# Anti-cathepsin D antibody-drug conjugates for the targeted delivery of monomethyl auristatin F and E elicit potent anticancer activity in breast cancer

Diane Nishimwe, Gaëlle Guillon, Aude Mallavialle, Camille Martin, Steve Huvelle, Lindsay B Alcaraz, Andréa Romero, Cynthia Garapon, Véronique Garambois, Hanane Mansouri, et al.

► To cite this version:

Diane Nishimwe, Gaëlle Guillon, Aude Mallavialle, Camille Martin, Steve Huvelle, et al. Anti-cathepsin D antibody-drug conjugates for the targeted delivery of monomethyl auristatin F and E elicit potent anticancer activity in breast cancer. Biomedicine and Pharmacotherapy, 2025, 193, pp.118775. 10.1016/j.biopha.2025.118775. inserm-05413705

HAL Id: inserm-05413705

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Submitted on 12 Dec 2025

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Anti-cathepsin D antibody-drug conjugates for the targeted delivery of monomethyl auristatin F and E elicit potent anticancer activity in breast cancer

Diane Nishimwe<sup>a,1</sup>, Gaëlle Guillon<sup>a,1</sup>, Aude Mallavialle<sup>a</sup>, Camille Martin<sup>b</sup>, Steve Huvelle<sup>b</sup>, Lindsay B. Alcaraz<sup>a</sup>, Andréa Romero<sup>a</sup>, Cynthia Garapon<sup>a</sup>, Véronique Garambois<sup>a</sup>, Hanane Mansouri<sup>a</sup>, Florence Boissière-Michot<sup>c</sup>, Ilona Berestjuk<sup>a</sup>, Marta Jarlier<sup>d</sup>, Andrei Turtoi<sup>a,h</sup>, Caroline Denevault-Sabourin<sup>b</sup>, Pascal Roger<sup>a,e</sup>, Séverine Guiu<sup>a,f</sup>, Thierry Chardès<sup>a,g</sup>, Nicolas Joubert<sup>b,2</sup>, Valérie Laurent-Matha<sup>a,2</sup>, Emmanuelle Liaudet-Coopman<sup>a,*,2</sup>

ARTICLE INFO

Keywords:

Breast cancer

ADC

Antibody-drug conjugate

Protease

Cathepsin

Tumor microenvironment

ABSTRACT

Breast cancer (BC) is the leading cause of cancer deaths in women. Patients with estrogen receptor-positive (ER+) and human epidermal growth factor receptor 2-negative or low (HER2; HER2<sup>low</sup>) BC exhibit a late recurrence pattern and resistance to endocrine therapy. Antibody-drug conjugates (ADCs) are new anticancer agents that currently show a remarkable acceleration in preclinical and clinical development. The aspartic protease cathepsin D (cath-D), a poor prognosis marker in BC, is a tumor cell-associated protein with pro-tumor activity that is highly and specifically secreted by BC cells. We recently showed using the human anti-cath-D antibody F1M1 that cath-D is a potent target for antibody-based therapy in BC. Here, we asked whether cath-D is a suitable target for ADC-based therapy in non-amplified ER<sup>+</sup>/HER2<sup>low</sup> BC. To this aim, we bioconjugated F1M1 to the cytotoxic payload monomethyl auristatin F (MMAF) via a non-cleavable linker (for the release of a non-cell permeable MMAF metabolite) and to its cell permeable derivative monomethyl auristatin E (MMAE) via a protease-sensitive cleavable linker, to form the ADCs F1M1-MMAF and F1M1-MMAE respectively, with a drug-to-antibody ratio close to four. These F1M1-based ADCs were specifically internalized and showed cytotoxic activity in the nanomolar range in the ER<sup>+</sup>/HER2<sup>low</sup> MCF-7 and endocrine therapy-resistant ER<sup>+</sup>/HER2<sup>low</sup> MCF-7/LCC2 BC cell lines. *In vivo*, three injections of F1M1-based ADCs led to long-lasting tumor growth inhibition in mice harboring MCF-7/LCC2 cell xenografts. In conclusion, we showed for the first time that cath-D is a potent target for ADC in BC.

<footnote><sup>a</sup> IRCM, INSERM U1194, University of Montpellier, ICM, Montpellier, France</footnote>

<footnote><sup>b</sup> UMR 1100 CEPR, INSERM, University of Tours, Tours F-37200, France</footnote>

<footnote><sup>c</sup> Translational Research Unit, ICM, Montpellier, France</footnote>

<footnote><sup>d</sup> Biometry Department, ICM, Montpellier, France</footnote>

<footnote><sup>e</sup> Department of Pathology, CHU Nîmes, Nîmes, France</footnote>

<footnote><sup>f</sup> Department of Medical Oncology, ICM, Montpellier, France</footnote>

<footnote><sup>g</sup> CNRS, Centre national de la recherche Scientifique, Paris F-75016, France</footnote>

<footnote><sup>h</sup> Gunma University Initiative for Advanced Research, Maebashi, Gunma 371-0034, Japan</footnote>

<footnote>* Correspondence to: IRCM - Inserm U1194, 208, rue des Apothicaires, Montpellier F-34298 Cedex 5, France.
E-mail address: emmanuelle.liaudet-coopman@inserm.fr (E. Liaudet-Coopman).</footnote>

<footnote n="1">¹ Diane Nishimwe and Gaëlle Guillon contributed equally to the work</footnote>

<footnote n="2">² Nicolas Joubert, Valérie Laurent-Matha and Emmanuelle Liaudet-Coopman contributed equally to the work</footnote>
# 1. Introduction

Breast cancer (BC) is one of the most commonly diagnosed cancers in the world and the leading cause of cancer mortality in women [1]. The estrogen receptor-positive (ER+) and human epidermal growth factor receptor 2-negative or low (HER2; HER2^low) BC subtype, known as luminal BC, represents ~70% of all BCs, and usually has favorable prognosis [1]. However, patients with luminal BC exhibit resistance to endocrine therapy and a late recurrence pattern [2]. Until recently, treatment options for patients with metastatic hormone sensitive (HR+)/HER2^-low BC resistant to endocrine therapy were limited to chemotherapy and targeted therapy [3-5]. Chemotherapy has been recently challenged and partly replaced by new targeted options, including antibody-drug conjugates (ADCs) [6,7]. ADCs are compounds that consist of three key components: antibody, linker (cleavable or not), and small-molecule cytotoxic payload. Therefore, they combine the tumor-targeting properties of the antibody moiety with the potency of cytotoxic agents [6,7]. In 2013, the US Food and Drug Administration (FDA) approved the first ADC, trastuzumab (anti-HER2 antibody) emtansine (T-DM1, Kadycya®), to be used as monotherapy against metastatic (or locally advanced unresectable) HER2+ BC [8]. This was followed by the approval of the ADC trastuzumab deruxtecan (T-DXd, Enhertu®) for unresectable or metastatic HER2+ BC in 2019 and for HER2^low BC in 2022 [9-11]. In 2022, FDA approved also the ADC sacituzumab (anti-trophoblast cell surface antigen 2, TROP2, antibody) govitecan (Trodelvy®) for metastatic HER2+ BC [12]. In 2025, the ADC datopotamab (anti-TROP2, antibody) deruxtecan (Datroway®) was approved in the US for patients with previously treated metastatic HER2-/HER2^low HR+ BC [13]. In China, the National Medical Products Administration (NMPA) approved the ADC disitamab (anti-HER2 antibody) vedotin (Aidixi®) for HER2+ and HER2^low metastatic BC in 2021 [12,14], and the ADC sacituzumab (anti-TROP2 antibody) tirumotecan (Jiatailai®) for advanced triple-negative BC (TNBC) in 2024 [13]. In September 2025, the ADC trastuzumab (anti-HER2 antibody) rezetecan (Aiweida®) received a breakthrough therapy designation for BC in China [13], indicating the growing place of ADCs in BC management.

However, resistance against ADCs can be acquired by tumor cells through many mechanisms, including lowering the expression of the targeted antigen on the cancer cell surface. Therefore, in addition to the HER2 and TROP2 tumor antigens, new cell surface-associated tumor antigens need to be urgently identified to develop ADCs against BC, particularly HER2+ and HER2^low BC resistant to endocrine therapy.

