{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,15]],"date-time":"2026-07-15T17:44:37Z","timestamp":1784137477015,"version":"3.55.0"},"reference-count":50,"publisher":"BMJ","issue":"11","license":[{"start":{"date-parts":[[2023,11,24]],"date-time":"2023-11-24T00:00:00Z","timestamp":1700784000000},"content-version":"unspecified","delay-in-days":23,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"name":"American Society of Hematology Research Training Award for Fellows","award":["N\/A"],"award-info":[{"award-number":["N\/A"]}]},{"name":"Chan Zuckerberg Biohub Physician-Scientist Fellowship Program","award":["N\/A"],"award-info":[{"award-number":["N\/A"]}]},{"name":"National Institute of Health T32 Research Training in Childhood Cancer","award":["T32CA128583"],"award-info":[{"award-number":["T32CA128583"]}]},{"name":"UCSF Living Therapeutics Initiative","award":["N\/A"],"award-info":[{"award-number":["N\/A"]}]},{"DOI":"10.13039\/100000090","name":"Department of Defense Congressionally Directed Medical Research Programs","doi-asserted-by":"crossref","award":["W81XWH2210807"],"award-info":[{"award-number":["W81XWH2210807"]}],"id":[{"id":"10.13039\/100000090","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Alex\u2019s Lemonade Stand Foundation Center of Excellence","award":["N\/A"],"award-info":[{"award-number":["N\/A"]}]},{"name":"NIH\/NCI","award":["P30 CA082103"],"award-info":[{"award-number":["P30 CA082103"]}]},{"DOI":"10.13039\/100007557","name":"California Institute of Regenerative Medicine","doi-asserted-by":"publisher","award":["TRAN1-12987"],"award-info":[{"award-number":["TRAN1-12987"]}],"id":[{"id":"10.13039\/100007557","id-type":"DOI","asserted-by":"publisher"}]},{"name":"BP28 Foundation","award":["N\/A"],"award-info":[{"award-number":["N\/A"]}]}],"content-domain":{"domain":["bmj.com"],"crossmark-restriction":true},"short-container-title":["J Immunother Cancer"],"accepted":{"date-parts":[[2023,10,17]]},"published-print":{"date-parts":[[2023,11]]},"abstract":"<jats:sec>\n                  <jats:title>Background<\/jats:title>\n                  <jats:p>Approximately 50% of patients who receive anti-CD19 CAR-T cells relapse, and new immunotherapeutic targets are urgently needed. We recently described CD72 as a promising target in B-cell malignancies and developed nanobody-based CAR-T cells (nanoCARs) against it. This cellular therapy design is understudied compared with scFv-based CAR-T cells, but has recently become of significant interest given the first regulatory approval of a nanoCAR in multiple myeloma.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>We humanized our previous nanobody framework regions, derived from llama, to generate a series of humanized anti-CD72 nanobodies. These nanobody binders were inserted into second-generation CD72 CAR-T cells and were evaluated against preclinical models of B cell acute lymphoblastic leukemia and B cell non-Hodgkin\u2019s lymphoma in vitro and in vivo. Humanized CD72 nanoCARs were compared with parental (\u201cNbD4\u201d) CD72 nanoCARs and the clinically approved CD19-directed CAR-T construct tisangenlecleucel. RNA-sequencing, flow cytometry, and cytokine secretion profiling were used to determine differences between the different CAR constructs. We then used affinity maturation on the parental NbD4 construct to generate high affinity binders against CD72 to test if higher affinity to CD72 improved antitumor potency.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>Toward clinical translation, here we humanize our previous nanobody framework regions, derived from llama, and surprisingly discover a clone (\u201cH24\u201d) with enhanced potency against B-cell tumors, including patient-derived samples after CD19 CAR-T relapse. Potentially underpinning improved potency, H24 has moderately higher binding affinity to CD72 compared with a fully llama framework. However, further affinity maturation (K<jats:sub>D<\/jats:sub>&lt;1\u2009nM) did not lead to improvement in cytotoxicity. After treatment with H24 nanoCARs, in vivo relapse was accompanied by CD72 antigen downregulation which was partially reversible. The H24 nanobody clone was found to have no off-target binding and is therefore designated as a true clinical candidate.