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IRCCS Ospedale Pediatrico Bambino Gesu, Rome, Italy
Elena Morrocchi, Chiara Pighi, Giuseppe Rubens Pascucci, Nicola Cotugno, Chiara Medri, Donato Amodio, Luna Colagrossi, Alessandra Ruggiero, Emma Concetta Manno, Chiara Casamento Tumeo, Stefania Bernardi, Kinga K Smolen, Carlo Federico Perno, Al Ozonoff, Paolo Rossi, Ofer Levy, and Paolo Palma
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, eISSN: 15376591, Pages: S51-S60, Published: 15 August 2022 Oxford University Press (OUP)
Abstract Background Immunization of vulnerable populations with distinct immunity often results in suboptimal immunogenicity, durability, and efficacy. Methods Safety and immunogenicity profiles of BNT162b2 messenger RNA coronavirus disease 2019 (COVID-19) vaccine, among people living with human immunodeficiency virus (HIV), were evaluated in 28 perinatally HIV-infected patients under antiretroviral therapy (ART) and 65 healthy controls (HCs) with no previous history of COVID-19. Thus, we measured severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific humoral and CD4+ T cell responses. Samples were collected before vaccination (baseline, day [D] 0), at the second dose (D21), and at 4 weeks (D28) and 6 months (D180) after D0. Proteomic profiles at D0 and D28 were assessed with a multiplexed proximity extension assay (Olink) on plasma samples. Results All HIV-infected patients mounted similar anti–SARS-CoV-2 humoral responses to those of HCs, albeit with lower titers of anti-trimeric S at D28 (P = .01). Only peripheral blood mononuclear cells of HIV-infected patients demonstrated at D28 an impaired ability to expand their specific (CD40L+) CD4+ T-cell populations. Similar humoral titers were maintained between the 2 groups at 6-months follow-up. We additionally correlated baseline protein levels to either humoral or cellular responses, identifying clusters of molecules involved in immune response regulation with inverse profiles between the 2 study groups. Conclusions Responses of ART-treated HIV-infected patients, compared to those of HCs, were characterized by distinct features especially within the proteomic compartment, supporting their eligibility to an additional dose, similarly to the HC schedule.
Nicola Cotugno, Enrica Franzese, Giulia Angelino, Donato Amodio, Erminia Francesca Romeo, Francesca Rea, Simona Faraci, Renato Tambucci, Elisa Profeti, Emma Concetta Manno, Veronica Santilli, Gioacchino Andrea Rotulo, Chiara Pighi, Chiara Medri, Elena Morrocchi, Luna Colagrossi, Giuseppe Rubens Pascucci, Diletta Valentini, Alberto Villani, Paolo Rossi, Paola De Angelis, and Paolo Palma
Vaccines, eISSN: 2076393X, Published: July 2022 MDPI AG
Patients affected by Inflammatory Bowel Disease (IBD) present higher risk for infection and suboptimal response upon vaccination. The immunogenicity of SARS-CoV2 vaccination is still largely unknown in adolescents or young adults affected by IBD (pIBD). We investigated the safety and immunogenicity of the BNT162B2 mRNA COVID-19 vaccine in 27 pIBD, as compared to 30 healthy controls (HC). Immunogenicity was measured by anti-SARS-CoV2 IgG (anti-S and anti-trim Ab) before vaccination, after 21 days (T21) and 7 days after the second dose (T28). The safety profile was investigated by close monitoring and self-reported adverse events. Vaccination was well tolerated, and short-term adverse events reported were only mild to moderate. Three out of twenty-seven patients showed IBD flare after vaccination, but no causal relationship could be established. Overall, pIBD showed a good humoral response upon vaccination compared to HC; however, pIBD on anti-TNFα treatment showed lower anti-S Ab titers compared to patients receiving other immune-suppressive regimens (p = 0.0413 at first dose and p = 0.0301 at second dose). These data show that pIBD present a good safety and immunogenicity profile following SARS-CoV-2 mRNA vaccination. Additional studies on the impact of specific immune-suppressive regimens, such as anti TNFα, on immunogenicity should be further investigated on larger cohorts.
