Pablo Scodeller

Traumatic brain injury (TBI) is a major clinical problem because of the high incidence and the severity of the subsequent sequelae. Despite extensive efforts, there are no therapeutic drugs clinically approved for treating acute TBI patients. To address this unmet need, we assessed the activity of the tetrapeptide, CAQK, in mice. When administered intravenously shortly after moderate or severe TBI, CAQK accumulates in the injured brain in mice and pigs. CAQK binds to an extracellular matrix glycoprotein complex that is upregulated in injured brain. Treatment of TBI mice with CAQK resulted in reduction in the size of the injury compared to control mice. There was reduced upregulation of the glycoprotein complex, less apoptosis, and lower expression of inflammatory markers in the injured area, indicating that CAQK alleviates neuroinflammation and the ensuing secondary injury. CAQK treatment also improved functional deficit in TBI mice, with no overt toxicity. Our findings suggest that CAQK may have therapeutic applications in TBI.

In triple‐negative breast cancer (TNBC), pro‐tumoral macrophages promote metastasis and suppress the immune response. To target these cells, a previously identified CD206 (mannose receptor)‐binding peptide, mUNO was engineered to enhance its affinity and proteolytic stability. The new rationally designed peptide, MACTIDE, includes a trypsin inhibitor loop, from the Sunflower Trypsin Inhibitor‐I. Binding studies to recombinant CD206 revealed a 15‐fold lower K_D for MACTIDE compared to parental mUNO. Mass spectrometry further demonstrated a 5‐fold increase in MACTIDE's half‐life in tumor lysates compared to mUNO. Homing studies in TNBC‐bearing mice shows that fluorescein (FAM)‐MACTIDE precisely targeted CD206⁺ tumor‐associated macrophages (TAM) upon intravenous, intraperitoneal, and even oral administration, with minimal liver accumulation. MACTIDE was conjugated to Verteporfin, an FDA‐approved photosensitizer and YAP/TAZ pathway inhibitor to create the conjugate MACTIDE‐V. In the orthotopic 4T1 TNBC mouse model, non‐irradiated MACTIDE‐V‐treated mice exhibited anti‐tumoral effects comparable to those treated with irradiated MACTIDE‐V, with fewer signs of toxicity, prompting further investigation into the laser‐independent activity of the conjugate. In vitro studies using bone marrow‐derived mouse macrophages showed that MACTIDE‐V excluded YAP from the nucleus, increased phagocytic activity, and upregulated several genes associated with cytotoxic anti‐tumoral macrophages. In mouse models of TNBC, MACTIDE‐V slowed primary tumor growth, suppressed lung metastases, and increased markers of phagocytosis and antigen presentation in TAM and monocytes, increasing the tumor infiltration of several lymphocyte subsets. MACTIDE‐V is proposed as a promising peptide‐drug conjugate for modulating macrophage function in breast cancer immunotherapy.

Disease-specific changes in tumors and other diseased tissues are an important target of research because they provide clues about the pathophysiology of the disease as well as uncover potentially useful markers for diagnosis and treatment. Here, we report a new cyclic peptide, CESPLLSEC (CES), that specifically accumulated (homed) in intracranial U87MG and the WT-GBM model of glioblastoma (GBM) from intravenous (IV) injection, and associated with the vasculature. Affinity chromatography of U87MG tumor extracts on insolubilized CES peptide identified Synaptosomal Associated Protein 25 (SNAP25) as a candidate target molecule (receptor) for CES. Several results supported the identification of SNAP25 as the CES receptor. IV-injected FAM-CES colocalized with SNAP25 in the tumors, and direct binding studies showed specific binding of the CES peptide to recombinant human SNAP25. A CES peptide-drug conjugate designed for photodynamic therapy showed selective cytotoxicity to SNAP25⁺ glioblastoma cell lines. Specific accumulation of systemically injected anti-SNAP25 antibody in U87MG glioblastoma and labeling of intact U87MG cells with anti-SNAP in flow cytometry showed that SNAP25 is available from the circulation but not in normal tissues and that it is present at the cell surface. Using an array of ECM proteins and surface plasmon resonance revealed that SNAP25 binds moderately to collagen V and strongly to collagen VI. Modeling studies suggested that CES and collagen VI compete for the same binding site on SNAP25. Our results introduce CES as a valuable targeting peptide for drug delivery and its receptor SNAP25 as a possible molecular marker of interest for glioblastoma.