The aspartic protease cathepsin D (cath-D), a poor prognostic marker in BC [15-19], ER+/HER2+ BC [20] and TNBC [21,22], is overproduced by BC cells and aberrantly secreted in the tumor microenvironment [23, 24]. Cath-D affects the tumor and its microenvironment by increasing BC cell proliferation [23,25-29] and by stimulating mammary fibroblast outgrowth [30,31], angiogenesis [25,32], immune evasion [33,34], and metastasis formation [28]. In BC, extracellular cath-D exhibits pro-tumor activities through proteolytic cleavage of stromal/tumor components [33,35-39], and/or by acting as a binding protein [30, 40-44]. Cath-D can modify the local extracellular matrix by cleaving chemokines, growth factors, collagens, fibronectin, proteoglycans, the matricellular protein SPARC, protease inhibitors (e.g., cystatin C, PAI 1), or by activating the cathepsin B and L precursors [33,35-39]. It can interact with the fibroblastic LDL receptor-related protein-1 (LRP1) and inhibits LRP1-regulated intramembrane proteolysis, leading to fibroblast outgrowth [30,43]. Moreover, extracellular cath-D can bind to and be internalized by the mannose-6-phosphate/insulin growth factor 2 receptors (M6P/IGF2-R) or other unknown receptors that are expressed at the surface of BC and stromal cells, such as fibroblasts [23,45]. These intrinsic properties of secreted cath-D, which is reinternalized by a receptor-mediated process, suggest that it may be a promising and innovative therapeutic extracellular target in the tumor microenvironment for antibody-mediated treatment strategies, such as ADCs. We recently reported that cath-D is tumor cell-associated and a suitable

target for antibody-based therapy in BC [46]. Moreover, we demonstrated that the fully-human anti-cath-D antibody F1M1, selected by phage display, inhibits tumor growth and promotes antitumor immunity in BC [47-50].

The aim of this study was to evaluate whether cath-D is a relevant target for ADC-based cancer therapy with the anti-cath-D F1M1 antibody in non-amplified ER+/HER2^low BC. F1M1 was bioconjugated, via a protease-cleavable valine-citrulline-PABC linker, with a drug-to-antibody ratio (DAR) of ~4, to the membrane-permeable payload monomethyl auristatin E (MMAE) that can exert bystander killing effects. MMAE is a highly potent antimitotic peptide that blocks tubulin polymerization, and is the most widely used payload for clinical and approved ADCs [51]. F1M1 was also bioconjugated to monomethyl auristatin F (MMAF), a derivative close to MMAE, through a non-cleavable linker (DAR of ~4). This linker allows the release of a MMAF metabolite with impaired cellular permeability, and consequently without bystander killing effects [52]. The F1M1-MMAF and F1M1-MMAE ADCs were internalized and exhibited cytotoxic activity in the ER+/HER2^low MCF-7 [53] and tamoxifen-resistant ER+/HER2^low MCF-7/LCC2 [54] BC cell lines. They also exerted potent antitumor activity in vivo in mice xenografted with MCF-7/LCC2 cells. This is the first proof of concept that cath-D is a suitable target for ADC-based therapy in BC.

# 2. Materials and methods

## 2.1. Materials/reagents

The mouse monoclonal antibody against the 52-, 48-, and 34-kDa forms of human cath-D (#610801) was purchased from BD Transduction Laboratories (San Jose, CA). The anti-human cath-D mouse monoclonal antibody M2E8 recognizes only the 52-kDa form of cath-D [55]. The monoclonal antibodies M1G8 and D7E3 against the 52-, 48-, and 34-kDa forms of human cath-D were previously described [56]. The rabbit polyclonal antibody (H-75; sc-10725) to detect the 52-kDa human pro-cath-D was from Santa Cruz Biotechnology (Dallas, TX). The anti-human cath-D (clone C-5) and anti-HSC70 (clone B-6, sc-7298) mouse monoclonal antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). The anti-β actin polyclonal antibody (#A2066) was from Sigma-Aldrich (Saint-Louis, MO). Hoechst (#33342) was from Thermo Fisher Scientific (Waltham, MA). The anti-human HER2 rabbit polyclonal antibody (#2242) was from Cell Signaling Technology. The F1M1 IgG1 (anti-human cath-D) was constructed from the scFv F1M1 sequences using gene synthesis, expressed in the Chinese hamster ovary (CHO) cell line, and purified on protein-A HiTrap columns (GE Healthcare) by Evitria AG (Schlieren, Switzerland). The anti-human CD20 chimeric IgG1 antibody rituximab (RTX; Rituxan®) was from Roche. The goat anti-human IgG (H+L) cross-adsorbed secondary antibody Alexa Fluor™ 488 (A11013) was from Thermo Fisher Scientific. The anti-human Fc goat polyclonal antibody conjugated to HRP (A0170) and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Sigma Aldrich (Saint-Louis, MO). The substrate reagent for HRP was purchased from Bio-Techne (R&D Systems, #DY999). Recombinant human pro-cath-D was purchased from R&D Systems. The Incucyte® Human Fabfluor-pH Red Antibody Labeling Dye was from Sartorius (#4722).

## 2.2. ADC generation

Unless otherwise noted, materials were obtained from commercial suppliers at the highest available purity grade and used without further purification. MMAF- and MMAE-linkers were synthesized according to previously reported procedures [57]. RTX (Rituxan®, Roche) was obtained from the Hospital Pharmacy of Tours Teaching Hospital and from ICM Hospital of Montpellier. For the production of the F1M1-MMAE and F1M1-MMAF ADCs, F1M1 (450 µg, 100 µL, 3.0 nmol) in borate buffered
saline pH 8 was mixed with tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 18 µL, 6 eq.) and incubated at 37 °C for 75 min. Then, MMAE-linker or MMAF-linker (10 mM in dimethyl sulfoxide, DMSO, 18 µL, 6 eq.) was added and incubated at 4 °C under agitation (600 rpm) for 17 h. TCEP was added again (10 mM, 9 µL, 3 eq.) and incubated at 4 °C for 1.25 h, followed by a second addition of MMAE-linker or MMAF-linker (10 mM in DMSO, 9 µL, 3 eq.) at 4 °C under agitation (600 rpm) for 17 h. The mixtures were purified using Sephadex G25 PD10 columns (10 kDa MWCO, from Sigma Aldrich) in phosphate-buffered saline (PBS) to obtain the desired ADCs (F1M1-MMAE and F1M1-MMAF). The same procedure was used with RTX to generate the control RTX-MMAE and RTX-MMAF ADCs.

## 2.3. ADC characterization

The prepared ADCs were analyzed by mass spectrometry (MS) using a Bruker maXis mass spectrometer coupled to a Dionex Ultimate 3000 RSLC system. Before MS analysis, samples (~5 µg) were desalted on a MassPREP desalting cartridge (2.1 × 10 mm, Waters) heated at 80 °C using 0.1 % formic acid as solvent A and 0.1 % formic acid in acetonitrile as solvent B at 500 µL/min. After 1 min, a linear gradient from 5 % to 90 % B in 1.5 min was applied; the first 1.5 min were diverted to waste. High resolution MS (HRMS) data were acquired in positive mode with electrospray ionization over the m/z range from 900 up to 5000 at 1 Hz and processed using the DataAnalysis 4.4 software (Bruker) and the MaxEnt algorithm for spectral deconvolution. Deconvolution was carried out in the 20–180 kDa range, and results were recorded for the full antibodies and also for the antibody fragments L, LH, HH, LHH due to antibody dissociation in MS. Not all species were observed for all ADCs. The DAR was approximated for each species as an average of the percentage abundance of each species present, with the quantities calculated by peak integration of the first glycosylation peak. The DAR<sub>average</sub> was then calculated using the percentage peak areas combined with their respective drug load numbers, according to the following general formula:

$$DAR_{average} = \frac{1}{4} * [DAR_{LHHL} + 2 * DAR_{LH} + (DAR_{LHH} + DAR_{L}) + (DAR_{HH} + 2 * DAR_{L})]$$

The prepared ADCs were analyzed by size-exclusion chromatography (SEC) analysis. ADCs were diluted to 1 mg/ml with PBS pH 7.4 and filtered through a 0.22 µm PVDF membrane. A sample of 40 µg was injected on an AdvanceBio SEC (7.8 × 300 mm, 2.7 µm) from Agilent Technologies, connected to a Waters Alliance (e2695) apparatus equipped with a photodiode array detector (2998) set for detection at 280 nm. Samples were run with an isocratic gradient (1 mM potassium phosphate monobasic, 155 mM sodium chloride, 3 mM sodium phosphate dibasic, 3 mM sodium azide, pH 7.0) at a flow rate of 1 ml/min for 24 min. The column oven temperature was maintained at 25 °C.