A. Ruggiero, G. Pascucci, N. Cotugno, S. Domínguez-Rodríguez, S. Rinaldi, A. Tagarro, P. Rojo, C. Foster, A. Bamford, A. De Rossi, E. Nastouli, N. Klein, E. Morrocchi, B. Fatou, K. Smolen, A. Ozonoff, M. Di Pastena, K. Luzuriaga, H. Steen, C. Giaquinto, P. Goulder, P. Rossi, O. Levy, S. Pahwa, P. Palma and Mark Shaun Thanyawee Louise Andrew Avy Kennedy Paula Ma Cotton Barnabas Puthanakit Kuhn Yates Violari Otwo
Frontiers in Immunology, eISSN: 16643224, Published: 31 March 2022
Background Despite a successful antiretroviral therapy (ART), adolescents living with perinatally acquired HIV (PHIV) experience signs of B-cell hyperactivation with expansion of ‘namely’ atypical B-cell phenotypes, including double negative (CD27-IgD-) and termed age associated (ABCs) B-cells (T-bet+CD11c+), which may result in reduced cell functionality, including loss of vaccine-induced immunological memory and higher risk of developing B-cells associated tumors. In this context, perinatally HIV infected children (PHIV) deserve particular attention, given their life-long exposure to chronic immune activation. Methods We studied 40 PHIV who started treatment by the 2nd year of life and maintained virological suppression for 13.5 years, with 5/40 patients experiencing transient elevation of the HIV-1 load in the plasma (Spike). We applied a multi-disciplinary approach including immunological B and T cell phenotype, plasma proteomics analysis, and serum level of anti-measles antibodies as functional correlates of vaccine-induced immunity. Results Phenotypic signs of B cell hyperactivation were elevated in subjects starting ART later (%DN T-bet+CD11c+ p=0.03; %AM T-bet+CD11c+ p=0.02) and were associated with detectable cell-associated HIV-1 RNA (%AM T-bet+CD11c+ p=0.0003) and transient elevation of the plasma viral load (spike). Furthermore, B-cell hyperactivation appeared to be present in individuals with higher frequency of exhausted T-cells, in particular: %CD4 TIGIT+ were associated with %DN (p=0.008), %DN T-bet+CD11c+ (p=0.0002) and %AM T-bet+CD11c+ (p=0.002) and %CD4 PD-1 were associated with %DN (p=0.048), %DN T-bet+CD11c+ (p=0.039) and %AM T-bet+CD11c+ (p=0.006). The proteomic analysis revealed that subjects with expansion of these atypical B-cells and exhausted T-cells had enrichment of proteins involved in immune inflammation and complement activation pathways. Furthermore, we observed that higher levels of ABCs were associated a reduced capacity to maintain vaccine-induced antibody immunity against measles (%B-cells CD19+CD10- T-bet+, p=0.035). Conclusion We identified that the levels of hyperactivated B cell subsets were strongly affected by time of ART start and associated with clinical, viral, cellular and plasma soluble markers. Furthermore, the expansion of ABCs also had a direct impact on the capacity to develop antibodies response following routine vaccination.
Nicola Cotugno, Chiara Pighi, Elena Morrocchi, Alessandra Ruggiero, Donato Amodio, Chiara Medri, Luna Colagrossi, Cristina Russo, Silvia Di Cesare, Veronica Santilli, Emma Concetta Manno, Paola Zangari, Carmela Giancotta, Stefania Bernardi, Luciana Nicolosi, Marta Ciofi Degli Atti, Massimiliano Raponi, Salvatore Zaffina, Sara Alfieri, Richard Kirk, Carlo Federico Perno, Paolo Rossi, Antonio Amodeo, and Paolo Palma
Transplantation, ISSN: 00411337, Volume: 106, Pages: E158-E160, Published: 1 February 2022 Ovid Technologies (Wolters Kluwer Health)
Nicola Cotugno, Alessandra Ruggiero, Giuseppe Rubens Pascucci, Francesco Bonfante, Maria Raffaella Petrara, Chiara Pighi, Loredana Cifaldi, Paola Zangari, Stefania Bernardi, Laura Cursi, Veronica Santilli, Emma Concetta Manno, Donato Amodio, Giulia Linardos, Livia Piccioni, Maria Antonietta Barbieri, Daniela Perrotta, Andrea Campana, Daniele Donà, Carlo Giaquinto, Carlo Concato, Petter Brodin, Paolo Rossi, Anita De Rossi, Paolo Palma, Lorenza Romani, Paola Pansa, Sara Chiurchiu, Andrea Finocchi, Caterina Cancrini, Laura Lancella, Maia De Luca, Renato Cutrera, Alberto Villani, Elena Morrocchi, Sonia Zicari, Lorenza Putignani, Francesca Calò Carducci, Maria A. De Ioris, Patrizia D'Argenio, Marta Ciofi degli Atti, Carmen D'Amore, and