Disease-specific changes in tumors and other diseased tissues are an important target of research because they provide clues on the pathophysiology of the disease as well as uncovering potentially useful markers for diagnosis and treatment. Here, we report a new cyclic peptide, CESPLLSEC (CES), that specifically accumulated (homed) in intracranial U87MG and the WT-GBM model of glioblastoma from intravenous (IV) injection, associating with the vasculature. Affinity chromatography of U87MG tumor extracts on insolubilized CES peptide identified Synaptosomal Associated Protein 25 (SNAP25) as a candidate target molecule (receptor) for CES. Several results supported the identification of SNAP25 as the CES receptor. IV-injected FAM-CES colocalized with SNAP25 in the tumors, and direct binding studies showed specific CES peptide binding to recombinant human SNAP25. A CES peptide-drug conjugate designed for photodynamic therapy showed selective cytotoxicity to SNAP25+ glioblastoma cell lines. Specific accumulation of systemically injected anti-SNAP25 antibody in U87MG glioblastoma, and labeling of intact U87MG cells with anti-SNAP in flow cytometry showed that SNAP25 is available from the circulation but not in normal tissues and that it is present at the cell surface. Using an array of ECM proteins and surface plasmon resonance revealed that SNAP25 binds moderately to collagen V and strongly to collagen VI. Modeling studies suggested that CES and collagen VI compete for the same binding site on SNAP25. Our results introduce CES as a valuable targeting peptide for drug delivery, and its receptor SNAP25 as a possible molecular marker of interest for glioblastoma.

In triple negative breast cancer (TNBC), pro-tumoral macrophages promote metastasis and suppress the immune response. To target these cells, we engineered a previously identified CD206 (mannose receptor)-binding peptide, mUNO, to enhance its affinity and proteolytic stability. The new rationally designed peptide, MACTIDE, includes a trypsin inhibitor loop, from the Sunflower Trypsin Inhibitor-I. Binding studies to recombinant CD206 revealed a 15-fold lower K _D for MACTIDE compared to parental mUNO. Additionally, mass spectrometry showed a 5-fold increase in half-life in tumor lysate for MACTIDE compared to mUNO. Homing studies in TNBC-bearing mice showed that fluorescein (FAM)-MACTIDE precisely targeted CD206 ⁺ tumor-associated macrophages (TAMs) upon intravenous, intraperitoneal and even oral administration, with no significant accumulation in liver. We coupled MACTIDE to the FDA-approved drug Verteporfin, an established photosensitizer for photodynamic therapy and inhibitor of the YAP/TAZ pathway, to generate a conjugate here referred to as MACTIDE-V. In the orthotopic 4T1 TNBC mouse model, non-irradiated MACTIDE-V-treated mice unexpectedly showed a similar anti-tumoral effect and fewer signs of toxicity as irradiated MACTIDE-V-treated mice, leading to subsequent studies on the laser-independent activity of this conjugate. In vitro studies using bone-marrow derived mouse macrophages showed that MACTIDE-V excluded YAP from the nucleus, increased the phagocytic activity and upregulated several genes associated with cytotoxic anti-tumoral macrophages. In mouse models of TNBC, MACTIDE-V slowed primary tumor growth, suppressed lung metastases, increased markers of phagocytosis and antigen presentation in TAMs and monocytes, increasing the tumor infiltration of several lymphocyte subsets. We therefore propose MACTIDE-V as a useful peptide-drug conjugate to modulate macrophage function in the context of breast tumor immunotherapy.

Although many studies have explored the depletion of tumor-associated macrophages (TAM) as a therapeutic strategy for solid tumors, currently available compounds suffer from poor efficacy and dose-limiting side effects. Here, we developed a novel TAM-depleting agent (“OximUNO”) that specifically targets CD206+ TAMs and demonstrated efficacy in a triple-negative breast cancer (TNBC) mouse model. OximUNO comprises a star-shaped polyglutamate (St-PGA) decorated with the CD206-targeting peptide mUNO that carries the chemotherapeutic drug doxorubicin (DOX). In the TNBC model, a fluorescently labeled mUNO-decorated St-PGA homed to CD206+ TAMs within primary lesions and metastases. OximUNO exhibited no acute liver or kidney toxicity in vivo. Treatment with OximUNO reduced the progression of primary tumor lesions and pulmonary metastases, significantly diminished the number of CD206+ TAMs and increased the CD8/FOXP3 expression ratio (indicating immunomodulation). Our findings suggest the potential benefit of OximUNO as a TAM-depleting agent for TNBC treatment. Importantly, our studies also represent a novel design of a peptide-targeted St-PGA as a targeted therapeutic nanoconjugate.Significance:A peptide-targeted nanoformulation of DOX exclusively eliminates mannose receptor+ TAMs in breast cancer models, generating response without off-target effects (a drawback of many TAM-depleting agents under clinical study).

