Scopus Publications
- CXCR4, CXCR7 and PBRM1 are responsible for everolimus and cabozantinib resistance in human renal cancer cells
Federica Auletta, Caterina Ieranò, Dario Guido Di Febbraro, Anna Maria Bello, Giuseppina Rea, et al.
Cell Death Discovery, 2026
The mTOR inhibitor everolimus (RAD001), previously used in first-line treatment of metastatic renal cancer (mRCC), is currently reserved for the following lines of therapy. However, many patients eventually develop resistance to RAD001. To shed new light on the mechanism of RAD001 resistance, A498 cells resistant to 1–5–10 µM of RAD001 were developed (A498-RAD 1-5-10). A498-RAD-resistant cells overexpressed the chromatin remodeling factor PBRM1 and mTOR and downregulated the chemokine receptors CXCR4 and CXCR7. To reverse RAD001 resistance, PBRM1 knockdown was conducted in A498-RAD10 cells. PBRM1 knockdown partially restored sensitivity to RAD001 while inducing CXCR7 but not CXCR4 expression. In A498-RAD10 cells, the CXCR7 transcriptional repressor YY1 is overexpressed and bound to the CXCR7 promoter. As CXCR4 was robustly downregulated in A498-RAD10, and the PBRM1 knockdown only partially restored RAD001 sensitivity, CXCR4 was transfected into A498-RAD10 (A498-RAD10-CXCR4). CXCR4 completely restored RAD001 sensitivity in resistant cells while reducing PBRM1 expression, implying negative feedback. Interestingly, A498-RAD10 cells were cross-resistant to cabozantinib, the tyrosine kinase inhibitor used in first-line treatment of mRCC with nivolumab. Cabozantinib resistance of A498-RAD10 cells was reversed by PBRM1 knockdown or CXCR4 re-expression, mimicking the RAD001 resistance. A498-CABO4-resistant cells were developed and showed PBRM1 overexpression and downregulated CXCR4 and CXCR7. In silico data supported a context‑dependent role of PBRM1 in ccRCC patients. To the best of our knowledge, this is the first description of a mechanism of RAD001 and cabozantinib resistance through PBRM1 overexpression and CXCR7/CXCR4 downregulation and suggest new therapeutic perspective for cabozantinib-resistant patients. - Comprehensive structural investigation of a potent and selective CXCR4 antagonist via crosslink modification
Anna Maria Trotta, Vincenzo Mazzarella, Michele Roggia, Antonia D'Aniello, Alessandra Del Bene, et al.
European Journal of Medicinal Chemistry, 2024 - Disulfide bond replacement with non-reducible side chain to tail macrolactamization for the development of potent and selective CXCR4 peptide antagonists endowed with flanking binding sites
Anna Maria Trotta, Stefano Tomassi, Gaetana Di Maiolo, Caterina Ieranò, Cinzia Vetrei, et al.
European Journal of Medicinal Chemistry, 2024 - Novel combinations of human immunomodulatory mAbs lacking cardiotoxic effects for therapy of TNBC
Cinzia Vetrei, Margherita Passariello, Guendalina Froechlich, Rosa Rapuano Lembo, Emanuele Sasso, et al.
Cancers, 2022
Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer characterized by a higher mortality rate among breast cancer subtypes. Poly(ADP-ribose) polymerase (PARP) inhibitors are used in clinics to treat a subgroup of TNBC patients, but other targeted therapies are urgently needed. Programmed death-ligand 1 (PD-L1), involved in tumor immune escape, was recently identified as a target for TNBC; accordingly, the anti-PD-L1 monoclonal antibody (mAb), atezolizumab, has been approved by FDA in combination with Paclitaxel for the therapy of metastatic TNBC. Here, we tested novel combinations of fully human immunomodulatory mAbs, including anti-PD-L1 mAbs generated in our laboratory and atezolizumab, on TNBC and other tumor cell lines. We evaluated their anti-tumor efficacy when used as single agents or in combinatorial treatments with anti-CTLA-4 mAbs in in vitro co-cultures of hPBMCs with tumor cells, by measuring tumor cell lysis and IL-2 and IFNγ cytokines secretion by lymphocytes. In parallel, by using co-cultures of hPBMCs and cardiomyocytes, we analyzed the potential cardiotoxic adverse side effects of the same antibody treatments by measuring the cardiac cell lysis and the secretion of pro-inflammatory cytokines. We identified novel combinations of immunomodulatory mAbs endowed with more potent anti-cancer activity on TNBC and lower cardiotoxic side effects than the combination of atezolizumab and ipilimumab. - Novel human neutralizing mAbs specific for Spike-RBD of SARS-CoV-2
Margherita Passariello, Chiara Gentile, Veronica Ferrucci, Emanuele Sasso, Cinzia Vetrei, et al.
