Small molecule–drug conjugates (SMDCs) represent an alternative to conventional antitumor chemotherapeutic agents, with the potential to improve the therapeutic window of cytotoxic payloads through active delivery at the site of the disease. In this article, we describe novel combination therapies consisting of anti-carbonic anhydrase IX SMDCs combined with different immunomodulatory products. The therapeutic effect of the SMDCs was potentiated by combination with PD-1 blockade and with tumor-homing antibody–cytokine fusions in mouse models of renal cell carcinoma and colorectal cancer. The combination with L19-IL12, a fusion protein specific to the alternatively spliced EDB domain of fibronectin containing the murine IL12 moiety, was also active against large established tumors. Analysis of the microscopic structures of healthy organs performed 3 months after tumor eradication confirmed absence of pathologic abnormalities in the healthy kidney, liver, lung, stomach, and intestine. Our findings may be of clinical significance as they provide motivation for the development of combinations based on SMDCs and immunotherapy for the treatment of renal cell carcinoma and hypoxic tumors.
The majority of patients with cancer receive chemotherapy as part of combination treatments (1). The clinical efficacy of conventional chemotherapeutic drugs is often limited by their unfavorable biodistribution profile (2). The use of antibodies, specific to certain tumor-associated antigens and serving as drug delivery vehicles, may improve the therapeutic window of potent cytotoxic agents (3–5). This approach has led to the marketing authorization of eight antibody–drug conjugates (ADCs; Mylotarg, Kadcyla, Adcetris, Besponsa, Polivy, Padcev, Enhertu and Trodelvy; refs. 4, 6–9). Although the clinical benefit of ADCs has become evident, the therapeutic window of these biopharmaceuticals is narrower than what had initially been estimated on the basis of preclinical studies (10, 11). The toxicity profile of ADC products often involves organs that do not express the target antigen and may be substantial when approaching the MTD (12, 13). The implementation of site-specific ADCs has been recently described as a strategy to improve their therapeutic index (14), but a clear proof of their clinical benefit over old-generation products still lacks.
ADC products often extravasate slowly over the blood vessel walls and may exhibit a heterogeneous distribution within solid tumor masses (15, 16). Small molecule–based targeting agents may represent an alternative to antibodies. Excellent biodistribution profiles in mouse and man have been reported for small organic ligands specific to folate receptor (17), prostate-specific membrane antigen (18), fibroblast activation protein (19, 20), and carbonic anhydrase IX (CAIX; ref. 21). The chemical conjugation of a small organic tumor-targeting moiety to a cytotoxic drug leads to the generation of a novel class of therapeutic compounds named small molecule–drug conjugates (SMDCs; refs. 22–24), which may represent an alternative to ADCs. Small ligands extravasate within seconds and may reach tumor cells far away from blood vessels (22). In a recent direct comparison of an ADC and an SMDC directed against CAIX, the small drug conjugate exhibited a better and more efficient tumor uptake, but both conjugates were efficacious in controlling tumor growth (25).
CAIX is a transmembrane homodimeric protein and a validated target for drug delivery applications (26). CAIX is overexpressed in the majority of clear-cell renal cell carcinomas and in hypoxic tumors, whereas its expression in normal tissues is mainly confined to certain structures of the gastrointestinal tract (27). Both antibodies and small molecules have been successfully used to target CAIX-positive tumors in vivo (28–30). Acetazolamide (AAZ), a small organic nanomolar binder of CAIX, represents a “portable” moiety for the active delivery of radionuclides, fluorophores, and cytotoxic drugs (21, 30–32).
We have recently described the generation of new antitumor SMDC products based on acetazolamide and its affinity-matured derivative, called acetazolamide-plus (AAZ+; ref. 33). In this study, the two compounds were conjugated through the cleavable valine-citrulline linker (Val-Cit) to the potent tubulin inhibitor, monomethyl auristatin E (MMAE), also used in Adcetris (34, 35).
We observed a potentiation of anti-CAIX SMDC products using antibody-IL2 and with antibody-IL12 fusion proteins, which target the alternatively spliced EDB domain of fibronectin (36). Moreover, we studied the combination of SMDCs with a PD-1 immune checkpoint inhibitor (37). All combinations were found to be safe over a prolonged period of time, as demonstrated by histopathologic analysis performed on organs derived from cured animals. Our results provide a rationale for the clinical development of combination treatments based on the acetazolamide SMDC products, ideally in combination with tumor-homing antibody–cytokine fusions or with anti–PD-1 antibodies.
