{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T01:14:29Z","timestamp":1781140469343,"version":"3.54.1"},"reference-count":51,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,4,30]],"date-time":"2025-04-30T00:00:00Z","timestamp":1745971200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Vet. Sci."],"abstract":"<jats:p>Alpacas belong to the Camelidae family. Antibodies produced through alpaca immunization are called nanoantibodies. Compared to traditional antibodies, nanoantibodies have several characteristics, including smaller molecular weight, stable structure, high homology with human antibodies, and suitability for prokaryotic expression. Malignant melanoma (MM) is a severe and aggressive form of cancer that affects both humans and animals. It commonly arises in the mucous membranes of the skin, nose, mouth, anus, and digestive tract, as well as in the choroid of the eyes. Multiple factors contribute to melanocyte carcinogenesis, including ultraviolet (UV) radiation, endocrine disorders, viral infections, immune deficiencies, and chemical carcinogens. At present, surgical resection remains to be the primary treatment for MM, although the prognosis is generally poor. However, targeted therapy and immune checkpoint inhibitors (ICIs) are increasingly employed in the clinical treatment of melanoma. NKG2A is an inhibitory receptor protein found on the surface of CD8<jats:sup>+<\/jats:sup> T cells and natural killer (NK) cells. HLA-E ligands expressed on the surface of malignant melanoma cells can facilitate immune evasion by binding to the NKG2A receptor complex on immune cells. This interaction suppresses immune responses, enabling tumor cells to escape immune surveillance. Immunosuppressive antibody drugs work by blocking this recognition mechanism, thereby reactivating immune cells to target and destroy tumor cells. As such, NKG2A has emerged as a novel target for immunotherapeutic intervention. In this study, an alpaca-derived nanoantibody targeting NKG2A with high affinity was obtained from a melanoma-specific phage library display. Through induced expression and protein purification, a monoclonal nanobody strain expressing NKG2A was successfully isolated. This NKG2A-targeting nanobody demonstrates the potential for application in both the detection and treatment of MM.<\/jats:p>","DOI":"10.3389\/fvets.2025.1571857","type":"journal-article","created":{"date-parts":[[2025,5,1]],"date-time":"2025-05-01T06:40:25Z","timestamp":1746081625000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["A novel NKG2A alpaca nanobody targeting immune checkpoint blockade for the treatment of malignant melanoma"],"prefix":"10.3389","volume":"12","author":[{"given":"Xiang","family":"Guo","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Congfang","family":"Guo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dongxiao","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuting","family":"Bai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mureed","family":"Abbas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruiwen","family":"Fan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yiyan","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,4,30]]},"reference":[{"key":"ref1","first-page":"253","article-title":"New advances in therapeutic use of monoclonal antibodies","volume":"64","author":"Scheen","year":"2009","journal-title":"Rev Med Liege"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"446","DOI":"10.1038\/363446a0","article-title":"Naturally occurring antibodies devoid of light chains","volume":"363","author":"Hamers-Casterman","year":"1993","journal-title":"Nature"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"13","DOI":"10.1016\/j.jim.2007.04.008","article-title":"Alpaca (Lama pacos) as a convenient source of recombinant camelid heavy chain antibodies (VHHs)","volume":"324","author":"Maass","year":"2007","journal-title":"J Immunol Methods"},{"key":"ref4","doi-asserted-by":"publisher","first-page":"47","DOI":"10.1038\/nrg2703","article-title":"Origin and evolution of the adaptive immune system: genetic events and selective pressures","volume":"11","author":"Flajnik","year":"2010","journal-title":"Nat Rev Genet"},{"key":"ref5","doi-asserted-by":"publisher","first-page":"2853","DOI":"10.1158\/0008-5472.CAN-03-3935","article-title":"Efficient cancer therapy with a nanobody-based conjugate","volume":"64","author":"Cortez-Retamozo","year":"2004","journal-title":"Cancer