Brentuximab and derivatives show high affinity binding to CD30 and strongly internalize into CD30-positive cells

TUB-010 is a next-generation antibody-drug conjugate (ADC) targeting CD30 expressed on various hematopoietic malignancies such as Hodgkin lymphoma. Among the therapeutic options for patients with relapsed and refractory CD30-positive cancers is brentuximab vedotin (Adcetris), a monomethyl auristatin E (MMAE)-delivering anti-CD30 ADC with a mean drug-to-antibody ratio of 4. Adcetris exhibits a high response rate at the cost of significant toxicities, likely driven by the payload MMAE and the instability of the maleimide conjugation chemistry. TUB-010 uses the same antibody and payload as Adcetris but is based on the Tub-tag conjugation strategy, which stably attaches MMAE to the hydrophilic Tub-tag peptides on the light chains via chemoenzymatic conjugation. This new technology enables the generation of a homogeneous and site-specific drug-to-antibody ratio 2 ADC with unique biophysical properties. TUB-010 demonstrates similar binding and lysosomal release characteristics as Adcetris, which translates into comparable in vitro cytotoxicity on CD30-positive cell lines when normalized to the MMAE concentration. Importantly, TUB-010 exhibits higher stability with negligible premature deconjugation in circulation and reduced aggregation, as well as lower nonspecific cytotoxicity on target-negative cells compared with Adcetris. As a consequence, TUB-010 induces superior tumor control compared with Adcetris when dosed at equal MMAE concentrations in vivo and also lower toxicity and higher tolerability in rodents and nonhuman primates. Taken together, TUB-010 is a novel, potential best-in-class anti-CD30 ADC with improved biophysical properties designed to deliver MMAE with higher precision and a wider therapeutic window than Adcetris using Tub-tag technology. Therefore, TUB-010 may increase the clinical benefit of anti-CD30 ADC therapies.

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. Approved antibody–drug conjugates (ADCs) remain constrained by a limited repertoire of payloads with restricted modes of action. Here, the authors present phosphoramidate-based self-immolative linker units that facilitate stable attachment in serum and traceless drug release in the target cell from aliphatic and aromatic alcohols with various modes of action.

Heterobifunctional small molecules recruiting the proteasomal machinery enable the targeted protein degradation (TPD) of selected proteins and considerably broaden the druggable space for therapeutic use. Many such molecules have seen fantastic success in preclinical and clinical developments, e.g., for targeted degradation of specific oncoproteins that were previously inaccessible for cancer therapy. Even though early hits are quickly generated by adapting the modular concept of bifunctional degraders, most of such molecules are against the central rules of a “drug-like” molecule: they are usually larger in size (800-1500 Da) and often lack sufficient solubility. These early bifunctional degraders hits require exhaustive structure optimization in each case to optimize pharmacokinetics (PK) profiles and bioavailability. Here, we report Tubulis’ antibody-degrader conjugates that exploit bifunctional degraders as high-potency bioactive payloads to be released specifically inside cells that are targeted by the antibody. Our linker technology ensures high degrader-to-antibody ratios (DARs) and excellent linker stability in blood plasma. Moreover, we liberate the degrader after receptor-mediated uptake of the ADC and show high efficiencies in target protein degradation. Most importantly, our conjugates exhibit antibody-like PK properties that ensure long-lasting plasma circulation of high DAR constructs over weeks, even when bifunctional degraders are employed that have not been optimized for PK properties. Taken together, these factors allow impressive in vivo efficacy of the conjugates after single dose application in preclinical animal models. Jan G. Felber, Anil P. Jagtap, Philipp Ochtrop, Saskia Schmitt, Isabelle Mai, Sarah Herterich, Paul Machui, Dietmar König, Dominik simon vogt, Schumacher, Jonas Helma-Smets, Annette Vogl, Marc-André Kasper. Antibody-degrader conjugates unlock the therapeutic potential of heterobifunctional degraders through antibody-like pharmacokinetics and excellent in vivo efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7018.

Supplementary Figure 7 shows absent cytotoxic activity of TUB-040 on target-negative primary human cells