Materials and Methods
Detailed synthetic procedures and characterization of the presented compounds are described in the Supplementary Information.
Preparation of SMDCs
AAZ-ValCit-MMAE was prepared following well-established synthetic procedures (31). Briefly, commercially available MC-ValCit-PAB-MMAE (1 eq.) was reacted with compound 1 (4 eq. dissolved in 100 μL of DMF) in freshly degassed PBS (50 mmol/L phosphate and 100 mmol/L NaCl, pH 7.4; 900 μL). The reaction was stirred at room temperature until completion (monitored by LC/MS). The crude was directly purified through reverse-phase high-performance liquid chromatography (RP-HPLC) and solvents were removed by lyophilization. The identity and purity (98%) of the final product was assessed by LC/MS.
AAZ+-ValCit-MMAE was prepared following well-established synthetic procedures (31). Briefly, commercially available MC-ValCit-PAB-MMAE (1 eq.) was reacted with compound 2 (4 eq. dissolved in 100 μL of DMF) in freshly degassed PBS (50 mmol/L phosphate and 100 mmol/L NaCl, pH 7.4; 900 μL). The reaction was stirred at room temperature until completion (monitored by LC/MS). The crude was directly purified through RP-HPLC and solvents were removed by lyophilization. The identity and purity (98%) of the final product was assessed by LC/MS.
Preparation of L19-IL12
The immunocytokine L19-IL12 was cloned as described previously (36). Briefly, the fusion protein was produced in CHO-S mammalian cells and purified from the cell culture medium by affinity chromatography using a Protein A (Sino Biological) affinity column, as described previously (36). After dialysis into PBS pH 7.4, the quality of the protein was assessed by size-exclusion chromatography and by SDS-PAGE (Supplementary Fig. S1). Binding to the cognate EDB antigen was assessed by surface plasmon resonance (Supplementary Fig. S1) following procedures described previously (36).
The human renal cell carcinoma cell line, SKRC-52, was kindly provided by Professor E. Oosterwijk (Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands). SKRC-52 cells were cultured in RPMI Medium (Invitrogen), supplemented with FCS (10%, Invitrogen) and Antibiotic-Antimycotic (1%, Invitrogen), at 37°C, 5% CO2. Cells at 90%–100% confluence were detached using 0.05% Trypsin-EDTA (Invitrogen) and reseeded at a dilution of 1:6.
The murine colorectal carcinoma CT26-3E10–transfected cells (31) were cultured in RPMI Medium (Invitrogen), supplemented with FCS (10%, Invitrogen) and Antibiotic-Antimycotic (1%, Invitrogen), at 37°C, 5% CO2. Cells at 90%–100% confluence were detached using 0.05% Trypsin-EDTA (Invitrogen) and reseeded at a dilution of 1:6.
All animal experiments were conducted in accordance with Swiss animal welfare laws and regulations under the license number ZH004/18 granted by the Veterinäramt des Kantons Zuärich.
Implantation of subcutaneous tumors
SKRC-52 cells were grown to 80%–100% confluence and detached with 0.05% Trypsin-EDTA (Invitrogen). Cells were washed once with Hank’s Balanced Salt Solution (HBSS, Thermo Fisher Scientific, pH 7.4), counted, and resuspended in HBSS. Aliquots of 5–10 × 106 cells were resuspended in 150 μL of HBSS and injected subcutaneously in the right flank of female athymic BALB/c nu/nu mice (8–10 weeks of age, Janvier Labs).
CT26-3E10 cells were grown to 80%–100% confluence and detached with 0.05% Trypsin-EDTA (Invitrogen). Cells were washed once with HBSS (Thermo Fisher Scientific, pH 7.4), counted, and resuspended in HBSS. Aliquots of 6 × 106 cells were resuspended in 150 μL of HBSS and injected subcutaneously in the right flank of female athymic BALB/c nu/nu mice (8–10 weeks of age, Janvier Labs).
Quantification of MMAE in SKRC-52 tumors
SKRC-52 tumor cells were implanted into female BALB/c athymic nu/nu mice and allowed to grow to an average volume of 100 mm3. AAZ-ValCit-MMAE was injected through tail vein injection (250 nmol/kg) and animals were euthanized at different time points (i.e., 10 minutes, 1 hour, 3 hours, 6 hours, and 24 hours after the administration). The tumors were collected, frozen, and stored at −80°C prior to sample preparation and LC/MS quantification of MMAE. Blood was collected and incubated for 20 minutes in microtainer tubes containing lithium heparin (BD Microtainer Tube). Plasma was obtained by centrifugation for 15 minutes at 3,000 rpm using a refrigerated centrifuge and stored at −80°C prior to sample preparation and LC/MS quantification of MMAE. The detailed protocol for processing the samples is described in the Supplementary Information.