## 2.4. BC cell lines, cath-D conditioned medium, western blotting, and ELISA

The ER<sup>+</sup> MCF-7 human breast carcinoma cell line [58] was originally supplied by the Michigan Cancer Foundation, Detroit, MI. The 4-hydroxytamoxifen (OHTam)-resistant MCF-7/LCC2 human BC cell line was obtained by growing the parental MCF-7/LCC1 cell line [54] in the presence of increasing OH-Tam concentrations and is routinely maintained in the presence of 1 pmol/L OHTam. MCF7/LCC2 cells retain similar ER expression levels as MCF-7/LCC1 and MCF-7 cells [54]. The HER2-amplified SK-BR-3 BC cell line was obtained from SIRIC Montpellier Cancer. The ER<sup>+</sup> MDA-MB-231 BC cell line was previously described [46]. Cells were cultured in DMEM with 10 % fetal calf serum (FCS, Eurobio). To produce conditioned medium, cells grown to 90 % confluence were incubated in the absence or in the presence of FCS for

24–48 h. Then, conditioned medium was centrifuged at 800 x g for 5 min. Cell lysates were prepared in lysis buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 10 % glycerol, 1 % Triton X-100, 1.5 mM MgCl₂, 1 mM EGTA) containing cOmplete™ Protease Inhibitor Cocktail (Roche, Switzerland) and centrifuged at 13,000 x g for 15 min. For western blotting experiments, proteins (10 µg) from cell lysates and conditioned media without FCS (40 µl) were separated on 13.5 % SDS-PAGE and analyzed by immunoblotting using standard techniques. For the sandwich ELISA against secreted human pro-cath-D, 96-well plates were coated with the M2E8 antibody in PBS (250 ng/well) at 4 °C overnight. After blocking non-specific sites with PBS/0.1 % Tween-20/1 % BSA, cath-D-containing conditioned medium with FCS from MCF-7 or MCF-7/LCC2 cells was added at 4 °C for 2 h. After washes in PBS/0.1 % Tween_20, serial dilutions of F1M1 or ADCs (F1M1-MMAE, F1M1-MMAF) were added at 4 °C for 2 h, and interactions revealed by incubation with an anti-human Fc antibody conjugated to HRP (1/2000; 355 ng/well) at 37 °C for 1 h. Total cath-D was quantified in human ER<sup>+</sup> BC cytosols by sandwich ELISA, as described above, after plate coating with the D7E3 antibody in PBS (200 ng/well) and with the HRP-conjugated M1G8 antibody (1/80), and using recombinant pro-cath-D (1.25–15 ng/ml) as reference [28].

## 2.5. Immunohistochemistry

For cath-D immunostaining, ER<sup>+</sup> BC biopsy sections were incubated with the anti-cath-D mouse antibody (clone C-5) at 0.4 µg/ml for 20 min after heat-induced antigen retrieval using the PTLink pre-treatment (Dako) and the High pH Buffer (Dako) and endogenous peroxidase quenching with Flex Peroxidase Block (Dako). After two rinses in EnVision™ Flex Wash buffer (Dako), sections were incubated with a HRP-labeled polymer coupled to a secondary anti-mouse antibody (Flex® system, Dako) for 20 min, followed by 3,3'-diaminobenzidine as chromogen. Sections were counterstained with Flex Hematoxylin (Dako) and mounted after dehydration. Images were digitalized with a NanoZoomer slide scanner (Hamamatsu) and analyzed with the Aperio Imagescope software.

## 2.6. Fluorescence microscopy

Paraffin-embedded MCF-7/LCC2 cell xenograft sections were deparaffined, rehydrated, rinsed and saturated in PBS/5 % FCS at 4 °C overnight. Sections were incubated with 0.4 µg/ml anti-cath-D mouse monoclonal antibody (clone C-5) followed by incubation with Alexa-Fluor 488-conjugated anti-mouse IgG (1/400). Nuclei were stained with 0.5 µg/ml Hoechst 33342. Sections were imaged with a 100X Plan-Apochromat objective on z stacks with a Zeiss Axio Imager light microscope equipped with Apotome to eliminate out-of-focus fluorescence.

## 2.7. Internalization assay by immunofluorescence

Internalization assays were performed as previously described [45]. Cells were grown to near confluence on glass coverslips in 6-multiwell plates with complete medium. After 3 × 5 min washing with DMEM without serum, cells were incubated (37 °C for 3 h) with the indicated antibodies or ADCs in 100 µl of a 48h-conditioned medium from MCF-7 cells without FCS. At the incubation end, cells were washed 3 times with DMEM and fixed in 4 % paraformaldehyde in PBS, pH 7.4, for 30 min. Cells were washed with PBS (3 × 3 min) and incubated with 50 mM NH₄Cl in PBS for 5 min. After washes with PBS (3 × 3 min), cells were incubated with PBS/0.1 % Triton X-100 for 5 min. Before saturation with PBS/5 % goat serum for 30 min, cells were washed again with PBS/0.02 % Tween 20. Internalized antibodies were detected by incubation with an anti-human Fc antibody (dilution 1/400 in PBS/0.5 % goat serum) conjugated to Alexa Fluor™ 488 (green fluorescence) at room temperature for 90 min. Before mounting, cells were washed with PBS/0.02 % Tween 20 in the presence of DAPI (1/10,000), pH 7.4
(3 ×10 min), and once with double distilled water to remove excess secondary antibody. Slides were analyzed with a Zeiss Axio Imager light microscope equipped with Apotome to eliminate out-of-focus fluorescence at a 100x magnification.

## 2.8. Internalization assay by live imaging

Antibodies and ADCs were labeled with the Incucyte® Human Fabfluor-pH Red Antibody Labeling Dye (Sartorius #4722) to monitor their internalization. 10,000 MCF-7/LLC2 cells or 4,500 MCF-7 cells per well were seeded in 96-well plates. After 48 h, cells were incubated with Fabfluor-labeled antibodies or Fabfluor-labeled ADCs in DMEM without FCS. Cells were scanned every hour for 24 h and the red fluorescence intensity was measured using the Sartorius Incucyte® device by live imaging using user-informed algorithms that are part of the Incucyte® software package. The Incucyte® Live-Cell Analysis System allows the real-time, kinetic evaluation of antibody or ADC internalization at a 20x magnification.

## 2.9. 2D cell viability assay

Cell viability was assessed with the mitochondrial dehydrogenase enzymatic assay using MTT (Sigma-Aldrich). MCF-7 (250 cells/well) and MCF-7/LCC2 (2,000 cells/well) cells were seeded in 96-well plates in complete culture medium. At day 3, medium was changed and cells were incubated with F1M1, ADCs, MMAE or MMAF at the indicated concentration in triplicate in complete culture medium for 7 days. At the incubation end, cells were incubated with 50 µl of MTT solution (0.5 mg/ml MTT in serum-free medium) for 2 h. After removal of the MTT solution, 100 µl DMSO was added to the wells for 10 min and the absorption value at 570 nm was measured using a multi-plate reader (PHERAstar® BMG Labtech, Ortenberg, Germany). Experiments were performed in triplicate/quadruplicate and the half maximal inhibitory concentration (IC₅₀) values were calculated using GraphPad Prism 10.

## 2.10. 3D cell viability assay in spheroids

Cells were seeded in 96-well plates (10,000 cells/well) precoated with 50 µl agarose to form spheroids. After 3 days, spheroids were incubated with F1M1, ADCs, MMAE or MMAF in complete culture medium for 7 days. Spheroids were analyzed using the Sartorius Incucyte® device by live imaging. At the incubation end, cytotoxicity was analyzed using the CellTiter-Glo® 3D Cell Viability assay (Promega).