Scientific Reports, 2021
Among the therapies against the pandemic SARS-CoV-2 virus, monoclonal Antibodies (mAbs) targeting the Spike glycoprotein represent good candidates to interfere in the Spike/ACE2 interaction, preventing virus cell entry. Since anti-spike mAbs, used individually, might be unable to block the virus entry in the case of resistant mutations, we designed an innovative strategy for the isolation of multiple novel human scFvs specific for the binding domain (RBD) of Spike. By panning a large phage display antibody library on immobilized RBD, we obtained specific binders by eluting with ACE2 in order to identify those scFvs recognizing the epitope of Spike interacting with its receptor. We converted the novel scFvs into full size IgG4, differently from the previously isolated IgG1 mAbs, to avoid unwanted potential side effects of IgG1 potent effector functions on immune system. The novel antibodies specifically bind to RBD in a nanomolar range and interfere in the interaction of Spike with ACE2 receptor, either used as purified protein or when expressed on cells in its native conformation. Furthermore, some of them have neutralizing activity for virus infection in cell cultures by using two different SARS-CoV-2 isolates including the highly contagious VOC 202012/01 variant and could become useful therapeutic tools to fight against the SARS-CoV-2 virus. - Interactions of spike‐rbd of sars‐cov‐2 and platelet factor 4: New insights in the etiopathogenesis of thrombosis
Margherita Passariello, Cinzia Vetrei, Felice Amato, Claudia De Lorenzo
International Journal of Molecular Sciences, 2021
The rare but dangerous adverse events evidenced after massive vaccination against SARS-CoV-2 are represented by thrombosis and thrombocytopenia. The patients diagnosed with severe COVID-19 may develop a pro-thrombotic state with a much higher frequency, thus we decided to investigate the role of Spike protein (the only common product of the two conditions) or the anti-Spike antibodies in the etiopathogenesis of thrombosis. A pathogenic Platelet Factor 4 (PF4)-dependent syndrome, unrelated to the use of heparin therapy, has been reported after the administration of vaccines in the patients manifesting acute thrombocytopenia and thrombosis. Thus, we aimed at shedding light on the structural similarities of Spike of SARS-CoV-2 and PF4 on their eventual biochemical interactions and on the role of their specific antibodies. The similarities between PF4 and Spike-RBD proteins were evaluated by a comparison of the structures and by testing the cross-reactivity of their specific antibodies by ELISA assays. We found that the anti-Spike antibodies do not recognize PF4, on the contrary, the anti-PF4 antibodies show some cross-reactivity for Spike-RBD. More interestingly, we report for the first time that the PF4 and Spike-RBD proteins can bind each other. These data suggest that the interaction of the two proteins could be involved in the generation of anti-PF4 antibodies, their binding to Spike-RBD, which could lead to platelets aggregation due also to their high expression of ACE2. - Immunomodulatory mabs as tools to investigate on cis-interaction of pd-1/pd-l1 on tumor cells and to set up methods for early screening of safe and potent combinatorial treatments
Cinzia Vetrei, Margherita Passariello, Guendalina Froechlich, Rosa Rapuano Lembo, Nicola Zambrano, et al.
Cancers, 2021
Antibodies targeting Immune Checkpoints (IC) on tumor infiltrating lymphocytes improve immune responses against cancer. Recently, the expression of some ICs has also been reported on cancer cells. We used the clinically validated Ipilimumab and Nivolumab and other novel human antibodies targeting Cytotoxic T- lymphocyte-antigen 4 (CTLA-4), Programmed Death receptor-1 (PD-1) and Programmed Death Ligand 1 (PD-L1) to shed light on the functions of these ICs in cancer cells. We show here for the first time that all these antagonistic mAbs are able to reduce Erk phosphorylation and, unexpectedly, to induce a significant increase of ICs expression on tumor cells, involving a hyperphosphorylation of NF-kB. On the contrary, agonistic PD-L1 and PD-1 recombinant proteins showed opposite effects by leading to a significant reduction of PD-1 and PD-L1, thus also suggesting the existence of a crosstalk in tumor cells between multiple ICs. Since the immunomodulatory mAbs show their higher anti-tumor efficacy by activating lymphocytes against cancer cells, we also investigated whether it was possible to identify the most efficient combinations of immunomodulatory mAbs for achieving potent anti-tumor efficacy associated with the lowest adverse side effects by setting up novel simple and predictive in vitro models based on co-cultures of tumor cells or human fetal cardiomyocytes with lymphocytes. We demonstrate here that novel combinations of immunomodulatory mAbs with more potent anti-cancer activity than Ipilimumab and Nivolumab combination can be identified with no or lower cardiotoxic side effects. Thus, we propose these co-cultures-based assays as useful tools to test also other combinatorial treatments of emerging immunomodulatory mAbs against different ICs for the early screening of most potent and safe combinatorial therapeutic regimens. - Isolation of two novel human anti-ctla-4 mabs with intriguing biological properties on tumor and nk cells
Margherita Passariello, Cinzia Vetrei, Emanuele Sasso, Guendalina Froechlich, Chiara Gentile, et al.