Dose-escalation study with L19-IL12
A dose-escalation study with L19-IL12 was performed in athymic BALB/c nu/nu mice (8–10 weeks of age, Janvier Labs). Four different doses of the immunocytokine were tested following the schedule reported in the Supplementary Information (three intravenous injections/animal at 0.6, 0.8, 1, or 1.2 mg/kg). Animals were weighed daily to assess acute toxicity of the immunocytokine at the different doses.
SKRC-52 or CT26-3E10 tumor cells were implanted into female BALB/c nu/nu mice and allowed to grow to an average volume of 100 or 200 mm3. Mice were randomly assigned into therapy groups of three, four, or five animals. Intravenous injections of AAZ-ValCit-MMAE (250 nmol/kg), AAZ+-ValCit-MMAE (250 nmol/kg), L19-IL2 (2.5 mg/kg), anti–PD-1 (10 mg/kg), L19-IL12 (1.2 mg/kg), or vehicle were performed with the schedules indicated in the text and in Figs. 2–4. Compounds 1 and 2 were injected as sterile PBS solution with 1% of DMSO. L19-IL12 and anti–PD-1 were injected as sterile PBS solution. L19-IL2 was injected in sterile formulation buffer (Philogen). Tumors were measured with an electronic caliper and animals were weighted daily. Tumor volume (mm3) was calculated with the formula (long side, mm) × (short side, mm) × (short side, mm) × 0.5. Animals were sacrificed when one or more termination criteria indicated by the experimental license were reached (e.g., weight loss > 15%). Prism 6 Software (GraphPad Software) was used for data analysis (regular two-way ANOVA followed by Bonferroni test).
Evaluation of chronic toxicity by plasma analysis and ex vivo histology
SKRC-52–bearing mice treated with vehicle, SMDC products, and immunocytokines as single agents or their combinations (n = 3/group) were sacrificed 90 days after treatment. The plasma was collected for each mouse and analyzed to detect the level of creatinine and urea. A complete gross postmortem examination was performed on each mouse, gross abnormalities were recorded, and all tissues were collected in 10% buffered formalin for histologic analysis. The tissue was trimmed, dehydrated, and embedded in paraffin wax. Sections of 3–4 μm thickness were prepared, mounted on glass slides, deparaffinized in xylene, and rehydrated through graded alcohols, before staining with hematoxylin and eosin for the histologic examination. Samples containing bone (bone marrow) were demineralized after fixation with EDTA for 48 hours prior to further processing, as described above. The microscopic morphology of different organs (kidney, liver, lung, stomach, bone marrow, and spleen) was blindly analyzed by a board-certified pathologist. Separately and afterward, they were compared with corresponding control samples collected from a nontreated animal to highlight structural changes deriving from the different treatments.
Production and characterization of SMDCs and immunocytokines
The structure of AAZ-ValCit-MMAE and AAZ+-ValCit-MMAE is shown in Fig. 1A. The peptide precursor compounds 1 and 2, featuring, respectively, acetazolamide or AAZ+ as CAIX-targeting moiety (31), were prepared by solid-phase peptide synthesis following a conventional Fmoc strategy and purified by reverse-phase HPLC. Compounds 1 and 2 were coupled in solution to the linker-payload module used for Adcetris, composed by the cleavable linker, Val-Cit, a self-immolative para-aminobenzyl carbamate moiety, and the potent anti-tubulin poison, MMAE. The resulting AAZ-ValCit-MMAE and AAZ+-ValCit-MMAE products were used for therapy studies in tumor-bearing mice.
The immunocytokine L19-IL12, recently described by our group, is a fusion protein consisting of the L19 antibody in tandem diabody format fused to a single chain of the murine IL12 by the (GGGGS)3 15-amino acid linker (Fig. 1B; ref. 36). We have also reported previously the fusion of the L19 antibody with IL2 (Fig. 1C; ref. 31). Structural and analytic information for all products is reported in the Supplementary Information and Supplementary Fig. S1.
