{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T16:20:00Z","timestamp":1787242800775,"version":"build-2736575974"},"reference-count":41,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,1,13]],"date-time":"2025-01-13T00:00:00Z","timestamp":1736726400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Italian Ministry of Health","award":["GR-2010-2306432"],"award-info":[{"award-number":["GR-2010-2306432"]}]},{"name":"Italian Ministry of Health","award":["PE-2016-02364927"],"award-info":[{"award-number":["PE-2016-02364927"]}]},{"name":"Istituto Superiore di Sanit\u00e0","award":["GR-2010-2306432"],"award-info":[{"award-number":["GR-2010-2306432"]}]},{"name":"Istituto Superiore di Sanit\u00e0","award":["PE-2016-02364927"],"award-info":[{"award-number":["PE-2016-02364927"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>In chronic lymphocytic leukemia (CLL), natural killer (NK) cells show a dysfunctional phenotype that correlates with disease progression. Our aim was to restore NK cell functionality in CLL through a specifically targeted IL15-stimulating activity; IL15 targeting could, in fact, potentiate the activity of NK cells and reduce off-target effects. We designed and developed a cis-acting immunocytokine composed of an anti-CD56 single-chain Fragment variable (scFv) and IL15, labeled scFvB1IL15. scFvB1IL15 was tested in vitro on peripheral blood mononuclear cells (PBMCs) obtained from both different healthy donors (HDs) and CLL patients in order to evaluate its ability to target NK cells and enhance their activation and NK-mediated directed cytotoxicity. scFvB1IL15 specifically induced strong degranulation and cytokine and chemokine production in NK cells in both HD- and CLL patient-derived PBMC samples. Furthermore, compared to IL15 alone, it was able to induce higher levels of NKG2D- and NKp30-activating receptors and restore NK-mediated direct killing in the CLL patient-derived samples. The preliminary data presented in this work suggest that IL15\u2019s targeting of NK cells via scFvB1 potentiates the effects of IL15 and that scFvB1IL15 can be a useful agent for overcoming NK functional gaps and contribute to NK-cell-based immunotherapies.<\/jats:p>","DOI":"10.3390\/biom15010117","type":"journal-article","created":{"date-parts":[[2025,1,13]],"date-time":"2025-01-13T09:49:17Z","timestamp":1736761757000},"page":"117","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["A New Antibody\u2013Cytokine Construct Targeting Natural Killer Cells: An Immunotherapeutic Approach to Chronic Lymphocytic Leukemia"],"prefix":"10.3390","volume":"15","author":[{"given":"Michela","family":"Flego","sequence":"first","affiliation":[{"name":"National Center for Global Health, Italian Institute of Health, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5168-8624","authenticated-orcid":false,"given":"Mauro","family":"Andreotti","sequence":"additional","affiliation":[{"name":"National Center for Global Health, Italian Institute of Health, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2425-9474","authenticated-orcid":false,"given":"Francesca Romana","family":"Mauro","sequence":"additional","affiliation":[{"name":"Hematology, Department of Translational and Precision Medicine, \u2018Sapienza\u2019 University, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9777-2327","authenticated-orcid":false,"given":"Maria Beatrice","family":"Arasi","sequence":"additional","affiliation":[{"name":"Department of Oncology and Molecular Medicine, Italian Institute of Health, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Silvia","family":"Zamboni","sequence":"additional","affiliation":[{"name":"Department of Neuroscience, Italian Institute of Health, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6841-7396","authenticated-orcid":false,"given":"Zuleika","family":"Michelini","sequence":"additional","affiliation":[{"name":"National Center for Global Health, Italian Institute of Health, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sara","family":"Pepe","sequence":"additional","affiliation":[{"name":"Hematology, Department of Translational and Precision Medicine, \u2018Sapienza\u2019 University, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Clementina Maria","family":"Galluzzo","sequence":"additional","affiliation":[{"name":"National Center for Global Health, Italian Institute of Health, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roberta","family":"Amici","sequence":"additional","affiliation":[{"name":"National Center for Global Health, Italian Institute of