All commercially available reagents were purchased from Sigma-Aldrich, Fluorochem, Tokyo Chemical Industries, Alfa Aesar, Carlo Erba, VWR Chemicals and used as received without further purifications. All manipulations requiring anhydrous conditions were carried out in flame-dried glassware with magnetic stirring under nitrogen atmosphere. Anhydrous solvents were withdrawn from the container by syringe, under a slight positive pressure of nitrogen. Reactions were checked by analytical thin-layer chromatography (TLC) using silica gel pre-coated ALUGRAM Xtra SIL G/UV₂₅₄ plates (0.60 mm thickness) purchased from Macherey-Nagel. TLCs were examined by visualization under UV light (λ = 254 nm) and/or staining with ninhydrin, ceric ammonium-molybdate or potassium permanganate alkaline solutions. Details of the synthetic procedures can be found in the Supplementary Material S1 document. Purifications by flash-chromatography were performed using 60 Å, 230–400 mesh, 40–63 μm silica gel. Purifications by preparative HPLC were carried using a SHIMADZU LC-20AP prominence apparatus equipped with a FRC-10A fraction collector, SPD-M20A diode-array detector, CBM-20A system controller and a Sepachrom Robusta 100 Å C18 5 μm 250 × 21.2 mm column (flow 15 mL/min). All HPLC solvents were degassed for 90 min under ultrasonic treatment. Pure freeze-dried compounds were obtained from frozen aqueous solutions using a Telstar Lyo Quest −55 lyophiliser. Purities of synthetized compounds were analyzed by analytical HPLC SHIMADZU LC-20AP equipped with diode array UV detector and LiChrosorb RP-18 (5 μm) C18 column and by Waters 600 HPLC System (equipped with Phenomenex LC column 150 × 4.6 mm Synergi 4 μm Fusion RP 80 Å) coupled with MS Waters Micromass ZQ, ESI source (flow 1 mL/min). All employed HPLC-MS solvents were degassed for 20 min under a 100 mL/min helium flow. Solution ¹H and ¹³C NMR spectra were recorded using a Bruker Avance 400 spectrometer operating respectively at 400.16 MHz and at 100.63 MHz. ¹H and ¹³C chemical shifts are reported in ppm (δ) relative to TMS (internal standard). Coupling constants are reported in Hz and spin multiplicity is described as follow: s = singlet, d = doublet, t = triplet, dd = doublet of doublets, m = multiplet. High resolution mass spectra (HRMS) were obtained with Thermo Fisher Scientific Orbitrap Exploris 120 equipped with UHPLC and C18 column.

Peptide sequences were prepared by manual Solid Phase Peptide Synthesis (SPPS) by the Fmoc-strategy on Fmoc-Gly preloaded Wang resin (commercially reported loading 0.4–0.8 mmol/g). The synthesis was performed using polypropylene syringes equipped with PTFE frits as reaction vessels; stirring was accomplished by a shaking plate. Each coupling step was checked through LC-MS analysis of a small-cleavage sample. Details of the synthetic procedures are reported in the Supplementary Material S1.

U87MG human malignant glioma, SK-MEL-28 human melanoma, SK-OV-3 human ovarian cancer, and A549 human lung cancer cell lines were purchased from ATCC. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Biosera, Nuaille, France), supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS; Biosera), and with 1% Penicillin/Streptomycin (Biosera). Cells were cultured in sterile T75 flasks with ventilation cap (Sarstedt, Nümbrecht, Germany) at 37 °C in a humidified atmosphere with 5% CO₂ in ESCO CelCulture Incubator (ESCO, Friedberg, Germany). Manipulations with the cells were performed in biosafety cabinet (laminar) ESCO Sentinel Gold class II model AC2-4E8 (ESCO).

