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Publications

Journal Article Preprint Epublish

PubMed: 40463123 DOI: 10.1101/2025.05.09.653155

Affinity-matured CD72-targeting Nanobody CAR T-cells Enhance Elimination of Antigen-Low B-cell Malignancies.

bioRxiv

Authors: Adila Izgutdina, Tasfia Rashid, William C Temple, Bonell Patiño-Escobar, Sujata Walunj, Huimin Geng, Hiroyuki Takamatsu, Daniel Gil-Alós, Amrik S Kang, Emilio Ramos, Szu-Ying Chen, Haley Johnson, Matthew A Nix, Akul Naik, Constance M Yuan, Hao-Wei Wang, Sarah Aminov, Srabani Sahu, Rebecca C Larson, Christopher Carpenter, Fernando Salangsang, Paul Phojanakong, Juan Antonio Camara Serrano, Isa Tariq, Ons Zakraoui, Veronica Steri, Antonio Valeri, Joaquin Martinez-Lopez, Marcela V Maus, Samir Parekh, Amit Verma, Nirali N Shah, Arun P Wiita

Affiliations

  • Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA.
  • Department of Pediatrics, Division of Oncology, UCSF Benioff Children's Hospitals, University of California, San Francisco, San Francisco, CA.
  • Department of Pediatrics, Division of Allergy, Immunology, and Bone Marrow Transplantation, UCSF Benioff Children's Hospitals, University of California, San Francisco, San Francisco, CA.
  • Department of Hematology, Kanazawa University, Kanazawa, Japan.
  • 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.
  • Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, MD.
  • Department of Oncology, Blood Cancer Institute, Montefiore Einstein Comprehensive Cancer Center, Bronx, NY.
  • Cellular Immunotherapy Program, Cancer Center, Massachusetts General Hospital, Boston, MA.
  • Harvard Medical School, Boston, MA.
  • Arc Institute, Palo Alto, CA.
  • Preclinical Therapeutics Core, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA.
  • Blood and Marrow Transplant Program, Massachusetts General Hospital, Boston, MA.
  • Dept. of Medicine, Division of Hematology and Medical Oncology; Dept. of Oncological Sciences; and Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY.
  • Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD.
  • Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA.
  • Chan Zuckerberg Biohub San Francisco, San Francisco, CA.
  • Parker Institute for Cancer Immunotherapy, San Francisco, CA.

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. Here, 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. 1 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- or patient-derived xenografts implanted in NOD scid gamma (NSG) 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 chimeric antigen receptor (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 scFv-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.

MeSH

Cells, Precursor Cell Lymphoblastic Leukemia-Lymphoma, Antigens, Surface, Neoplasms, Immunotherapy, Cell Line, Receptors, Antigen, Therapeutics, B-Lymphocytes, Car-T Cell