## 2.11. Study approval

Mouse experiments were performed in compliance with the French regulations and ethical guidelines for experimental animal studies in an accredited establishment (Agreement No. #31135-2021042212479661). For immunohistochemistry, BC biopsy samples were provided by the biological resource center (Biobank number BB-0033-00059) after approval by the Montpellier Cancer Institute Institutional Review Board, following the French national Ethics and Legal dispositions for patients' information and consent. For BC cytosols, patient samples were processed according to the French Public Health Code (law n°2004-800, articles L. 1243-4 and R. 1243-61), and the biological resources center is authorized (authorization number: AC-2008-700; Val d'Aurelle, ICM, Montpellier) to deliver human samples for scientific research. All patients were informed before surgery that their surgical specimens might be used for research purposes.

## 2.12. In vivo tumor growth and survival

MCF-7/LCC2 cells (5 ×10⁶; mixed 1:1 with Matrigel) were injected subcutaneously in 6-week-old female athymic mice (NU(NCr)-Foxn1nu) (Charles River Laboratory, France). When MCF-7/LCC2 cell xenografts

reached a volume of 100 mm³, tumor-bearing mice were randomized and treated intravenously three times with F1M1-MMAF, RTX-MMAF, F1M1-MMAE, RTX-MMAE or NaCl at day 17 (10 mg/kg), 24 (5 mg/kg) and 31 (5 mg/kg) post-graft. Tumors were measured using a caliper and volume was calculated with the formula $V = (tumor length × tumor width × tumor depth)/2$, until the tumor volume reached 1000 mm³.

## 2.13. Meta-analysis of proteomics and spatial transcriptomics datasets

Previously published proteomic [59] and spatial transcriptomic [60] datasets were used to determine cath-D expression in ER⁺ BC. Proteomic data were re-analyzed using the ProteinLynx Global Server software (v. 3.0.3; Waters, Milford, MA, USA) and transcriptomic data set using Seurat (v. 4.3.0) in the R computational environment [61].

## 2.14. Statistical analysis

A linear mixed regression model was used to determine the relationship between tumor growth and number of days post-xenograft. Number of days post-xenograft and treatment groups were the variables included in the fixed part of the model. Their interaction also was evaluated. Random intercepts and random slopes were included to take into account the time effect. The model coefficients were estimated by maximum likelihood. Survival rates were estimated using the Kaplan-Meier method and compared with the log-rank test. The non-parametric Kruskal-Wallis test was used to compare more than two groups and the Mann-Whitney test for pairwise comparisons. Statistical significance was set at the 0.05 level. All statistical analyses were done with STATA 16.0.

# 3. Results

## 3.1. Cath-D is a tumor microenvironment antigen eligible for ADC-based therapy in ER⁺ BC

Efficient ADC uptake/internalization is required for their activity. Indeed, ADCs undergo antigen-mediated internalization and intracellular processing to release their cytotoxic agent or metabolite in the cancer cell cytosol. ADC internalization requires the presence of antibody-antigen complexes at the cancer cell surface. Therefore, to assess whether cath-D in ER⁺ BC was an accessible molecular target for ADC-based therapy, we analyzed cath-D expression status in previously published datasets used for biotin-based affinity isolation and proteomic analysis of accessible protein biomarkers in human BC tissue samples [62]. We found that extracellular and/or membrane-associated cath-D was detected only in ER⁺ BC samples and not in the adjacent normal breast tissues (Fig. 1A). We validated these proteomic data by immunochemistry analysis of ER⁺ BC samples. We detected cath-D as vesicular intracellular staining and also perimembranous staining at the cancer cell periphery (Fig. 1B). Spatial transcriptomic analysis of ER⁺ BC biopsies revealed a relatively high level of cath-D mRNA expression in the tumor part (in situ and invasive), particularly in areas with infiltrating immune cells (Supplementary Figures 1 and 2). Quantification of cath-D expression in cytosols of primary ER⁺ BC samples revealed a mean cath-D level of 43.5 pmol/mg protein in the ER⁺ BC subtype (Fig. 1C), which was in the range of the cut-off values reported in clinical studies on all combined BC subtypes [15–19]. We then examined cath-D expression in three ER⁺ BC cell lines (MCF-7, OH-Tam-resistant MCF-7/LLC2, SK-BR-3) and in the MDA-MB-231 ER⁺ BC cell line (Fig. 1D). We detected the fully mature cellular lysosomal cath-D (34-kDa heavy chain) in all tested BC cell lines as well as the 52-kDa cath-D precursor (pro-cath-D), which was secreted in the culture medium of all cell lines (Fig. 1D), in agreement with its cell surface association after secretion (Fig. 1A-B). As previously described [63], we validated the low HER2 expression status of MCF-7 and MCF-7/LLC2 cells, compared with HER2-amplified SK-BR-3 cells (Fig. 1D).
Moreover, we detected the 52-kDa pro-cath-D in the cytosols (i.e. whole tumor lysates) of 31 ER⁺ BC samples and in MCF-7/LCC2 cell xenografts (Fig. 1E). Immunofluorescence analysis of cath-D in ER⁺ MCF-7/LCC2 cell xenografts showed intense, punctate staining at the tumor periphery and in cell membrane protrusions, potentially areas of exocytosis (Fig. 1F), in agreement with cath-D expression profile in ER⁺ BC biopsy sample (Fig. 1B). Together with previously published clinical data [15–19], these observations strongly suggest that cath-D is a tumor

microenvironment antigen highly expressed in ER⁺ BC, and therefore, a potential good candidate for ADC-based therapy in ER⁺ BC.

Next, we assessed whether the human anti-cath-D antibody F1M1 [48] could bind to cath-D secreted by ER⁺ BC cell lines and be intercalized. Sandwich ELISA with cath-D secreted from MCF-7/LCC2 cells and MCF-7 cells (Fig. 2A) showed that F1M1 had good binding capacity (half maximal effective concentration, EC₅₀ = 0.19 nM and 0.14 nM, respectively). Moreover, F1M1 binding to cath-D was similar at pH

<figure><img src="image_2.png" /></figure>

(caption on next page)
**Fig. 1.** Cath-D is a tumor microenvironment antigen relevant for ADC therapy in ER⁺ BC. (A) Detection of extracellular and cell-surface cath-D in ER⁺ BC by proteomics. Two ER⁺ BC biopsies and paired normal breast tissue samples were biotinylated followed by protein digestion. The corresponding glycoprotein fractions containing proteins located in the extracellular matrix (light gray) or at the exterior face of the plasma membrane (dark gray) were analyzed by proteomics. ND, not detectable. (B) Representative image of cath-D expression in an ER⁺ BC biopsy. Cath-D expression was analyzed in an ER⁺ BC biopsy by immuno-histochemistry using an anti-cath-D antibody (C-5; sc-377127). Scale bar, 50 µm (left panel). Higher magnifications of the boxed region showing extracellular cath-D expression (middle and right panels). Staining is prominent in BC cells and is also detected in the tumor stroma. Arrows (right panel) show cath-D localization at the cancer cell surface. (C) Cath-D quantification in the cytosol of ER⁺ BC samples. Total cath-D expression was determined in 80 whole cytosols from primary ER⁺ BC biopsies by sandwich ELISA with the immobilized anti-human cath-D D7E3 antibody and the anti-human cath-D M1G8 antibody coupled to HRP. Data are the mean ± SEM. (D) Cath-D expression and secretion in BC cell lines. Whole cell extracts (cell lysates, 20 µg proteins) and 24 h conditioned media in the absence of serum (40 µl) were separated on 13.5 % SDS-PAGE and analyzed by immunoblotting with the anti-cath-D mouse monoclonal antibody (#610801) (to detect mature cath-D) and rabbit polyclonal antibody (H-75) (to detect pro-cath-D), and anti-HER2 rabbit polyclonal antibody (#2242). β-actin (#A2066) was used as loading control. MW, molecular weight (kDa). These data are exploratory. (E) Cath-D expression in ER⁺ BC cytosols. Cath-D expression was determined in 31 cytosols from primary ER⁺ BC samples. Whole cytosols (10 µg proteins) were immunoblotted with the mouse monoclonal (#610801) (to detect mature cath-D), and rabbit polyclonal (H-75; sc-10725) (to detect pro-cath-D) anti-cath-D antibodies. HSC70 (clone B-6) was used as loading control. A MCF-7/LCC2 BC cell xenograft was used as a positive control for cath-D expression. MW, molecular weight (kDa). These data are exploratory. (F) Cath-D expression and localization in OH-Tam-resistant MCF-7/LCC2 BC cell xenografts. Representative image of MCF-7/LCC2 BC cell xenograft sections incubated with an anti-human cath-D monoclonal antibody (clone C-5) (red). Nuclei were stained with Hoechst 33342 (blue). Scale bar, 5 µm (left panel). The higher magnification of the boxed region shows cath-D localization at the cancer cell surface (arrows). OH-Tam, 4-hydroxytamoxifen.

values from 7.2 to 5.8 (Supplementary Figure 3), suggesting that it is active also in the highly acidic tumor microenvironment. By immuno-fluorescence analysis of living cells, we observed that F1M1, but not the control RTX antibody, was internalized in MCF-7/LCC2 cells and MCF-7 cells (Fig. 2B) after incubation for 3 h. We confirmed F1M1 internali-zation by 24-h live imaging with Incucyte® (Fig. 2C for MCF-7/LCC2 cells). Overall, our findings indicate that cath-D is a potential target for F1M1-based ADCs with an intracellular mechanism of drug release in ER⁺ BC.

