Extracellular vesicles isolated from adipose tissue-derived mesenchymal stromal cells as carriers for Paclitaxel delivery Angela Marcianti, Eleonora Spampinato, Sara Nava, Giulia Maria Stella, Paola Perego, Simona Pogliani, Simona Frigerio, Luca Mirra, Paola Gagni, Fabio Moda, Federico Angelo Cazzaniga, Giovanni Luca Beretta, Guido Maronati, Giuseppe Paglia, Angelo Guido Corsico, Catia Traversari, Daniela Lisini Stem Cell Research and Therapy, 2025 BACKGROUND: Mesenchymal Stromal Cells (MSC)-derived Extracellular Vesicles (EV) represent innovative tools for drug delivery systems. However, their clinical use is limited by the lack of standardized good manufacturing practice (GMP)-compliant isolation and conservation protocols. In this study, we developed a GMP-compliant protocol for the preparation of MSC-EVs and investigated the feasibility of producing EVs loaded with paclitaxel (PTX) for clinical application as drug products. METHODS: Adipose tissues from 13 donors were used to obtain MSC-EVs via culture supernatant ultracentrifugation. EVs loaded with PTX were manufactured by adding the drug to the culture medium of MSCs before supernatant collection. EV identity was verified in terms of concentration/size, protein content, morphology, and expression of EV surface markers. The anti-proliferative activity, accumulation ability in tumor cells and PTX content, as well as their stability over time, were also evaluated. RESULTS: High numbers of EV/EV-PTX compliant in terms of integrity/identity were obtained and can be successfully stored for up to one year at -80 °C. Cellular studies have shown that EVs are capable of accumulating in tumor cells and, when loaded with PTX, inhibiting the proliferation of a pleural mesothelioma cell line. CONCLUSIONS: These results support the potential future clinical use of EVs as carriers for drug delivery to improve cancer treatment strategies.
Polyclonal expansion of functional tumor-reactive lymphocytes infiltrating glioblastoma for personalized cell therapy Martina Maffezzini, Silvia Musio, Natalia Di Ianni, Agnese Rumolo, Monica Patanè, Andrea Galluzzo, Irene Sambruni, Arianna Berlendis, Domenico Aquino, Giacomo Baso, Manuela Zingarelli, Manuel Facciolla, Luisa Maddaloni, Rossella Valentino, Rosina Paterra, Fabio Agistri, Mariangela Farinotti, Luca Mattei, Paola Coluccia, Francesco Acerbi, Francesco DiMeco, Bianca Pollo, Antonio Silvani, Marica Eoli, Catia Traversari, Daniela Montagna, Serena Pellegatta Nature Communications, 2025 Tumor-infiltrating lymphocyte (TIL)-therapy has received FDA approval for the treatment of advanced melanoma and shows potential for broader applications in solid tumors, including glioblastoma. In this study, tumor-reactive TILs (tr-TILs) are isolated and enriched for CD137 expression from cavitron ultrasonic aspirator (CUSA) emulsions of 161 adult patients diagnosed with diffuse gliomas. Tr-TILs are successfully expanded in 87 out of the 161 patients, reflecting an expansion rate of 54%. Notably, the presence of IDH1 mutation and the cumulative dose of steroids are identified as significant negative predictors of expansion efficacy. The expanded tr-TILs exhibit distinct phenotypic and molecular dysfunctional features yet show upregulated expression of progenitor/memory-like markers and polyclonal T-cell receptors. Importantly, these tr-TILs demonstrate specific antitumor reactivity against autologous tumor cells in both in vitro and in vivo xenograft models. These findings provide a compelling background for a personalized immunotherapeutic approach while tackling one of the most significant challenges in oncology. Tumor-infiltrating lymphocyte (TIL) therapy has demonstrated clinical benefit in patients with solid tumors. Here the authors propose a protocol for isolating and enriching TILs from cavitron ultrasonic aspirator emulsions of patients with glioma, showing the successful expansion of functional tumor-reactive TILs for personalized cell therapy.