Supplementary Figure 1 shows healthy tissue expression of NaPi2b

TUB-040 is a highly homogeneous and hydrophilic antibody-drug conjugate (ADC) targeting sodium-dependent phosphate transport protein 2b (NaPi2b), a surface receptor overexpressed in ovarian cancer and non-small cell lung adenocarcinoma. Previous NaPi2b-directed therapies have shown target-mediated and expression-dependent clinical activity. However, none of the previous tubulin inhibitor-based ADCs have been able to leverage the full therapeutic potential of the target. TUB-040 was constructed with a drug-to-antibody ratio of 8 using the Tubutecan linker-payload technology based on ethynylphosphonamidates (P5 conjugation chemistry), a protease cleavage site, and exatecan, a potent topoisomerase 1 inhibitor. TUB-040 induces potent antigen-specific cytotoxicity against NaPi2b-expressing cancer cells and demonstrates strong bystander activity. It displays favorable pharmacokinetic behavior, showing dose proportionality and superimposable total antibody and intact ADC curves, as well as low free payload levels, reflecting the high stability of TUB-040 enabled by the P5 conjugation platform. This specific feature also ensures sustained delivery of exatecan to tumor sites, which translates into excellent in vivo efficacy and tolerability. In cell line- and patient-derived xenograft models, including those with low target expression, single-dose TUB-040 administration leads to prolonged tumor growth inhibition and significant rates of complete remission, with a minimally effective dose level of 1 mg/kg in the OVCAR-3 model. Repeated-dose toxicologic assessment in rats indicates that TUB-040 is well tolerated, with no evidence of lung toxicity or thrombocytopenia. Taken together, TUB-040 is designed to enable long-lasting, durable tumor responses and to optimize both efficacy and tolerability, supporting the advancement of TUB-040 into clinical trials.

Supplementary Methods 1 includes descriptions about chemicals, LC/MS methods, sample preparation for MS, DARav determination, TUB-040 conjugation and free exatecan quantification

Supplementary Figure 4 shows no binding of NaPi2b mAb and TUB-040 ADC to NaPi2a and NaPi2c

Supplementary Table 1 shows binding affinities of NaPi2b mAb and TUB-040 ADC to NaPi2b

Supplementary Table 2 shows toxicokinetic parameters of TUB-040 in rats

Supplementary Figure 3 shows high-affinity binding of NaPi2b mAb and TUB-040 ADC to HCC-78 cells

Hydrophobic interaction chromatography measurements. Comparison of trastuzumab, trastuzumab-LP3 DAR8, trastuzumab-LP5 DAR8, Adcetris and Enhertu. Shown are chromatograms of Adcetris in black (Homogenous ADC, brentuximab conjugated to MC-VC-PAB-MMAE in DAR0, DAR2, DAR4, DAR6 and DAR8). Enhertu in grey, trastuzumab in light orange, trastuzumab-LP5 in orange and brentuximab-LP3 in purple. Hydrophilicity of all shown DAR8 VC-PAB-Exatecan constructs are in the range of a DAR2 of Adcetris.

Analytical SEC chromatograms of trastuzumab-LP3 DAR8, trastuzumab-LP5 DAR8 and Enhertu at day 0 and after storage for four weeks at 4°C and 37°C in the dark.

Organic Synthesis procedures for the linker payloads and characterization of products and intermediates.

Optimization of the conjugation reaction of an antibody with LP5. A) Variation of equivalents of 3 and TCEP in the conjugation reaction and analysis of the DAR by MS. Constant conditions: 10 mg/mL antibody, buffer at pH 8.3, 16 hours. B) Variation of the pH in the conjugation reaction and analysis of the DAR by MS over the course of 24 hours. Constant conditions: 10 mg/mL antibody, 10 equivalents of LP5 with respect to the mAb, 5 eq. of TCEP.

Abstract TUB-040 is a novel Antibody-Drug Conjugate (ADC) targeting Napi2b, a highly overexpressed target in ovarian cancer and lung adenocarcinoma. Napi2b is a member of the solute-carrier transporter family with eight transmembrane domains and an antibody-accessible extracellular loop, that regulates sodium-dependent phosphate homeostasis. Ethynylphosphonamidate conjugation chemistry was used to build TUB-040 with a homogenous DAR (drug-antibody-ratio) of 8 and the Topoisomerase I inhibitor Exatecan as payload, which is connected to a humanized, Fc-silenced IgG1 antibody targeting Napi2b using a cleavable linker system. In vitro, TUB-040 is characterized by sub-nanomolar affinity and specific binding to Napi2b, efficient target-mediated internalization and high cytotoxicity towards Napi2b-expressing ovarian and lung cancer cell lines as well as bystander activity against co-cultured target-negative cells. TUB-040 induces DNA damage and apoptosis in a dose-dependent fashion as well as markers of immunogenic cell death. In addition, no unspecific uptake and cytotoxicity in healthy, target-negative human cells could be detected, reducing the risk of target-independent toxicities. Pharmacokinetic analysis showed that - in contrast to maleimide-conjugated ADCs - TUB-040 is highly stable - and does not lose or transfer its linker-payload to serum proteins during blood circulation, thus enabling most efficient and continued delivery of Exatecan to the tumor. Across a variety of cell line derived xenograft (CDX) and patient derived xenograft (PDX) models of ovarian and non-small cell lung cancer, including models with low target expression, single dose administration of TUB-040 results in long-lasting tumor growth inhibition with high complete remission rates in vivo. The minimally effective dose (MED) leading to complete remission is 1 mg/kg. Preliminary repeat-dose toxicological assessment of TUB-040 in the pharmacologically relevant species cynomolgus monkey demonstrated that TUB-040 is well tolerated with no signs of lung toxicity and thrombocytopenia. Allometric scaling and PK/PD modelling predicts a human serum half-life greater 10 days and a therapeutic index in humans up to 55. Based on these results, TUB-040, designed with differentiating technology for optimal efficacy and tolerability, is ready for testing in clinical trials. Citation Format: Saskia Schmitt, Isabelle Mai, Paul Machui, Sarah Herterich, Danila Hauswald, Philipp Ochtrop, Philipp Cyprys, Izabela Kozlowska, Annabel Kitowski, Marcus Gerlach, Olivier Marcq, Pamela A. Trail, Dominik Schumacher, Marc-André Kasper, Günter Fingerle-Rowson, Björn Hock, Jonas Helma-Smets, Annette M. Vogl. TUB-040, a novel Napi2b-targeting ADC built with ethynylphosphonamidate conjugation chemistry, demonstrates high and long-lasting anti-tumor efficacy via Topoisomerase-I inhibition and excellent tolerability predictive of a wide therapeutic window in humans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2622.

