MARIA CATERINA ROTIROTI

All Publications

• Co-opting signalling molecules enables logic-gated control of CAR T cells. Nature Tousley, A. M., Rotiroti, M. C., Labanieh, L., Rysavy, L. W., Kim, W. J., Lareau, C., Sotillo, E., Weber, E. W., Rietberg, S. P., Dalton, G. N., Yin, Y., Klysz, D., Xu, P., de la Serna, E. L., Dunn, A. R., Satpathy, A. T., Mackall, C. L., Majzner, R. G. 2023

  Abstract

  Although chimeric antigen receptor (CAR) T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumour toxicity has hampered their development for solid tumours because most target antigens are shared with normal cells1,2. Researchers have attempted to apply Boolean-logic gating to CAR T cells to prevent toxicity3-5; however, a truly safe and effective logic-gated CAR has remained elusive6. Here we describe an approach to CAR engineering in which we replace traditional CD3ζ domains with intracellular proximal T cell signalling molecules. We show that certain proximal signalling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumours in vivo while bypassing upstream signalling proteins, including CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for signal propagation. We exploited the cooperative role of LAT and SLP-76 to engineer logic-gated intracellular network (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and prevention of on-target, off-tumour toxicity. LINK CAR will expand the range of molecules that can be targeted with CAR T cells, and will enable these powerful therapeutic agents to be used for solid tumours and diverse diseases such as autoimmunity7 and fibrosis8. In addition, this work shows that the internal signalling machinery of cells can be repurposed into surface receptors, which could open new avenues for cellular engineering.

  View details for DOI 10.1038/s41586-023-05778-2

  View details for PubMedID 36890224

  View details for PubMedCentralID 7433347

• IL3-zetakine combined with a CD33 costimulatory receptor as a Dual CAR approach for safer and selective targeting of AML. Blood advances Perriello, V. M., Rotiroti, M. C., Pisani, I., Galimberti, S., Alberti, G., Pianigiani, G., Ciaurro, V., Marra, A., Sabino, M., Tini, V., Spinozzi, G., Mezzasoma, F., Morena, F., Martino, S., Salerno, D., Ashby, J. F., Wingham, B., Serafini, M., Martelli, M. P., Falini, B., Biondi, A., Tettamanti, S. 2022

  Abstract

  Acute Myeloid Leukemia (AML) still represents an unmet clinical need for adult and pediatric patients. Adoptive cell therapy by chimeric antigen receptor (CAR)-engineered T cells demonstrated a high therapeutic potential, but further development is required to ensure a safe and durable disease remission in AML, especially in elderly patients. To date, translation of CAR T cell therapy in AML is limited by the absence of an ideal tumor-specific antigen. CD123 and CD33 are the two most widely overexpressed LSCs biomarkers but their shared expression with endothelial and hematopoietic stem and progenitor cells (HSPCs) increases the risk of undesired vascular and hematologic toxicities. To counteract this issue, we established a balanced Dual CAR strategy aimed at reducing off-target toxicities while retaining full functionality against AML. Cytokine-Induced Killer (CIK) cells, co-expressing a first-generation low affinity anti-CD123 IL3-zetakine and an anti-CD33 as costimulatory receptor (CCR) without activation signaling domains, demonstrated a powerful antitumor efficacy against AML targets without any relevant toxicity on HSPCs and endothelial cells. The proposed optimized Dual CAR CIK strategy could offer the opportunity to unleash the potential of specifically target CD123+/CD33+ leukemic cells while minimizing toxicity against healthy cells.

  View details for DOI 10.1182/bloodadvances.2022008762

  View details for PubMedID 36521101

• Lineage plasticity dictates responsiveness to anti-GD2 therapy in neuroblastoma. Mabe, N. W., Huang, M., Schaefer, D. A., Dalton, G. N., Digiovanni, G., Alexe, G., Geraghty, A. C., Khalid, D., Mader, M. M., Sheffer, M., Linde, M. H., Ly, N., Rotiroti, M., Smith, B. H., Wernig, M., Bertozzi, C. R., Monje, M., Mitsiades, C., Majeti, R., Satpathy, A. T., Stegmaier, K., Majzner, R. G. AMER ASSOC CANCER RESEARCH. 2022