Cells were harvested using Accutase (Sigma Aldrich, St. Louis, MO, United States), and one million cells from each cell line were used for measuring the level of integrin α_Vβ₃ receptor on the cell surface. The cells were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature (rt), washed with phosphate buffered saline (PBS, Biosera) and exposed to 3% Bovine Serum Albumin (BSA; Sigma Aldrich) in PBS for 20 min at rt. Afterwards, integrin α_Vβ₃ antibody (Anti-Integrin α_Vβ₃ antibody, clone LM609, mouse; MAB 1976, Merck) was used in a concentration of 3 µg/million cells, diluted in PBS and 3% BSA solution and incubated for 2 h at rt. A fluorescent secondary antibody was used for detection (Cell Signaling Technology, AlexaFluor^® 488-conjugated anti-mouse IgG Fab fragment, CST 4408, 1:1000) and incubated at rt for 30 min. As control, samples only exposed to secondary antibody were used. The fluorescence was detected using the FITC-A channel of FACSVerseTM Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, United States). The FCSalyzer 0.9.22-alpha free software (SourceForge, San Diego, CA, United States) was applied to determine and evaluate gate percentage, mean fluorescence intensity (MFI) and median fluorescence intensity (MDFI).

After standard trypsinization and harvesting cells by trypsin (Biosera), ethylenediaminetetraacetic acid (EDTA; Biosera) and PBS solution, 6×10³ cells per well were seeded in serum containing (10%) growth medium to 96-well plates with flat bottom (Eppendorf, Hamburg, Germany), in a 100 μL volume per well, and incubated at 37°C.

After 24 h, cells were treated with 100 µL of 9 different concentrations of the conjugate 2, the free ligand and free drug MMAE (1.19 p.m.–20 μM, final volume in the well was 200 µL), dissolved in serum free medium and incubated for 72 h continuously (5% serum final) in the no-wash out experiment. In the wash out experiment, cells were incubated with the compounds (5% serum final) for 30 min, then they were washed out, and additionally incubated in growth medium (5% serum final) up to 72 h. The control wells were treated only with serum free medium (5% serum final).

For the evaluation of the in vitro antiproliferative activity of compounds, the cell viability was determined by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) obtained from Duchefa Biochemie (Haarlem, Netherlands).

22 µL of MTT solution (5 mg/mL in PBS, 0.5 mg/mL final) was added to each well and after 2 h incubation at 37°C, the supernatant was removed. The precipitated purple formazan crystals were dissolved in 100 µL of a 1:1 solution of dimethylsulfoxide (DMSO; Merck, St. Louis, MO, United States): 96% Ethanol (Molar Chemicals Kft., Halásztelek, Hungary) and the absorbance was measured after 15 min at λ = 570 nm by using microplate reader CLARIOstar Plus (BMG Labtech, Ortenberg, Germany). Average background absorbance (DMSO-Ethanol) was subtracted from absorbance values of control and treated wells, and cell viability was determined relative to untreated (control) wells where cell viability was arbitrarily set to 100%. Absorbance values of treated samples were normalized versus untreated control samples and interpolated by nonlinear regression analysis with GraphPad Prism 6 software (GraphPad, La Jolla, San Diego, CA, United States) to generate sigmoidal dose-response curves from which the 50% inhibitory concentration (IC₅₀) values of the compounds were calculated and presented as nanomolar (nM) units. The experiments were done in triplicate and each experiment was repeated three times.

The conjugate 2 and the free MMAE were tested in presence of the MMP-2 inhibitor cis-9-octadecenoyl-N-hydroxylamine (Merck) dissolved in DMSO (You et al., 2018). The three α_Vβ₃ integrin and MMP-2 overexpressing cancer cell lines (U87MG, SK-MEL-28, SK-OV-38) were seeded with serum containing medium (Ellijimi et al., 2018; Shim et al., 2019) and, after 8 h, were washed-out. Then, 100 µL per well of MMP-2 inhibitor (10 µM final concentration) were added and cells were incubated for 16 h, prior to the treatment. Later, the conjugate 2 and free MMAE were dissolved in 10% serum containing medium and added to reach the same concentrations evaluated in the previous test in a final volume of 100 µL per well and incubated (5% serum final) continuously for 72 h of the treatment. The experiments were performed in triplicate.