Harnessing the reverse cholesterol transport pathway to favor differentiation of monocyte-derived APCs and antitumor responses Laura Raccosta, Maura Marinozzi, Susan Costantini, Daniela Maggioni, Lorena Maria Ferreira, Gianfranca Corna, Paola Zordan, Angela Sorice, Diego Farinello, Silvia Bianchessi, Michela Riba, Dejan Lazarevic, Paolo Provero, Matthias Mack, Attilio Bondanza, Ivan Nalvarte, J-A Gustafsson, Valeria Ranzani, Francesco De Sanctis, Stefano Ugel, Silvère Baron, Jean-Marc A. Lobaccaro, Lorenzo Pontini, Manuela Pacciarini, Catia Traversari, Massimiliano Pagani, Vincenzo Bronte, Giovanni Sitia, Per Antonson, Andrea Brendolan, Alfredo Budillon, Vincenzo Russo Cell Death and Disease, 2023 Lipid and cholesterol metabolism play a crucial role in tumor cell behavior and in shaping the tumor microenvironment. In particular, enzymatic and non-enzymatic cholesterol metabolism, and derived metabolites control dendritic cell (DC) functions, ultimately impacting tumor antigen presentation within and outside the tumor mass, dampening tumor immunity and immunotherapeutic attempts. The mechanisms accounting for such events remain largely to be defined. Here we perturbed (oxy)sterol metabolism genetically and pharmacologically and analyzed the tumor lipidome landscape in relation to the tumor-infiltrating immune cells. We report that perturbing the lipidome of tumor microenvironment by the expression of sulfotransferase 2B1b crucial in cholesterol and oxysterol sulfate synthesis, favored intratumoral representation of monocyte-derived antigen-presenting cells, including monocyte-DCs. We also found that treating mice with a newly developed antagonist of the oxysterol receptors Liver X Receptors (LXRs), promoted intratumoral monocyte-DC differentiation, delayed tumor growth and synergized with anti-PD-1 immunotherapy and adoptive T cell therapy. Of note, looking at LXR/cholesterol gene signature in melanoma patients treated with anti-PD-1-based immunotherapy predicted diverse clinical outcomes. Indeed, patients whose tumors were poorly infiltrated by monocytes/macrophages expressing LXR target genes showed improved survival over the course of therapy. Thus, our data support a role for (oxy)sterol metabolism in shaping monocyte-to-DC differentiation, and in tumor antigen presentation critical for responsiveness to immunotherapy. The identification of a new LXR antagonist opens new treatment avenues for cancer patients.
Characterization and functional analysis of CD44v6.CAR T cells endowed with a new low-affinity nerve growth factor receptor-based spacer Anna Stornaiuolo, Barbara Valentinis, Camilla Sirini, Cinzia Scavullo, Claudia Asperti, Dan Zhou, Yeny Martinez De La Torre, Stefano Corna, Monica Casucci, Simona Porcellini, Catia Traversari Human Gene Therapy, 2021 Effectiveness of adoptively transferred chimeric antigen receptor (CAR) T cells strongly depends on the quality of CAR-mediated interaction of the effector cells with the target antigen on tumor cells. A major role in this interaction is played by the affinity of the single-chain variable fragment (scFv) for the antigen, and by the CAR design. In particular, the spacer domain may impact on the CAR T cell function by affecting the length and flexibility of the resulting CAR. This study addresses the need to improve the manufacturing process and the antitumor activity of CD44v6-specific CAR T cells by defining the optimal structure of a spacer region derived from the extracellular domain of the human low-affinity nerve growth factor receptor (LNGFR). We tailored the LNGFR spacer to modulate CAR length to efficiently recognize distal or proximal epitopes and to allow selection of transduced CAR T cells by the use of clinical-grade validated manufacturing systems. The different LNGFR spacers investigated in this study are responsible for the generation of CAR T cells with a different memory phenotype, which is mainly related to the level of CAR expression and the extent of the associated tonic signaling. In particular, the CD44v6-NWN2.CAR T cells are enriched in central memory cells and show improved in vitro functions in terms of killing capability, and in vivo antitumor activity against hematological and solid tumors. Clinical Trial Registration numbers: clinicaltrial.gov NCT04097301; ClinicalTrials.gov, NCT00423124.