Pediatric Allergy and Immunology, ISSN: 09056157, eISSN: 13993038, Pages: 1833-1842, Published: November 2021 Wiley
BACKGROUND Despite SARS-CoV-2 immunizations have started in most countries, children are not currently included in the vaccination programs, thus it remains crucial to define their anti-SARS-CoV-2 immune response in order to minimize the risk for other epidemic waves. This study seeks to provide a description of the virology ad anti-SARS-CoV-2 immunity in children with distinct symptomatology. METHODS Between March and July 2020, we recruited 15 SARS-CoV-2 asymptomatic (AS) and 51 symptomatic children (SY), stratified according to WHO clinical classification. We measured SARS-CoV-2 viral load using ddPCR and qPCR in longitudinally collected nasopharyngeal swabs samples. To define anti-SARS-CoV-2 antibodies we measured neutralization activity and total IgG load (Diasorin). We also evaluated antigen-specific B and CD8+T-cells, using a labelled S1+S2 protein and ICAM expression, respectively. Plasma protein profiling was performed with Olink. RESULTS Virological profiling showed that AS had lower viral load at diagnosis (p=0.004) and faster virus clearance (p=0.0002) compared to SY. Anti-SARS CoV-2 humoral and cellular response did not appear to be associated with the presence of symptoms. AS and SY showed similar titers of SARS-CoV-2 IgG, levels of neutralizing activity, and frequency of Ag-specific B and CD8+T-cells. Whereas pro-inflammatory plasma protein profile was associated to symptomatology. CONCLUSION We demonstrated the development of anti-SARS-CoV-2 humoral and cellular response with any regards to symptomatology, suggesting the ability of both SY and AS to contribute towards herd immunity. The virological profiling of AS suggested that they have lower virus load associated with faster virus clearance.
Donato Amodio, Alessandra Ruggiero, Mayla Sgrulletti, Chiara Pighi, Nicola Cotugno, Chiara Medri, Elena Morrocchi, Luna Colagrossi, Cristina Russo, Salvatore Zaffina, Gigliola Di Matteo, Cristina Cifaldi, Silvia Di Cesare, Beatrice Rivalta, Lucia Pacillo, Veronica Santilli, Carmela Giancotta, Emma Concetta Manno, Marta Ciofi Degli Atti, Massimiliano Raponi, Paolo Rossi, Andrea Finocchi, Caterina Cancrini, Carlo Federico Perno, Viviana Moschese, and Paolo Palma
Frontiers in Immunology, eISSN: 16643224, Published: 4 October 2021 Frontiers Media SA
Mass SARS-Cov-2 vaccination campaign represents the only strategy to defeat the global pandemic we are facing. Immunocompromised patients represent a vulnerable population at high risk of developing severe COVID-19 and thus should be prioritized in the vaccination programs and in the study of the vaccine efficacy. Nevertheless, most data on efficacy and safety of the available vaccines derive from trials conducted on healthy individuals; hence, studies on immunogenicity of SARS-CoV2 vaccines in such populations are deeply needed. Here, we perform an observational longitudinal study analyzing the humoral and cellular response following the BNT162b2 mRNA COVID-19 vaccine in a cohort of patients affected by inborn errors of immunity (IEI) compared to healthy controls (HC). We show that both IEI and HC groups experienced a significant increase in anti-SARS-CoV-2 Abs 1 week after the second scheduled dose as well as an overall statistically significant expansion of the Ag-specific CD4+CD40L+ T cells in both HC and IEI. Five IEI patients did not develop any specific CD4+CD40L+ T cellular response, with one of these patients unable to also mount any humoral response. These data raise immunologic concerns about using Ab response as a sole metric of protective immunity following vaccination for SARS-CoV-2. Taken together, these findings suggest that evaluation of vaccine-induced immunity in this subpopulation should also include quantification of Ag-specific T cells.