The cleavage test with the MMP-2 enzyme was performed using recombinant human MMP-2 protein (Active) purchased from Abcam (ab81550). A buffer solution Tris∙HCl (50 mM), NaCl (0.2 M), CaCl₂ (10 mM), Brij-35 (0.05%), ZnSO₄ (50 µM), pH 7.4 was prepared in accordance with the literature (Tauro et al., 2008). A solution of EDTA (50 mM) was prepared for stopping the MMP-2 activity. For the assay, the MMP-2 enzyme (10 µg) was diluted with 100 µL Milli-Q Water, accordingly to the data sheet. A solution of the H₂N-GPLG-PABC-MMAE 7 was prepared (0.105 mg in 0.881 mL of buffer solution). To start the assay, 25 µL MMP-2 enzyme was added to 475 µL of the linker solution to have 1:20 (w/w) enzyme: linker ratio. The final solution was stirred and incubated at 37 °C. 100 μL-aliquots were removed at 0, 6 and 24 h, and the enzymatic activity was quenched by adding 350 µL EDTA solution. The resulting solution was diluted with 180 µL Milli-Q Water and 70 µL of acetonitrile (MeCN) and analyzed by analytical HPLC SHIMADZU LC-20AP equipped with diode array UV detector and LiChrosorb RP-18 (5 μm) C18 column. A linear gradient elution was used from 10% to 90% B in 30 min (eluent A: H₂O, 0.1% trifluoroacetic acid, TFA; eluent B: MeCN) at a flow rate of 1 mL/min and the column temperature was set to 30 °C (vide Supplementary Material S5). Based on previous literature data (Butowska et al., 2020), the peptide Fmoc-GPLGLAGG-OH was used as positive control for the evaluation of the MMP-2 enzymatical activity, and it was synthesized by SPPS following the general procedure reported in the Supplementary Material S1. For this, 25 µL of MMP-2 enzyme solution was added to 475 µL of Fmoc-GPLGLAGG-OH solution (0.118 mg in 1.3 mL buffer solution), and the assay was carried out with the same procedure. To establish the release of the payload, the HPLC trace of free MMAE was used for comparison.

The enzymatically cleavage test was performed by incubating the conjugate 2 with rat liver lysosomal homogenate and analyzed by LC–MS. In particular, the conjugate 2 (2.35 μg/μL in DMSO, 5 μL) was diluted with 0.2 M NaOAc solution (pH 5.05, 495 μL) to 0.024 μg/μL. The lysosomal homogenate was prepared from rat liver (Gomena et al., 2023), and contained proteins in 71.76 μg/μL concentration. 8.6 μL of this lysosomal homogenate was diluted with 66.4 μL 0.2 M NaOAc solution (pH 5.05), to have a protein concentration of 8.27 μg/μL. To start the assay, 15 μL lysosome homogenate (8.27 μg/μL) was added to 500 μL conjugate solution (0.025 μg/μL), to have a conjugate: lysosomal protein = 1:10 (w/w) ratio. Furthermore, a control reaction mixture was prepared, containing the conjugate (2.35 μg/μL in DMSO, 5 μL) and 510 μL of 0.2 M NaOAc solution (pH 5.05). The samples were stirred at 300 rpm, 37°C and 50 µL aliquots were taken out at 0 min, 15 min, 30 min, 1 h, 2 h, 6 h, 24 h, and 72 h. The enzymatic activity was quenched by adding 5 μL formic acid (FA) to the samples. After this procedure, samples were frozen immediately at −25°C. Control samples were collected at 0 min, 1 h, 6 h, 24 h and 72 h. The composition of the samples was determined by HPLC-MS. The LC-MS analysis was carried out on an UltiMate 3000 UHPLC system (Thermo Fisher Scientific, Bremen, Germany) coupled to a Q Exactive^TM Focus, high resolution and high mass accuracy, hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Compounds were separated on a Supelco Ascentis C18 column (150 × 2.1 mm, 3 µm) (Hesperia, CA) using a linear gradient elution from 2% to 90% B in 16 min (eluent A: H₂O, 0.1% FA; eluent B: 80% MeCN, 0.1% FA) at a flow rate of 0.2 mL/min and the column temperature was set to 40°C. High-resolution mass spectra were acquired in the 200–2000 m/z range. LC-MS data were analyzed by XcaliburTM software (Thermo Fisher Scientific).