Health, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Diego","family":"Moricoli","sequence":"additional","affiliation":[{"name":"Diatheva s.r.l., Via Sant\u2019Anna 131\/135, 61030 Cartoceto, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chiara","family":"Mazzei","sequence":"additional","affiliation":[{"name":"National Center for Global Health, Italian Institute of Health, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alessandro","family":"Ascione","sequence":"additional","affiliation":[{"name":"National Center for Global Health, Italian Institute of Health, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2392-0769","authenticated-orcid":false,"given":"Alessandra","family":"Mallano","sequence":"additional","affiliation":[{"name":"National Center for Global Health, Italian Institute of Health, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,1,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1038\/s41571-023-00754-1","article-title":"Long-term outcomes following CAR T cell therapy: What we know so far","volume":"20","author":"Cappell","year":"2023","journal-title":"Nat. Rev. Clin. Oncol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"5641","DOI":"10.1182\/bloodadvances.2021005883","article-title":"Evaluation of allogeneic and autologous membrane-bound IL-21-expanded NK cells for chronic lymphocytic leukemia therapy","volume":"25","author":"Yano","year":"2022","journal-title":"Blood Adv."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2451","DOI":"10.1182\/blood.2021015184","article-title":"Dual antigen-targeted off-the-shelf NK cells show durable response and prevent antigen escape in lymphoma and leukemia","volume":"140","author":"Cichocki","year":"2022","journal-title":"Blood"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Douka, S., Papamoschou, V., Raimo, M., Mastrobattista, E., and Caiazzo, M. (2024). Harnessing the Power of NK Cell Receptor Engineering as a New Prospect in Cancer Immunotherapy. Pharmaceutics, 16.","DOI":"10.3390\/pharmaceutics16091143"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"e308","DOI":"10.1097\/HS9.0000000000000308","article-title":"Natural Killer Cell Hypo-responsiveness in Chronic Lymphocytic Leukemia can be Circumvented In Vitro by Adequate Activating Signaling","volume":"3","author":"Hofland","year":"2019","journal-title":"Hemasphere"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"68513","DOI":"10.18632\/oncotarget.12097","article-title":"NK cell function is markedly impaired in patients with chronic lymphocytic leukaemia but is preserved in patients with small lymphocytic lymphoma","volume":"7","author":"Parry","year":"2016","journal-title":"Oncotarget"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"897","DOI":"10.1182\/bloodadvances.2018029371","article-title":"Novel CD19-targeted TriKE restores NK cell function and proliferative capacity in CLL","volume":"3","author":"Felices","year":"2019","journal-title":"Blood Adv."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"202","DOI":"10.1159\/000497208","article-title":"Dysregulated Expression of Tim-3 and NKp30 Receptors on NK Cells of Patients with Chronic Lymphocytic Leukemia","volume":"42","author":"Hadadi","year":"2019","journal-title":"Oncol. Res. Treat."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"704","DOI":"10.4103\/jcrt.jcrt_52_21","article-title":"Tim-3 and PD-1 blocking cannot restore the functional properties of natural killer cells in early clinical stages of chronic lymphocytic leukemia: An in vitro study","volume":"18","author":"Astaneh","year":"2022","journal-title":"J. Cancer Res. Ther."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e1330235","DOI":"10.1080\/2162402X.2017.1330235","article-title":"NK cell dysfunction in chronic lymphocytic leukemia is associated with loss of the mature cells expressing inhibitory killer cell Ig-like receptors","volume":"6","author":"MacFarlane","year":"2017","journal-title":"Oncoimmunology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1111\/j.1365-2567.2011.03521.x","article-title":"Expression of natural killer cell activating receptors in patients with chronic lymphocytic leukaemia","volume":"135","author":"Costello","year":"2012","journal-title":"Immunology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"632","DOI":"10.1007\/s10875-011-9624-5","article-title":"Primary B-CLL resistance to NK cell cytotoxicity can be overcome in vitro and in vivo by priming NK cells and monoclonal antibody therapy","volume":"32","author":"Veuillen","year":"2012","journal-title":"J. Clin. Immunol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Huergo-Zapico, L., Acebes-Huerta, A., Gonzalez-Rodriguez, A.P., Contesti, J., Gonzalez-Garc\u00eda, E., Payer, A.R., Villa-Alvarez, M., Fern\u00e1ndez-Guiz\u00e1n, A., L\u00f3pez-Soto, A., and Gonzalez, S. (2014). Expansion of NK cells and reduction of NKG2D expression in chronic lymphocytic leukemia. Correlation with progressive disease. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0108326"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Yano, M., Byrd, J.C., and Muthusamy, N. (2022). Natural Killer Cells in Chronic Lymphocytic Leukemia: Functional Impairment and Therapeutic Potential. Cancers, 14.","DOI":"10.3390\/cancers14235787"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"4097","DOI":"10.1158\/0008-5472.CAN-15-0781","article-title":"Anti-CD20 Therapy Acts via Fc\u03b3RIIIA to Diminish Responsiveness of Human Natural Killer Cells","volume":"75","author":"Capuano","year":"2015","journal-title":"Cancer Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1064","DOI":"10.1016\/j.exphem.2011.08.006","article-title":"Interleukin-15 enhances rituximab-dependent cytotoxicity against chronic lymphocytic leukemia cells and overcomes transforming growth factor beta-mediated immunosuppression","volume":"39","author":"Moga","year":"2011","journal-title":"Exp. Hematol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"675","DOI":"10.3892\/etm.2021.10107","article-title":"Therapeutic potential of interleukin 15 in cancer (Review)","volume":"22","author":"Isvoranu","year":"2021","journal-title":"Exp. Ther. Med."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Cai, M., Huang, X., Huang, X., Ju, D., Zhu, Y.Z., and Ye, L. (2023). Research progress of interleukin-15 in cancer immunotherapy. Front. Pharmacol., 14.","DOI":"10.3389\/fphar.2023.1184703"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Zhou, Y., Husman, T., Cen, X., Tsao, T., Brown, J., Bajpai, A., Li, M., Zhou, K., and Yang, L. (2022). Interleukin 15 in Cell-Based Cancer Immunotherapy. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23137311"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Sindaco, P., Pandey, H., Isabelle, C., Chakravarti, N., Brammer, J.E., Porcu, P., and Mishra, A. (2023). The role of interleukin-15 in the development and treatment of hematological malignancies. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1141208"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2924","DOI":"10.4049\/jimmunol.1801142","article-title":"Mechanism for IL-15-Driven B Cell Chronic Lymphocytic Leukemia Cycling: Roles for AKT and STAT5 in Modulating Cyclin D2 and DNA Damage Response Proteins","volume":"15","author":"Gupta","year":"2019","journal-title":"J. Immunol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"6676","DOI":"10.4049\/jimmunol.173.11.6676","article-title":"Follicular dendritic cells produce IL-15 that enhances germinal center b cell proliferation in membrane-bound form","volume":"173","author":"Park","year":"2004","journal-title":"J. Immunol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.jbiotec.2016.02.034","article-title":"Design and construction of a new human naive single-chain fragment variable antibody library, IORISS1","volume":"224","author":"Pasello","year":"2016","journal-title":"J. Biotechnol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"63","DOI":"10.3233\/HAB-200431","article-title":"Isolation and preliminary characterization of a human \u2018phage display\u2019-derived antibody against neural adhesion molecule-1 antigen interfering with fibroblast growth factor receptor-1 binding","volume":"29","author":"Flego","year":"2021","journal-title":"Hum. Antibodies"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1038\/nprot.2009.2","article-title":"Protein structure prediction on the Web: A case study using the Phyre server","volume":"4","author":"Kelley","year":"2009","journal-title":"Nat. Protoc."