Nicola Cotugno, Alessandra Ruggiero, Francesco Bonfante, Maria Raffaella Petrara, Sonia Zicari, Giuseppe Rubens Pascucci, Paola Zangari, Maria Antonietta De Ioris, Veronica Santilli, E.C. Manno, Donato Amodio, Alessio Bortolami, Matteo Pagliari, Carlo Concato, Giulia Linardos, Andrea Campana, Daniele Donà, Carlo Giaquinto, Petter Brodin, Paolo Rossi, Anita De Rossi, Paolo Palma, Stefania Bernardi, Lorenza Romani, Paola Pansa, Sara Chiurchiú, Andrea Finocchi, Caterina Cancrini, Laura Lancella, Laura Cursi, Maia De Luca, Renato Cutrera, Libera Sessa, Elena Morrocchi, Chiara Medri, Lorenza Putignani, F.I. Calò Carducci, Patrizia D’Argenio, Marta Ciofi degli Atti, Carmen D’Amore, Livia Piccioni, Martina Di Giuseppe, Alessandro Jenkner, Carmela Giancotta, and Andrzej Krzysztofiak
Cell Reports, eISSN: 22111247, Published: 16 March 2021 Elsevier BV
As the global COVID-19 pandemic progresses, it is paramount to gain knowledge on adaptive immunity to SARS-CoV-2 in children to define immune correlates of protection upon immunization or infection. We analyzed anti-SARS-CoV-2 antibodies and their neutralizing activity (PRNT) in 66 COVID-19-infected children at 7 (±2) days after symptom onset. Individuals with specific humoral responses presented faster virus clearance and lower viral load associated with a reduced in vitro infectivity. We demonstrated that the frequencies of SARS-CoV-2-specific CD4+CD40L+ T cells and Spike-specific B cells were associated with the anti-SARS-CoV-2 antibodies and the magnitude of neutralizing activity. The plasma proteome confirmed the association between cellular and humoral SARS-CoV-2 immunity, and PRNT+ patients show higher viral signal transduction molecules (SLAMF1, CD244, CLEC4G). This work sheds lights on cellular and humoral anti-SARS-CoV-2 responses in children, which may drive future vaccination trial endpoints and quarantine measures policies.
Guzman Sanchez-Schmitz, Elena Morrocchi, Mitchell Cooney, Dheeraj Soni, Rahima Khatun, Paolo Palma, David J. Dowling, and Ofer Levy
Scientific Reports, eISSN: 20452322, Published: 1 December 2020 Springer Science and Business Media LLC
Abstract Infections are most frequent at the extremes of life, especially among newborns, reflecting age-specific differences in immunity. Monocytes maintain tissue-homeostasis and defence-readiness by escaping circulation in the absence of inflammation to become tissue-resident antigen presenting cells in vivo. Despite equivalent circulating levels, neonates demonstrate lower presence of monocytes inside peripheral tissues as compared to adults. To study the ability of monocytes to undergo autonomous transendothelial extravasation under biologically accurate circumstances we engineered a three-dimensional human vascular-interstitial model including collagen, fibronectin, primary endothelial cells and autologous untreated plasma. This microphysiological tissue construct enabled age-specific autonomous extravasation of monocytes through a confluent human endothelium in the absence of exogenous chemokines and activation. Both CD16− and CD16+ newborn monocytes demonstrated lower adherence and extravasation as compared to adults. In contrast, pre-activated tissue constructs were colonized by newborn monocytes at the same frequency than adult monocytes, suggesting that neonatal monocytes are capable of colonizing inflamed tissues. The presence of autologous plasma neither improved newborn homeostatic extravasation nor shaped age-specific differences in endothelial cytokines that could account for this impairment. Newborn monocytes demonstrated significantly lower surface expression of CD31 and CD11b, and mechanistic experiments using blocking antibodies confirmed a functional role for CD31 and CD54 in neonatal homeostatic extravasation. Our data suggests that newborn monocytes are intrinsically impaired in extravasation through quiescent endothelia, a phenomenon that could contribute to the divergent immune responsiveness to vaccines and susceptibility to infection observed during early life.