## 3.2. ADC design, generation and characterization

We conjugated F1M1 (human anti-cath-D antibody) and RTX (human anti-CD20 antibody; control) to MMAF-linker or MMAE-linker in two steps to obtain the four ADCs of this study: F1M1-MMAF, F1M1-MMAE, RTX-MMAF and RTX-MMAE. First, we mildly reduced the antibody interchain disulfide bridges using TCEP to release the free thiols, and then we added MMAF-linker and MMAE-linker to bridge the cysteine residues (Fig. 3A) [64]. MMAF-linker is a non-cleavable diphenylthio-maleimido-caproic acid-MMAF (diSPhM-C-MMAF) struc-ture that after lysosomal ADC degradation, allows the release of an active MMAF metabolite, which is a potent tubulin polymerization in-hibitor (Fig. 3B) [52]. This charged metabolite at physiological pH cannot cross biological membranes to exert its bystander effect on neighboring cells [65]. MMAE-linker is a cleavable diphenylth-io-maleimido-caproic-valine-citrulline-para-aminobenzylcarbonate-MM AE (diSPhM-C-VC-PABC-MMAE) structure. The VC-PABC sequence is a cathepsin B-sensitive trigger in which PABC acts as a self-immolative spacer to free unchanged cytotoxic MMAE in the lysosomal compart-ment of the targeted tumor cells, after cleavage by cathepsin B (Fig. 3C). MMAE, a highly potent antimitotic drug that inhibits tubulin polymer-ization [66,67], can diffuse through biological membranes to achieve a bystander killing effect. We conjugated MMAF-linker and MMAE-linker to F1M1 or RTX using a site-specific conjugation method and a diSPhMC head, for disulfide stapling on cysteine residues [64,68–70]. This methodology allows the rebridging of previously reduced interchain disulfide bonds of the antibody to obtain stable and site-specific immunoconjugates with a DAR close to 4 [64,70,71]. We previously tested several conditions, including a higher number of equivalents of reducer and linker-auristatin in a single addition sequence [72,73]. However, in accordance with our previous study [64] in which hydro-phobic auristatin-based linker-payloads were used to target ADCs with DAR of ~ 4, two successive additions of both reducer and linker-payload were necessary to obtain the desired DAR of ~4. We determined the purity and DAR of the prepared ADCs using HRMS and we calculated the average DAR values from the deconvoluted HRMS spectra (Supplementary information). They ranged from 3.4 to 3.8, as expected

when performing disulfide stapling with a bioconjugation head. These are generally considered as optimal DAR values for auristatin-containing ADCs. In addition, we evaluated the aggregation profile of each ADC by SEC (Supplementary information), and did not find any obvious aggre-gation, suggesting that each immunoconjugate was composed mainly (>90 %) of monomers.

## 3.3. *In vitro* binding, internalization and inhibition of cell viability of F1M1-based ADCs in ER⁺ BC cells

First, we compared the binding capacity of F1M1-MMAF, F1M1-MMAE and naked F1M1 to cath-D secreted by BC ER⁺ cell lines using sandwich ELISA. The binding capacity of F1M1-MMAF and F1M1-MMAE was reduced by 2.5- to 5-fold compared with F1M1: EC₅₀ = 0.45 nM, and 0.50 nM vs 0.18 nM, respectively, to cath-D secreted by MCF-7/LCC2 cells (Fig. 4A, left panel), and EC₅₀ = 0.77 nM and 0.67 nM vs 0.15 nM, respectively, to cath-D secreted by MCF-7 cells (Fig. 4A, right panel). Binding of F1M1-MMAF and F1M1-MMAE to cath-D was similar at pH values from 7.2 to 5.8 (Supplementary Figure 3), indicating that F1M1-based ADCs should be cytotoxic also in the acidic tumor microenvironment. Immunofluorescence analysis confirmed F1M1-MMAF and F1M1-MMAE internalization in MCF-7/LCC2 (Fig. 4B, left panel) and MCF-7 (Fig. 4B, right panel) living cells after incubation for 3 h. Conversely, RTX-MMAF and RTX-MMAE remained undetect-able. Furthermore, live imaging after labeling with Incucyte® Human Fabfluor-pH Red Antibody Labeling Dye showed similar internalization levels for F1M1-MMAF, F1M1-MMAE and naked F1M1 (Fig. 4C).

Then, we analyzed the *in vitro* cytotoxic activity of the F1M1-MMAF and F1M1-MMAE ADCs by measuring cell viability in 2D and in 3D cultures of MCF-7/LCC2 and MCF-7 cells (Fig. 5). Compared with RTX-MMAF (control; IC₅₀ > 50 nM), the F1M1-MMAF ADC (payload conju-gated via a non-cleavable linker) strongly inhibited the viability of MCF-7/LCC2 cells (IC₅₀ = 21.3 nM) (Fig. 5A, top panel) and MCF-7 cells (IC₅₀ = 9.8 nM) (Fig. 5B, top panel). Conversely, naked F1M1 did not affect cell viability (Supplementary Figures 4 A and 5 A). As expected, free MMAF, which is a non-cell-permeable drug, showed almost no cyto-toxicity in MCF-7/LCC2 and MCF-7 cells (Supplementary Figures 4B and 5B).

The IC₅₀ values of F1M1-MMAE ADC (payload conjugated via a cleavable linker) were 13.8 nM in MCF-7/LCC2 cells (Fig. 5A, bottom panel) and 8.7 nM in MCF-7 cells (Fig. 5B, bottom panel). RTX-MMAE also decreased cell viability, particularly in MCF-7 cells, but to a lesser extent compared with F1M1-MMAE. This could be due to extracellular cleavage of the protease-cleavable linker of RTX-MMAE and release of the cell-permeable MMAE drug. In agreement, free MMAE alone was very cytotoxic *in vitro* in MCF-7/LCC2 and MCF-7 cells (Supplementary Figures 4 C and 5 C).
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Cath-D is a potential target for ADC-based therapy with the anti-human cath-D antibody F1M1 in MCF-7/LCC2 (OHTam-resistant) and MCF-7 BC cells. (A) Binding of F1M1 to human cath-D secreted from MCF-7/LCC2 and MCF-7 cells. Sandwich ELISA in which human cath-D from conditioned medium of MCF-7/LCC2 (left panel) and MCF-7 cells (right panel) was added to wells pre-coated with the anti-human cath-D M2E8 mouse monoclonal antibody in the presence of increasing concentrations of the F1M1 antibody. F1M1 binding to secreted human cath-D was revealed with an anti-human Fc HRP-conjugated antibody. The EC₅₀ values are shown. (B) **Internalization of F1M1 in MCF-7/LCC2 and MCF-7 cells.** MCF-7/LCC2 and MCF-7 cells were incubated with 50 µg/ml of F1M1 or rituximab (RTX; negative control) and a 48 h serum-free conditioned medium from MCF-7 cells at 37°C for 3 h. F1M1 internalization was visualized with a Alexa Fluor 488-labeled anti-human Fc secondary antibody (green). Nuclei were stained with Hoechst 33342 (blue). Bars, 10 µm. (C) **F1M1 internalization in MCF-7/LCC2 cells by live imaging.** MCF-7/LCC2 cells were incubated with 10 µg/ml of Fabfluor-labeled F1M1 or rituximab (RTX) in serum-free DMEM at 37°C for 24 h. Cells were scanned every hour for 24 h and the red fluorescence intensity was measured using the Sartorius Incucyte® device by live imaging. Values are the mean ± SD.</figcaption></figure>

We confirmed these results in 3D cultures. In MCF7/LCC2 and MCF-7 spheroids (Fig. 5C), F1M1-MMAF and F1M1-MMAE inhibited cell viability more significantly than RTX-MMAF and RTX-MMAE.