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2608","DOI":"10.1093\/bioinformatics\/bts493","article-title":"Application of asymmetric statistical potentials to antibody-protein docking","volume":"28","author":"Brenke","year":"2012","journal-title":"Bioinformatics"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1016\/0022-1759(88)90204-9","article-title":"An inhibition enzyme immunoassay for estimating relative antibody affinity and affinity heterogeneity","volume":"106","author":"Rath","year":"1988","journal-title":"J. Immunol. Methods"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"380","DOI":"10.1089\/hyb.2004.23.380","article-title":"Isolation and characterization of the human monoclonal antibodies C10 in single-chain fragment variable (scFv) format to glucose oxidase from Aspergillus niger","volume":"23","author":"Ascione","year":"2004","journal-title":"Hybrid. Hybridomics"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"610789","DOI":"10.3389\/fimmu.2021.610789","article-title":"Transcription Factors Associated With IL-15 Cytokine Signaling During NK Cell Development","volume":"18","author":"Wang","year":"2021","journal-title":"Front. Immunol"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/j.jim.2007.05.013","article-title":"A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: Comparisons to a 4 h 51Cr-release assay","volume":"325","author":"Kim","year":"2007","journal-title":"J. Immunol. Methods"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1111\/j.1365-3083.2011.02667.x","article-title":"Evaluation of a flow cytometry-based assay for natural killer cell activity in clinical settings","volume":"75","author":"Valiathan","year":"2012","journal-title":"Scand. J. Immunol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1007\/s10719-015-9627-1","article-title":"Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation","volume":"33","author":"Chertova","year":"2016","journal-title":"Glycoconj. J."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"4563","DOI":"10.1111\/bph.15222","article-title":"NKp30\u2014A prospective target for new cancer immunotherapy strategies","volume":"177","author":"Pinheiro","year":"2020","journal-title":"Br. J. Pharmacol."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Kim, N., Yi, E., Lee, E., Park, H.J., and Kim, H.S. (2024). Interleukin-2 is required for NKp30-dependent NK cell cytotoxicity by preferentially regulating NKp30 expression. Front. Immunol., 18.","DOI":"10.3389\/fimmu.2024.1388018"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2167","DOI":"10.1182\/blood-2009-08-238469","article-title":"Regulation of human NK-cell cytokine and chemokine production by target cell recognition","volume":"115","author":"Fauriat","year":"2010","journal-title":"Blood"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1189\/jlb.0611308","article-title":"Natural killer cell-produced IFN-\u03b3 and TNF-\u03b1 induce target cell cytolysis through up-regulation of ICAM-1","volume":"91","author":"Wang","year":"2012","journal-title":"J. Leukoc. Biol."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Wurzer, H., Filali, L., Hoffmann, C., Krecke, M., Biolato, A.M., Mastio, J., De Wilde, S., Fran\u00e7ois, J.H., Largeot, A., and Berchem, G. (2021). Intrinsic Resistance of Chronic Lymphocytic Leukemia Cells to NK Cell-Mediated Lysis Can Be Overcome in vitro by Pharmacological Inhibition of Cdc42-Induced Actin Cytoskeleton Remodeling. Front. Immunol., 24.","DOI":"10.3389\/fimmu.2021.619069"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1046\/j.1365-2567.2001.01248.x","article-title":"Systematic characterization of human CD8+ T cells with natural killer cell markers in comparison with natural killer cells and normal CD8+ T cells","volume":"103","author":"Ohkawa","year":"2001","journal-title":"Immunology"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"833","DOI":"10.1016\/j.it.2022.08.004","article-title":"Harnessing IL-15 signaling to potentiate NK cell-mediated cancer immunotherapy","volume":"43","author":"Ma","year":"2022","journal-title":"Trends Immunol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"e8","DOI":"10.4110\/in.2023.23.e8","article-title":"Human CD8+ T-Cell Populations That Express Natural Killer Receptors","volume":"20","author":"Koh","year":"2023","journal-title":"Immune Netw."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Almeida, J.S., Casanova, J.M., Santos-Rosa, M., Tarazona, R., Solana, R., and Rodrigues-Santos, P. (2023). Natural Killer T-like Cells: Immunobiology and Role in Disease. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24032743"}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/15\/1\/117\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T10:27:59Z","timestamp":1759919279000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/15\/1\/117"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,1,13]]},"references-count":41,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,1]]}},"alternative-id":["biom15010117"],"URL":"https:\/\/doi.org\/10.3390\/biom15010117","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,1,13]]}}}