  View details for Web of Science ID 000924848300026

• Lineage plasticity dictates responsiveness to anti-GD2 therapy in neuroblastoma. Mabe, N. W., Huang, M., Schaefer, D. A., Dalton, G. N., Digiovanni, G., Alexe, G., Geraghty, A. C., Khalid, D., Mader, M. M., Sheffer, M., Linde, M. H., Ly, N., Rotiroti, M., Smith, B. H., Wernig, M., Bertozzi, C. R., Monje, M., Mitsiades, C., Majeti, R., Satpathy, A. T., Stegmaier, K., Majzner, R. G. AMER ASSOC CANCER RESEARCH. 2022: 3

  View details for Web of Science ID 000920540300012

• Tuned IL3-Zetakine Coupled to a CD33 Costimulatory Receptor As a Dual CAR for Safer and Selective Targeting of Acute Myeloid Leukemia Perriello, V., Rotiroti, M., Pisani, I., Galimberti, S., Alberti, G., Pianigiani, G., Ciaurro, V., Marra, A., Sabino, M., Tini, V., Mezzasoma, F., Morena, F., Martino, S., Salerno, D., Ashby, J., Wingham, B., Serafini, M., Martelli, M., Falini, B., Biondi, A., Tettamanti, S. AMER SOC HEMATOLOGY. 2022: 10237-10238

  View details for DOI 10.1182/blood-2022-166555

  View details for Web of Science ID 000893230303109

• IKAROS MEDIATES ANTIGEN ESCAPE FOLLOWING CD19 CAR T CELL THERAPY IN R/R B-ALL Domizi, P., Sarno, J., Jager, A., Rotiroti, M., Baskar, R., Reynolds, W., Sahaf, B., Bendall, S., Mullighan, C., Leahy, A., Myers, R., Grupp, S., Majzner, R., Sotillo, E., Barrett, D., Davis, K. WILEY. 2022

  View details for Web of Science ID 000859203900105

• Transition to a mesenchymal state in neuroblastoma confers resistance to anti-GD2 antibody via reduced expression of ST8SIA1. Nature cancer Mabe, N. W., Huang, M., Dalton, G. N., Alexe, G., Schaefer, D. A., Geraghty, A. C., Robichaud, A. L., Conway, A. S., Khalid, D., Mader, M. M., Belk, J. A., Ross, K. N., Sheffer, M., Linde, M. H., Ly, N., Yao, W., Rotiroti, M. C., Smith, B. A., Wernig, M., Bertozzi, C. R., Monje, M., Mitsiades, C. S., Majeti, R., Satpathy, A. T., Stegmaier, K., Majzner, R. G. 2022

  Abstract

  Immunotherapy with anti-GD2 antibodies has advanced the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse, and little is known about mechanisms of resistance to anti-GD2 therapy. Here, we show that reduced GD2 expression was significantly correlated with the mesenchymal cell state in neuroblastoma and that a forced adrenergic-to-mesenchymal transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2-expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.

  View details for DOI 10.1038/s43018-022-00405-x

  View details for PubMedID 35817829

• Lenalidomide enhances CD23.CAR T cell therapy in chronic lymphocytic leukemia. Leukemia & lymphoma Tettamanti, S., Rotiroti, M. C., Giordano Attianese, G. M., Arcangeli, S., Zhang, R., Banerjee, P., Galletti, G., McManus, S., Mazza, M., Nicolini, F., Martinelli, G., Ivan, C., Veliz Rodriguez, T., Barbaglio, F., Scarfò, L., Ponzoni, M., Wierda, W., Gandhi, V., Keating, M., Biondi, A., Caligaris-Cappio, F., Biagi, E., Ghia, P., Bertilaccio, M. T. 2022: 1-14

  Abstract

  Chimeric antigen receptors (CAR)-modified T cells are an emerging therapeutic tool for chronic lymphocytic leukemia (CLL). However, in patients with CLL, well-known T-cell defects and the inhibitory properties of the tumor microenvironment (TME) hinder the efficacy of CAR T cells. We explored a novel approach combining CARs with lenalidomide, an immunomodulatory drug that tempers the immunosuppressive activity of the CLL TME. T cells from patients with CLL were engineered to express a CAR specific for CD23, a promising target antigen. Lenalidomide maintained the in vitro effector functions of CD23.CAR+ T cells effector functions in terms of antigen-specific cytotoxicity, cytokine release and proliferation. Overall, lenalidomide preserved functional CAR T-CLL cell immune synapses. In a Rag2-/-γc-/--based xenograft model of CLL, we demonstrated that, when combined with low-dose lenalidomide, CD23.CAR+ T cells efficiently migrated to leukemic sites and delayed disease progression when compared to CD23.CAR+ T cells given with rhIL-2. These observations underline the therapeutic potential of this novel CAR-based combination strategy in CLL.