CAR T Cells Redirected to CD44v6 Control Tumor Growth in Lung and Ovary Adenocarcinoma Bearing Mice Simona Porcellini, Claudia Asperti, Stefano Corna, Eleonora Cicoria, Veronica Valtolina, Anna Stornaiuolo, Barbara Valentinis, Claudio Bordignon, Catia Traversari Frontiers in Immunology, 2020 The main challenge of adoptive therapy with Chimeric Antigen Receptor modified T cells (CAR T) is the application to the field of solid tumors, where the identification of a proper antigen has emerged as one of the major drawbacks to CAR T cell treatment success. CD44 is a glycoprotein involved in cell-cell and cell-matrix interactions. The isoform containing the variant domain 6 of CD44 gene (CD44v6) has been implicated in tumorigenesis, tumor cell invasion and metastasis and represents an attractive target for CAR T cell therapies. Targeting CD44v6 antigen has been shown to control tumor growth in acute myeloid leukemia and multiple myeloma mouse models. While CAR T approach for the treatment of B cell malignancies has shown great success, response rates among patients with solid cancer are less favorable. The purpose of our study was to test the efficacy of CD44v6.CAR T cells, produced in compliance with Good Manufacturing Practice (GMP), in adenocarcinoma tumor models. We generated a bicistronic retroviral vector containing the CD44v6 CAR and the HSV-TK Mut2 suicide gene to enhance the safety of the proposed CAR T cell therapy. CD44v6 transduced CAR T cells were homogeneously positive for ΔLNGFR selection marker, were enriched in T central memory (TCM) and T memory stem cells (TSCM) and displayed a highly activated phenotype. In vitro assays revealed antigen-specific activation and cytotoxicity of human CD44v6.CAR T cells against CD44v6 expressing tumor cell lines. When infused in immunodeficient tumor bearing mice, human CD44v6.CAR T cells were able to reach, infiltrate and proliferate at tumor sites, finally resulting in tumor growth control. Next, we checked if cells produced in compliance with GMP grade standards retained the same antitumor activity of those produced with research grade materials and protocols. Noteworthy, no differences in the potency of the CAR T obtained with the two manufacturing processes were observed. In conclusion, our preclinical results suggest that CD44v6.CAR T based adoptive therapy could be a promising strategy in solid cancer treatment.
Mechanism of action of the tumor vessel targeting agent NGR-hTNF: Role of both NGR peptide and hTNF in cell binding and signaling Barbara Valentinis, Simona Porcellini, Claudia Asperti, Manuela Cota, Dan Zhou, Paola Di Matteo, Gianpiero Garau, Chiara Zucchelli, Nilla Roberta Avanzi, Gian Paolo Rizzardi, Massimo Degano, Giovanna Musco, Catia Traversari International Journal of Molecular Sciences, 2019 NGR-hTNF is a therapeutic agent for a solid tumor that specifically targets angiogenic tumor blood vessels, through the NGR motif. Its activity has been assessed in several clinical studies encompassing tumors of different histological types. The drug’s activity is based on an improved permeabilization of newly formed tumor vasculature, which favors intratumor penetration of chemotherapeutic agents and leukocyte trafficking. This work investigated the binding and the signaling properties of the NGR-hTNF, to elucidate its mechanism of action. The crystal structure of NGR-hTNF and modeling of its interaction with TNFR suggested that the NGR region is available for binding to a specific receptor. Using 2D TR-NOESY experiments, this study confirmed that the NGR-peptides binds to a specific CD13 isoform, whose expression is restricted to tumor vasculature cells, and to some tumor cell lines. The interaction between hTNF or NGR-hTNF with immobilized TNFRs showed similar kinetic parameters, whereas the competition experiments performed on the cells expressing both TNFR and CD13 showed that NGR-hTNF had a higher binding affinity than hTNF. The analysis of the NGR-hTNF-triggered signal transduction events showed a specific impairment in the activation of pro-survival pathways (Ras, Erk and Akt), compared to hTNF. Since a signaling pattern identical to NGR-hTNF was obtained with hTNF and NGR-sequence given as distinct molecules, the inhibition observed on the survival pathways was presumably due to a direct effect of the NGR-CD13 engagement on the TNFR signaling pathway. The reduced activation of the pro survival pathways induced by NGR-hTNF correlated with the increased caspases activation and reduced cell survival. This study demonstrates that the binding of the NGR-motif to CD13 determines not only the homing of NGR-hTNF to tumor vessels, but also the increase in its antiangiogenic activity.
Succinimide-Based Conjugates Improve IsoDGR Cyclopeptide Affinity to αvβ3 without Promoting Integrin Allosteric Activation Francesca Nardelli, Cristina Paissoni, Giacomo Quilici, Alessandro Gori, Catia Traversari, Barbara Valentinis, Angelina Sacchi, Angelo Corti, Flavio Curnis, Michela Ghitti, Giovanna Musco Journal of Medicinal Chemistry, 2018 The isoDGR sequence is an integrin-binding motif that has been successfully employed as a tumor-vasculature-homing molecule or for the targeted delivery of drugs and diagnostic agents to tumors. In this context, we previously demonstrated that cyclopeptide 2, the product of the conjugation of c(CGisoDGRG) (1) to 4-( N-maleimidomethyl)cyclohexane-1-carboxamide, can be successfully used as a tumor-homing ligand for nanodrug delivery to neoplastic tissues. Here, combining NMR, computational, and biochemical methods, we show that the succinimide ring contained in 2 contributes to stabilizing interactions with αvβ3, an integrin overexpressed in the tumor vasculature. Furthermore, we demonstrate that various cyclopeptides containing the isoDGR sequence embedded in different molecular scaffolds do not induce αvβ3 allosteric activation and work as pure integrin antagonists. These results could be profitably exploited for the rational design of novel isoDGR-based ligands and tumor-targeting molecules with improved αvβ3-binding properties and devoid of adverse integrin-activating effects.