Quantification study of MMAE in SKRC-52 tumor
We quantified the amount of the MMAE cytotoxic payload released in tumors and in plasma after injection of a single dose of AAZ-ValCit-MMAE prodrug (250 nmol/kg) in SKRC-52 tumor–bearing mice (Fig. 1D). A high and stable accumulation of free MMAE was observed in tumor samples. The highest concentration was obtained in tumor samples collected 3 hours after the intravenous injection, with values corresponding to 270 pmol/g of tissue (i.e., to 5.4% of the injected dose/g). Free MMAE levels in circulation quickly went down to values lower than 1% of the injected dose per mL of plasma already 1 hour after injection of the AAZ-ValCit-MMAE prodrug.
Therapy experiment of combination of SMDCs and L19-IL2 in SKRC-52 renal cell carcinoma model
We had reported previously that L19-IL2 potentiates the anticancer activity of AAZ+-ValCit-MMAE in two mouse models of cancer (31) and that the AAZ+ moiety targets tumors more efficiently than acetazolamide (38). Here, we performed a comparison of AAZ-ValCit-MMAE and AAZ+-ValCit-MMAE in combination with L19-IL2, to study the activity and tolerability of the two drugs in BALB/c athymic nude mice bearing subcutaneously grafted SKRC-52 tumors.
AAZ-ValCit-MMAE and AAZ+-ValCit-MMAE were administered at 250 nmol/kg, a dose that was identified previously to be compatible with selective accumulation of acetazolamide derivatives in tumors and which appeared to be safe and effective in the SKRC-52 renal cell carcinoma model (21, 25, 31). The antibody fusion protein, L19-IL2, was administered at 2.5 mg/kg, identified previously as an efficacious and well-tolerated dose (31). Therapy started when the average volume of established tumors reached 100 mm3. We compared the simultaneous administration of SMDC and L19-IL2 with a modified schedule, featuring a 24-hour delay between the two injections (Fig. 2A and C), as it has been suggested previously that immunotherapy may benefit from the damage caused to the tumor by cytotoxic agents (39). A potent in vivo anticancer activity of both SMDCs in combination with L19-IL2 was observed in both schedules (Fig. 2A and C). AAZ+-ValCit-MMAE was more active in this study, leading to four of four cures in combination with L19-IL2 in the sequential administration schedule and two of four cures upon simultaneous administration (Fig. 2C). All treatments were well tolerated at the administered doses (Fig. 2B and D).
In a second therapy experiment, we tested the anticancer activity of the combination AAZ+-ValCit-MMAE and L19-IL2 on larger established SKRC-52 tumors starting at an average volume of 200 mm3. The administration of AAZ+-ValCit-MMAE as single agents resulted in a partial tumor growth retardation (Fig. 2E). Interestingly, only the combination of AAZ+-ValCit-MMAE and L19-IL12 induced potent tumor regression in this setting (i.e., three of the four animals treated were cured). The combination treatment was well tolerated and no sign of acute toxicity was observed (Fig. 2F).
Therapy experiment of combination of SMDCs and anti–PD-1 in CT26-3E10 colorectal cell carcinoma model
The anticancer activity of AAZ-ValCit-MMAE, AAZ+-ValCit-MMAE, anti–PD-1, and their combination was assessed in fully immunocompetent BALB/c mice subcutaneously bearing CT26-3E10 colorectal cell carcinoma. AAZ-ValCit-MMAE and AAZ+-ValCit-MMAE were administered at 250 nmol/kg, as mentioned previously. The anti–PD-1 antibody was administered at the dose of 10 mg/kg, as described previously (37). Therapy started when the average volume of established tumors had reached 100 mm3. The compounds were administered the same day, with a 6-hour interval between the two injections, starting with the SMDC (Fig. 3A). A potent anticancer activity of AAZ-ValCit-MMAE, AAZ+-ValCit-MMAE, and anti–PD-1 was observed in vivo when the products were given as combination, while the single-agent treatment with anti–PD-1 was ineffective (Fig. 3A). All treatments were well tolerated at the doses administered (Fig. 3B).
