selectivity compared to the isotype ADC (Fig. 4d). Like for SN38, a phosphoramidate release handle in 3-DXd was superior to phosphonamidates in 1-DXd (Fig. 4d), displaying a higher potency (IC50 = 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. 4c). 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 that possesses a terminal carboxy group instead of the tert-butyl ester as shown in Fig. 5c. The terminal carboxy group is also desired, since it adds additional hydrophilicity to the linker system, as shown for SN38 in Fig. 3d.

To better understand whether Dxd release involves an attack of the alanine carboxy group on the central phosphorus 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. 4d), underlining the mechanism that is shown in Fig. 1b, 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 allow 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. 4e, we assessed ADC-stability in rat serum and demonstrated 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. 2f). 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 (Fig. S4). 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. 4f). 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. 4f). This might be attributed to increased hydrophilicity of tras-15-DXd in comparison to TD, as evidenced by HIC retention time (Fig. S4c). Increased ADC hydrophilicity has been previously shown to lower ADC clearance in vivo18. 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 hydroxy-containing antiproliferative agents. 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. To confirm the suitability of these two linker systems we again investigated both intracellular cleavability and extracellular stability in circulation. In addition to the esterase cleavage experiments on isolated linker payloads 3, 9 and 10-SN38, we also demonstrated that other proteases such as cathepsin A (CatA) are able to release the payload from 3-SN38, 9-SN38, but importantly also from 15-DXd. Figure S5 This data suggests that both, the esterase cleavage handle in linker 9 and the protease cleavage handle in linker 15 can be cleaved by multiple enzymes in the endosomal-lysosomal system, similar as previously described for more traditional linker systems63. We believe that the systems described herein achieve cancer specificity by the targeting antibody rather than a tumor-selective release of the attached drug. To further assess the extracellular linker stability, we submitted the isolated linker-payloads 9-SN38 and 15-DXd to a neutrophile elastase assay (Fig. S6a–c). Susceptibility to neutrophile elastase cleavage has been identified as a potential cause of premature drug release leading to neutropenia64. Interestingly, both linker systems behaved differently, showing drug release only from the esterase cleavable 9-SN38, while 15-DXd showed no signs of DXd release in the presence of human neutrophile elastase. To study this further, we also tested the ADCs from 9-SN38 and 15-DXd revealing only minor or no signs of enzymatic linker cleavage, respectively, while a control ADC utilizing mc-VC-PAB-MMAE as linker-payload underwent extensive degradation, releasing 90% of the expected NH₂-Citrulline-PAB-MMAE fragment over 24 hours as previously reported (Fig. S6d)64. This data suggests that the protease cleavable linker 15 is fully stable, while there is a potential liability for the esterase based system 9 towards neutrophile elastase. However, in the much more relevant ADC format, we were able to demonstrate that the severeness of this instability is significantly lower compared to approved Vedotin-based ADCs such as Adcetris. This could be confirmed also in human serum stability assays of ADCs from 9 and 15 (Fig. S6e).

Next, we employed the HSP90 inhibitor ganetespib65 and the elongation factor inhibitor ON01310066 as examples for aromatic payloads; the nucleoside analogue gemcitabine67 and the dihydroorotate dehydrogenase (DHODH) inhibitors BAY-240223468 and DHODH-IN-1669, as primary alcohols; the HSP90 inhibitor SNX211270 and the tubulin inhibitor paclitaxel71 as secondary alcohols and the nicotinamid phosphoribosyltransferase (NAMPT) inhibitor (LSN3154567)72 as example for a tertiary alcohol (Fig. 5b). All linker-drugs were conjugated to the three different antibodies trastuzumab (HER2-targeting), datopotamab (Trop2-targeting) and brentuximab (CD30-targeting) (Fig. 5a). Even though all these drugs differ in hydrophobicity and more hydrophobic payloads lead to ADCs with a higher HIC retention time, it was still possible to generate homogenous DAR8 constructs from the inhibitors described herein (Fig. 5c and Fig. S7). 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 MDA-MB-468, and brentuximab-based ADCs on the CD30-positive lymphoma cancer cell lines L-540 and Karpas-299 (Fig. S8). This in vitro evaluation revealed successful target-dependent delivery of all chosen payloads as can be seen by the IC50 values and selectivity factors in Fig. 5d. 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 HSP90-73, NAMPT-inhibitors72 and paclitaxel74 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 potency7. Potency generally ranged between IC50 values 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 10, paclitaxel and LSN315456 demonstrated only modest selectivity for the targeted cell line (Fig. 5d). 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 effectiveness of the targeted ADCs (Fig. 5e). 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 predictions4,75. However, to the best of our knowledge, this is the first time that this has been 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 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 time76. Antibody-mediated drug delivery could overcome those pharmacokinetic limitations, unlocking gemcitabines’ full therapeutic potential. ADCs with both regioisomers of 15-gemcitabine, connected via the primary 5’-hydroxy or the secondary 3’-hydroxy, were synthesized from trastuzumab (Fig. 6a) 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. 6b). As depicted in Fig. 6a, 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. 6c). This effect proved to be tras 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. 6b), 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 regression77, 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. 6c. 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 vivo78. Tras-15-5’-gemcitabine has also been evaluated for toxicity in rats in doses up to 150 mg/kg (Fig. S9). Not a single sign of toxicity was observed at the highest dose tested, which is 7.5 times higher than the effective dose used in the in vivo efficacy experiment, clearly pointing towards large therapeutic indices that can be reached with the linker systems described herein.

To conclude, 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. While several functional groups have been previously used to conjugate hydroxy-containing payloads to antibodies, a broadly applicable solution for stable attachment in circulation and traceless release intracellularly is still unprecedented. Here, we introduce cleavable phosphoramidates as a self-immolative entity that can be broadly applied to alcohols of very different kinds (primary, secondary, tertiary and phenolic) with different electronic properties. 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 linkers 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 IC50 values 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 an ADC based on gemcitabine. The antibody-like 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.

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 h. The reaction mixtures were purified by preparative size-exclusion chromatography with a 25 ml Superdex™ 200 Increase 10/300 GL (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- 2 mL MWCO: 30 kDa, Merck, Germany).

In vitro cytotoxicity on cancer cells
To investigate the cytotoxicity of ADCs and unconjugated cytotoxins, 5000 cells per well were incubated for 7 days or 4 days, respectively, with increasing concentrations of the ADCs (0.18–12,000 ng/ml = 0.0012–80 nM) or small molecules (0.015–1000 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 analyzed 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 nm 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 (5000 cells/well) and incubated for 5 days. Resazurin readout was performed as described for the in vitro toxicity evaluation.