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Conclusion<\/jats:title>\n                  <jats:p>This work supports translation of H24 CD72 nanoCARs for refractory B-cell malignancies, reveals potential mechanisms of resistance, and unexpectedly demonstrates that nanoCAR potency can be improved by framework alterations alone. These findings may have implications for future engineering of nanobody-based cellular therapies.<\/jats:p>\n               <\/jats:sec>","DOI":"10.1136\/jitc-2023-006985","type":"journal-article","created":{"date-parts":[[2023,11,25]],"date-time":"2023-11-25T00:55:10Z","timestamp":1700873710000},"page":"e006985","update-policy":"https:\/\/doi.org\/10.1136\/crossmarkpolicy","source":"Crossref","is-referenced-by-count":23,"title":["Framework humanization optimizes potency of anti-CD72 nanobody CAR-T cells for B-cell malignancies"],"prefix":"10.1136","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3375-2703","authenticated-orcid":false,"given":"William C","family":"Temple","sequence":"first","affiliation":[{"name":"Department of Pediatrics, Division of Hematology\/Oncology, University of California, UCSF Benioff Children's Hospital, San Francisco, California, USA"},{"name":"Department of Pediatrics, Division of Allergy, Immunology, and Bone Marrow Transplantation, University of California, UCSF Benioff Children's Hospital, San Francisco, California, USA"},{"name":"Department of Laboratory Medicine, University of California, San Francisco, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew A","family":"Nix","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, University of California, San Francisco, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Akul","family":"Naik","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, University of California, San Francisco, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adila","family":"Izgutdina","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, University of California, San Francisco, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin J","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Hematology\/Oncology, University of California, UCSF Benioff Children's Hospital, San Francisco, California, USA"},{"name":"Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gianina","family":"Wicaksono","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, University of California, San Francisco, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul","family":"Phojanakong","sequence":"additional","affiliation":[{"name":"Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan Antonio Camara","family":"Serrano","sequence":"additional","affiliation":[{"name":"Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeth P","family":"Young","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Hematology\/Oncology, University of California, UCSF Benioff Children's Hospital, San Francisco, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emilio","family":"Ramos","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, University of California, San Francisco, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fernando","family":"Salangsang","sequence":"additional","affiliation":[{"name":"Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Veronica","family":"Steri","sequence":"additional","affiliation":[{"name":"Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simayijiang","family":"Xirenayi","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Hematology\/Oncology, University of California, UCSF Benioff Children's Hospital, San Francisco, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michelle","family":"Hermiston","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Hematology\/Oncology, University of California, UCSF Benioff Children's Hospital, San Francisco, California, USA"},{"name":"Department of Pediatrics, Division of Allergy, Immunology, and