Therapy experiments of combination of SMDC and L19-IL12 in SKRC-52 renal cell carcinoma model
The anticancer activity of AAZ+-ValCit-MMAE, L19-IL12, and their combination was assessed in BALB/c athymic nude mice bearing subcutaneously grafted SKRC-52 renal cell carcinoma. AAZ+-ValCit-MMAE was administered at 250 nmol/kg. The antibody fusion protein, L19-IL12, was administered at the dose of 1.2 mg/kg. This dose was considered to be safe on the basis of a preliminary dose escalation experiment in nude mice (Supplementary Information). Therapy started when the average volume of established tumors reached 100 mm3. The two compounds were administered according to the schedule described in Fig. 4A. A potent anticancer activity for AAZ+-ValCit-MMAE and L19-IL12 was observed in vivo when products were given as monotherapy. Moreover, all animals treated with the combination of the two products experienced a complete and durable tumor eradication (Fig. 4A). AAZ+-ValCit-MMAE, L19-IL12, and their combination treatment were well tolerated at the administered doses (Fig. 4B). Cured animals were kept alive and tumor free for 90 days after the administration of the first dose of SMDC (Fig. 4A).
In a second therapy experiment, we tested the anticancer activity of the combination AAZ+-ValCit-MMAE and L19-IL12 on larger established SKRC-52 tumors (average volume of 200 mm3) implanted in BALB/c nude mice. The administration of AAZ+-ValCit-MMAE and L19-IL12 as single agents resulted in a partial tumor growth retardation, compared with saline-treated animals used as controls (Fig. 4C). Interestingly, only the combination of AAZ+-ValCit-MMAE and L19-IL12 induced tumor regression in this setting. All treatments were well tolerated and no sign of acute toxicity was observed (Fig. 4D). Mice in the combination group received a second cycle of injections (i.e., AAZ+-ValCit-MMAE and L19-IL12) on therapy day 52. During this second round of treatments, an acute and reversible body weight loss was observed as sign of toxicity (Fig. 4D). Mice treated with the combination were kept alive for more than 60 days (Fig. 4C).
Analysis of chronic toxicity of combination treatment of SMDCs with L19-IL2
Gross examination of mice revealed a slightly to moderately enlarged spleen of all animals treated with L19-IL2 alone or in combination with SMDCs compared with the control group. Histologically, none of the treatments caused cellular infiltration and/or connective tissue (fibrosis) in liver and kidney of the treated animal in comparison with specimens from control animals. No signs of toxicity were found in other healthy organs, including lung and stomach that are known to be exposed to higher concentration of acetazolamide-based derivatives (Fig. 5A; ref. 30). Urea and creatinine plasma levels measured 3 months after treatment with SMDCs, L19-IL2, and combinations (blood chemistry for kidney chronic toxicity) revealed no significant differences between treatment groups and compared with the control group (Fig. 5B and C).
Analysis of chronic toxicity of combination treatment of SMDCs with L19-IL12
Gross examination of mice revealed a moderately enlarged spleen in all animals treated with L19-IL12 alone or in combination with AAZ+-ValCit-MMAE compared with the control group. In the liver, both treatment groups exhibited a mild (40) periportal infiltrate of mononuclear cells (Fig. 6), rarely associated with increased connective tissue. Both changes were not noted in the control animal and did not differ significantly between the groups. The histopathologic examination of kidney, lung, and stomach did not show signs of inflammation and/or fibrosis (Fig. 6). In the spleen, a marked increase in extramedullary hematopoiesis in the red pulp correlated macroscopically with an enlarged spleen. The bone marrow of both treatment groups revealed a high cellularity with all three cell lines: erythropoiesis, myelopoiesis, and thrombocytopoiesis were present with maturation in all three lines and did not seem to be altered compared with the control animals (Supplementary Information).
We have recently demonstrated that the anticancer activity of AAZ+-ValCit-MMAE, an SMDC targeting CAIX, is potently enhanced by the combination with targeted IL2 (31). In this article we have extended our previous findings, describing the in vivo anticancer activity and safety profile of novel therapies consisting of SMDC products targeting CAIX combined with PD-1 blockade and with the antibody–cytokine fusions, L19-IL2 and L19-IL12. Combination with all immunomodulatory drugs tested enhanced the therapeutic activity of SMDCs. The result of a systematic histopathologic evaluation in treated mice shows that curative doses of SMDCs in combination with L19-IL2 and with L19-IL12 do not cause chronic toxicity in kidney and other healthy organs.