Cell lines
Cell lines were purchased from the “German Collection of Microorganisms and Cell Cultures” (DSMZ, Leibniz Institute, Germany), American Type Culture Collection (ATCC, USA), Japanese Collection of Research Bioresources (JCRB) via Tebubio (France), Merck (Germany) or Thermo Fisher Scientific (USA): SR-786 (ACC 369, DSMZ; RRID:CVCL_1711; sex: male), L-540 (ACC 72, DSMZ; RRID:CVCL_1362; sex: female), Karpas-299 (Merck, RRID:CVCL_1324); sex: male), NCI-N87 (CRL-5822, ATCC; RRID:CVCL_1603; sex: male), SK-BR-3 (ACC 736, DSMZ; RRID:CVCL_0033; sex: female), HCC-1569 (CRL-2330, ATCC; RRID:CVCL_1255; sex: female), H441 (HTB-174, ATCC; RRID:CVCL_1561; sex: male), MDA-MB-468 (ACC 738, DSMZ; RRID:CVCL_0419; sex: female), HCC78 (ACC 563, DSMZ; RRID:CVCL_2061; sex: male), SW620 (300466, Tebubio; RRID:CVCL_0547; sex: male), ExpiCHO-S (A29127, Thermo Fisher Scientific; RRID:CVCL_5J31; sex: female). Cells were cultured according to the manufacturer’s instructions in either RPMI 1640 or DMEM medium supplemented with GlutaMAX (Gibco, Thermo Fisher Scientific, USA) and 10–20% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, USA). Cell lines for antigen or antibody production were cultivated in their proprietary medium (Thermo Fisher Scientific, USA). For passage or for assay setup, the adherent cells were detached by TrypLE Express Enzyme (Thermo Fisher Scientific, USA) according to manufacturer’s instructions and splitted or seeded accordingly.

A mycoplasma test is performed using MycoAlert® Mycoplasma Detection Kit (Lonza, Switzerland) according to the manufacturer’s instructions every 6 month (latest test: 04/2025). Cell line authentication was performed at the cell bank and is conducted internally every 6 months via SNP typing (latest test: 04/2025). Cell lines are used at a maximum passage of 30 and then re-thawed for further experiments.

In vivo efficacy
All animal experiments were conducted in accordance with German animal welfare law and approved by local authorities. Mice were housed at room temperature (24 ± 2 °C) with a relative humidity of 50 ± 10% at artificial light with a 12 h dark and 12 h light rhythm. Mice were 8 weeks old at the start of the studies.

For the SN38 study, 1×107 MDA-MB-468 cells (50 µl + 50 µl Matrigel) were subcutaneously injected to female CB-17/Icr-Prkdcscid/scid/Rj mice. Treatment was initiated when tumors reached a tumor volume of about 0.1 cm3 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, 2×106 N87 cells were subcutaneously injected to female CB-17/Icr-Prkdcscid/scid/Rj mice. Treatment was initiated when tumors reached a tumor volume of about 0.1 cm3 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 PK analysis, blood samples were drawn from one mouse per group of the tras-15-DXd and the Enhertu group after 5 min, 4 h, 1 day, 2 days, 7 days, 14 days and 21 days and analyzed for ADC exposure by ELISA as described below.

For the gemcitabine study, 2×106 N87 cells were subcutaneously injected to female CB-17/Icr-Prkdcscid/scid/Rj mice. Treatment was initiated when tumors reached a tumor volume of about 0.1 cm3 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 20 mg/kg tras and 0.28 mg/kg gemcitabine and compared to a vehicle treated group. For PK analysis, blood samples were drawn from one mouse per group of the tras-15-gemcitabine and the tras + gemcitabine group after 5 min, 4 h, 1 day, 2 days, 7 days, 14 days and 21 days and analyzed for ADC exposure by ELISA as described below. Mice were treated via intravenous injection after randomization into treatment and control groups. Tumor volumes, body weights and general health conditions were recorded throughout the whole study.

In vivo toxicity
All animal experiments were conducted in accordance with Hungarian animal welfare law and approved by local authorities. Sprague-Dawley rats (Charles River Laboratories) were housed at room temperature (22 ± 3 °C) with a relative humidity of 30–70% at artificial light with a 12 h dark and 12 hours light rhythm. 7-week-old male rats were treated once at day 0 with either 50, 100 or 150 mg/kg tras-15-gemcitabine. The animals were observed for 22 days. Body weight and food consumption was monitored throughout the study. Blood samples for the PK analysis were drawn after 1 h, 7 days, 14 days and 21 days. The samples were analyzed for ADC exposure as described below. Hematology and clinical chemistry was measured 5 days before dosing and 7 days, 14 days and 21 days after dosing.

Analysis of the in vivo samples by ELISA to assess the pharmacokinetics of the ADCs
To evaluate the PK of the ADCs in vivo, the total antibody concentration was measured at different timepoints in serum of ADC-treated mice or rats from the in vivo studies described above. Total trastuzumab- and derived ADC levels were analyzed in mouse- and rat serum serum over the range 2000–15.6 ng/mL. Clear Nunc flat bottom MaxiSorp 96-well plate (Thermo Fisher Scientific, USA) was coated with recombinant human HER2 antigen diluted in DPBS (Thermo Fisher Scientific, USA) to a final concentration of 1 µg/mL and incubated overnight at 4 °C. Excess antigen was removed by washing with PBST (DPBS + 0.05% Tween 20, Merck, Germany) and plates were blocked for 1 h at room temperature in blocking solution (2% albumin in PBST; Merck, Germany). Prepared standards (2000–15.6 ng/ml) of the respective ADCs, QCs and serum test samples were incubated at room temperature for 1 h. Samples were detected using HRP-conjugated goat anti-human kappa light chain secondary antibody (Thermo Fisher Scientific, USA, Cat# A18853, RRID:AB_2535630) at a 1:12,000 dilution (final concentration 0.083 µg/mL) for 1 h at room temperature. Antibodies were detected using Ultra-TMB substrate (Thermo Fisher Scientific, USA) for 10 min at room temperature followed by stopping the reaction with 1 M sulfuric acid (Merck, Germany). The absorbance at a wavelength of 450 nm was measured on an Infinite 200 Pro plate reader (Tecan, USA).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Kasper and colleagues have compiled an excellent manuscript describing exciting results that are broadly applicable to the field of targeted therapy, as well as specifically transformative to the area of antibody-drug conjugates. They have demonstrated a linker system that is applicable to phenols and alkyl alcohols, regardless of the leaving group ability or sterics of the alcohol. There are very few existing technologies that can accomplish this and the few that do are more specific (less general) and are synthetically difficult, therefore limiting the broad applicability. The authors have demonstrated this with around 10 different drugs with a variety of properties as well as types of hydroxy functional group. They have demonstrated in vitro as well as in vivo POC demonstrating efficacy, stability and PK. They have demonstrated that the linker can potentially be further tuned to improve potency, stability and selectivity. Finally, kudos to the authors for what was perhaps the most clear figures I've ever observed. These systems are super complex, yet the authors use of structure, color allows the reader to easily understand (without referring to the legend or the main body) what structure is associated with what data.\nA few small corrections/suggested edits:\n- In several of the figures the authors refer to what R is for the vinyl phosphonate. If there is room, perhaps also add this\nR=PEG24 to Fig. 3a. and 4a\n- summerized (typo)\n- as shown for SN38 in Fig. 4d. (think this should be 3d)\n- consider showing HIC for TD and/or comment on why is the PK worse for TD vs. T-15-Dxd\n- obtaining DAR8 ADCs for all of these new payloads is quite impressive. Perhaps comment on (or provide range) of logDs.