Abstract 4820: Anti-CD72 nanobody-based CAR-T affinity maturation leads to improved in vitro efficacy versus low antigen density lymphoma models, but limited benefit in vivo

Abstract Introduction: CD19 CAR T-cells have markedly improved the survival of patients with B-cell malignancies. However, over half of patients relapse after this therapy, many due to CD19 antigen loss. Our laboratory discovered CD72, a B cell specific surface antigen widely expressed on B-cell malignancies. We developed an effective humanized nanobody-based CAR-T (“H24 nanoCAR”) against this target (Nix et al. Cancer Discov 2021, Temple et al. JITC 2023) that we are moving to clinical translation. However, we observed reduced efficacy of H24 nanoCAR versus tumor with low CD72 antigen density. We hypothesized that affinity matured CD72 nanoCAR-Ts could target lower antigen thresholds in lymphoma models. Methods: Affinity maturation and humanization of CAR-Ts was described in Temple et al. 2023. Humanized affinity matured NbD4.13-H24 CAR was generated by altering the framework region of clone NbD4.13 (KD = 0.8 nM) to that in clone H24 (KD = 33 nM). We previously described engineered JeKo1-CD72 low cell line (Izgutdina et al. ASH 2023). Results: Flow cytometry confirmed ubiquitous CD72 expression on 20 primary B-NHL samples. Two B-ALL patient samples relapsed after CD19 CAR therapy showed downregulation of both surface CD19 and CD22, but surface CD72 was retained, raising the hypothesis that CD72 is a preferential second-line antigen after CD19 relapse. To evaluate efficacy versus CD72 antigen low model, we tested affinity matured NbD4.13 CAR and humanized affinity matured NbD4.13-H24, in comparison to H24 (clinical candidate), against an engineered JeKo1-CD72 low cell line. NbD4.13 and NbD4.13-H24 demonstrated enhanced cytokine secretion compared to H24. Upon repetitive restimulation with JeKo1-CD72 low, NbD4.13 CAR showed more rapid elimination of tumor than H24. However, NbD4.13 and H24 demonstrated equal efficacy in vivo versus mantle cell lymphoma PDX. In an in vivo experiment with JeKo1-low, NbD4.13 showed improved early tumor control than H24, but no survival benefit, while NbD4.13-H24 showed a modest survival benefit (p=0.0442 by Log-rank test). As an alternate strategy to address low antigen density, we showed that the protein kinase C inhibitor bryostatin can lead to increased surface CD72 when applied to the CD19 resistant model Nalm6-R. To understand the mechanism of action, we performed bulk RNAseq of CD72 antigen-low lymphoma cell line SC1 treated with bryostatin. GSEA showed that bryostatin broadly increases B cell lineage specific expression programs. Conclusion: CD72 is a potential second line antigen post-CD19 relapse in B-cell malignancies. We found that affinity maturation did not demonstrably improve CD72 nanoCAR efficacy. Our results support findings demonstrated for scFv-based CARs, that moderate affinity for surface antigen may lead to optimal CAR performance in vivo, even when nanobodies are used as the recognition element. Citation Format: Adila Izgutdina, Tasfia Rashid, Bonell Patino-Escobar, Sujata Walunj, Huimin Geng, Amrik Kang, Szu-Ying Chen, Haley Johnson, William C. Temple, Matthew A. Nix, Akul Naik, Emilio Ramos, Hiroyuki Takamatsu, Daniel Gil-Alós, Joaquin Martinez-Lopez, Antonio Valeri, Constance Yuan, Hao-Wei Wang, Sarah Aminov, Srabani Sahu, Christopher Carpenter, Fernando Salangsang, Paul Phojanakong, Juan Antonio Camara Serrano, Isa Tariq, Veronica Steri, Samir Parekh, Amit Verma, Nirali N. Shah, Arun P. Wiita. Anti-CD72 nanobody-based CAR-T affinity maturation leads to improved in vitro efficacy versus low antigen density lymphoma models, but limited benefit in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4820.