Nicola Cotugno, Sonia Zicari, Elena Morrocchi, Lesley R. de Armas, Suresh Pallikkuth, Stefano Rinaldi, Alessandra Ruggiero, Emma Concetta Manno, Paola Zangari, Maria Chiriaco, Stefania Bernardi, Sarah F. Andrews, Alberto Cagigi, Paolo Rossi, Adrian B. McDermott, Savita Pahwa, and Paolo Palma
Clinical Immunology, ISSN: 15216616, eISSN: 15217035, Volume: 215, Published: June 2020 Elsevier BV
Perinatally HIV-infected children (PHIV), despite successful antiretroviral therapy, present suboptimal responses to vaccinations compared to healthy-controls (HC). Here we investigated phenotypic and transcriptional signatures of H1N1-specific B-cells (H1N1-Sp) in PHIV, differentially responding to trivalent-influenza-vaccine (TIV), and HC. Patients were categorized in responders (R) and non-responders (NR) according to hemagglutination-inhibition-assay at baseline and 21 days after TIV. No differences in H1N1-Sp frequencies were found between groups. H1N1-Sp transcriptional analysis revealed a distinct signature between PHIV and HC. NR presented higher PIK3C2B and NOD2 expression compared to R, confirmed by downregulation of PIK3C2B in resting-memory of R after H1N1 in-vitro stimulation. In conclusion this study confirms that qualitative rather than quantitative analyses are needed to characterize immune responses in PHIV. These results further suggest that higher PIK3C2B in H1N1-Sp of NR is associated with lower H1N1 immunogenicity and may be targeted by future modulating strategies to improve TIV responses in PHIV.
Nicola Cotugno, Elena Morrocchi, Stefano Rinaldi, Salvatore Rocca, Ilaria Pepponi, Silvia di Cesare, Stefania Bernardi, Paola Zangari, Suresh Pallikkuth, Lesley de Armas, Ofer Levy, Paolo Rossi, Savita Pahwa, and Paolo Palma ISSN: 02699370, eISSN: 14735571, Pages: 669-680, Published: 1 April 2020 Ovid Technologies (Wolters Kluwer Health)
OBJECTIVE To investigate long-term persistence of HIV-specific lymphocyte immunity in perinatally HIV-infected children treated within the first year of life. DESIGN Twenty perinatally HIV-infected children who received ART therapy within the first year of life (early treated) and with stable viral control (>5 years) were grouped according to their serological response to HIV. METHODS Western blot analysis and ELISA defined 14 HIV-seropositive and 6 seronegative patients. Frequencies of gp140-specific T-cell and B-cell, and T-cell cytokine production were quantified by flow cytometry in both seronegatives and seropositives. Transcriptional signatures in purified gp140-specific B-cell subsets, in response to in-vitro stimulation with HIV peptides was evaluated by multiplex RT-PCR. RESULTS Gp140-specific T cells and B cells persist at similar levels in both groups. A higher production of IL-21 in gp140-specific T cells was found in seropositives vs. seronegatives (P = 0.003). Gene expression in switched IgM-IgD- gp140-specific memory B cells after stimulation with HIV peptides in vitro demonstrated a differential expression of genes involved in signal transduction and activation after BCR/TLR triggering and B-cell activation. Genes relating to antibody production (PRDM1) and T-B cognate stimulation (CXCR4, IL21R) were differentially induced after in-vitro stimulation in seronegatives vs. seropositives suggesting a truncated process of B-cell maturation. CONCLUSION HIV-specific memory B and T cells persist in early treated regardless their serological status. Seronegatives and seropositives are distinguished by gp140-specific T-cell function and by distinct transcriptional signatures of gp140-specific B cells after in-vitro stimulation, presumably because of a different antigen exposure. Such qualitative insights may inform future immunotherapeutic interventions.
Sonia Zicari, Libera Sessa, Nicola Cotugno, Alessandra Ruggiero, Elena Morrocchi, Carlo Concato, Salvatore Rocca, Paola Zangari, Emma Manno, and Paolo Palma
Viruses, eISSN: 19994915, Published: March 2019 MDPI AG
Despite effective antiretroviral therapy (ART), people living with HIV (PLWH) still present persistent chronic immune activation and inflammation. This condition is the result of several factors including thymic dysfunction, persistent antigen stimulation due to low residual viremia, microbial translocation and dysbiosis, caused by the disruption of the gut mucosa, co-infections, and cumulative ART toxicity. All of these factors can create a vicious cycle that does not allow the full control of immune activation and inflammation, leading to an increased risk of developing non-AIDS co-morbidities such as metabolic syndrome and cardiovascular diseases. This review aims to provide an overview of the most recent data about HIV-associated inflammation and chronic immune exhaustion in PLWH under effective ART. Furthermore, we discuss new therapy approaches that are currently being tested to reduce the risk of developing inflammation, ART toxicity, and non-AIDS co-morbidities.