\nTable of HIC values for the resulting conjugates would also be informative\n- Fig. 7e (typo)\nCongrats on a really fantastic manuscript!", "reviewer_2": "This manuscript presents a study utilizing a phosphoramidate-based self-immolative linker to expand the applicability of\npayloads in antibody-drug conjugates (ADCs) beyond conventional designs. Specifically, the authors claim superior in vivo\nstability, pharmacokinetics (PK), and efficacy compared to existing approved ADCs. Additionally, the study explores the\nversatility and tunability of various payload-linker combinations, which is a commendable effort. However, the manuscript\nhas several critical concerns and gaps that need to be addressed to be suitable for publication in a high-impact journal like\nNature Communications. Below are detailed comments:\n1. Lack of Comprehensive Background Information\nComment: While the Introduction briefly mentions the importance of linker design, it lacks sufficient background to\ncontextualize the study's significance. The impact of linker design on ADC stability and efficacy could be strengthened by\nciting more recent and relevant literature. For instance, the report on Exo-linkers (J. Med. Chem. 2024, 67, 20, 18124–\n18138), which shares similarities with the current study's compact and externalized linker design, should be cited.\nAdditionally, including references to tandem-cleavable linkers and Glu-Val-Cit peptides designed for carboxylesterase\nresistance would further enhance the relevance and impact of the Introduction.\n2. Inadequate Stability Testing\nComment: Given that the linker relies on ester hydrolysis as a trigger, stability testing against key enzymes such as\ncarboxylesterase and human neutrophil elastase is essential. Without this data, the reliability of the linker design in vivo\nremains unsubstantiated. Comprehensive enzyme susceptibility assays should be included to support the claim of linker\nstability.\n3. Additional Data for Figure 3\nComment: While Figure 3 illustrates differences in degradation rates among various esters, additional supporting data is\nrequired to substantiate these observations. Including time-course LC-MS data to track degradation would provide more\nrobust evidence for the claims made in the manuscript.\n4. Lack of Consideration for Alternative Cleavage Mechanisms\nComment: The study focuses solely on the targeted cleavage mechanism of the linker but fails to rule out alternative\npathways. Proteases such as cathepsins (e.g., Cathepsin B, L, K, and S), known for their broad substrate specificity, could\npotentially cleave the linker. Comprehensive protease susceptibility testing would help establish the specificity of the\ndesigned linker.\n5. Unclear Intracellular Mechanism\nComment: The manuscript does not clarify the intracellular compartment (e.g., lysosome, late endosome) where linker\ncleavage and payload release occur. Imaging studies using fluorescent probes could provide insights into the timing and\nlocation of payload release, adding depth to the mechanistic understanding of the linker.\n6. Insufficient Comparison with T-DXd\nComment: The claim that the presented ADC is more hydrophilic than T-DXd lacks quantitative validation. Comparative\ndata, such as HIC retention times, would provide a clear basis for this assertion. Including such data in Figure 3d or as a\nsupplementary figure is recommended.\n7. Ambiguities in SI HPLC Data\nComment: The HPLC data in the supplementary information (SI) for antibodies such as Sacituzumab and Datopotomab\ncontain unclear peaks, suggesting potential impurities. The extra peaks observed in Brentuximab-ADC may indicate non-\nspecific conjugation. Additional analyses, such as LC-MS or improved HPLC separation, are necessary to resolve these\nambiguities.\n8. Absence of Safety Evaluations\nComment: The lack of safety evaluations is the most significant concern in this manuscript. The study does not include data\non normal cell uptake or off-target toxicity, both of which are critical for assessing the specificity and safety of the linker and\npayload. To align with the standards of Nature Communications, dose-limiting toxicity (DLT) assessments using non-human\nprimate models (e.g., monkeys) are recommended. These data would substantiate claims of safety and tolerability.\n9. Validation of Novelty and Practicality\nComment: While the authors emphasize the novelty of the phosphoramidate-based linker derived from ProTide technology,\nits distinctiveness compared to existing technologies remains unclear. Furthermore, while the application of diverse\npayloads is intriguing, more detailed evaluation of clinically relevant examples, including toxicity profiles and target\nspecificity, is needed to validate the practicality of this approach.\nThis manuscript addresses an important topic in ADC design and demonstrates promising features of the phosphoramidate-\nbased linker system. However, significant gaps in stability testing, safety evaluation, and mechanistic clarity must be\naddressed. Moreover, the claims of novelty and superiority require more robust validation through comprehensive\nexperiments and comparisons. Addressing these points will significantly enhance the scientific rigor and impact of the study,\nmaking it more suitable for publication in a high-impact journal.", "reviewer_3": "The manuscript by Ochtrop and colleagues introduces a series of phosphoramidate-based self-immolative linkers for\nantibody-drug conjugates (ADCs). These linkers enable stable attachment and traceless release of both aliphatic and\naromatic alcohol-containing payloads. The research team, with significant experience in ADC development, presents a\ndetailed study, showcasing the targeted delivery of 10 different hydroxy-containing cytotoxins with various intracellular\nmechanisms of action. This excellent work highlights the potential of the phosphoramidate-based linker system to expand\nthe range of payloads for ADCs. The methodology used in the study is sound, with appropriate techniques employed to\nassess linker stability. fully support the publication of this work, pending a few revisions and clarifications noted below:\n1. The authors are leveraging the clinically validated ProTide technology; however, they should cite recent relevant works\non ProTides, such as Jin et al., Acta Pharmaceutica Sinica B (2024) and J. Am. Chem. Soc. (2024, 146, 1, 521–531). A more\nthorough literature review is recommended to strengthen the manuscript's foundation.\n2. The authors highlight two key distinctions between their design and clinically validated ProTides:\nA) Their design incorporates a conjugation handle into the core phosphorus(V) [P(V)] structure, allowing covalent\nattachment to targeting moieties such as antibodies. To demonstrate the superiority of their design, the authors synthesized\nmultiple linker systems conjugated to antibodies and conducted thorough studies to evaluate their properties and\nperformance. I agree with their claim and strongly believe this innovation significantly advances the ProTide concept into the\nrealm of ADC development. I would like to congratulate the team on conducting such a comprehensive study to support their\nclaim.\nB) The authors claim that, unlike traditional nucleoside-based ProTides—which rely exclusively on esterase-mediated\ncleavage following passive cytosolic uptake—their phosphoramidate-based system allows for payload release via multiple\nintracellular and endosomal triggers. Specifically, their lead linker system, based on Ala-Ala for traceless release of\ngemcitabine, is supported by extensive data demonstrating protease-mediated cleavage. However, it is essential for the\nauthors to revise their manuscript and appropriately cite J. Am. Chem. Soc. (2024, 146, 1, 521–531), which was the first\nstudy to utilize carboxypeptidases to enhance ProTide technology. While that study employed fluorescence probes for\ntargeting purposes, it was the first to report the foundational use of carboxypeptidases in this context. The authors must\nclarify this in their manuscript and properly acknowledge the prior contribution.\n3. Page 2- The statement “ available linker systems for alcohol attachment …….none of these functional groups combine\nbroad applicability, stability in serum, and efficient traceless release” requires further contextual support, particularly given\nthe team's expertise in ADC development. For instance, the FDA022-BB05 s-amino carbamate self-immolative spacers\nused for conjugating aliphatic alcohol-based payloads have demonstrated a manageable safety profile and promising\nantitumor activity in metastatic breast cancer (Breast Cancer, Metastatic, Volume 35, Supplement 4, S1420–S1421,\nDecember 2024). Additionally, I recommend citing J. Med. Chem. (2024, 67, 21, 19852–19873).