{ "abstract": "Background Chimeric antigen receptor (CAR) T-cell therapies are highly efficacious for several different hematologic cancers. However, for most CAR T targets it is observed that low surface antigen density on tumors can significantly reduce therapeutic efficacy. In this study, we explore this dynamic in the context of CD72, a surface antigen we recently found as a promising target for refractory B-cell cancers, but for which CD72 low antigen density can lead to therapeutic resistance in preclinical models.Methods Primary samples were accessed via institutional review board-approved protocols. Affinity-matured and humanized nanobody clones were previously described in Temple et al. (2023). CAR T cells were generated via lentiviral transduction. In vitro cytotoxicity assays were performed using luciferase-labeled cell lines. In vivo studies were performed using cell line-derived or patient-derived xenografts implanted in NOD scid gamma mice.Results We first confirmed ubiquitous CD72 expression across a range of primary B-cell non-Hodgkin lymphomas. We further found that after resistance to CD19-directed therapies, across both B-cell acute lymphoblastic leukemia (B-ALL) models and primary tumor samples, surface CD72 expression was largely preserved while CD22 expression was significantly diminished. Affinity maturation of a nanobody targeting CD72, when incorporated into CAR T cells, led to more effective elimination in vitro of isogenic models of CD72 low-expressing tumors. These results suggested that nanobody-based CAR T cells (nanoCARs) may exhibit a similar relationship between binder affinity, antigen expression, and efficacy as previously demonstrated only for single chain variable fragment-based CAR T cells. Surprisingly, however, this significantly improved in vitro efficacy only translated to modest in vivo survival benefit. As a parallel strategy to enhance CAR T function, we found that the small molecule bryostatin could also significantly increase CD72 surface antigen density on B-cell malignancy models. Structural modeling and biochemical analysis identified critical residues improving CD72 antigen recognition of our lead affinity-matured nanobody.Conclusions Together, these findings support affinity-matured CD72 nanoCARs as a potential immunotherapy product for CD19-refractory B-cell cancers. Our results also suggest that for B-ALL in particular, CD72 may be a preferable second-line immunotherapy target over CD22.", "authors": [ { "affiliations": [ "Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA" ], "name": "Adila Izgutdina" }, { "affiliations": [ "Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA" ], "name": "Tasfia Rashid" }, { "affiliations": [ "Department of Pediatrics, Division of Oncology, UCSF Benioff Children’s Hospital, University of California San Francisco, San Francisco, California, USA", "Department of Pediatrics, Division of Allergy, Immunology, and Bone Marrow Transplantation, UCSF Benioff Children’s Hospital, University of California San Francisco, San Francisco, California, USA" ], "name": "William C Temple" }, { "affiliations": [ "Department of Oncology, Blood Cancer Institute, Albert Einstein College of Medicine, Bronx, New York, USA" ], "name": "Sarah Aminov" }, { "affiliations": [ "Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA" ], "name": "Bonell Patiño-Escobar" }, { "affiliations": [ "Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA" ], "name": "Sujata Walunj" }, { "affiliations": [ "Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA" ], "name": "Huimin Geng" }, { "affiliations": [ "Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA", "Department of Hematology, Kanazawa University, Kanazawa, Japan" ], "name": "Hiroyuki Takamatsu" }, { "affiliations": [ "Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA", "Hospital Universitario 12 de Octubre-Centro Nacional de Investigaciones Oncológicas (H12O-CNIO) Haematological Malignancies Clinical Research Unit, Spanish National Cancer Research Centre, Madrid, Spain", "Department of Hematology, Hospital Universitario 12 de Octubre-Universidad Complutense, Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12), CIBERONC, Madrid, Spain" ], "name": "Daniel Gil-Alós" }, { "affiliations": [ "Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA" ], "name": "Amrik S Kang" }, { "affiliations": [ "Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA" ], "name": "Emilio Ramos" }, { "affiliations": [ "Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA" ], "name": "Szu-Ying Chen" }, { "affiliations": [ "Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA" ], "name": "Haley Johnson" }, { "affiliations": [ "Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA" ], "name": "Matthew A Nix" }, { "affiliations": [ "Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA" ], "name": "Akul Naik" }, { "affiliations": [ "Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA" ], "name": "Mingcheng Li" }, { "affiliations": [ "Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA" ], "name": "Constance M Yuan" }, { "affiliations": [ "Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA" ], "name": "Hao-Wei Wang" }, { "affiliations": [ "Department of Oncology, Blood Cancer Institute, Albert Einstein College of Medicine, Bronx, New York, USA" ], "name": "Srabani Sahu" }, { "affiliations": [ "Cellular Immunotherapy Program, Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, USA", "Harvard Medical School, Boston, Massachusetts, USA" ], "name": "Rebecca C Larson" }, { "affiliations": [ "Arc Institute, Palo Alto, California, USA" ], "name": "Christopher Carpenter" }, { "affiliations": [ "Preclinical Therapeutics Core, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California, USA" ], "name": "Fernando Salangsang" }, { "affiliations": [ "Preclinical Therapeutics Core, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California, USA" ], "name": "Paul Phojanakong" }, { "affiliations": [ "Preclinical Therapeutics Core, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California, USA" ], "name": "Juan Antonio Camara Serrano" }, { "affiliations": [ "Preclinical Therapeutics Core, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California, USA" ], "name": "Isa Tariq" }, { "affiliations": [ "Preclinical Therapeutics Core, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California, USA" ], "name": "Ons Zakraoui" }, { "affiliations": [ "Preclinical Therapeutics Core, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California, USA" ], "name": "Veronica Steri" }, { "affiliations": [ "Hospital Universitario 12 de Octubre-Centro Nacional de Investigaciones Oncológicas (H12O-CNIO) Haematological Malignancies Clinical Research Unit, Spanish National Cancer Research Centre, Madrid, Spain", "Department of Hematology, Hospital Universitario 12 de Octubre-Universidad Complutense, Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12), CIBERONC, Madrid, Spain" ], "name": "Antonio Valeri" }, { "affiliations": [ "Hospital Universitario 12 de Octubre-Centro Nacional de Investigaciones Oncológicas (H12O-CNIO) Haematological Malignancies Clinical Research Unit, Spanish National Cancer Research Centre, Madrid, Spain", "Department of Hematology, Hospital Universitario 12 de Octubre-Universidad Complutense, Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12), CIBERONC, Madrid, Spain" ], "name": "Joaquin Martinez-Lopez" }, { "affiliations": [ "Cellular Immunotherapy Program, Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, USA", "Harvard Medical School, Boston, Massachusetts, USA", "Blood and Marrow Transplant Program, Massachusetts General Hospital, Boston, Massachusetts, USA" ], "name": "Marcela V Maus" }, { "affiliations": [ "Department of Medicine, Division of Hematology and Medical Oncology", "Department of Oncological Sciences", "and Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA" ], "name": "Samir Parekh" }, { "affiliations": [ "Department of Oncology, Blood Cancer Institute, Albert Einstein College of Medicine, Bronx, New York, USA" ], "name": "Amit Verma" }, { "affiliations": [ "Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA" ], "name": "Nirali N Shah" }, { "affiliations": [ "Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA", "Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California, USA", "Chan Zuckerberg Biohub, San Francisco, California, USA", "Parker Insitute for Cancer Immunotherapy, San Francisco, California, USA" ], "name": "Arun P Wiita" } ], "title": "Affinity-matured CD72-targeting nanobody CAR T cells enhance elimination of antigen-low B-cell malignancies", "uid": "210b836b-787a-5862-8fe6-71924c6c72b6" }