The synthesis of the conjugate cyclo[DKP-isoDGR]-PEG₄-GPLG-PABC-MMAE 2 started from the preparation of the GPLG peptide sequence (Scheme 1). Fmoc-GPLG-OH 3 was prepared by manual SPPS starting from Fmoc-Gly preloaded Wang resin (commercially reported loading 0.4–0.8 mmol/g), which was subsequently coupled to Fmoc-Leu (5 eq) using (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU) as coupling reagent (5 eq) and N,N-diisopropylethylamine (DIPEA, 8.5 eq) as base, after removal of the Fmoc-protecting group. The same procedure was used for the successive coupling reactions with Fmoc-Pro and Fmoc-Gly, respectively. After cleavage from the resin with TFA/H₂O/triisopropyl silane (TIS) 95:2.5:2.5, the peptide was obtained with quantitative yield after purification by flash-chromatography column (Scheme 1).

The synthesis of Fmoc-GPLG-PABC-MMAE fragment (6) was performed as described in Scheme 2. After treatment of Fmoc-GPLG-OH 3 with 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) and PAB-OH (82% yield), compound 4 was treated with 4-nitrophenyl chloroformate and pyridine in THF for 6 h, affording product 5 in 47% yield. MMAE was then conjugated to the carbonate derivative 5 using DIPEA as base and 1-hydroxy-7-azabenzotriazole (HOAt) in N,N-dimethylformamide (DMF), leading to the desired Fmoc-GPLG-PABC-MMAE (6) in 73% yield. Sequential Fmoc-cleavage in the presence of piperidine, which afforded the free amine 7, and subsequent reaction with 4-pentynoic acid-NHS ester led to alkyne 8 in moderate yield (36%). A copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) «click » reaction was finally used to conjugate 8 and the functionalized ligand cyclo[DKP-isoDGR]-PEG₄-azide 9, prepared as previously reported (Bodero et al., 2018), which gave the SMDC 2 in 30% yield (Scheme 2).

To evaluate the cytotoxicity of our novel SMDC 2, we chose U87MG, SK-MEL-28, SK-OV-3 cancer cell lines, which are reported to overexpress both the α_Vβ₃ integrin receptor and the MMP-2 enzyme (Brooks et al., 1996). Additionally, we chose the A549 cancer cell line as low expressing α_Vβ₃ integrin receptor to validate the targeting. The expression of α_Vβ₃ integrin was anyway measured by flow cytometry, confirming a higher content in the case of U87MG, SK-MEL-28, SK-OV-3 compared to A549 cancer cell lines as shown in Figure 2.

In a first instance we performed an in vitro antiproliferative assay in which the different cell lines were incubated with different concentrations of the conjugate 2 for 72 h. As reported in Figure 3, 2 showed a potent antitumor activity in a low nanomolar range, from 3.7 to 70.4 nM depending on the cancer cell line, whereas the treatment with the free drug MMAE led to an antitumor activity of 0.03 nM for SK-OV-3 and of 0.23 nM for U87MG, and in very low nanomolar range on SK-MEL-28 cell line (Figure 3).