\n4. on page 6, paragraph 2, the phrase “the free carboxylic acid in dialanine linker 12” appears to be incorrect. I believe the\nauthors meant dialanine linker 11.\n5. In Fig. 4e, the authors compare the stability of trastuzumab deruxtecan, which uses a cleavable peptide-based linker\n(GGFG), with construct 15, which utilizes a phosphorus-based linker system. Their data demonstrate that, in terms of ex vivo\nstability (rat serum, Fig. 4e), Tras-15-DXd is more stable than the FDA-approved Enhertu. Just out of curiosity, is GGFG\nmore stable than the Valine-Citrulline (Val-Cit) linker in the presence of carboxylesterase 1C (Ces1C) in mouse and rat\nplasma? If not, as carboxylesterase 1C in mouse and rat plasma is known to affect the stability of peptide-based\nlinkers, can this ex vivo study be considered a valid measure of stability of these two linker systems?\nExperimental section also well written and comprehensive.\nCongratulations again to the team involved in this project. I thoroughly enjoyed reading this work and am confident that it will\ngenerate significant interest among those actively involved in the drug delivery research field."}, "author_response": "We sincerely thank the reviewer for the positive and encouraging feedback, as well as the thoughtful\nsuggestions that helped to improve the clarity and impact of our manuscript. We have addressed each\npoint in detail below.\nA few small corrections/suggested edits:\n1. Comment: In several of the figures the authors refer to what R is for the vinyl phosphonate. If there\nis room, perhaps also add this R=PEG24 to Fig. 3a. and 4a\nResponse: We thank the reviewer for this suggestion. We have added “PEG24” directly to the labels\nin all concerning figures, as requested, to enhance clarity.\n2. Comment: Typo: \"summerized\"\nResponse: Corrected. The word has been changed to “summarized” in the revised manuscript.\n3. Comment: Incorrect figure reference: “Fig. 4d” for SN38, likely meant 3d\nResponse: The reviewer is correct. We have revised the reference to correctly cite Figure 3d, showing\nthe table of HIC retention times for different SN38 LPs.\n4. Comment: consider showing HIC for TD and/or comment on why is the PK worse for TD vs. T-15-\nDxd\nResponse: We thank the reviewer for this suggestion. We now included a brief explanation in the\nResults section addressing the pharmacokinetic differences between TD and T-15-DXd, referring to\ndifferences in payload hydrophobicity and linker composition. Additionally, comparative HIC traces for\nboth constructs have been added to Fig S4 for transparency.\n5. Comment: obtaining DAR8 ADCs for all of these new payloads is quite impressive. Perhaps\ncomment on (or provide range) of logDs. Table of HIC values for the resulting conjugates would also\nbe informative\nResponse: We appreciate the reviewer’s interest. We have included a new supplementary figure (Fig.\nS7) summarizing the CLogP values and HIC retention times for all payloads and resulting ADCs\ndescribed in this section of the manuscript. For the sake of simple comparability, we decided to include\nthe CLogP values instead of the logD suggested by the reviewer. We still believe that the outcome\nwould be very similar. The result helps contextualizing the impact of the linker and payload properties\non conjugate hydrophobicity. We show that linker 9 and 15 are capable of producing ADCs from\npayloads with a range of different CLogP values. Interestingly, even though no clear correlation between\nHIC retention and CLogP is seen, we still observe the expected trend of more hydrophobic payloads\nleading to more hydrophobic DAR 8 ADCs, judged by the HIC retention. A supporting figure as well as\na sentence to the main manuscript has been added.\n6. Comment: Typo in Fig. 7e\nResponse: Thank you for spotting this. The typo has been corrected, and we now correctly reference\nFig. 5d\nWe thank Reviewer #2 for the detailed and constructive feedback. We appreciate the recognition of the\nstudy’s potential and have carefully addressed each of the reviewer’s concerns below. We have revised\nthe manuscript accordingly to enhance clarity and completeness.\n1. Comment: Lack of Comprehensive Background Information\nWhile the Introduction briefly mentions the importance of linker design, it lacks sufficient background to\ncontextualize the study's significance. The impact of linker design on ADC stability and efficacy could\nbe strengthened by citing more recent and relevant literature. For instance, the report on Exo-linkers\n(J. Med. Chem. 2024, 67, 20, 18124–18138), which shares similarities with the current study's compact\nand externalized linker design, should be cited. Additionally, including references to tandem-cleavable\nlinkers and Glu-Val-Cit peptides designed for carboxylesterase resistant linkers would further enhance the\nrelevance and impact of the Introduction.\nResponse: We thank the reviewer for this valuable suggestion. We have expanded the Introduction to\nprovide more comprehensive background on linker technologies. Specifically, we now included the\nfollowing studies in the introduction of the manuscript:\n• recent work on Exo-linkers that reduce undesired linker cleavage and payload release (J. Med.\nChem. 2024, 67, 20, 18124–18138)\n• Work on tandem-cleavable linkers for enhanced stability and selective release (Bioconjugate\nChemistry 2021 32 (4), 746-754)\n• Work on Glu-Val-Cit-based peptides, engineered for carboxylesterase resistant linkers (Nat.\nCom. Nat Commun 9, 2512 (2018)\n2. Comment: Inadequate Stability Testing\nGiven that the linker relies on ester hydrolysis as a trigger, stability testing against key enzymes such\nas carboxylesterase and human neutrophil elastase is essential. Without this data, the reliability of the\nlinker design in vivo remains unsubstantiated. Comprehensive enzyme susceptibility assays should be\nincluded to support the claim of linker stability.\nResponse: We thank the reviewer for this valuable suggestion. While we did not perform direct enzyme\nsusceptibility assays in the initial submission, we conducted serum stability and in vivo pharmacokinetic\nanalyses that provide insights into the stability of the linker system under physiologically relevant\nconditions. Specifically, linker 3–SN38 demonstrated higher stability in rat serum over a 7-day\nincubation period, as shown in Figure 2f, compared to current linker systems for SN38, as that used in\nTrodelvy (also shown in Figure 2f). Similarly, linker 15–DXd showed robust stability in rat serum over\n7 days, investigated by MS, and maintained its integrity throughout a 21-day PK study in mice, as\nassessed by intact ADC ELISA (Figures 4e and 4f). These findings support the in vivo robustness of\nour linker designs and suggest limited susceptibility to premature degradation under physiological\nconditions.\nTo further strengthen this dataset as suggested by the reviewer, we have now included further ex vivo\nstability data in human serum.\nMoreover, we performed enzyme susceptibility assays on the lead linker-payloads 3–SN38, 9–SN38,\nand 15–DXd using human neutrophil elastase. We also tested the corresponding ADCs (Sacituzumab-\n3–SN38, sacituzumab-9–SN38, trastuzumab-15–DXd and trastuzumab-15-gemcitabine) for\ndegradation upon exposure to neutrophil elastase. In these assays, linker–payloads 3–SN38 and 9–\nSN38 were found to be susceptible to ester cleavage by neutrophil elastase, resulting in measurable\nSN38 release. This indicates that esterase-cleavable linkers, while stable under serum and PK\nconditions, may still be vulnerable to specific enzymatic activity under certain conditions. In contrast,\nLP 15–DXd exhibited no detectable degradation in the presence of human neutrophil elastase, even at\nconcentrations where the positive control (NAcCys-MC-VC-PAB-MMAE) was efficiently cleaved to\nrelease the expected NH -Citrulline-PAB-MMAE fragment. Still, it should be noted that the ADCs tested\n2\nin the neutrophil elastase susceptibility assay demonstrated markedly higher stability against esterase-\nmediated cleavage compared to the isolated linker-payloads. The approved ADC Adcetris, which\ncontains the MC-VC-PAB-MMAE linker, known to be labile under these conditions, underwent extensive\ndegradation, releasing ~90% of the expected NH₂-Citrulline-PAB-MMAE fragment over 24 hours. In\ncomparison, the phosphoramidate-linked ADCs sacituzumab-3–SN38, trastuzumab-15–DXd, and\ntrastuzumab-15-gemcitabine remained fully intact under identical conditions. Only sacituzumab-9–\nSN38 showed partial susceptibility, with moderate isopropyl ester cleavage and ~25% SN38 release.