Bone Marrow Transplantation, University of California, UCSF Benioff Children's Hospital, San Francisco, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aaron C","family":"Logan","sequence":"additional","affiliation":[{"name":"Department of Medicine, Division of Hematology and Blood and Marrow Transplantation, University of California, San Francisco, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elliot","family":"Stieglitz","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Hematology\/Oncology, University of California, UCSF Benioff Children's Hospital, San Francisco, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arun P","family":"Wiita","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, University of California, San Francisco, California, USA"},{"name":"Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California, USA"},{"name":"Chan Zuckerberg Biohub, San Francisco, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"239","published-online":{"date-parts":[[2023,11,24]]},"reference":[{"key":"2025100310473916000_11.11.e006985.1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1407222"},{"key":"2025100310473916000_11.11.e006985.2","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1709866"},{"key":"2025100310473916000_11.11.e006985.3","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-019-0564-6"},{"key":"2025100310473916000_11.11.e006985.4","doi-asserted-by":"crossref","first-page":"354","DOI":"10.1200\/JCO.22.01076","article-title":"Outcomes after Nonresponse and relapse post-Tisagenlecleucel in children, adolescents, and young adults with B-cell acute Lymphoblastic leukemia","volume":"41","author":"Schultz","year":"2023","journal-title":"JCO"},{"key":"2025100310473916000_11.11.e006985.5","doi-asserted-by":"crossref","first-page":"531","DOI":"10.1182\/blood.2020009515","article-title":"Interventions and outcomes of adult patients with B-ALL progressing after Cd19 Chimeric antigen receptor T-cell therapy","volume":"138","author":"Wudhikarn","year":"2021","journal-title":"Blood"},{"key":"2025100310473916000_11.11.e006985.6","doi-asserted-by":"publisher","DOI":"10.1002\/ajh.25505"},{"key":"2025100310473916000_11.11.e006985.7","doi-asserted-by":"publisher","DOI":"10.1200\/JCO.19.03279"},{"key":"2025100310473916000_11.11.e006985.8","doi-asserted-by":"publisher","DOI":"10.1158\/2159-8290.CD-20-0242"},{"key":"2025100310473916000_11.11.e006985.9","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1080\/25785826.2019.1660038","article-title":"Inhibitory B cell Co-receptors and autoimmune diseases","volume":"42","author":"Tsubata","year":"2019","journal-title":"Immunol Med"},{"key":"2025100310473916000_11.11.e006985.10","doi-asserted-by":"crossref","first-page":"238","DOI":"10.3390\/biom11020238","article-title":"The application of Nanobody in CAR-T therapy","volume":"11","author":"Bao","year":"2021","journal-title":"Biomolecules"},{"key":"2025100310473916000_11.11.e006985.11","doi-asserted-by":"crossref","first-page":"842","DOI":"10.1038\/s41591-021-01326-5","article-title":"Antigen-independent activation enhances the efficacy of 4-1Bb-Costimulated Cd22 CAR T cells","volume":"27","author":"Singh","year":"2021","journal-title":"Nat Med"},{"key":"2025100310473916000_11.11.e006985.12","doi-asserted-by":"crossref","first-page":"4936","DOI":"10.4049\/jimmunol.1000173","article-title":"Evidence for a TCR affinity threshold delimiting maximal Cd8 T cell function","volume":"184","author":"Schmid","year":"2010","journal-title":"The Journal of Immunology"},{"key":"2025100310473916000_11.11.e006985.13","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2010-12-326736"},{"key":"2025100310473916000_11.11.e006985.14","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-019-0549-5"},{"key":"2025100310473916000_11.11.e006985.15","doi-asserted-by":"publisher","DOI":"10.1016\/s0140-6736(21)00933-8"},{"key":"2025100310473916000_11.11.e006985.16","doi-asserted-by":"crossref","DOI":"10.1186\/s40364-022-00371-7","article-title":"Nanobody-based CAR-T cells for cancer Immunotherapy","volume":"10","author":"Safarzadeh