  View details for DOI 10.1080/10428194.2022.2043299

  View details for PubMedID 35259043

• GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas. Nature Majzner, R. G., Ramakrishna, S., Yeom, K. W., Patel, S., Chinnasamy, H., Schultz, L. M., Richards, R. M., Jiang, L., Barsan, V., Mancusi, R., Geraghty, A. C., Good, Z., Mochizuki, A. Y., Gillespie, S. M., Toland, A. M., Mahdi, J., Reschke, A., Nie, E., Chau, I. J., Rotiroti, M. C., Mount, C. W., Baggott, C., Mavroukakis, S., Egeler, E., Moon, J., Erickson, C., Green, S., Kunicki, M., Fujimoto, M., Ehlinger, Z., Reynolds, W., Kurra, S., Warren, K. E., Prabhu, S., Vogel, H., Rasmussen, L., Cornell, T. T., Partap, S., Fisher, P. G., Campen, C. J., Filbin, M. G., Grant, G., Sahaf, B., Davis, K. L., Feldman, S. A., Mackall, C. L., Monje, M. 2022

  Abstract

  Diffuse intrinsic pontine glioma (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMG) are universally lethal paediatric central nervous system tumours1. We previously discovered that the disialoganglioside GD2 is highly expressed on H3K27M-mutant glioma cells and demonstrated promising preclinical efficacy of GD2-directed chimeric antigen receptor (CAR) T cells2, providing the rationale for a first-in-human Phase 1 clinical trial (NCT04196413). Because CAR T-cell-induced brainstem inflammation can result in obstructive hydrocephalus, increased intracranial pressure, and dangerous tissue shifts, neurocritical care precautions were incorporated. Here we present the clinical experience from the first four patients with H3K27M-mutant DIPG/DMG treated with GD2-CAR T cells (GD2-CART) at dose level 1 (1e6 GD2-CAR T cells/kg administered intravenously). Patients who exhibited clinical benefit were eligible for subsequent GD2-CAR T infusions administered intracerebroventricularly3. Toxicity was largely related to tumor location and reversible with intensive supportive care. On-target, off-tumor toxicity was not observed. Three of four patients exhibited clinical and radiographic improvement. Proinflammatory cytokines were increased in plasma and cerebrospinal fluid (CSF). Transcriptomic analyses of 65,598 single cells from CAR T cell products and CSF elucidate heterogeneity in response between subjects and administration routes. These early results underscore the promise of this approach for H3K27M+ DIPG/DMG therapy.

  View details for DOI 10.1038/s41586-022-04489-4

  View details for PubMedID 35130560

• Optimization of therapeutic T cell expansion in G-Rex device and applicability to large-scale production for clinical use. Cytotherapy Gotti, E., Tettamanti, S., Zaninelli, S., Cuofano, C., Cattaneo, I., Rotiroti, M. C., Cribioli, S., Alzani, R., Rambaldi, A., Introna, M., Golay, J. 2022

  Abstract

  Our center performs experimental clinical studies with advanced therapy medicinal products (ATMPs) based on polyclonal T cells, all of which are currently expanded in standard T-flasks. Given the need to increase the efficiency and safety of large-scale T cell expansion for clinical use, we have optimized the method to expand in G-Rex devices both cytokine-induced killer cells (CIKs) from peripheral or cord blood and blinatumomab-expanded T cells (BETs). We show that the G-Rex reproducibly allowed the expansion of >30 × 106 CD3+ cells/cm2 of gas-permeable membrane in a mean of 10 to 11 days in a single unit, without manipulation, except for addition of cytokines and sampling of supernatant for lactate measurement every 3 to 4 days. In contrast, 21 to 24 days, twice-weekly cell resuspension and dilution into 48 to 72 T-flasks were required to complete expansions using the standard method. We show that the CIKs produced in G-Rex (CIK-G) were phenotypically very similar, for a large panel of markers, to those expanded in T-flasks, although CIK-G products had lower expression of CD56 and higher expression of CD27 and CD28. Functionally, CIK-Gs were strongly cytotoxic in vitro against the NK cell target K562 and the REH pre-B ALL cell line in the presence of blinatumomab. CIK-Gs also showed therapeutic activity in vivo in the Ph+ pre-B ALL-2 model in mice. The expansion of both CIKs and BETs in G-Rex was validated in good manufacturing practices (GMP) conditions, and we plan to use G-Rex for T cell expansion in future clinical studies.