\nThese results suggest that while isolated linker–payloads containing cleavable esters such as in LPs 3\nor 9 may be susceptible to degradation by certain esterases, the dipeptide-based LP 15 exhibits greater\nresistance. Furthermore, the corresponding ADCs demonstrate even higher stability in the presence of\nneutrophil elastase, highlighting an improved enzymatic robustness of our phosphoramidate linker\ndesigns compared to conventional VC-PAB linkers.\nThe additional serum stability studies and experimental setup for the human neutrophil elastase assay\nare included in Fig. S6 and in the experimental section 3.7. A paragraph that discusses the susceptibility\ntowards cleavage by these enzymes has been added to the manuscript.\n3. Comment: Additional Data for Figure 3\nWhile Figure 3 illustrates differences in degradation rates among various esters, additional supporting\ndata is required to substantiate these observations. Including time-course LC-MS data to track\ndegradation would provide more robust evidence for the claims made in the manuscript.\nResponse: We thank the reviewer for this constructive suggestion. To further support the observations\npresented in Figure 3, we conducted additional experiments in which the Linker-Payloads 3–SN38, 9-\nSN38, and 10–SN38 were incubated with porcine liver esterase (PLE), and SN38 release was\nquantitatively monitored over time using LC-MS coupled with UV detection. The resulting time-course\ndata are now included in the revised Supporting Information as Fig. S3 and a sentence for discussion\nhas been added to the main manuscript.\nThe results clearly demonstrate that SN38 release is enzyme (esterase) dependent. The isopropyl\nesters 9–SN38 and 10–SN38 are undergoing more rapid enzymatic cleavage by PLE compared to the\ntert-butyl ester 3–SN38 in line with previous literature reports on ProTides. The observed differences in\nrelease rates highlight the tunability of the linker design, allowing to optimize stability and release rates\nthrough rational structural variation. The favorable release rates observed for 9–SN38 support our\nrational to prioritize this design as the lead linker candidate for aromatic payloads, as it achieves a\nbeneficial balance between chemical stability and enzyme-triggered release.\n4. Comment: Lack of Consideration for Alternative Cleavage Mechanisms\nThe study focuses solely on the targeted cleavage mechanism of the linker but fails to rule out\nalternative pathways. Proteases such as cathepsins (e.g., Cathepsin B, L, K, and S), known for their\nbroad substrate specificity, could potentially cleave the linker. Comprehensive protease susceptibility\ntesting would help establish the specificity of the designed linker.\nResponse: We thank the reviewer for raising this important point. To clarify, our study does not propose\na single, specific enzyme-targeted cleavage mechanism. We believe that cancer specificity in the ADCs\ndescribed herein originates from the tumor targeting antibody and not from a tumor-specific release\nfrom the phosphoramidates. A clarifying statement has been added to the manuscript.\nLinkers 3–10 were designed to undergo cleavage via ester hydrolysis, following a ProTide-like self-\nimmolation mechanism. This design rationale is supported by our updated experiments, now included\nin the revised SI, which demonstrate esterase-mediated cleavage of these linkers (Fig. S3). In contrast,\nlinkers 14 and 15 were purposefully engineered for cleavage by proteolytic enzymes, while control\nlinkers 16 and 17 contain cleavable motifs that do not result in payload release, as shown in Figure 4d.\nIn response to the reviewer’s suggestion regarding enzymatic pathway identification, we performed\nadditional experiments with Cathepsin A (CatA), a lysosomal carboxylesterase known for its role in\nProTide activation. We evaluated the CatA-mediated cleavage of the Linker-Payloads 3–SN38, 9–\nSN38, and 15–DXd. The results, included in the revised SI (Fig. S5), revealed clear differences in CatA-\nmediated cleavage efficiency among the tested LPs. LP 9–SN38 and LP 15-DXd were efficiently\ncleaved by CatA. In contrast, LP 3–SN38, exhibited minimal cleavage, releasing only 7% SN38,\nindicating it is a poorer substrate for CatA.\nTaken together, we believe that the additional experiments with PLE (Fig. S3) and Cathepsin A (Fig.\nS5) clearly underline our proposed mechanism of ester/amide cleavage that is absolutely needed to\nsubsequently release the payloads from the phosphoramidate. However, this initial cleavage is not\nspecific to a certain esterase or protease and can be triggered by several enzymes. Very similar\nobservations have been made for classical ADC cleavage sites such as VC-PAB\n(https://doi.org/10.1158/0008-5472.CAN-17-2391).\n5. Comment: Unclear Intracellular Mechanism of Payload Release\nThe manuscript does not clarify the intracellular compartment (e.g., lysosome, late endosome) where\nlinker cleavage and payload release occur. Imaging studies using fluorescent probes could provide\ninsights into the timing and location of payload release, adding depth to the mechanistic understanding\nof the linker.\nResponse: We appreciate the reviewer’s thoughtful suggestion. While imaging studies, to pinpoint the\nintracellular site of linker cleavage, would indeed provide interesting mechanistic insights, we think that\nsuch investigations are outside the scope of the current study. As shown in the following examples, the\ndesign of such fluorescent probes is by far not trivial and warrant a full-paper publication by itself\n(https://doi.org/10.1016/j.chembiol.2019.09.008 or https://doi.org/10.1021/jacs.4c16842). Our primary\nobjective in the current study was to identify and characterize novel linkers that allow conjugation of\nnovel ADC payloads with alcohol functions for the generation of potent and selective ADCs that are\ncapable of targeted payload release, as demonstrated by in vitro and in vivo efficacy and stability\nassessments. Detailed localization studies could form the basis for future follow-up studies.\n6. Comment: Insufficient Comparison with T-DXd\nThe claim that the presented ADC is more hydrophilic than T-DXd lacks quantitative validation.\nComparative data, such as HIC retention times, would provide a clear basis for this assertion. Including\nsuch data in Figure 3d or as a supplementary figure is recommended.\nResponse: We thank the reviewer for this valuable suggestion. In response, we have now included Fig\nS4c, which presents comparative hydrophobic interaction chromatography (HIC) traces for tras-15-DXd\nand T-DXd under identical conditions. These data clearly show a shorter retention time for tras-15–\nDXd, confirming its increased hydrophilicity relative to T-DXd. We believe this directly supports our\nclaim and provides the quantitative basis requested by the reviewer.\n7. Comment Ambiguities in SI HPLC Data\nThe HPLC data in the supplementary information (SI) for antibodies such as Sacituzumab and\nDatopotomab contain unclear peaks, suggesting potential impurities. The extra peaks observed in\nBrentuximab-ADC may indicate non-specific conjugation. Additional analyses, such as LC-MS or\nimproved HPLC separation, are necessary to resolve these ambiguities.\nResponse: We thank the reviewer for carefully looking at our characterization data. We would like to\nclarify that the additional peaks observed in the HPLC chromatograms of sacituzumab, datopotomab,\nand brentuximab ADCs are already present in the parent monoclonal antibodies prior to conjugation.