Framework humanization optimizes potency of anti-CD72 nanobody CAR-T cells for B-cell malignancies.

Nov 27, 2023

Experts: William C Temple,Matthew A Nix,Akul Naik,Adila Izgutdina,Benjamin J Huang,Gianina Wicaksono,Paul Phojanakong,Juan Antonio Camara Serrano,Elizabeth P Young,Emilio Ramos,Fernando Salangsang,Veronica Steri,Simayijiang Xirenayi,Michelle Hermiston,Aaron C Logan,Elliot Stieglitz,Arun P Wiita

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.
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’s lymphoma in vitro and in vivo. Humanized CD72 nanoCARs were compared with parental (“NbD4”) 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.
Toward clinical translation, here we humanize our previous nanobody framework regions, derived from llama, and surprisingly discover a clone (“H24”) 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<1 nM) 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.
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.

© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

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ABOUT THE EXPERTS

William C Temple,Matthew A Nix,Akul Naik,Adila Izgutdina,Benjamin J Huang,Gianina Wicaksono,Paul Phojanakong,Juan Antonio Camara Serrano,Elizabeth P Young,Emilio Ramos,Fernando Salangsang,Veronica Steri,Simayijiang Xirenayi,Michelle Hermiston,Aaron C Logan,Elliot Stieglitz,Arun P Wiita

William C Temple

Department of Pediatrics, Division of Hematology/Oncology, University of California, UCSF Benioff Children’s Hospital, San Francisco, California, USA.

Department of Pediatrics, Division of Allergy, Immunology, and Bone Marrow Transplantation, University of California, UCSF Benioff Children’s Hospital, San Francisco, California, USA.

Department of Laboratory Medicine, University of California, San Francisco, California, USA.

Matthew A Nix

Department of Laboratory Medicine, University of California, San Francisco, California, USA.

Akul Naik

Department of Laboratory Medicine, University of California, San Francisco, California, USA.

Adila Izgutdina

Department of Laboratory Medicine, University of California, San Francisco, California, USA.

Benjamin J Huang

Department of Pediatrics, Division of Hematology/Oncology, University of California, UCSF Benioff Children’s Hospital, San Francisco, California, USA.

Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA.

Gianina Wicaksono

Department of Laboratory Medicine, University of California, San Francisco, California, USA.

Paul Phojanakong

Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA.

Juan Antonio Camara Serrano

Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA.

Elizabeth P Young

Department of Pediatrics, Division of Hematology/Oncology, University of California, UCSF Benioff Children’s Hospital, San Francisco, California, USA.

Emilio Ramos

Department of Laboratory Medicine, University of California, San Francisco, California, USA.

Fernando Salangsang

Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA.

Veronica Steri

Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA.

Simayijiang Xirenayi

Department of Pediatrics, Division of Hematology/Oncology, University of California, UCSF Benioff Children’s Hospital, San Francisco, California, USA.

Michelle Hermiston

Department of Pediatrics, Division of Hematology/Oncology, University of California, UCSF Benioff Children’s Hospital, San Francisco, California, USA.

Department of Pediatrics, Division of Allergy, Immunology, and Bone Marrow Transplantation, University of California, UCSF Benioff Children’s Hospital, San Francisco, California, USA.

Aaron C Logan

Department of Medicine, Division of Hematology and Blood and Marrow Transplantation, University of California, San Francisco, California, USA.

Elliot Stieglitz

Department of Pediatrics, Division of Hematology/Oncology, University of California, UCSF Benioff Children’s Hospital, San Francisco, California, USA.

Arun P Wiita

Department of Laboratory Medicine, University of California, San Francisco, California, USA arun.wiita@ucsf.edu.

Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California, USA.

Chan Zuckerberg Biohub, San Francisco, California, USA.