### 3.4. F1M1-ADCs inhibit tumor growth and improve survival in mice xenografted with MCF-7/LCC2 cells

We used athymic Foxn1<sup>nu</sup> nude mice xenografted subcutaneously with MCF-7/LCC2 (ER<sup>+</sup> BC) cells to study the anti-tumor properties of

the F1M1-ADCs. When tumors reached 100 mm³, we treated mice with F1M1-MMAF, RTX-MMAF, F1M1-MMAE, or RTX-MMAE at day 17 (10 mg/kg), 24 (5 mg/kg) and 31 (5 mg/kg) post-graft, or with NaCl (control for the non-specific activity of RTX-based ADCs). We sacrificed mice when tumor volume reached 1000 mm³. Treatment with F1M1-MMAF and F1M1-MMAE significantly delayed tumor growth compared with control RTX-ADCs (Fig. 6A; *P* = 0.004 for F1M1-MMAF vs RTX-MMAF, *P* = 0.014 for F1M1-MMAE vs RTX-MMAE). Tumor growth was similar in mice treated with RTX-MMAF and with NaCl.
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Structure and average DAR of the F1M1-MMAF and F1M1-MMAE ADCs. (A) Bioconjugation for F1M1-based ADC production: two bioconjugation sequences of (i) mild reduction of F1M1 antibody interchain disulfide bridges followed by (ii) MMAE/F-linker addition were used to obtain the desired F1M1-based ADCs. (B) Chemical structure of the F1M1-MMAF ADC: antibody, non-cleavable linker, and payload, and lysosomal antibody digestion to release the MMAF metabolite. (C) Chemical structure of the F1M1-MMAE ADC: antibody, cleavable linker and payload, and linker proteolysis to release free MMAE.</figcaption></figure>

Conversely, tumor growth was significantly inhibited in mice treated with RTX-MMAE, certainly due to the non-specific extracellular cleavage of the linker and release of the cell-permeable MMAE into the tumor microenvironment (see Fig. 5), allowing its bystander toxic activity. At day 64, tumor volume was reduced by 65.5 % in the F1M1-MMAF group (*P* < 0.0001 vs RTX-MMAF) and by 68.7 % in the F1M1-MMAE group (*P* = 0.0053 vs RTX-MMAE) compared with the RTX ADCs (Fig. 6B). Moreover, overall survival (time to reach a tumor volume of ~1000 mm³) was significantly longer in F1M1-MMAF- and F1M1-MMAE-treated mice than in RTX-ADC-treated mice (median survival = 104.5

and 153 days for the F1M1-MMAF and F1M1-MMAE groups vs 80.5 and 123.5 days for RTX-MMAF and RTX-MMAE groups) (Fig. 6C; Kaplan-Meier survival analysis, *P* < 0.0001 for F1M1-MMAF vs RTX-MMAF, *P* < 0.0001 for F1M1-MMAE vs RTX-MMAE). F1M1-MMAE inhibited tumor growth more strongly with a longer survival compared with F1M1-MMAF. However, some of this antitumor activity was due to the non-specific extracellular release of cell-permeable MMAE, as observed in mice treated with RTX-MMAE (Fig. 6A-C). Although the F1M1 antibody cross-reacts with murine cath-D [47,48], all treated mice gained weight and had normal activities, suggesting no apparent toxicity of
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Binding and internalization of the F1M1-MMAF and F1M1-MMAE ADCs in MCF-7/LCC2 (OH-Tam-resistant) and MCF-7 BC cells. (A) Binding of F1M1-MMAF and F1M1-MMAE to human cath-D secreted from MCF-7/LCC2 and MCF-7 cells. Sandwich ELISA in which human cath-D from conditioned medium of MCF-7/LCC2 (left panel) and MCF-7 cells (right panel) was added to wells pre-coated with the anti-human cath-D M2E8 mouse monoclonal antibody in the presence of increasing concentrations of F1M1, F1M1-MMAF, or F1M1-MMAE. Binding of F1M1, F1M1-MMAF, and F1M1-MMAE to secreted human cath-D was revealed with an anti-human Fc HRP-conjugated antibody. The EC₅₀ values are shown. (B) **Internalization of F1M1-MMAF and F1M1-MMAE in MCF-7/LCC2 and MCF-7 cells.** MCF-7/LCC2 cells (left panels) and MCF-7 cells (right panels) were incubated with 50 µg/ml (333 nM) of F1M1-MMAF, RTX-MMAF, F1M1-MMAE, or RTX-MMAE and a 48 h serum-free conditioned medium from MCF-7 cells at 37°C for 3 h. ADC internalization was visualized with an Alexa Fluor 488-labeled anti-human Fc secondary antibody (green). Nuclei were stained with Hoechst 33342 (blue). Bars, 10 µm. (C) **Internalization of F1M1-MMAF and F1M1-MMAE in MCF-7/LCC2 cells by live imaging.** MCF-7/LCC2 and MCF-7 cells were incubated with 10 µg/ml (66.6 nM) of Fabfluor-labeled F1M1, F1M1-MMAF or F1M1-MMAE in serum-free DMEM at 37°C for 24 h. Cells were scanned every hour for 24 h and the red fluorescence intensity was measured using the Sartorius Incucyte® device by live imaging. Values are the mean ± SD.</figcaption></figure>

F1M1-based ADCs (Fig. 6D). Overall, these results indicate that anti-cath-D F1M1 bioconjugated to the MMAF or MMAE payload can hinder tumor growth in nude mice xenografted with MCF-7/LCC2 cells.

This is the first *in vivo* proof of concept that cath-D is a relevant target to develop ADCs for BC, particularly ER⁺/HER2low BC resistant to endocrine therapy.
<figure><img src="image_6.png" /><figcaption>Fig. 5. *In vitro*, F1M1-MMAF and F1M1-MMAE specifically inhibit cell viability in MCF-7/LCC2 (OHTam-resistant) and MCF-7 cells. (A) Inhibition of cell viability by F1M1-MMAF and F1M1-MMAE in MCF-7/LCC2 cells. MCF-7/LCC2 cells were incubated with increasing concentrations (from 1 to 50 nM) of F1M1-MMAF or RTX-MMAF (top panel), or F1M1-MMAE or RTX-MMAE (bottom panel) in DMEM/10 % FCS for 7 days. Cell viability was evaluated with the MTT assay. Results are expressed as the percentage of cell viability relative to control (untreated cells). Values are the mean ± SD (n = 3). The IC₅₀ value are shown. Similar results were obtained in four independent experiments. (B) Inhibition of cell viability by F1M1-MMAF and F1M1-MMAE in MCF-7 cells. MCF-7 cells were incubated with increasing concentrations (from 1 to 50 nM) of F1M1-MMAF or RTX-MMAF (top panel), or F1M1-MMAE or RTX-MMAE (bottom panel) in DMEM/10 % FCS for 7 days. Cell viability was evaluated with the MTT assay. Results are expressed as the percentage of cell viability relative to control (untreated cells). Values are the mean ± SD (n = 3). The IC₅₀ value are shown. Similar results were obtained in four independent experiments. (C) Inhibition of cell viability by F1M1-MMAF and F1M1-MMAE in MCF-7/LCC2 and MCF-7 cell spheroids. MCF-7/LCC2 (left panel) and MCF-7 cell spheroids (right panel) were incubated with RTX, F1M1, F1M1-MMAF, RTX-MMAF, F1M1-MMAE, RTX-MMAF (all 100 nM), MMAE (33 nM), or MMAF (33 nM) in DMEM/10 % FCS for 8 days. Cell viability was evaluated with CellTiter-Glo® and the Sartorius Incucyte® device by live imaging. Values are the mean ± SD (n = 4). MCF-7/LCC2 cell spheroids: **P = 0.0049 for F1M1-MMAE vs RTX-MMAE; ****P < 0.0001 for F1M1-MMAF vs RTX-MMAF. MCF-7 cell spheroids: **P = 0.0022 for F1M1-MMAE vs RTX-MMAE; ****P < 0.0001 for F1M1-MMAF vs RTX-MMAF. Similar results were obtained in two independent experiments using MCF-7/LCC2 and MCF-7 cell spheroids.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 6.** F1M1-MMAF and F1M1-MMAE inhibit tumor growth and increase survival in mice xenografted with OH-Tam-resistant MCF-7/LCC2 cells. (A) Effect of F1M1-MMAF and F1M1-MMAE on tumor growth. MCF-7/LCC2 cells (5 ×10⁶ cells), were subcutaneously injected in nude mice. When tumor volume reached 100 mm³, mice were treated (intravenous injection) with F1M1-MMAF (n = 10), RTX-MMAF (n = 10), F1M1-MMAE (n = 10), or RTX-MMAE (n = 10) at day 17 (10 mg/kg), 24 (5 mg/kg) and 31 (5 mg/kg) post-graft, or with NaCl (n = 10). Mice were sacrificed when tumor volume reached 1000 mm³. Tumor volume (in mm³) is shown as the mean ± SEM. \*\*P = 0.004 for F1M1-MMAF vs RTX-MMAF; \*P = 0.014 for F1M1-MMAE vs RTX-MMAE (mixed-effects multiple linear regression test). (B) Mean tumor volume at day 64 post-graft in the different treatment groups. n = 10 for F1M1-MMAF; n = 10 for RTX-MMAF; n = 10 for F1M1-MMAE; n = 10 for RTX-MMAE; n = 10 for NaCl; \*\*\*\*P < 0.0001 for F1M1-MMAF vs RTX-MMAF; \*\*P = 0.0053 for F1M1-MMAE vs RTX-MMAE (Student's t-test); data are the mean ± SEM. (C) Kaplan-Meier survival analysis in the different treatment groups. n = 10 for F1M1-MMAF; n = 10 for RTX-MMAF; n = 10 for F1M1-MMAE; n = 10 for RTX-MMAE; n = 10 for NaCl; \*\*\*\*\*P < 0.0001 for F1M1-MMAF vs RTX-MMAF; \*\*\*\*\*P < 0.0001 for F1M1-MMAE vs RTX-MMAE (Log-rank Mantel-Cox test). (D) Mouse weight monitoring in the F1M1-MMAF-, RTX-MMAF-, F1M1-MMAE-, RTX-MMAE- and NaCl-treated groups. n = 10 for F1M1-MMAF, n = 10 for RTX-MMAF, n = 10 for F1M1-MMAE, n = 10 for RTX-MMAE, n = 10 for NaCl. Data are the mean ± SEM.</figcaption></figure>