Interestingly, the relative potency (RP) of the conjugate, normalized with respect to MMAE (i.e., the ratio of the IC₅₀ values of 2 vs. MMAE) in U87MG cancer cell line after 72 h was much lower than the one observed with the previously reported cyclo[DKP-isoDGR]-PEG₄-VA-PABC-MMAE (33-fold loss of potency vs. 151-fold loss of potency, respectively) bearing the VA linker (Dias et al., 2019). This data could suggest a positive effect of the GPLG cleavable linker on the release of the MMAE. Furthermore, a drop of potency between the free drug and the conjugate can be noticed in A549 from the RP value, where the integrin receptor level is significantly lower than in the other cancer cell lines. To better explore the targeting ability of the conjugate 2 towards different level of integrin expression, we decided to further evaluate the antiproliferative activity of the GPLG-conjugate by changing the incubation time.

Recently, a new protocol for evaluating the antiproliferative activity and the targeting ability of integrin-ligand based conjugates was developed by the group of Prof. Neundorf and ourselves (Feni et al., 2019). In this procedure, which we named “kiss-and-run” experiment, after very short initial contact times (15–30 min) of the toxin with the cells in vitro, the solution is drained, and cells are resuspended in the culture medium for a 72 h incubation. This protocol is aimed at simulating the rapid clearance of the drugs that occurs in vivo in the extracellular tumor environment, thus highlighting the ability of the conjugate to bind the integrin receptor on the cell surface.

Thus, the 4 cell cultures were incubated with conjugate 2 or free MMAE for 30 min, followed by a washout of the treatment and a further incubation of the cells up to 72 h. As hypothesized, our conjugate showed higher antiproliferative activity than free MMAE on U87MG, SK-OV-3 and SK-MEL-28 integrin α_Vβ₃ receptor overexpressing cancer cell lines in comparison to low expressing cell line A549, with nanomolar activity (Figure 4).

The results clearly indicate that cyclo[DKP-isoDGR]-PEG₄-GPLG-PABC-MMAE (2) is able to bind the surface of those cancer cells showing higher level of integrin overexpression (U87, SK-MEL-28 and SK-OV-3), and that the washout process does not remove it from the cell culture media. By contrast, the low level of integrin expression in A549 led to less amount of bound conjugate to the surface, and thus the washout experiment tends to eliminate it, eventually leading to a more significant decrease of the toxicity (Figure 4). Remarkably, the conjugate revealed higher antiproliferative activity compared to free drug MMAE on U87MG and SK-MEL-28, and very similar on SK-OV-3, whereas the free drug was more potent compared to the conjugate in the low integrin α_Vβ₃ receptor expressing cancer cell line A549. The relative potency (RP) of the conjugate in the different cell lines shows a good correlation with the α_Vβ₃ integrin expression. This important result confirms the efficacy of the cyclo[DKP-isoDGR] α_Vβ₃ integrin ligand in retaining the SMDC thus highlighting its targeting role. The final issue to be confirmed was whether the drug release mechanism relied on the extracellular enzymatic cleavage of the GPLG linker by the MMP-2, and subsequent internalization of MMAE by simple passive diffusion. To better elucidate the MMP-2 ability to recognize and cleave the linker, we performed a further antiproliferative bioassay adding a commercially available MMP-2 inhibitor to the cell culture in the presence of the conjugate 2.

To determine whether release of the MMAE from the conjugate depends on the action of MMP-2 enzyme by cleaving the GPLG-linker, the conjugate 2 was incubated with the α_Vβ₃ integrin positive cancer cell lines in the presence of the MMP-2-inhibitor cis-9-octadecenoyl-N-hydroxylamide. This strategy has been already reported by You and co-workers to evaluate the influence of an MMP-2 cleavable linker in the observed cytotoxicity (You et al., 2018). Surprisingly, the IC₅₀ values obtained after 72 h no-washout experiments were 7.4 nM, 28.7 nM and 3.2 nM for U87MG, SK-MEL-28, SK-OV-3, respectively (Supplementary Table S4.1), which are comparable to those obtained without adding any MMP-2 inhibitor to the cell cultures (Figure 3).