Res"},{"key":"ref6","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1016\/S0168-1656(99)00228-X","article-title":"Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae","volume":"78","author":"Frenken","year":"2000","journal-title":"J Biotechnol"},{"key":"ref7","doi-asserted-by":"publisher","first-page":"11893","DOI":"10.1038\/s41598-018-30338-4","article-title":"CEACAM1 promotes melanoma metastasis and is involved in the regulation of the EMT associated gene network in melanoma cells","volume":"8","author":"Wicklein","year":"2018","journal-title":"Sci Rep"},{"key":"ref8","article-title":"Corrigendum: melanoma of the dog and cat: consensus and guidelines","author":"Polton","year":"2024","journal-title":"Front. Vet. Sci"},{"key":"ref9","doi-asserted-by":"publisher","first-page":"290","DOI":"10.6004\/jnccn.2024.0036","article-title":"NCCN guidelines\u00ae insights: melanoma: cutaneous, version 2.2024","volume":"22","author":"Swetter","year":"2024","journal-title":"J Natl Compr Cancer Netw"},{"key":"ref10","doi-asserted-by":"publisher","first-page":"529","DOI":"10.1093\/ced\/llae465","article-title":"Cutaneous melanoma in situ: a review","volume":"50","author":"Karponis","year":"2025","journal-title":"Clin Exp Dermatol"},{"key":"ref11","doi-asserted-by":"publisher","first-page":"23","DOI":"10.1038\/s41572-025-00603-8","article-title":"Cutaneous melanoma","volume":"11","author":"Tasdogan","year":"2025","journal-title":"Nat Rev Dis Primers"},{"key":"ref12","doi-asserted-by":"publisher","first-page":"517","DOI":"10.1590\/abd1806-4841.20186803","article-title":"Prevalence and reactivity of anti-melanoma differentiation-associated gene 5 (anti-MDA-5) autoantibody in Brazilian patients with dermatomyositis","volume":"93","author":"Borges","year":"2018","journal-title":"An Bras Dermatol"},{"key":"ref13","doi-asserted-by":"publisher","first-page":"27","DOI":"10.1186\/2052-8426-2-27","article-title":"Gene therapy for cancer: present status and future perspective","volume":"2","author":"Amer","year":"2014","journal-title":"Mol Cell Ther"},{"key":"ref14","doi-asserted-by":"publisher","first-page":"366","DOI":"10.6004\/jnccn.2012.0036","article-title":"Melanoma","volume":"10","author":"Coit","year":"2012","journal-title":"J Natl Compr Cancer Netw"},{"key":"ref15","doi-asserted-by":"publisher","first-page":"8984","DOI":"10.3390\/ijms21238984","article-title":"Overcoming immune evasion in melanoma","volume":"21","author":"Eddy","year":"2020","journal-title":"Int J Mol Sci"},{"key":"ref16","doi-asserted-by":"publisher","first-page":"549","DOI":"10.1016\/j.immuni.2007.05.006","article-title":"Good news, bad news for missing-self recognition by NK cells: autoimmune control but viral evasion","volume":"26","author":"Vivier","year":"2007","journal-title":"Immunity"},{"key":"ref17","doi-asserted-by":"publisher","first-page":"1720","DOI":"10.1016\/j.cell.2018.11.048","article-title":"NKG2A, a new kid on the immune checkpoint block","volume":"175","author":"Haanen","year":"2018","journal-title":"Cell"},{"key":"ref18","doi-asserted-by":"publisher","first-page":"1744","DOI":"10.1016\/j.cell.2018.10.028","article-title":"NKG2A blockade potentiates CD8 T cell immunity induced by cancer vaccines","volume":"175","author":"van Montfoort","year":"2018","journal-title":"Cell"},{"key":"ref19","doi-asserted-by":"publisher","first-page":"277","DOI":"10.1038\/s41571-019-0182-8","article-title":"The NKG2A immune checkpoint \u2014 a new direction in cancer immunotherapy","volume":"16","author":"Creelan","year":"2019","journal-title":"Nat Rev Clin Oncol"},{"key":"ref20","doi-asserted-by":"publisher","first-page":"904","DOI":"10.1007\/s00262-004-0517-9","article-title":"The selection of tumor variants with altered expression of classical and nonclassical MHC class I molecules: implications for tumor immune escape","volume":"53","author":"Algarra","year":"2004","journal-title":"Cancer Immunol Immunother"},{"key":"ref21","doi-asserted-by":"publisher","first-page":"628","DOI":"10.1158\/2159-8290.Cd-18-1489","article-title":"Molecular profiling reveals unique immune and metabolic features of melanoma brain metastases","volume":"9","author":"Fischer","year":"2019","journal-title":"Cancer Discov"},{"key":"ref22","doi-asserted-by":"publisher","first-page":"353","DOI":"10.1038\/s41591-021-01623-z","article-title":"T cell characteristics associated with toxicity to immune checkpoint blockade in patients with melanoma","volume":"28","author":"Lozano","year":"2022","journal-title":"Nat Med"},{"key":"ref23","doi-asserted-by":"publisher","first-page":"1390","DOI":"10.1038\/s43018-024-00810-4","article-title":"A