## 4. Discussion

Here, using the anti-human cath-D antibody F1M1 [47–50], we showed for the first time that cath-D is an eligible target for ADC-based cancer therapy in ER⁺/HER2<sup>low</sup> BC and endocrine therapy-resistant ER⁺/HER2<sup>low</sup> BC. We focused on the luminal BC type because new targets for innovative ADC therapy are urgently required due to its high frequency (70 % of all BC), resistance to endocrine therapy and late recurrence [2,3]. The combination of the CDK4/6 inhibitor and endocrine therapy is effective in advanced ER⁺/HER2<sup>-/low</sup> BC and is currently used as first-line treatment in patients with ER⁺/HER2<sup>-/low</sup> metastatic BC [74]. However, despite the great clinical benefit compared to endocrine therapy alone, almost all patients will progress after a median of 24 months. After failure of this combination, second- and third-line treatments are based on chemotherapy, conjugated antibodies or targeted therapy combined with other endocrine therapy types. Novel therapeutic opportunities for endocrine-resistant metastatic ER⁺/HER2<sup>-/low</sup> BC could come from innovative ADCs, for instance the anti-cath-D F1M1-ADCs described in this study. We chose the MCF-7 and MCF-7/LCC2 BC cell lines for the current study for the following

reasons: (1) these cell lines represent the luminal BC, which is ER⁺ with non-amplified, normal HER2 levels (HER2<sup>low</sup>) [53]; (2) MCF-7/LCC2 cells are an anti-estrogen (tamoxifen)-resistant BC model [54]; (3) using these cell lines bridges the gap between basic research and clinical application by exploring whether ADCs against cath-D, which is highly expressed in ER⁺/HER2<sup>low</sup> BC, could represent a new therapeutic option for patients with metastatic ER⁺/HER2<sup>-/low</sup> BC.

Cath-D is a well-validated marker of poor prognosis in all BC subtypes [15–19], in ER⁺ BC [20], and TNBC [21,22]. In this study, we quantified cath-D tumor expression in 80 ER⁺ BC biopsies, and the mean cath-D expression was 45.8 pmol/mg protein at the tumor site, which is in line with the cut-off observed for cath-D prognostic value in all BC subtypes [15–19]. We recently reported that cath-D is a tumor cell-associated marker, and a suitable target for antibody-based therapy in BC [46–50]. Specifically, we showed that cath-D expressed at the cancer cell surface can be targeted by the F1M1 antibody to mediate NK cell-mediated antibody-dependent cellular cytotoxicity in TNBC cells [48–50]. Moreover, cath-D displays important biological activities in BC: (1) its expression is specifically upregulated in BC cells and is hypersecreted in the BC microenvironment [23,24]; (2) cath-D locally
secreted at the tumor site can be partly tethered to the cell surface through interaction with different cell surface receptors, such as LRP1, or through association with caveola sites [26,30,43,75]; (3) secreted cath-D is endocytosed/internalized by BC cells and is targeted to the lysosomal pathways [23,43,45]; (4) extracellular cath-D bound to anti-cath-D mouse monoclonal antibodies (M2E8 or M1G8 generated by hybridoma approach) is internalized with the murine antibody by BC cells [45]. On the basis of our previous results with F1M1 [46–50] and cath-D biological activities in BC, we hypothesized that cath-D could be an eligible target for ADC therapy in BC. Our re-analysis of previously published proteomic data highlighted the extracellular and cell-surface expression of cath-D in $ER^+$ BC samples, but not in matched normal breast samples. Furthermore, we validated the presence of cath-D on the surface of cancer cells in $ER^+$ biopsies by immunohistochemistry and in MCF-7/LCC2 cell xenografts by immunofluorescence. In addition, we showed that extracellular cath-D is specifically internalized with the human antibody F1M1 in MCF-7 and MCF-7/LCC2 BC cells, as previously demonstrated with mouse M2E8 and M1G8 antibodies [45]. These findings indicate that F1M1-based ADCs could be internalized in $ER^+$ BC cells.

To generate anti-cath-D F1M1 ADCs, we conjugated two auristatin derivatives to F1M1 *via* a cleavable or a non-cleavable linker. Classical ADCs target internalizing antigens that are expressed at the surface of cancer cells. After specific antigen binding, these conjugates undergo clathrin- and antigen-mediated internalization and release in the lysosomal compartment (i) their cytotoxic agent (after proteolysis of the cleavable linker) or an active metabolite (after complete ADC digestion for ADCs bearing a non-cleavable linker). Non-cleavable linkers are associated with high plasma stability, but they also rely on efficient internalization and intracellular ADC degradation to release the active metabolite [76]. Moreover, this active metabolite is poly-charged at physiological pH and cannot cross biological membranes to achieve a bystander killing effect of neighboring cells. Therefore, ADCs with non-cleavable linkers kill mainly antigen-positive tumor cells [77,78], and their use remains restricted to tumors that strongly and homogeneously express the targeted antigen. On the other hand, cleavable linkers exploit endogenous stimuli by the tumor endosomal or lysosomal environment (e.g. high glutathione, glucuronidase or protease levels, acidic conditions) for the efficient intracellular release of the free cytotoxic agent (for review, see [79]). The capacity of the released free cytotoxic agent to achieve a bystander killing effect depends on its permeability and ability to diffuse through biological membranes outside the cancer cell and to enter the surrounding cells, leading to the death of both antigen-positive and antigen-negative neighboring cells [80]. This property allows the treatment of tumors with heterogeneous target antigen expression.