These data were further confirmed by the enzymatic test performed incubating the H₂N-GPLG-PABC-MMAE (7) with the active form of MMP-2 enzyme and analyzing the final solution by HPLC. Indeed, by comparison of the HPLC retention time of 7 and the free MMAE, the latter was not observed even after 74 h of incubation at 37°C (Supplementary Figure S5.3).

The lack of MMAE release upon incubation with MMP-2 enzyme, as well as the lack of significant differences in cytotoxicity when an MMP-2 inhibitor was added into the cell media, clearly indicate that the GPLG-PABC linker used in 2 is not sensitive to the extracellular MMP-2 enzyme. Although our GPLG-PABC linker is sharing a Gly at P1 and a Pro at P3 position with the most commonly used MMP-2 recognized sequences (GPLG-IAGQ, PLG-LAG and GPVG-LIGK, where hyphen indicates the cleavage site), the PABC moiety is probably not able to replace the interactions within the active site of the enzyme usually generated by the branched amino acid at the P’ position and, more in general, by the longer aminoacidic sequence (Poreba, 2020). Thus, the linker is not recognized by the MMP-2 and consequently cannot be hydrolyzed. Nevertheless, the increase in potency observed in the non-washout experiment compared with the VA-based conjugate, and the remarkably higher potency of 2 than the free MMAE in the washout experiment, suggest that an efficient release of the drug is occurring by the action of different proteolytic agents.

Although the poor ligand-mediated internalization of small integrin ligands is well documented (Liu et al., 2018; Kemker et al., 2020), the similarity of the GPLG sequence with the well-known lysosomal recognized GFLG sequence (Ruan et al., 2015), led us to hypothesize that the cleavage might occur by lysosomal enzymes, too. Indeed, the possibility to have an efficient payload release from a drug conjugate through the action of intracellular proteolytic enzymes has been reported also for non-internalizing mAbs. For example, Neri and co-workers reported that the use of the Cathepsin B sensitive Val-Cit linker in the non-internalizing IgG (F16)−MMAE conjugate led to a selective and highly cytotoxic compound both in vitro and in vivo experiments, whereas similar products based on non-cleavable linkers were not active (Dal Corso et al., 2017b). Similar evidences were also reported for non-internalizing SMDCs: in this context, a Carbonic Anhydrase IX (CAIX) small ligand and MMAE were successful conjugated through a series of Cathepsin B sensitive peptide sequences (Cazzamalli et al., 2017). In these cases, the cleavage of such sequences has been ascribed to the presence of the lysosomal Cathepsin B also in the tumor stroma, where is devoted to degrading extracellular matrix components (Sloane, 1990). The released drug may then accumulate in the extracellular environment and internalize into cancer cells by passive diffusion. Finally, the consequent cell death may induce the release of additional proteolytic enzyme (by-stander killing effect), which could cleave further cytotoxic payload from the non-internalizing conjugate (Li et al., 2016).

Based on these considerations, we decided to determine the enzymatic susceptibility of conjugate 2 containing GPLG-PABC linker by studying its degradation in the presence of rat liver lysosomal homogenate. The cleavage of the conjugate, along with the release of the free MMAE was monitored by UPLC-HRMS. As shown in Figure 5C, the cyclo[DKP-isoDGR]-PEG₄-GPLG-PABC-MMAE 2 was rapidly cleaved in the presence of lysosomal enzymes, and, upon the 1,6-elimination of the PABC spacer, the free MMAE was detectable already 15 min after incubation (Figure 5; Supplementary Figure S6.1).

Interestingly, the conjugate was not fully consumed by lysosomal enzymes even after 72 h, probably due to the decreasing activity or amount of the specific enzyme responsible for the cleavage of this specific linker type. Although further work is needed to better investigate this experimental evidence, and assuming that in the cell culture assays a similar behavior was occurring, the amount of the released MMAE was sufficient for the potent cytotoxic activity observed in the in vitro assays.