Cancers, 2020
The cytotoxic T lymphocyte-antigen 4 (CTLA-4) has been considered an IC exclusively expressed on T cells, where it counteracts the co-stimulatory CD28 receptor, by competing for its binding to CD-80 and CD-86. We recently found that it is expressed also on tumor and NK cells, suggesting other possible unknown roles of CTLA-4. To shed light on these novel aspects of CTLA-4, we used Ipilimumab, the first FDA approved human antibody targeting CTLA-4, in parallel studies with two novel human mAbs we isolated by using an efficient phage display selection strategy on live activated lymphocytes and purified mouse and human CTLA-4. The selection for cross-reactive mAbs was guaranteed by a high throughput sequencing to identify the sequences commonly enriched by two parallel pannings on human and mouse CTLA-4. Two isolated antibodies were found to bind with high affinity to both human and mouse CTLA-4 and lymphocytes, showing nanomolar or sub-nanomolar Kd values. They were able to kill Treg cells by ADCC, and to activate both human and mouse PBMCs, by strongly increasing cytokines secretion. Interestingly, they activated NK cells, exhibited cytotoxicity against cancer cells by inducing ADCC and inhibited tumor cell growth by affecting CTLA-4 downstream pathways in a similar fashion to CD-80 and CD-86 ligands and differently from Ipilimumab. Moreover, the novel mAbs showed a reduced ability to interfere in the binding of CD-80 ligands to CTLA-4 on T cells with respect to Ipilimumab, suggesting that they could allow for anti-tumor effects without the irAEs associated with the potent antagonistic activity of Ipilimumab. - Ipilimumab and its derived EGFR aptamer-based conjugate induce efficient NK cell activation against cancer cells
Margherita Passariello, Simona Camorani, Cinzia Vetrei, Stefania Ricci, Laura Cerchia, et al.
Cancers, 2020
The immune checkpoint CTLA-4 (cytotoxic T-lymphocyte-antigen 4), which inhibits the co-stimulatory CD28 signal on T cells, has been recently found expressed on other cell populations, such as tumor and natural killer (NK) cells. We tested for the first time the effects of ipilimumab, the human anti-CTLA4 mAb in clinical use, on these cells and found that it inhibits the growth of tumor cells expressing CTLA-4 also in the absence of lymphocytes, and efficiently activates NK cells, thus suggesting an important unexplored role of NK cells in ipilimumab-modulated immune responses. Interestingly, the epidermal growth factor receptor (EGFR) has been shown to play a key role in tumor cell escape from immune surveillance, and in cytotoxic T lymphocyte inhibition. Thus, we tested combinatorial treatments of ipilimumab with an anti-EGFR aptamer endowed with anti-tumor activity, and constructed for the first time a novel bispecific immunoconjugate, made up of these two compounds. The novel immunoconjugate binds to the target cells, induces the activation of lymphocytes, including NK cells, and inhibits the growth of tumor target cells more efficiently than the parental compounds, by strongly enhancing the cytotoxic activity of both human peripheral blood mononuclear cells and NK cells against tumor cells. - Novel Human Anti-PD-L1 mAbs Inhibit Immune-Independent Tumor Cell Growth and PD-L1 Associated Intracellular Signalling
Margherita Passariello, Anna Morena D’Alise, Annachiara Esposito, Cinzia Vetrei, Guendalina Froechlich, et al.
Scientific Reports, 2019
The novel antibody-based immunotherapy in oncology exploits the activation of immune system mediated by immunomodulatory antibodies specific for immune checkpoints. Among them, the programmed death ligand-1 (PD-L1) is of particular interest as it is expressed not only on T-cells, but also on other immune cells and on a large variety of cancer cells, such as breast cancer cells, considering its high expression in both ErbB2-positive and Triple Negative Breast Cancers. We demonstrate here that PD-L1_1, a novel anti-PD-L1 T -cell stimulating antibody, inhibits PD-L1-tumor cell growth also by affecting the intracellular MAPK pathway and by activating caspase 3. Similar in vitro results were obtained for the first time here also with the clinically validated anti-PD-L1 mAb Atezolizumab and in vivo with another validated anti-mouse anti-PD-L1 mAb. Moreover, we found that two high affinity variants of PD-L1_1 inhibited tumor cell viability more efficiently than the parental PD-L1_1 by affecting the same MAPK pathways with a more potent effect. Altogether, these results shed light on the role of PD-L1 in cancer cells and suggest that PD-L1_1 and its high affinity variants could become powerful antitumor weapons to be used alone or in combination with other drugs such as the anti-ErbB2 cAb already successfully tested in in vitro combinatorial treatments. - Novel human bispecific aptamer–antibody conjugates for efficient cancer cell killing
Margherita Passariello, Simona Camorani, Cinzia Vetrei, Laura Cerchia, Claudia De Lorenzo
Cancers, 2019 - Wee1 rather than plk1 is inhibited by AZD1775 at therapeutically relevant concentrations
Angela Flavia Serpico, Giuseppe D’Alterio, Cinzia Vetrei, Rosa Della Monica, Luca Nardella, et al.
Cancers, 2019 - Evidence that PP2A activity is dispensable for spindle assembly checkpoint-dependent control of Cdk1
Nando Cervone, Rosa Della Monica, Angela Flavia Serpico, Cinzia Vetrei, Mario Scaraglio, et al.
Oncotarget, 2018