targetable type III immune response with increase of IL-17A expressing CD4+ T\u202fcells is associated with immunotherapy-induced toxicity in melanoma","volume":"5","author":"Dimitriou","year":"2024","journal-title":"Nat Cancer"},{"key":"ref24","doi-asserted-by":"publisher","first-page":"1582","DOI":"10.1016\/j.ccell.2024.08.007","article-title":"Combination anti-PD-1 and anti-CTLA-4 therapy generates waves of clonal responses that include progenitor-exhausted CD8+ T cells","volume":"42","author":"Wang","year":"2024","journal-title":"Cancer Cell"},{"key":"ref25","doi-asserted-by":"publisher","first-page":"582","DOI":"10.1093\/annonc\/mdz011","article-title":"Five-year survival outcomes for patients with advanced melanoma treated with pembrolizumab in KEYNOTE-001","volume":"30","author":"Hamid","year":"2019","journal-title":"Ann Oncol"},{"key":"ref26","doi-asserted-by":"publisher","first-page":"e2410545","DOI":"10.1002\/advs.202410545","article-title":"Co-delivery of aPD-L1 and CD73 inhibitor using calcium phosphate nanoparticles for enhanced melanoma immunotherapy with reduced toxicity","volume":"12","author":"Liu","year":"2024","journal-title":"Adv Sci"},{"key":"ref27","doi-asserted-by":"publisher","first-page":"1731","DOI":"10.1016\/j.cell.2018.10.014","article-title":"Anti-NKG2A mAb is a checkpoint inhibitor that promotes antitumor immunity by unleashing both T and NK cells","volume":"175","author":"Andr\u00e9","year":"2018","journal-title":"Cell"},{"key":"ref28","first-page":"1294","article-title":"Construction and identification of a single domain antibody library of melanoma cells","volume":"35","author":"Ji","year":"2019","journal-title":"Chinese J Biochem Mol Biol"},{"key":"ref29","doi-asserted-by":"publisher","first-page":"8397","DOI":"10.4238\/2015.July.28.6","article-title":"Construction of a natural phage antibody library of human anaplastic thyroid carcinoma","volume":"14","author":"Xi","year":"2015","journal-title":"Genet Mol Res"},{"key":"ref30","doi-asserted-by":"publisher","first-page":"381","DOI":"10.3390\/vetsci11080381","article-title":"Preparation and application of Clostridium perfringens alpha toxin nanobodies","volume":"11","author":"Jia","year":"2024","journal-title":"Vet Sci"},{"key":"ref31","doi-asserted-by":"publisher","first-page":"2487","DOI":"10.1002\/eji.1830261032","article-title":"CD94 functions as a natural killer cell inhibitory receptor for different HLA class I alleles: identification of the inhibitory form of CD94 by the use of novel monoclonal antibodies","volume":"26","author":"Sivori","year":"1996","journal-title":"Eur J Immunol"},{"key":"ref32","doi-asserted-by":"publisher","first-page":"707","DOI":"10.1016\/j.cell.2017.01.017","article-title":"Primary, adaptive, and acquired resistance to cancer immunotherapy","volume":"168","author":"Sharma","year":"2017","journal-title":"Cell"},{"key":"ref33","doi-asserted-by":"publisher","first-page":"3609","DOI":"10.1172\/JCI45816","article-title":"Human breast cancer cells enhance self tolerance by promoting evasion from NK cell antitumor immunity","volume":"121","author":"Mamessier","year":"2011","journal-title":"J Clin Invest"},{"key":"ref34","doi-asserted-by":"crossref","first-page":"1801","DOI":"10.1084\/jem.190.12.1801","article-title":"Recognition of the class Ib molecule Qa-1(b) by putative activating receptors CD94\/NKG2C and CD94\/NKG2E on mouse natural killer cells","volume":"190","author":"Vance","year":"1999","journal-title":"J Exp Med"},{"key":"ref35","doi-asserted-by":"publisher","first-page":"239","DOI":"10.1038\/nri3174","article-title":"Targeting natural killer cells and natural killer T cells in cancer","volume":"12","author":"Vivier","year":"2012","journal-title":"Nat Rev Immunol"},{"key":"ref36","doi-asserted-by":"publisher","first-page":"150","DOI":"10.1016\/S0923-2494(97)84216-2","article-title":"HLA class-I-specific inhibitory receptor in human T lymphocytes: interference with T-cell functions","volume":"148","author":"Mingari","year":"1997","journal-title":"Res Immunol"},{"key":"ref37","doi-asserted-by":"publisher","first-page":"301","DOI":"10.1146\/annurev-med-012017-043208","article-title":"CD8 T cell exhaustion in chronic infection and cancer: opportunities for interventions","volume":"69","author":"Hashimoto","year":"2018","journal-title":"Annu Rev Med"},{"key":"ref38","doi-asserted-by":"publisher","first-page":"109","DOI":"10.1006\/smim.2000.0213","article-title":"NK cell recognition of nonclassical