Extracellular NGFR spacers allow efficient tracking and enrichment of fully functional car-t cells co-expressing a suicide gene Monica Casucci, Laura Falcone, Barbara Camisa, Margherita Norelli, Simona Porcellini, Anna Stornaiuolo, Fabio Ciceri, Catia Traversari, Claudio Bordignon, Chiara Bonini, Attilio Bondanza Frontiers in Immunology, 2018 Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting.
24-hydroxycholesterol participates in pancreatic neuroendocrine tumor development Matias Soncini, Gianfranca Corna, Marta Moresco, Nadia Coltella, Umberto Restuccia, Daniela Maggioni, Laura Raccosta, Chin-Yo Lin, Francesca Invernizzi, Roberto Crocchiolo, Claudio Doglioni, Catia Traversari, Angela Bachi, Rosa Bernardi, Claudio Bordignon, Jan-Åke Gustafsson, Vincenzo Russo Proceedings of the National Academy of Sciences of the United States of America, 2016
Haploidentical HSCT: A 15-year experience at San Raffaele C Bonini, J Peccatori, M T L Stanghellini, L Vago, A Bondanza, N Cieri, R Greco, M Bernardi, C Corti, G Oliveira, E Zappone, C Traversari, C Bordignon, F Ciceri Bone Marrow Transplantation, 2015
Anti-metastatic activity of the tumor vascular targeting agent NGR-TNF Paola Di Matteo, Patrizia Mangia, Elena Tiziano, Barbara Valentinis, Simona Porcellini, Claudio Doglioni, Francesca Sanvito, Claudio Bordignon, Gian-Paolo Rizzardi, Catia Traversari Clinical and Experimental Metastasis, 2015
The oxysterol-cxcr2 axis plays a key role in the recruitment of tumor-promoting neutrophils Laura Raccosta, Raffaella Fontana, Daniela Maggioni, Claudia Lanterna, Eduardo J. Villablanca, Aida Paniccia, Andrea Musumeci, Elena Chiricozzi, Maria Letizia Trincavelli, Simona Daniele, Claudia Martini, Jan-Ake Gustafsson, Claudio Doglioni, Safiyè Gonzalvo Feo, Andrea Leiva, Maria Grazia Ciampa, Laura Mauri, Cristina Sensi, Alessandro Prinetti, Ivano Eberini, J. Rodrigo Mora, Claudio Bordignon, Knut R. Steffensen, Sandro Sonnino, Silvano Sozzani, Catia Traversari, Vincenzo Russo Journal of Experimental Medicine, 2013
Clinical and immunologic responses in melanoma patients vaccinated with MAGE-A3-genetically modified lymphocytes Vincenzo Russo, Lorenzo Pilla, Francesca Lunghi, Roberto Crocchiolo, Raffaella Greco, Fabio Ciceri, Daniela Maggioni, Raffaella Fontana, Sylvain Mukenge, Licia Rivoltini, Gianluigi Rigamonti, Santo Raffaele Mercuri, Roberto Nicoletti, Alessandro Del Maschio, Luigi Gianolli, Ferruccio Fazio, Alfonso Marchianò, Annabella Di Florio, Michele Maio, Monica Salomoni, Corrado Gallo-Stampino, Matteo Del Fiacco, Antonio Lambiase, Pierre G. Coulie, Roberto Patuzzo, Giorgio Parmiani, Catia Traversari, Claudio Bordignon, Mario Santinami, Marco Bregni International Journal of Cancer, 2013
De Novo design of a tumor-penetrating peptide Luca Alberici, Lise Roth, Kazuki N. Sugahara, Lilach Agemy, Venkata R. Kotamraju, Tambet Teesalu, Claudio Bordignon, Catia Traversari, Gian-Paolo Rizzardi, Erkki Ruoslahti Cancer Research, 2013
HMGB1 is an endogenous immune adjuvant released by necrotic cells Patrizia Rovere‐Querini, Annalisa Capobianco, Paola Scaffidi, Barbara Valentinis, Federica Catalanotti, Marta Giazzon, Ingrid E Dumitriu, Susanne Müller, Matteo Iannacone, Catia Traversari, Marco E Bianchi, Angelo A Manfredi EMBO Reports, 2004
Prognostic significance of cancer-testis gene expression in resected non-small cell lung cancer patients Oncology Reports, 2004