\nThis indicates that these peaks are not a result of non-specific conjugation or linker instability but rather\nreflect inherent heterogeneity in the native antibody preparations. This heterogeneity is likely to originate\nfrom the transient expression of the antibody leading to differences in post-translational modifications\nincluding glycosylation patterns leading to differences in the parental antibody and the ADC. While the\nuse of such transient material for such a discovery study is broadly accepted, the quality and\nhomogeneity of the antibody would certainly be optimized for clinical use by generating stably\nexpressing single clones. To further support the specificity and homogeneity of the conjugation process,\nwe have now included mass spectrometry (MS) spectra for the ADCs in the revised SI (Table 2). This\nMS data confirms clean and specific conjugation, yielding well-defined ADC species with a drug-to-\nantibody ratio (DAR) of 8. We believe this new data directly addresses the reviewer’s concern and\nreinforces the integrity of our conjugation strategy.\n8. Comment: Absence of Safety Evaluations\nThe lack of safety evaluations is the most significant concern in this manuscript. The study does not\ninclude data on normal cell uptake or off-target toxicity, both of which are critical for assessing the\nspecificity and safety of the linker and payload. To align with the standards of Nature Communications,\ndose-limiting toxicity (DLT) assessments using non-human primate models (e.g., monkeys) are\nrecommended. These data would substantiate claims of safety and tolerability.\nResponse:\nWe agree with the reviewer and concur with the importance of safety evaluations in ADC development.\nImportantly, all Linker-Payloads in the manuscript were evaluated for non-specific cytotoxicity in vitro\nusing isotype control antibodies, and these results are presented as selectivity in Figures 3c, 4d, and\n5d as well as in SI fig. S1, S2, S4 and S8. Additionally, all in vivo efficacy studies included isotype-ADC\ncontrols, which showed no activity further supporting the specificity of our constructs. To further support\nthe manuscript with safety relevant data, body weights of all treated mice that were monitored in the in\nvivo efficacy experiments, indicating good tolerability of the ADCs, have been added to the SI (fig. S10).\nWhile we agree that comprehensive safety assessments, including DLT studies in non-human primates,\nare valuable for late-stage development, we do believe that in vivo toxicity experiments with\ncamptothecin-based ADCs extend significantly beyond the exploratory scope of the current manuscript\nfor the following reasons:\n1. According to the directive of the European Medicines Agency (EMA) for ethical use of animals in\nmedicine testing and the 3R principles of replacing, reducing and refining the use of animals in\npreclinical studies, we would only apply non-human primate models if absolutely necessary to enable\nclinical testing in human. However, since the current manuscript does not describe a novel drug\ncandidate, soon to be enter into clinical testing, but a novel methodology to conjugate drugs to\nantibodies, we do not deem NHP studies necessary to support our claims.\n2. The primary aim of our work is to present novel linker technology that enables the efficient and stable\nconjugation of hydroxy-functionalized payloads to antibodies. We believe that this approach significantly\nbroadens the range of usable payloads in ADC development, as conventional linker systems are mostly\nlimited to amine-containing payloads. In this regard, our central claim is not to demonstrate superiority\nof “bio-better” candidates over approved ADCs such as Enhertu or Trodelvy, but rather to showcase a\ntechnological innovation that enables conjugation of payloads previously incompatible with standard\nlinker technologies. Hence, SN38 and DXd have only been used in our studies to benchmark our novel\nlinker technology that we would like to apply finally to broaden the scope of conjugatable drugs beyond\ncamptothecins, as showcased by the 8 novel payloads within this study.\n3. Camptothecin ADCs are tolerated in very high doses in rodents, making H2H comparisons\nquestionable due to the large disconnect to tolerability in human and since it is almost impossible to\ndefine the MTD for an ADC in rats or mice.\nNevertheless, in response to the reviewer’s concern, we have strengthened the safety evaluation of our\nADCs and included a comparison of Saci-3–SN38 and Saci-9–SN38 against the clinically approved\nADC Trodelvy on a panel of normal human primary cell lines to the revised SI (Fig. S1C). This\ncomparison provides a direct benchmark for assessing off-target cytotoxicity and demonstrates the\nimproved selective activity of our linker system relative to that of an established therapeutic. This in vitro\ncell panel provides in our view a suitable replacement for an in vivo toxicity study.\nStill, we understand potential limitations of in vitro toxicity studies and therefore the reviewers’ desire to\ninclude in vivo toxicity data into the manuscript. However instead of using a camptothecin-based ADC\nfor the three reasons outlined above, we performed a rodent toxicity study withtTras-15–Gemcitabine,\nan ADC that is novel in terms of both, Linker and Payload, and therefore warrants further toxicity\nevaluations in vivo according to the 3Rs. In this study, no signs of toxicity were observed at all tested\ndose levels as can be seen in the novel Fig. S9, giving a >7.5x therapeutic window in rodents, when\njust comparing the efficacious dose of 20 mg/kg with the highest dose tested in the toxicity study of 150\nmg/kg. These in vivo findings further support the tolerability of our linker-payload combination in a\npreclinical setting.\n9. Comment: Validation of Novelty and Practicality\nWhile the authors emphasize the novelty of the phosphoramidate-based linker derived from ProTide\ntechnology, its distinctiveness compared to existing technologies remains unclear. Furthermore, while\nthe application of diverse payloads is intriguing, more detailed evaluation of clinically relevant examples,\nincluding toxicity profiles and target specificity, is needed to validate the practicality of this approach.\nResponse: We appreciate the reviewer’s thoughtful comments. The phosphoramidate-based linker\ntechnology presented in this manuscript is distinct from existing linker platforms as it enables a general\nand modular conjugation of alcohol-containing payloads. This new self immolative moiety is generally\napplicable to a wide range of alcohol-functionalized drugs, including some with novel modes of action\nthat have previously not been explored in the ADC context. This flexibility represents a key innovation,\nas it significantly expands the chemical space of usable payloads beyond those compatible with\ntraditional linkers using other functional groups for hydroxy attachment. A sentence summarizing this\nhas been added to the conclusion for clarity.\nWe appreciate all comments of the reviewer that helped us to improve the quality of the manuscript\nmarkedly and hope that the additional ex vivo stability studies in human serum, the enzyme\nsusceptibility assays towards human neutrophile elastase and human Cathepsin A, the in vitro toxicity\nassessments with healthy human primary cells and finally the in vivo toxicity study with tras-15-\ngemcitabine in rats helps to validate the practicality of our approach.\nWe thank the reviewer for the thoughtful and encouraging feedback, as well as the insightful\nsuggestions to improve the manuscript. We are pleased that the reviewer recognizes the potential of\nour phosphoramidate-based linker system to expand the range of payloads in ADCs. Below, we\nrespond to each comment in detail.\n1. Comment: Additional citations from recent literature on ProTides\nThe authors are leveraging the clinically validated ProTide technology; however, they should cite recent\nrelevant works on ProTides, such as Jin et al., Acta Pharmaceutica Sinica B (2024) and J. Am. Chem.