HLA class I molecules","volume":"12","author":"L\u00f3pez-Botet","year":"2000","journal-title":"Semin Immunol"},{"key":"ref39","doi-asserted-by":"publisher","first-page":"417","DOI":"10.1007\/s00262-015-1750-0","article-title":"Age-related changes in natural killer cell repertoires: impact on NK cell function and immune surveillance","volume":"65","author":"Manser","year":"2016","journal-title":"Cancer Immunol Immunother"},{"key":"ref40","doi-asserted-by":"publisher","first-page":"813","DOI":"10.1093\/intimm\/dxm057","article-title":"PD-1 and PD-1 ligands: from discovery to clinical application","volume":"19","author":"Okazaki","year":"2007","journal-title":"Int Immunol"},{"key":"ref41","doi-asserted-by":"publisher","first-page":"e15184","DOI":"10.1371\/journal.pone.0015184","article-title":"Development and function of CD94-deficient natural killer cells","volume":"5","author":"Orr","year":"2010","journal-title":"PLoS One"},{"key":"ref42","doi-asserted-by":"publisher","first-page":"4245","DOI":"10.3390\/cancers13164245","article-title":"Identification and targeting of mutant peptide neoantigens in cancer immunotherapy","volume":"13","author":"Verdon","year":"2021","journal-title":"Cancers"},{"key":"ref43","doi-asserted-by":"publisher","first-page":"392","DOI":"10.1053\/j.gastro.2012.10.039","article-title":"Blocking the natural killer cell inhibitory receptor NKG2A increases activity of human natural killer cells and clears hepatitis B virus infection in mice","volume":"144","author":"Li","year":"2013","journal-title":"Gastroenterology"},{"key":"ref44","doi-asserted-by":"publisher","first-page":"1076","DOI":"10.1002\/(SICI)1521-4141(199904)29:04<1076::AID-IMMU1076>3.0.CO;2-Z","article-title":"Differential regulation of killer cell Ig-like receptors and CD94 lectin-like dimers on NK and T lymphocytes from HIV-1-infected individuals","volume":"29","author":"Andr\u00e9","year":"1999","journal-title":"Eur J Immunol"},{"key":"ref45","doi-asserted-by":"publisher","first-page":"539","DOI":"10.1146\/annurev-immunol-032414-112049","article-title":"Coinhibitory pathways in immunotherapy for cancer","volume":"34","author":"Baumeister","year":"2016","journal-title":"Annu Rev Immunol"},{"key":"ref46","doi-asserted-by":"publisher","first-page":"4324","DOI":"10.1182\/blood-2012-06-438028","article-title":"A phase 1 trial of the anti-KIR antibody IPH2101 in patients with relapsed\/refractory multiple myeloma","volume":"120","author":"Benson","year":"2012","journal-title":"Blood"},{"key":"ref47","doi-asserted-by":"publisher","first-page":"1069","DOI":"10.1016\/j.immuni.2016.04.023","article-title":"Targeting T cell co-receptors for cancer therapy","volume":"44","author":"Callahan Margaret","year":"2016","journal-title":"Immunity"},{"key":"ref48","doi-asserted-by":"publisher","first-page":"259","DOI":"10.1007\/s00262-016-1947-x","article-title":"Rejection versus escape: the tumor MHC dilemma","volume":"66","author":"Garrido","year":"2017","journal-title":"Cancer Immunol Immunother"},{"key":"ref49","doi-asserted-by":"publisher","first-page":"1460","DOI":"10.1126\/science.aat2184","article-title":"Natural killers join the fight against cancer an antibody overcomes cancer cell immune evasion and activates natural killer cells","volume":"359","author":"Cerwenka","year":"2018","journal-title":"Science"},{"key":"ref50","doi-asserted-by":"publisher","first-page":"463","DOI":"10.20517\/2394-4722.2016.62","article-title":"Targeting toll-like receptors against cancer","volume":"2","author":"Du","year":"2016","journal-title":"J Cancer Metastasis Treatment"},{"key":"ref51","doi-asserted-by":"publisher","first-page":"eaag1672","DOI":"10.1126\/sciimmunol.aag1672","article-title":"Class I HLA haplotypes form two schools that educate NK cells in different ways","volume":"1","author":"Horowitz","year":"2016","journal-title":"Sci Immunol"}],"container-title":["Frontiers in Veterinary Science"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fvets.2025.1571857\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,5,1]],"date-time":"2025-05-01T21:04:49Z","timestamp":1746133489000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fvets.2025.1571857\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,4,30]]},"references-count":51,"alternative-id":["10.3389\/fvets.2025.1571857"],"URL":"https:\/\/doi.org\/10.3389\/fvets.2025.1571857","relation":{},"ISSN":["2297-1769"],"issn-type":[{"value":"2297-1769","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,4,30]]},"article-number":"1571857"}}