Kozani","year":"2022","journal-title":"Biomark Res"},{"key":"2025100310473916000_11.11.e006985.17","doi-asserted-by":"crossref","first-page":"1779","DOI":"10.1080\/03007995.2021.1953456","article-title":"Matching-adjusted indirect comparison of efficacy outcomes for Ciltacabtagene Autoleucel in CARTITUDE-1 versus Idecabtagene Vicleucel in Karmma for the treatment of patients with Relapsed or refractory multiple myeloma","volume":"37","author":"Martin","year":"2021","journal-title":"Curr Med Res Opin"},{"key":"2025100310473916000_11.11.e006985.18","doi-asserted-by":"publisher","DOI":"10.1155\/2020\/4927120"},{"key":"2025100310473916000_11.11.e006985.19","doi-asserted-by":"publisher","DOI":"10.1021\/acs.molpharmaceut.8b00584"},{"key":"2025100310473916000_11.11.e006985.20","doi-asserted-by":"crossref","first-page":"3002","DOI":"10.1038\/s41375-021-01269-3","article-title":"Single VHH-directed BCMA CAR-T cells cause remission of Relapsed\/refractory multiple myeloma","volume":"35","author":"Han","year":"2021","journal-title":"Leukemia"},{"key":"2025100310473916000_11.11.e006985.21","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1038\/s41571-021-00476-2","article-title":"Immunogenicity of CAR T cells in cancer therapy","volume":"18","author":"Wagner","year":"2021","journal-title":"Nat Rev Clin Oncol"},{"key":"2025100310473916000_11.11.e006985.22","doi-asserted-by":"publisher","DOI":"10.1158\/2326-6066.CIR-13-0006"},{"key":"2025100310473916000_11.11.e006985.23","doi-asserted-by":"publisher","DOI":"10.1172\/JCI85309"},{"key":"2025100310473916000_11.11.e006985.24","doi-asserted-by":"publisher","DOI":"10.1158\/1535-7163.MCT-19-1109"},{"key":"2025100310473916000_11.11.e006985.25","doi-asserted-by":"crossref","unstructured":"Duan Y , Chen R , Huang Y , et al . Tuning the ignition of CAR: optimizing the affinity of scFv to improve CAR-T therapy. Cell Mol Life Sci 2022;79. doi:10.1007\/s00018-021-04089-x","DOI":"10.1007\/s00018-021-04089-x"},{"key":"2025100310473916000_11.11.e006985.26","doi-asserted-by":"publisher","DOI":"10.1038\/mt.2014.208"},{"key":"2025100310473916000_11.11.e006985.27","doi-asserted-by":"crossref","DOI":"10.1126\/scitranslmed.aaw2672","article-title":"Chlorotoxin-directed CAR T cells for specific and effective targeting of glioblastoma","volume":"12","author":"Wang","year":"2020","journal-title":"Sci Transl Med"},{"key":"2025100310473916000_11.11.e006985.28","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M806889200"},{"key":"2025100310473916000_11.11.e006985.29","doi-asserted-by":"crossref","first-page":"715","DOI":"10.1038\/s41571-021-00530-z","article-title":"A comparison of Chimeric antigen receptors containing Cd28 versus 4-1Bb Costimulatory domains","volume":"18","author":"Cappell","year":"2021","journal-title":"Nat Rev Clin Oncol"},{"key":"2025100310473916000_11.11.e006985.30","doi-asserted-by":"crossref","first-page":"696","DOI":"10.1038\/s43018-022-00376-z","article-title":"A local human V\u03b41 T cell population is associated with survival in Nonsmall-cell lung cancer","volume":"3","author":"Wu","year":"2022","journal-title":"Nat Cancer"},{"key":"2025100310473916000_11.11.e006985.31","doi-asserted-by":"publisher","DOI":"10.1158\/2159-8290.CD-20-1677"},{"key":"2025100310473916000_11.11.e006985.32","first-page":"3153","article-title":"Costimulatory domains direct distinct fates of CAR-driven T-cell dysfunction","volume":"141","author":"Selli","year":"2023","journal-title":"Blood"},{"key":"2025100310473916000_11.11.e006985.33","doi-asserted-by":"publisher","DOI":"10.1158\/2159-8290.CD-15-1020"},{"key":"2025100310473916000_11.11.e006985.34","doi-asserted-by":"crossref","first-page":"2191","DOI":"10.1038\/leu.2017.57","article-title":"Fully human Cd19-specific Chimeric antigen receptors for T-cell therapy","volume":"31","author":"Sommermeyer","year":"2017","journal-title":"Leukemia"},{"key":"2025100310473916000_11.11.e006985.35","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1215134"},{"key":"2025100310473916000_11.11.e006985.36","doi-asserted-by":"crossref","first-page":"575","DOI":"10.1182\/bloodadvances.2022007423","article-title":"Preinfusion