  View details for DOI 10.1016/j.jcyt.2021.11.004

  View details for PubMedID 35063359

• CD123 and CD33 Co-Targeting By Balanced Signaling on CAR-CIK Cells Reduces Potential Off-Target Toxicity While Preserving the Anti-Leukemic Activity of Acute Myeloid Leukemia Perriello, V., Rotiroti, M., Pisani, I., Alberti, G., Pianigiani, G., Rossi, R., Ciaurro, V., Serafini, M., Martelli, M., Falini, B., Biondi, A., Tettamanti, S. AMER SOC HEMATOLOGY. 2021

  View details for DOI 10.1182/blood-2021-150487

  View details for Web of Science ID 000736398806165

• CAR T cells with dual targeting of CD19 and CD22 in adult patients with recurrent or refractory B cell malignancies: a phase 1 trial. Nature medicine Spiegel, J. Y., Patel, S., Muffly, L., Hossain, N. M., Oak, J., Baird, J. H., Frank, M. J., Shiraz, P., Sahaf, B., Craig, J., Iglesias, M., Younes, S., Natkunam, Y., Ozawa, M. G., Yang, E., Tamaresis, J., Chinnasamy, H., Ehlinger, Z., Reynolds, W., Lynn, R., Rotiroti, M. C., Gkitsas, N., Arai, S., Johnston, L., Lowsky, R., Majzner, R. G., Meyer, E., Negrin, R. S., Rezvani, A. R., Sidana, S., Shizuru, J., Weng, W., Mullins, C., Jacob, A., Kirsch, I., Bazzano, M., Zhou, J., Mackay, S., Bornheimer, S. J., Schultz, L., Ramakrishna, S., Davis, K. L., Kong, K. A., Shah, N. N., Qin, H., Fry, T., Feldman, S., Mackall, C. L., Miklos, D. B. 2021

  Abstract

  Despite impressive progress, more than 50% of patients treated with CD19-targeting chimeric antigen receptor T cells (CAR19) experience progressive disease. Ten of 16 patients with large B cell lymphoma (LBCL) with progressive disease after CAR19 treatment had absent or low CD19. Lower surface CD19 density pretreatment was associated with progressive disease. To prevent relapse with CD19- or CD19lo disease, we tested a bispecific CAR targeting CD19 and/or CD22 (CD19-22.BB.z-CAR) in a phase I clinical trial ( NCT03233854 ) of adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL) and LBCL. The primary end points were manufacturing feasibility and safety with a secondary efficacy end point. Primary end points were met; 97% of products met protocol-specified dose and no dose-limiting toxicities occurred during dose escalation. In B-ALL (n=17), 100% of patients responded with 88% minimal residual disease-negative complete remission (CR); in LBCL (n=21), 62% of patients responded with 29% CR. Relapses were CD19-/lo in 50% (5 out of 10) of patients with B-ALL and 29% (4 out of 14) of patients with LBCL but were not associated with CD22-/lo disease. CD19/22-CAR products demonstrated reduced cytokine production when stimulated with CD22 versus CD19. Our results further implicate antigen loss as a major cause of CAR T cell resistance, highlight the challenge of engineering multi-specific CAR T cells with equivalent potency across targets and identify cytokine production as an important quality indicator for CAR T cell potency.

  View details for DOI 10.1038/s41591-021-01436-0

  View details for PubMedID 34312556

• Targeting CD33 in Chemoresistant AML Patient-Derived Xenografts by CAR-CIK Cells Modified with an Improved SB Transposon System MOLECULAR THERAPY Rotiroti, M., Buracchi, C., Arcangeli, S., Galimberti, S., Valsecchi, M., Perriello, V., Rasko, T., Alberti, G., Magnani, C., Cappuzzello, C., Lundberg, F., Pande, A., Dastoli, G., Introna, M., Serafini, M., Biagi, E., Izsvak, Z., Biondi, A., Tettamanti, S. 2020; 28 (9): 1974–86