Abstract TUB-030 is a novel Antibody-Drug Conjugate (ADC) targeting the oncofetal 5T4 antigen, a glycoprotein, that is functionally involved in regulation of the Wnt signalling pathway and promoting cellular motility and adhesion. Immunohistochemical analysis identified 5T4 overexpressed in many types of cancers, including bladder, lung, breast, stomach, esophagus, head and neck, colon, and ovarian. Analysis in fresh frozen healthy human tissue with the therapeutic mAb candidate of TUB-030 revealed limited cross reactivity. Ethynylphosphonamidate conjugation chemistry was used to build TUB-030 with a homogenous DAR (drug-antibody-ratio) of 8 and the Topoisomerase I inhibitor Exatecan as payload, which is connected to a humanized, Fc-silenced IgG1 antibody targeting 5T4 using a cleavable linker system. Overall, TUB-030 contrasts previous ADC designs targeting 5T4, implementing several layers of differentiation in terms of antibody, payload and conjugation technology. In vitro, TUB-030 is characterized by sub-nanomolar affinity to 5T4, efficient target-mediated internalization and strong cytotoxicity towards cancer cells from different tumor indications with a broad range of 5T4 expression levels. Differently to previous 5T4-targeting ADCs, TUB-030 has efficient bystander activity against co-cultured target-negative cells, which facilitates targeting of tumors with heterogenous 5T4 expression. Pharmacokinetic analysis showed that TUB-030 is highly stable - and does not lose or transfer its linker-payload to serum proteins during blood circulation and antibody-like clearance profile contrasting maleimide-conjugated ADCs and enabling most efficient and continued delivery of Exatecan to the tumor site. Single-dose treatment with TUB-030 results in long-lasting tumor regression with high complete remission rates across a variety of both cell-line derived xenograft (CDX) and patient-derived xenograft (PDX) cancer models of multiple tumor indications in vivo, including models with low target expression. Preliminary repeat-dose toxicological assessment of TUB-030 in a pharmacologically relevant non-human primate species demonstrated that TUB-030 is well tolerated. The preclinical results underline that TUB-030 with novel ADC technology has the potential to enable 5T4 as a therapeutic target and provide treatment options in numerous solid cancer indications with high unmet medical need supporting further development towards the clinic. Citation Format: Saskia Schmitt, Isabelle Mai, Paul Machui, Sarah Herterich, Danila Hauswald, Philipp Ochtrop, Philipp Cyprys, Izabela Kozlowska, Annabel Kitowski, Marcus Gerlach, Olivier Marcq, Pamela A. Trail, Dominik Schumacher, Marc-André Kasper, Günter Fingerle-Rowson, Björn Hock, Jonas Helma-Smets, Annette M. Vogl. Enabling 5T4 for targeted cancer therapy: TUB-030, a novel ADC built with ethynylphosphonamidate conjugation chemistry, shows long-lasting anti-tumor activity via Topoisomerase-I inhibition with an optimized therapeutic index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2623.

Dose-response curves for the ADC synthesized from trastuzumab and LP1 and LP3-LP3 on SKBR-3, HCC-78 and MDA-MB-468

Exatecan, DXd, Enhertu and trastuzumab-LP5 induce immunogenic cell death. Immunogenic cell death induced by exatecan, DXd, trastuzumab-LP5 DAR 8 and Enhertu. HER2-positive SKBR-3 and N-87 cells were treated for indicated time points with 20 nM of respective free payloads and ADCs. As markers for immunogenic cell death, ATP and HMGB1 release into the supernatant as well as calreticulin exposure on the cell surface were determined. Therefore, cells were stained with live/dead stain and anti-calreticulin and analyzed by flow cytometry. ATP and HMGB1 release into the supernatant were measured by luminescence-based readouts. Graphs show means of n = 2 ± SEM.