The clinical efficacy of targeted cytotoxics strictly depends on the amount of payload that can be delivered to the tumor cells, compatibly with a safe administration to the patients. While being designed to specifically deliver their toxic payload to tumors, both ADCs and SMDCs are found at significantly high concentrations in nontarget organs (41). ADCs are slowly cleared by the liver. Common clinical side effects of ADCs include liver damage, bone marrow toxicity, and hemorrhage (11). SMDCs are rapidly cleared by the kidney, with very short plasma half-lives and time of exposure in other healthy organs. In this article, we have shown how the fast clearance of SMDCs does not impair their therapeutic efficacy. Bioanalysis of tumor samples clearly shows that high and stable concentration of cytotoxic drugs can be delivered by acetazolamide conjugates over a time window of 24 hours after intravenous administration. Our data are consistent with recently published literature data on clearance of peptide–drug conjugates and in vivo delivery of MMAE to tumors (42). The toxicity profile of SMDCs has not been sufficiently investigated, with only few preclinical (42, 43) and clinical (44) examples currently available. We have demonstrated that anti-CAIX SMDC products are safe when administered at curative doses, with no signs of chronic damage in nontarget healthy organs and in those healthy structures known to express CAIX (27, 31). These findings are in strong contrast with what has been reported for small molecule–radio conjugates, for which administration of therapeutic doses typically results in significant kidney toxicity (45). Our results suggest that the delivery of cytotoxic drugs damaging cells in rapid proliferation may be advantageous over the use of therapeutic radionuclide payloads that damage indistinctively all type of cells (46).
Immunocytokines and checkpoint inhibitors have previously been found to potently enhance the anticancer activity of untargeted chemotherapy (39, 47) and ADCs (48). When given as combination, the administration of immunotherapy and of cytotoxic drugs can be harmonized to maximize therapeutic potential and minimize systemic toxicities. We could show that the different tumor residence time of AAZ-ValCit-MMAE and AAZ+-ValCit-MMAE has an impact on the therapeutic outcome of the combination treatment. Moreover, we found that SMDC products based on acetazolamide administered before immunotherapy were more efficacious than immunotherapy first followed by SMDCs. These findings are consistent with what has been observed previously in preclinical experiments performed with combinations of conventional chemotherapeutic agents and immunocytokines (39). We suggest that the initial damage induced by targeted cytotoxics can render the tumor microenvironment more susceptible to the action of immunotherapy, as cytotoxic agents increase the expression of stress proteins, such as Mic-A (39, 49), and cause immunogenic cell death (50).
We have demonstrated previously that the combination treatment based on AAZ+-ValCit-MMAE and L19-IL2 is able to promote the natural killer (NK)-cell infiltration in large tumors (31). Moreover, we have already shown that L19-IL12 is able to boost NK-cell tumor infiltration in monotherapy (36). The therapy experiments performed in large tumors presented in this article suggest that the immunosuppressive environment represent the major driver of the reduction of antitumor activity of immunocytokine products (i.e., NK cells are present in the tumor, but they are “immunosuppressed”) and that the combination with cytotoxic insults leads to potent anticancer activity in large lesions.
J. Millul reports employment with Philochem AG, a company active in the field of small molecule drug conjugates and targeted cytokines. A. Zana reports employment with Philochem AG, a company active in the field of small molecule drug conjugates and targeted cytokines. S. Dakhel Plaza reports employment with Philochem AG, a company in the field is small molecules drug conjugates and targeted cytokines. E. Puca reports employment with Philochem AG, a company active in the field of small molecule drug conjugates and targeted cytokines. A. Villa reports employment with Philochem AG, a company active in the field of small molecule drug conjugates and targeted cytokines. D. Neri reports board membership, chief scientific officer, and co-CEO of the Philogen group. S. Cazzamalli reports employment with Philochem AG, a company active in the field of small molecule drug conjugates and targeted cytokines. No other disclosures were reported.
J. Millul: Conceptualization, data curation, investigation, methodology, writing–original draft, writing–review and editing. C. Krudewig: Investigation, writing–review and editing. A. Zana: Investigation, writing–review and editing. S. Dakhel Plaza: Investigation, writing–review and editing. E. Puca: Investigation, writing–review and editing. A. Villa: Writing–review and editing. D. Neri: Conceptualization, resources, supervision, funding acquisition, writing–original draft, project administration, writing–review and editing. S. Cazzamalli: Conceptualization, supervision, investigation, writing–original draft, project administration, writing–review and editing.
D. Neri acknowledges funding from ETH Zurich. This project has received funding from the Swiss National Science Foundation (grant No., 310030_182003/1) and the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement 670603). The authors wish to thank Baptiste Gouyou and Tiziano Ongaro for their technical support during in vivo experiments and Theresa Pesch for technical support with necropsy and histology. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 861316.
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