Chiara Lonoce, Carla Marusic, Elena Morrocchi, Anna Maria Salzano, Andrea Scaloni, Flavia Novelli, Claudio Pioli, Mistianne Feeney, Lorenzo Frigerio, and Marcello Donini
Biotechnology Journal, ISSN: 18606768, eISSN: 18607314, Published: March 2019 Wiley
Hairy root (HR) cultures represent an attractive platform for the production of heterologous proteins, due to the possibility of secreting the molecule of interest in the culture medium. The main limitation is the low accumulation yields of heterologous proteins. The aim of this study is to enhance the accumulation of a tumor-targeting antibody with a human-compatible glycosylation profile in HR culture medium. To this aim, the authors produce Nicotiana benthamiana HR cultures expressing the red fluorescent protein (RFP) to easily screen for different auxins able to induce heterologous protein secretion in the medium. The hormone 2,4-dichlorophenoxyacetic acid (2,4-D) is found to induce high accumulation levels (334 mg L-1 ) of RFP in the culture medium. The same protocol is used to improve the secretion of the tumor-targeting, CD20-specific 2B8-FcΔXF recombinant antibody from glyco-engineered ΔXTFT N. benthamiana HR cultures. The addition of 2,4-D determine a 28-fold increase of the accumulation of fully functional 2B8-FcΔXF in the culture medium, at levels of ≈16 mg L-1 . Antibody N-glycosylation profiling reveal the prominent occurrence of GnGn structures and low levels of xylose- and fucose-containing counterparts. This result is the first example of the expression of an engineered anti-CD20 antibody with a scFv-Fc format at high levels in HR.
Carla Marusic, Claudio Pioli, Szymon Stelter, Flavia Novelli, Chiara Lonoce, Elena Morrocchi, Eugenio Benvenuto, Anna Maria Salzano, Andrea Scaloni, and Marcello Donini
Biotechnology and Bioengineering, ISSN: 00063592, eISSN: 10970290, Volume: 115, Pages: 565-576, Published: March 2018 Wiley
Anti‐CD20 recombinant antibodies are among the most promising therapeutics for the treatment of B‐cell malignancies such as non‐Hodgkin lymphomas. We recently demonstrated that an immunocytokine (2B8‐Fc‐hIL2), obtained by fusing an anti‐CD20 scFv‐Fc antibody derived from C2B8 mAb (rituximab) to the human interleukin 2 (hIL‐2), can be efficiently produced in Nicotiana benthamiana plants. The purified immunocytokine (IC) bearing a typical plant protein N‐glycosylation profile showed a CD20 binding activity comparable to that of rituximab and was efficient in eliciting antibody‐dependent cell‐mediated cytotoxicity (ADCC) of human PBMC against Daudi cells, indicating its fuctional integrity. In this work, the immunocytokine devoid of the typical xylose/fucose N‐glycosylation plant signature (IC‐ΔXF) and the corresponding scFv‐Fc‐ΔXF antibody not fused to the cytokine, were obtained in a glyco‐engineered ΔXylT/FucT N. benthamiana line. Purification yields from agroinfiltrated plants amounted to 20–35 mg/kg of leaf fresh weight. When assayed for interaction with FcγRI and FcγRIIIa, IC‐ΔXF exhibited significantly enhanced binding affinities if compared to the counterpart bearing the typical plant protein N‐glycosylation profile (IC) and to rituximab. The glyco‐engineered recombinant molecules also exhibited a strongly improved ADCC and complement‐dependent cytotoxicity (CDC). Notably, our results demonstrate a reduced C1q binding of xylose/fucose carrying IC and scFv‐Fc compared to versions that lack these sugar moieties. These results demonstrate that specific N‐glycosylation alterations in recombinant products can dramatically affect the effector functions of the immunocytokine, resulting in an overall improvement of the biological functions and consequently of the therapeutic potential.