  Abstract

  The successful implementation of chimeric antigen receptor (CAR)-T cell therapy in the clinical context of B cell malignancies has paved the way for further development in the more critical setting of acute myeloid leukemia (AML). Among the potentially targetable AML antigens, CD33 is insofar one of the main validated molecules. Here, we describe the feasibility of engineering cytokine-induced killer (CIK) cells with a CD33.CAR by using the latest optimized version of the non-viral Sleeping Beauty (SB) transposon system "SB100X-pT4." This offers the advantage of improving CAR expression on CIK cells, while reducing the amount of DNA transposase as compared to the previously employed "SB11-pT" version. SB-modified CD33.CAR-CIK cells exhibited significant antileukemic activity in vitro and in vivo in patient-derived AML xenograft models, reducing AML development when administered as an "early treatment" and delaying AML progression in mice with established disease. Notably, by exploiting an already optimized xenograft chemotherapy model that mimics human induction therapy in mice, we demonstrated for the first time that CD33.CAR-CIK cells are also effective toward chemotherapy resistant/residual AML cells, further supporting its future clinical development and implementation within the current standard regimens.

  View details for DOI 10.1016/j.ymthe.2020.05.021

  View details for Web of Science ID 000571942900005

  View details for PubMedID 32526203

  View details for PubMedCentralID PMC7474266

• Balance of Anti-CD123 Chimeric Antigen Receptor Binding Affinity and Density for the Targeting of Acute Myeloid Leukemia MOLECULAR THERAPY Arcangeli, S., Rotiroti, M., Bardelli, M., Simonelli, L., Magnani, C., Biondi, A., Biagi, E., Tettamanti, S., Varani, L. 2017; 25 (8): 1933–45

  Abstract

  Chimeric antigen receptor (CAR)-redirected T lymphocytes are a promising immunotherapeutic approach and object of pre-clinical evaluation for the treatment of acute myeloid leukemia (AML). We developed a CAR against CD123, overexpressed on AML blasts and leukemic stem cells. However, potential recognition of low CD123-positive healthy tissues, through the on-target, off-tumor effect, limits safe clinical employment of CAR-redirected T cells. Therefore, we evaluated the effect of context-dependent variables capable of modulating CAR T cell functional profiles, such as CAR binding affinity, CAR expression, and target antigen density. Computational structural biology tools allowed for the design of rational mutations in the anti-CD123 CAR antigen binding domain that altered CAR expression and CAR binding affinity without affecting the overall CAR design. We defined both lytic and activation antigen thresholds, with early cytotoxic activity unaffected by either CAR expression or CAR affinity tuning but later effector functions impaired by low CAR expression. Moreover, the anti-CD123 CAR safety profile was confirmed by lowering CAR binding affinity, corroborating CD123 is a good therapeutic target antigen. Overall, full dissection of these variables offers suitable anti-CD123 CAR design optimization for the treatment of AML.

  View details for DOI 10.1016/j.ymthe.2017.04.017

  View details for Web of Science ID 000406989700022

  View details for PubMedID 28479045

  View details for PubMedCentralID PMC5542631

• Acute Myeloid Leukemia Targeting by Chimeric Antigen Receptor T Cells: Bridging the Gap from Preclinical Modeling to Human Studies HUMAN GENE THERAPY Rotiroti, M., Arcangeli, S., Casucci, M., Perriello, V., Bondanza, A., Biondi, A., Tettamanti, S., Biagi, E. 2017; 28 (3): 231–41

  Abstract

  Acute myeloid leukemia (AML) still represents an unmet clinical need for adult and pediatric high-risk patients, thus demanding advanced and personalized therapies. In this regard, different targeted immunotherapeutic approaches are available, ranging from naked monoclonal antibodies (mAb) to conjugated and multifunctional mAbs (i.e., BiTEs and DARTs). Recently, researchers have focused their attention on novel techniques of genetic manipulation specifically to redirect cytotoxic T cells endowed with chimeric antigen receptors (CARs) toward selected tumor associated antigens. So far, CAR T cells targeting the CD19 antigen expressed by B-cell origin hematological cancers have gained impressive clinical results, leading to the possibility of translating the CAR platform to treat other hematological malignancies such as AML. However, one of the main concerns in the field of AML CAR immunotherapy is the identification of an ideal target cell surface antigen, being highly expressed on tumor cells but minimally present on healthy tissues, together with the design of an anti-AML CAR appropriately balancing efficacy and safety profiles. The current review focuses mainly on AML target antigens and the related immunotherapeutic approaches developed so far, deeply dissecting methods of CAR T cell safety improvements, when designing novel CARs approaching human studies.

  View details for DOI 10.1089/hum.2016.092

  View details for Web of Science ID 000397570400003

  View details for PubMedID 27967241