Safety of retroviral gene marking with a truncated NGF receptor [1] C. Bonini, M. Grez, C. Traversari, F. Ciceri, S. Marktel, G. Ferrari, M. Dinauer, M. Sadat, A. Aiuti, S. Deola, M. Radrizzani, A. Hagenbeek, J. Apperley, S. Ebeling, A. Martens, H.J. Kolb, M. Weber, F. Lotti, A. Grande, E. Weissinger, J.A. Bueren, M. Lamana, J.H.F. Falkenburg, M.H.M. Heemskerk, T. Austin, S. Kornblau, F. Marini, C. Benati, Z. Magnani, S. Cazzaniga, S. Toma, C. Gallo-Stampino, M. Introna, S. Slavin, P.D. Greenberg, M. Bregni, F. Mavilio, C. Bordignon Nature Medicine, 2003
Tumor-antigens recognised by T lymphocytes Minerva Biotecnologica, 1999
Identification of five MAGE-A1 epitopes recognized by cytolytic T lymphocytes obtained by in vitro stimulation with dendritic cells transduced with MAGE-A1 Journal of Immunology, 1999
Identification of a promiscuous T-cell epitope encoded by multiple members of the mage family Cancer Research, 1999
Major histocompatibility complex class I restricted cytotoxic T cells specific for natural melanoma peptides recognize unidentified shared melanoma antigen(s) Cancer Research, 1999
The DAM gene family encodes a new group of tumor-specific antigens recognized by human leukocyte antigen A2-restricted cytotoxic T lymphocytes Cancer Research, 1998
Gene therapy of lymphoid tumors Minerva Biotecnologica, 1997
Functional heterogeneity of HLA-A*02 subtypes revealed by presentation of a MAGE-3-encoded peptide to cytotoxic T cell clones Journal of Immunology, 1997
Multiple Melanoma-Associated Epitopes Recognized by HLA-A3-Restricted CTLs and Shared by Melanomas but Not Melanocytes Journal of Immunology, 1996
Multiple HLA-A alleles can present an immunodominant peptide of the human melanoma antigen Melan-A/MART-1 to a peptide-specific HLA-A*0201+ cytotoxic T cell line Journal of Immunology, 1996
Cytotoxic T-lymphocyte clones from different patients display limited T-cell-receptor variable-region gene usage in HLA-A2-restricted recognition of the melanoma antigen Melan-A/MART-1 Proceedings of the National Academy of Sciences of the United States of America, 1995
Transfer of the HSV-tk gene into donor peripheral blood lymphocytes for in vivo modulation of donor anti-tumor immunity after allogeneic bone marrow transplantation Human Gene Therapy, 1995
Structure, chromosomal localization, and expression of 12 genes of the MAGE family Etienne De Plaen, Catia Traversari, José J. Gaforio, Jean-Pierre Szikora, Charles De Smet, Francis Brasseur, Pierre van der Bruggen, Bernard Lethé, Christophe Lurquin, Patrick Chomez, Olivier De Backer, Thierry Boon, Karen Arden, Webster Cavenee, Robert Brasseur Immunogenetics, 1994
A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-E Journal of Experimental Medicine, 1992
Involvement of somatically acquired ecotropic viruses in the immunogenicity of nude-transplanted NIH/3T3 transformed cell lines Journal of Immunology, 1992
Expression of retrovirus-related, cytotoxic T lymphocyte- and transplantation-defined antigens in NIH/3T3 transfectants after a single passage in nude mice Journal of Immunology, 1989
Early lymphocyte activation molecule defined by the monoclonal antibody MLR-3: Biochemical and functional studies Immunology, 1988
Modulation of the human Harvey-ras oncogene expression by DNA methylation Oncogene Research, 1988
Monoclonal antibodies against NIH 3T3 cells transformed by human thyroid carcinoma DNA RACHELE ALZANI, MARCO A. PIEROTTI, SYLVIE MÉNARD, SILVANA CANEVARI, MARIA L. FERTONANI, DARIO BALLINARI, CATIA TRAVERSARI, GABRIELLA DELLA TORRE, PAOLO RADICE, SILVANA PILOTTI, ALFREDO FUSCO, MICHELE GRIECO, GIUSEPPE DELLA PORTA, MARIA I. COLNAGHI Hybridoma, 1988
Ly-5.185 molecule is associated with thymic maturation of lymphocytes but not with their cytotoxic activity Experimental and Clinical Immunogenetics, 1987