Although many studies have explored the depletion of tumor-associated macrophages (TAM) as a therapeutic strategy for solid tumors, currently available compounds suffer from poor efficacy and dose-limiting side effects. Here, we developed a novel TAM-depleting agent (“OximUNO”) that specifically targets CD206+ TAMs and demonstrated efficacy in a triple-negative breast cancer (TNBC) mouse model. OximUNO comprises a star-shaped polyglutamate (St-PGA) decorated with the CD206-targeting peptide mUNO that carries the chemotherapeutic drug doxorubicin (DOX). In the TNBC model, a fluorescently labeled mUNO-decorated St-PGA homed to CD206+ TAMs within primary lesions and metastases. OximUNO exhibited no acute liver or kidney toxicity in vivo. Treatment with OximUNO reduced the progression of primary tumor lesions and pulmonary metastases, significantly diminished the number of CD206+ TAMs and increased the CD8/FOXP3 expression ratio (indicating immunomodulation). Our findings suggest the potential benefit of OximUNO as a TAM-depleting agent for TNBC treatment. Importantly, our studies also represent a novel design of a peptide-targeted St-PGA as a targeted therapeutic nanoconjugate.Significance:A peptide-targeted nanoformulation of DOX exclusively eliminates mannose receptor+ TAMs in breast cancer models, generating response without off-target effects (a drawback of many TAM-depleting agents under clinical study).

One .docx word file containing the following information:Scheme S1. Synthetic approach for St-PGA-OG-mUNO.Scheme S2. Synthetic approach for OximUNO.Video S1. Fifty ns MD trajectory of St-PGA in solution.Table S1. St-PGA-OG-mUNO and St-PGA-OG dye loading, mUNO loading, size and charge.Table S2. OximUNO and St-PGA-DOX drug loading, mUNO loading, size and charge.Table S3. Hepatic and renal toxicity analysed following one dose of OximUNO.Fig. S1. Representative characterisation of St-PGA-OG and St-PGA-OG-mUNO.Fig. S2. The angle used to characterise the rotation of mUNO around PGA.Fig. S3. St-PGA-OG-mUNO shows low hepatic accumulation in the orthotopic TNBC.Fig. S4. Immunostaining showing CD206 expression in the liver compared to the saturated signal of CD206 in the orthotopic 4T1 tumour.Fig. S5. Immunostaining of endogenous IgG indicates leaky tumour vasculature in the orthotopic 4T1 tumours.Fig. S6. Distribution of St-PGA-OG-mUNO in the lung, spleen, sentinel lymph node (SLN) and kidney.Fig. S7. St-PGA-OG-mUNO shows low hepatic accumulation in the experimental metastasis of TNBC.Fig. S8. St-PGA-OG-mUNO shows higher receptor colocalisation than FAM-mUNO.Fig. S9. St-PGA-OG-mUNO shows high homing to M2 TAMs in the orthotopic TNBC but higher hepatic accumulation with a higher dose.Fig. S10. Representative characterisation of St-PGA-DOX and OximUNO.Fig. S11. LC-MS method development for the determination of DOX in drug release studies and stability studies of OximUNO in i.p. fluid and dPBS.Fig. S12. OximUNO shows no signs of hepatic or renal toxicity.Fig. S13. H&E on healthy Balb/c mouse lung.Fig. S14. Localisation of St-PGA-OG/St-PGA-OG-mUNO and tumour vessels.Fig. S15. H&E staining of hearts from the treatment of orthotopic TNBC.Fig. S16. A repetition of OximUNO treatment on the orthotopic TNBC monitoring tumour growth and survival.Fig. S17. Flow cytometry plots for M2 and M1 macrophages.Fig. S18. Flow cytometry gating for cytotoxic T lymphocytes (CTLs).Fig. S19. Flow cytometry gating for T regulatory cells (Tregs). Fig. S20. Flow cytometry analysis showing total cells.Fig. S21. Treatment with OximUNO does not affect splenic macrophages.