factors Impacting relapse Immunophenotype following Cd19 CAR T cells","volume":"7","author":"Lamble","year":"2023","journal-title":"Blood Adv"},{"key":"2025100310473916000_11.11.e006985.37","doi-asserted-by":"crossref","first-page":"2664","DOI":"10.3389\/fimmu.2019.02664","article-title":"Mechanisms of relapse after Cd19 CAR T-cell therapy for acute Lymphoblastic leukemia and its prevention and treatment strategies","volume":"10","author":"Xu","year":"2019","journal-title":"Front Immunol"},{"key":"2025100310473916000_11.11.e006985.38","doi-asserted-by":"crossref","DOI":"10.3389\/fonc.2019.01237","article-title":"Understanding the mechanisms of resistance to CAR T-cell therapy in malignancies","volume":"9","author":"Cheng","year":"2019","journal-title":"Front Oncol"},{"key":"2025100310473916000_11.11.e006985.39","doi-asserted-by":"crossref","DOI":"10.3389\/fimmu.2018.02593","article-title":"Making CAR T cells a solid option for solid tumors","volume":"9","author":"Schmidts","year":"2018","journal-title":"Front Immunol"},{"key":"2025100310473916000_11.11.e006985.40","doi-asserted-by":"publisher","DOI":"10.1038\/s41571-019-0184-6"},{"key":"2025100310473916000_11.11.e006985.41","first-page":"303","article-title":"Human gastric intrinsic factor expression is not restricted to Parietal cells","volume":"189","author":"Howard","year":"1996","journal-title":"J Anat"},{"key":"2025100310473916000_11.11.e006985.42","doi-asserted-by":"publisher","DOI":"10.1158\/1535-7163.MCT-20-0476"},{"key":"2025100310473916000_11.11.e006985.43","doi-asserted-by":"crossref","first-page":"4974","DOI":"10.1182\/blood-2022-169040","article-title":"Cd72 is a pan-tumor antigen associated with childhood acute leukemia","volume":"140","author":"Buldini","year":"2022","journal-title":"Blood"},{"key":"2025100310473916000_11.11.e006985.44","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-021-01621-1"},{"key":"2025100310473916000_11.11.e006985.45","doi-asserted-by":"publisher","DOI":"10.1038\/s41573-021-00189-2"},{"key":"2025100310473916000_11.11.e006985.46","doi-asserted-by":"crossref","DOI":"10.1038\/s41591-019-0360-3","article-title":"Publisher correction: calibration of CAR activation potential directs alternative T cell Fates and therapeutic potency","volume":"25","author":"Feucht","year":"2019","journal-title":"Nat Med"},{"key":"2025100310473916000_11.11.e006985.47","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-019-1054-1"},{"key":"2025100310473916000_11.11.e006985.48","doi-asserted-by":"crossref","first-page":"1943","DOI":"10.1038\/s41375-022-01585-2","article-title":"Low-affinity CAR T cells exhibit reduced Trogocytosis, preventing rapid antigen loss, and increasing CAR T cell expansion","volume":"36","author":"Olson","year":"2022","journal-title":"Leukemia"},{"key":"2025100310473916000_11.11.e006985.49","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2012.085"},{"key":"2025100310473916000_11.11.e006985.50","unstructured":"Temple WC , Huang BJ , Wiita AP . Framework Humanization increases the affinity and potency of anti-Cd72 Nanobody-based CAR-T cells for B-cell malignancies. Available: https:\/\/www.ncbi.nlm.nih.gov\/geo\/query\/acc.cgi?acc=GSE218791 [Accessed 29 Dec 2022]."}],"container-title":["Journal for ImmunoTherapy of Cancer"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1136\/jitc-2023-006985","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,3]],"date-time":"2025-10-03T17:47:52Z","timestamp":1759513672000},"score":1,"resource":{"primary":{"URL":"https:\/\/jitc.bmj.com\/lookup\/doi\/10.1136\/jitc-2023-006985"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,11]]},"references-count":50,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2023,11,24]]},"published-print":{"date-parts":[[2023,11]]}},"alternative-id":["10.1136\/jitc-2023-006985"],"URL":"https:\/\/doi.org\/10.1136\/jitc-2023-006985","relation":{},"ISSN":["2051-1426"],"issn-type":[{"value":"2051-1426","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,11]]}}}