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

in Journal for immunotherapy of cancer by 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

TLDR

  • Researchers develop novel CD72-targeted nanobody-based CAR-T cells with enhanced potency against B-cell tumors, including patient-derived samples after CD19 CAR-T relapse.

Abstract

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.

Overview

  • The study aims to develop new immunotherapeutic targets for B-cell malignancies by exploiting the CD72 antigen and using nanobody-based CAR-T cells.
  • The researchers humanized a series of llama-derived nanobody framework regions to generate humanized anti-CD72 nanobodies, which were then inserted into CD72 CAR-T cells.
  • The primary objective of the study is to evaluate the potency and efficacy of CD72 nanoCARs against B-cell tumors, including patient-derived samples after CD19 CAR-T relapse.

Comparative Analysis & Findings

  • The study compared the anti-tumor potency of humanized CD72 nanoCARs (H24) with parental (NbD4) CD72 nanoCARs and the clinically approved CD19-directed CAR-T construct tisagenlecleucel.
  • The results showed that H24 nanoCARs had enhanced potency against B-cell tumors, including patient-derived samples after CD19 CAR-T relapse, compared to parental CD72 nanoCARs.
  • The study also found that affinity maturation of the parental NbD4 construct did not lead to improvement in cytotoxicity, but the humanized H24 clone had moderately higher binding affinity to CD72.

Implications and Future Directions

  • The study suggests that nanoCARs can be improved by framework alterations alone, potentially expanding the efficacy of nanobody-based cellular therapies.
  • The findings may have implications for future engineering of nanobody-based cellular therapies.
  • Further studies are needed to explore the mechanisms of resistance and to determine the optimal binding affinity for CAR-T cells targeting CD72.
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