Thus, we first bioconjugated F1M1 *via* a non-cleavable linker to the tubulin polymerization inhibitor MMAF, a cytotoxic drug used to generate various ADCs currently tested in clinical trials [6]. MMAF has been mainly exploited for the development of ADC constructs with non-cleavable linkers that require internalization and lysosomal degradation to release into the cytoplasm an active metabolite, which is poly-charged at physiological pH, thus inhibiting its diffusion across membranes [81]. We chose this strategy to validate the cath-D target for an ADC approach because cath-D is not a conventional target (i.e. secreted and also associated with the cancer cell surface). Here we demonstrated that F1M1-MMAF binds to secreted cath-D, is specifically internalized in the MCF-7 and MCF-7/LCC2 BC cell lines and can kill effectively these cells. Conversely, MMAF alone was not toxic. Next, we showed that compared with RTX-MMAF (control), F1M1-MMAF significantly inhibited MCF-7/LCC2 cell xenograft growth and prolonged the survival of mice harboring such xenografts. Moreover, F1M1-MMAF was well-tolerated, without signs of systemic toxicity in the treated animals. Importantly, as F1M1 cross-reacts with murine cath-D [47,48], if secreted cath-D were present in normal/healthy mouse tissues, strong toxicity should have been observed, which was not

the case. The payload MMAF displays low bystander activity [52], and presumably the ADC lacks the ability to target cath-D-negative cells. However, as the MCF-7/LCC2 cell xenografts used in the present study were derived from a BC cell line, homogeneous expression of cath-D within the tumor mass was expected, as observed. Conversely, BC samples from patients are heterogeneous, and cath-D might be expressed at the cancer cell surface only in a fraction of tumor cells. Consequently, we can speculate that the therapeutic efficacy of an F1M1-based ADC could be enhanced by using payloads with bystander activity (e.g. MMAE), which might target also neighboring cath-D negative tumor cells. Furthermore, although ADC linkers can be non-cleavable, most ADCs in clinical development have specific release mechanisms that allow the linker controlled cleavage at the target site and therefore, are described as cleavable [82]. For this reason, we then generated the F1M1-MMAE ADC using a classic cleavable VC-PABC protease-sensitive linker, and compared its efficacy to that of F1M1-MMAF *in vitro* and *in vivo*. F1M1-MMAE bound to secreted cath-D, and was internalized in MCF-7 and MCF-7/LCC2 BC cells. F1M1-MMAE showed a stronger cytotoxic activity, compared with F1M1-MMAF, in MCF-7 and MCF-7/LCC2 BC cell lines. However, RTX-MMAE (control ADC) also displayed non-specific killing activity, certainly due to the linker extracellular cleavage and release of cell-permeable MMAE. Indeed, MMAE alone displayed very high cytotoxic activity in the same cell lines. Next, compared with F1M1-MMAF, F1M1-MMAE more potently inhibited tumor growth and prolonged the survival of mice harboring MCF-7/LCC2 cell xenografts. However, RTX-MMAE also displayed some non-specific inhibition of tumor growth compared with the NaCl-treated control group, reflecting the non-specific release of MMAE, certainly due to the extracellular cleavage of the protease-sensitive linker of this ADC by stromal proteases, allowing its bystander cytotoxic effect. Indeed, the tumor lysosomal compartment and microenvironment share characteristic features, such as high protease levels, allowing the active payload release from protease-sensitive linkers in the tumor microenvironment through an extracellular mechanism [83].

Many ADCs with MMAE (DAR=4) targeting the nectin-4 receptor [84], the RON receptor [85], the immunomodulatory protein B7-homologue 3 (B7-H3) [86], or the neural cell adhesion molecule CD56 [87] have been studied in different cancer types, demonstrating the ongoing interest of the MMAE payload. Our results add to the notion that a secreted protein which can be internalized, such as cath-D, could also be targeted with an ADC bearing MMAE as payload, using a cleavable linker.

Similar to cath-D, galectin-3-binding protein, which is secreted specifically at the tumor site, has been validated as a promising new target against melanoma, but with non-internalizing ADCs using maytansinoid payloads [88]. An interesting, alternative and complementary ADC approach is to target the tumor stromal compartment because tumor growth is determined by the dynamic interaction between tumor cells and the surrounding stromal cells [78,89,90]. This was demonstrated for various targets, such as the type II transmembrane serine protease fibroblast activating protein (FAP; a serine protease overexpressed on the surface of cancer-associated fibroblasts) [91], leucine-rich repeat-containing protein 15 (LRRC15; a receptor expressed by cancer-associated fibroblasts) [92] and tumor endothelial marker 8 (TEM8) [93], after bioconjugation of a specific antibody to the MMAE payload, *via* a cleavable linker. In addition, recently, MMAE and MMAF were delivered to the tumor microenvironment using small molecule-drug conjugates activated *in vivo* by cleavage of a FAP-specific linker [94,95]. As extracellular cath-D can bind to and be internalized by various cell surface receptors on the surface of BC cells and stromal cells, such as fibroblasts [23,43,45], we can speculate that anti-cath-D ADCs might also target stromal cells at the cancer site.

## 5. Conclusions

This study shows for the first time that cath-D is a potent target for
ADC therapy. As this protease is overexpressed and hypersecreted in many cancer types, such as ovarian cancer [44], melanoma [96], squamous cell carcinoma [97], pancreatic cancer [98], glioblastoma [99] and hepatocarcinoma [100], F1M1-based ADCs could be applicable to a wide range of tumor entities.

## Ethics approval

Mouse experiments were performed in compliance with the French regulations and ethical guidelines for experimental animal studies in an accredited establishment (Agreement No. #31135-2021042212479661). For immunohistochemistry, BC biopsy samples were provided by the biological resource center (Biobank number BB-0033-00059) after approval by the Montpellier Cancer Institute Institutional Review Board, following the French national Ethics and Legal dispositions for patients' information and consent. For BC cytosols, patient samples were processed according to the French Public Health Code (law n°2004-800, articles L. 1243-4 and R. 1243-61), and the biological resources center is authorized (authorization number: AC-2008-700; Val d'Aurelle, ICM, Montpellier) to deliver human samples for scientific research. All patients were informed before surgery that their surgical specimens might be used for research purposes.

## Funding

This work was supported by a public grant overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program (reference: LabEx MabImprove ANR-10-LABX-53-01), ACCelerating Research for Early Development of Innovative Antibodies – ACCREDIA (ANR-22-PEBI-0009), University of Montpellier, Inserm Transfert, Région Occitaine. This work was also supported by La Ligue contre le cancer (committees 18, 30, 34, 35, 36, 37, 41, 44, 53, 72, 79, 85), the Centre-Val de Loire Region and European Regional Development Fund (FEDER). This publication is based on work from COST Action ProteoCure, CA20113, supported by COST (European Cooperation in Science and Technology). S. Huvelle thanks Centre-Val de Loire Region for his post-doctoral fellowship. C. Martin thanks LabEx SynOrg (ANR-11-LABX-0029) for her PhD studentship.

## CRediT authorship contribution statement

**Gaëlle Guillon:** Methodology, Formal analysis, Conceptualization.
**Andrei Turtoi:** Methodology. **Diane Nishimwe:** Methodology, Formal analysis, Conceptualization. **Ilona Berestjuk:** Methodology. **Florence Boissière-Michot:** Methodology. **Hanane Mansouri:** Formal analysis.
**Véronique Garambois:** Methodology. **Emmanuelle Liaudet-Cooperman:** Writing - review & editing, Writing - original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Cynthia Garapon:** Methodology. **Valérie Laurent-Matha:** Writing - review & editing, Validation, Supervision, Project administration, Methodology, Conceptualization. **Andréa Romero:** Methodology. **Nicolas Joubert:** Writing - original draft, Supervision, Methodology, Conceptualization. **Lindsay B. Alcaraz:** Methodology, Data curation. **Thierry Chardès:** Writing - original draft, Methodology, Conceptualization. **Steve Huvelle:** Methodology, Conceptualization. **Séverine Guiu:** Supervision. **Camille Martin:** Methodology, Conceptualization. **Pascal Roger:** Supervision. **Aude Mallavialle:** Methodology. **Caroline Denevault-Sabourin:** Resources.

## Declaration of Competing Interest

The authors have no potential conflict of interest to disclose.

## Acknowledgments

We thank the IRCM animal facility staff (Biocampus RAM-PEFO, IRCM, Montpellier, France) for animal care and for in vivo studies. We thank the imaging facility MRI, member of the national France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INBS-04, Investments for the future). The authors would like to thank the Hospital Pharmacy of Tours Teaching Hospital for providing leftover rituximab (Rituxan®, Roche) and Dr Cyril Colas (ICOA UMR 7311 CNRS Université d'Orléans) for MS analysis.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biopha.2025.118775.

## Data availability

Data will be made available on request.

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