\nSoc. (2024, 146, 1, 521–531). A more thorough literature review is recommended to strengthen the\nmanuscript's foundation.\nResponse: We appreciate this recommendation. We have now included citations to the requested\nrecent works on ProTide technology, and expanded our literature review to strengthen the growing\nimportance of this technology and our manuscript specifically:\n• Jin et al., Acta Pharmaceutica Sinica B, 2024, was included in the introduction\nReferences for the three FDA approved ProTide anti viral drugs were included\n• sofosbuvir (J Med Chem. 2010;53(19):7202–7218)\n• tenofovir alafenamide (Antimicrob Agents Chemother. 2005;49(5):1898–1906)\n• remdesivir (Med Chem. 2017;60(5):1648–1661 )\nThree references were included to highlight three ProTide based anti cancer drugs that are under\nclinical investigation:\n• Biochem Pharmacol. 2001;61(2):179–189\n• J Med Chem. 2014;57 (4):1531–1542\n• Cancer Chemother Pharmacol. 2023 May;91(5):401-412\n• J. Am. Chem. Soc. 2024, 146, 1, 521–531, was included in the results section that discusses\nthe di-peptide release handles\n2. Comment: The authors highlight two key distinctions between their design and clinically\nvalidated ProTides\nA) Their design incorporates a conjugation handle into the core phosphorus(V) [P(V)] structure, allowing\ncovalent attachment to targeting moieties such as antibodies. To demonstrate the superiority of their\ndesign, the authors synthesized multiple linker systems conjugated to antibodies and conducted\nthorough studies to evaluate their properties and performance. I agree with their claim and strongly\nbelieve this innovation significantly advances the ProTide concept into the realm of ADC development.\nI would like to congratulate the team on conducting such a comprehensive study to support their claim.\nB) The authors claim that, unlike traditional nucleoside-based ProTides—which rely exclusively on\nesterase-mediated cleavage following passive cytosolic uptake—their phosphoramidate-based system\nallows for payload release via multiple intracellular and endosomal triggers. Specifically, their lead linker\nsystem, based on Ala-Ala for traceless release of gemcitabine, is supported by extensive data\ndemonstrating protease-mediated cleavage. However, it is essential for the authors to revise their\nmanuscript and appropriately cite J. Am. Chem. Soc. (2024, 146, 1, 521–531), which was the first study\nto utilize carboxypeptidases to enhance ProTide technology. While that study employed fluorescence\nprobes for targeting purposes, it was the first to report the foundational use of carboxypeptidases in this\ncontext. The authors must clarify this in their manuscript and properly acknowledge the prior\ncontribution.\nResponse: We thank the reviewer for summarizing the key distinctions so clearly. We have revised the\nResults section and now explicitly state that our design extends the ProTide concept into the ADC space\nwhile we acknowledge the carboxypeptidase-activated ProTide system reported by JACS (2024, 146,\n1, 521–531) as the first report utilizing carboxypeptidases in ProTide activation.\n3. Comment: Page 2- The statement “ available linker systems for alcohol attachment …….none of\nthese functional groups combine broad applicability, stability in serum, and efficient traceless release”\nrequires further contextual support, particularly given the team's expertise in ADC development. For\ninstance, the FDA022-BB05 s-amino carbamate self-immolative spacers used for conjugating aliphatic\nalcohol-based payloads have demonstrated a manageable safety profile and promising antitumor\nactivity in metastatic breast cancer (Breast Cancer, Metastatic, Volume 35, Supplement 4, S1420–\nS1421, December 2024). Additionally, I recommend citing J. Med. Chem. (2024, 67, 21, 19852–19873).\nResponse: We thank reviewer for the insightful comment and for suggesting these valuable references.\nWe agree that the FDA022-BB05 s-amino carbamate self-immolative spacer represents a noteworthy\nalternative to the GGFG-DXd linker in deruxtecan used in Enhertu, demonstrating both serum stability\nand efficient release of DXd in clinical settings. However, we would like to clarify that this carbamate-\nbased system is specifically tailored to the structural features of DXd, particularly its glycolic acid amide\nmoiety, which is essential for its successful release. As such, its applicability to a broader range of\nalcohol-containing payloads is limited. We incorporated the recommended references to provide better\nrepresentation of linkers for alcoholic payloads in the revised manuscript and added a sentence\nmentioning the successful conjugation of the primary alcohol of Dxd via hemiaminals to the introduction.\n4. Comment: on page 6, paragraph 2, the phrase “the free carboxylic acid in dialanine linker 12”\nappears to be incorrect. I believe the authors meant dialanine linker 11.\nResponse: Correct. We thank the reviewer for pointing this out. The error has been corrected, and\nthe reference now reads: “the free carboxylic acid in dialanine linker 11”.\n5. Comment: In Fig. 4e, the authors compare the stability of trastuzumab deruxtecan, which uses a\ncleavable peptide-based linker (GGFG), with construct 15, which utilizes a phosphorus-based linker\nsystem. Their data demonstrate that, in terms of ex vivo stability (rat serum, Fig. 4e), Tras-15-DXd is\nmore stable than the FDA-approved Enhertu. Just out of curiosity, is GGFG more stable than the Valine-\nCitrulline (Val-Cit) linker in the presence of carboxylesterase 1C (Ces1C) in mouse and rat plasma? If\nnot, as carboxylesterase 1C in mouse and rat plasma is known to affect the stability of peptide-based\nlinkers, can this ex vivo study be considered a valid measure of stability of these two linker systems?\nResponse: We thank the reviewer for the thoughtful question and for raising an important point\nregarding the interpretation of ex vivo stability data. In our study, the primary instability observed for\nEnhertu in rat serum appears to be related to the retro-Michael addition of the maleimide moiety in the\nGGFG linker, rather than enzymatic cleavage. While carboxylesterase 1C (Ces1C) is indeed known to\ncompromise the stability of certain peptide-based linkers, particularly Val-Cit linkers, in rodent plasma,\nwe cannot definitively conclude whether the GGFG linker itself is a substrate for Ces1C, as this has not\nbeen fully characterized to our knowledge. In contrast, our phosphoramidate-based linker system\ndemonstrated higher stability with only one major degradation where we detect the hydrolysis of DXd\nfrom the phosphoramidate.\nTo provide a more comprehensive view of the stability of these linker systems across species, we\nhave included additional human serum stability data in the revised Supporting Information. We hope\nthis offers a more complete perspective on the comparative robustness of the linker technologies\ndiscussed.\nExperimental section also well written and comprehensive.\nCongratulations again to the team involved in this project. I thoroughly enjoyed reading this work and\nam confident that it will generate significant interest among those actively involved in the drug delivery\nresearch field.\nWe thank the reviewer again for the kind and encouraging feedback. We truly appreciate the positive\nremarks."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The additional data and edits have thoroughly address my concerns, improved the manuscript and I support publication.", "reviewer_2": "The authors have appropriately addressed most of the concerns raised in the initial review, and the manuscript has\nimproved. However, the reviewer believes the issue of safety evaluation remains insufficiently resolved.\nWhile the reviewer appreciates the authors’ additional safety data, the current dataset still does not address the central\n the central\nconcerns raised in the initial review. The rodent toxicity study using a gemcitabine ADC is not mechanistically representative\nof the key payloads used in this work (SN-38, DXd), and thus fails to de-risk the core linker-payload combinations. I\nrecommend including at least one rodent tolerability study with a representative camptothecin-based ADC (e.g., Saci–SN38\nor Saci–DXd) to better support safety claims.