One .docx word file containing the following information:Scheme S1. Synthetic approach for St-PGA-OG-mUNO.Scheme S2. Synthetic approach for OximUNO.Video S1. Fifty ns MD trajectory of St-PGA in solution.Table S1. St-PGA-OG-mUNO and St-PGA-OG dye loading, mUNO loading, size and charge.Table S2. OximUNO and St-PGA-DOX drug loading, mUNO loading, size and charge.Table S3. Hepatic and renal toxicity analysed following one dose of OximUNO.Fig. S1. Representative characterisation of St-PGA-OG and St-PGA-OG-mUNO.Fig. S2. The angle used to characterise the rotation of mUNO around PGA.Fig. S3. St-PGA-OG-mUNO shows low hepatic accumulation in the orthotopic TNBC.Fig. S4. Immunostaining showing CD206 expression in the liver compared to the saturated signal of CD206 in the orthotopic 4T1 tumour.Fig. S5. Immunostaining of endogenous IgG indicates leaky tumour vasculature in the orthotopic 4T1 tumours.Fig. S6. Distribution of St-PGA-OG-mUNO in the lung, spleen, sentinel lymph node (SLN) and kidney.Fig. S7. St-PGA-OG-mUNO shows low hepatic accumulation in the experimental metastasis of TNBC.Fig. S8. St-PGA-OG-mUNO shows higher receptor colocalisation than FAM-mUNO.Fig. S9. St-PGA-OG-mUNO shows high homing to M2 TAMs in the orthotopic TNBC but higher hepatic accumulation with a higher dose.Fig. S10. Representative characterisation of St-PGA-DOX and OximUNO.Fig. S11. LC-MS method development for the determination of DOX in drug release studies and stability studies of OximUNO in i.p. fluid and dPBS.Fig. S12. OximUNO shows no signs of hepatic or renal toxicity.Fig. S13. H&E on healthy Balb/c mouse lung.Fig. S14. Localisation of St-PGA-OG/St-PGA-OG-mUNO and tumour vessels.Fig. S15. H&E staining of hearts from the treatment of orthotopic TNBC.Fig. S16. A repetition of OximUNO treatment on the orthotopic TNBC monitoring tumour growth and survival.Fig. S17. Flow cytometry plots for M2 and M1 macrophages.Fig. S18. Flow cytometry gating for cytotoxic T lymphocytes (CTLs).Fig. S19. Flow cytometry gating for T regulatory cells (Tregs). Fig. S20. Flow cytometry analysis showing total cells.Fig. S21. Treatment with OximUNO does not affect splenic macrophages.

Novel approaches are required to address the urgent need to develop lipid-based carriers of paclitaxel (PTX) and other hydrophobic drugs for cancer chemotherapy. Carriers based on cationic liposomes (CLs) with fluid (i.e., chain-melted) membranes (e.g., EndoTAG-1) have shown promise in preclinical and late-stage clinical studies. Recent work found that the addition of a cone-shaped poly(ethylene glycol)-lipid (PEG-lipid) to PTX-loaded CLs (CLs_PTX) promotes a transition to sterically stabilized, higher-curvature (smaller) nanoparticles consisting of a mixture of PEGylated CLs_PTX and PTX-containing fluid lipid nanodiscs (nanodiscs_PTX). These CLs_PTX and nanodiscs_PTX show significantly improved uptake and cytotoxicity in cultured human cancer cells at PEG coverage in the brush regime (10 mol % PEG-lipid). Here, we studied the PTX loading, in vivo circulation half-life, and biodistribution of systemically administered CLs_PTX and nanodiscs_PTX and assessed their ability to induce apoptosis in triple-negative breast-cancer-bearing immunocompetent mice. We focused on fluid rather than solid lipid nanodiscs because of the significantly higher solubility of PTX in fluid membranes. At 5 and 10 mol % of a PEG-lipid (PEG5K-lipid, molecular weight of PEG 5000 g/mol), the mixture of PEGylated CLs_PTX and nanodiscs_PTX was able to incorporate up to 2.5 mol % PTX without crystallization for at least 20 h. Remarkably, compared to preparations containing 2 and 5 mol % PEG5K-lipid (with the PEG chains in the mushroom regime), the particles at 10 mol % (with PEG chains in the brush regime) showed significantly higher blood half-life, tumor penetration, and proapoptotic activity. Our study suggests that increasing the PEG coverage of CL-based drug nanoformulations can improve their pharmacokinetics and therapeutic efficacy.