\nAdditionally, given that DXd-based ADCs such as T-DXd are associated with ILD through uptake by alveolar macrophages,\nthe authors should consider performing in vitro toxicity and drug release assays using THP-1–derived or primary human\nmacrophage-like cells to evaluate pulmonary off-target effects.\nFinally, to substantiate the claimed improvement in selectivity over Trodelvy, IC₅₀ values and selectivity indices for tumor vs.\nnormal cells should be provided. These targeted additions would strengthen the translational value of the manuscript and\nalign it with the standards of Nature Communications.", "reviewer_3": "I have reviewed the revised manuscript and I am satisfied with the corrections and clarifications provided by the authors. The\nmanuscript has been improved and addresses my concerns raised in the initial review. I support its acceptance for\npublication in its current form."}, "author_response": "We thank the reviewer for the positive assessment and the support for publication.\nWe thank the reviewer for acknowledging the improvements made to the manuscript and\nthe efforts to address the initial concerns. Regarding the safety evaluation, we believe that\nthe data, including in vivo tolerability studies with a gemcitabine-based ADC, adequately\naddress safety evaluation within the scope of this study. As outlined in detail below, our\nmanuscript focuses on the development and validation of a versatile linker platform for\nnovel alcohol-containing payloads, beyond the established topoisomerase-I-Inhibitors,\nthat we mainly used as proof-of-concept.\nWe thank the reviewer again for highlighting the importance of safety evaluations in the\ndevelopment of ADCs, which we do generally agree on. Still, we would like to stress that\nSN38 and Dxd are in our view not the key payloads in this study. Rather, they are used\nas established tool compounds for the first development steps, for example, to identify the\noptimal phosphoramidate linker among many possible variations. The primary focus of\nour work is to apply the modular linker platform for alcohol-containing cytotoxins, to a\nbroad range of next-generation ADC payloads. A goal, that we already defined in first\npart of the title of our Manuscript: “Expanding the payload scope in antibody-drug\nconjugates:...”. Hence, we respectfully disagree with the reviewer’s assertion that the\ngemcitabine-based ADC is not mechanistically representative of our linker technology,\nsince expanding the payload scope means expanding beyond the established SN38 and\nDxd.\nNotwithstanding the above, we do not consider additional supportive rodent tolerability\nstudies with a SN38- or Dxd-based ADCs as compliant with the 3R principles of replacing,\nreducing and refining the use of animals in preclinical studies. To support that opinion, we\nsummarized available tolerability data for SN38 and Dxd based ADCs in the table below.\n[Table omitted for brevity]\nLooking at the available toxicity data, this table highlights two important points in our\nopinion:\n1) The rodent toxicity studies are not predictive of the situation in the clinic\nFor the three ADCs in the table that are approved (meaning a reliable human dose is\navailable), the tolerated dose in rodents is much higher compared to the clinical dose.\nApplying well-established allometric scaling (doi: 10.4103/0976-0105.177703), we did\nassess the Following: The 750 mg/kg in mice for Sacituzumab-Govitecan correspond to\n~61 mg/kg in human, a dose that is 6-times higher than what is actually tolerated in the\nclinic. The 197 mg/kg and 200 mg/kg in rats for Trastuzumab-Deruxtecan and\nDatopotamab-Deruxtecan, respectively, correspond to ~32 mg/kg in human, a dose that\nis also 5-6-times higher than what is actually tolerated in the clinic.\n2) The tolerated dose in rodents is not influenced by the linker that is used in\nthe ADC\nAll SN38-based ADCs exhibit the same very high tolerability of 750 mg/kg in mice. The\nsame is true for the Dxd-based ADCs that are usually tolerated at doses of 200 mg/kg in\nrats. The only exception might be FZ-AD005 with 100 mg/kg, however, according to the\nliterature, this ADC has not been tested at higher doses. Importantly, in the case of SN38,\nthese high doses are tolerated with Govitecan, as a very unstable linker system and in\ncase of Dxd with more stable linker systems such as in Deruxtecan and in FZ-AD005.\nEven unconjugated SN38 and Dxd are tolerated in doses, that are even higher compared\nto the tolerable payload equivalent doses of the respective ADC in rodents. From this, we\nbelieve that Camptothecins, linked via any linker to an antibody, would always result in a\nvery high tolerable dose in rodents, making such studies not predictive. In addition, during\nour extensive literature search, we could not find any example in which a liability in the\nlinker system would change the tolerability of Camptothecin-based ADCs in rodents.\nSummarizing the arguments above, a rodent toxicity study with a SN38- and a Dxd-based\nADC would not be predictive for the situation in human and not be influenced by a novel\nlinker system that we describe herein. Hence, we cannot justify performing such study,\nsince it would not be compliant with the 3R principles of replacing, reducing and refining\nthe use of animals in preclinical studies, according to the directive of the European\nMedicines Agency (EMA) for ethical use of animals in medicine testing. We hope that this\nargumentation can be followed by the reviewer and appreciate again the thorough\nevaluation of our manuscript.\nWe appreciate the reviewer’s concern regarding T-Dxd associated ILD. However, we think\nthat the suggested experiments are outside the scope of our current manuscript. As\noutlined above, the goal is not to address or resolve the known toxicity profile of Dxd-\nbased ADCs such as T-Dxd, but to apply our novel linker to expand the payload space in\nADCs.\nMoreover, looking at very recently published explanations for the mechanistic rational\nbehind ILD cases following treatment with T-Dxd, there is not much evidence that the\nsuggested in vitro assays with macrophage-like cells could reveal an influence of our novel\nlinker system on the root causes of ILD.\nWei et al (doi.org/10.1158/0008-5472.CAN-24-2021), for instance, identified that off-target\nendocytosis of T-Dxd by perivascular niche–resident alveolar macrophages, followed by\nthe triggered release of free Dxd causes a bystander-killing effect, that serves as the main\nmechanism responsible for T-Dxd–induced ILD. Since we use the same antibody\n(trastuzumab) with the same unmodified Fc part and do not claim that our linker system\ncould improve off-target endocytosis of the whole ADC, mediated by Fc–FcγR\nengagement, as described by Wei et al. and Koganemaru et al. (doi.org/10.1158/1535-\n7163.MCT-24-0267), we would not expect any differences in the suggested assays H2H\nto T-Dxd.\nFor these reasons, we consider the suggested experiments valuable in the broader\ncontext of Dxd-based ADC safety, but not directly relevant to the objectives or claims of\nthis study.\nWe appreciate this suggestion and have now included side-by-side IC₅₀ values as\nmeasure of cellular activity and selectivity indices comparing Saci-3-SN38 and Trodelvy\nacross both tumor cell lines and representative non-tumorigenic healthy cells. The Saci-\n3-SN38 ADC demonstrated a 10-fold improvement in tumor vs. normal cell selectivity\nrelative to Trodelvy, consistent with improved linker stability and enhanced targeted\npayload delivery. New graphs were added as Supplementary figure 1d.\nWe thank the reviewer for the positive assessment and the support for publication."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The additional data and edits have thoroughly address my concerns, improved the\nmanuscript and I support publication."}, "author_response": ""}]

Decision letter