Abstract A032: Bispecific antibody dose optimization in a syngeneic lymphoma model and development of bispecific nanoshells (BINS) for enhanced immune targeting
Aiko Matsuda, Isabella M. Stathas, Irina V. Balyasnikova, Leo I. Gordon, Adam Y. LinAbstract
Introduction:
Bispecific antibody (BsAb) therapy redirects T cells to attack lymphomas, but the optimal dosing strategies and immune effects remain poorly understood. Previous studies using nanoparticle-based T cell engagers have shown that multispecific targeting is feasible (Alhallak et al., Leukemia 2021). However, liposomes have limitations. Gold nanoshells (NS) provide an alternative platform with the added benefit of photothermal therapy and optical properties. Here, we analyze the dose-response relationship of BsAb in a syngeneic lymphoma model, and present the development of Bispecific Nanoshells (BINS), which link anti-CD3 and anti-CD20 antibodies NS.
Methods:
For in vivo experiments, A20 lymphoma cells were injected subcutaneously into BALB/c mice. The anti-CD3/CD20 BsAbs were first validated in vitro using LDH and flow cytometry-based cytotoxicity assays. They were then administered intraperitoneally at doses of 5, 10, 15, or 20 µg once a week, with an additional group receiving 10 µg twice weekly. Tumor growth and the phenotype of splenic and tumor-infiltrating lymphocytes were analyzed via flow cytometry. BINS were produced using kit-based methods (kBINS; Nanocomposix NHS Gold Nanoshell Kit) and laboratory synthesis (synthBINS; 5 kDa carboxyl-PEG-thiol with EDC/sulfo-NHS). Their stability and conjugation efficiency were evaluated through absorbance spectroscopy and cytotoxicity testing.
Results:
Lymphoma growth inhibition was dose-dependent (20 > 15 > 10 > 5 µg). Notably, administering twice weekly was as effective as once weekly at the same dose, despite the total dose doubling. This suggests that the dose per injection, rather than the frequency of administration, determines the therapeutic effect. Immunophenotyping showed significant decreases in splenic CD3 T cells, alongside increases in tumor-infiltrating effector memory CD8+ T cells (CD44+CD62L−) and higher granzyme B expression, indicating T cell migration from the spleen to the tumor. Splenic CD8 T cell Tim-3 expression was increased, implying activation-induced exhaustion. For BINS, CD20 antibody aggregation during conjugation was a key stability issue on both platforms. Optimizing with 5% trehalose and 0.02% Tween-20 greatly improved the stability of dual-conjugated CD3/CD20 particles. Absorbance spectroscopy confirmed successful conjugation through a redshift in peak wavelength. In co-culture assays, CD3/CD20 kBINS at 1:1 and 2:1 ratios significantly surpassed free BsAb in killing A20 cells, whereas unconjugated NS showed no effect.
Conclusions:
The effectiveness of BsAb depends on the dose per injection rather than how often it is administered. This indicates that higher doses given less frequently might be enough, which could influence clinical scheduling for BsAb treatments. Additionally, BINS exhibited better in vitro cytotoxicity than free BsAb, creating a scalable nanoplatform for future in vivo studies and photothermal applications. Grammarly and Claude were used to assist with the editing of this abstract, but the authors completed the final versions.
Citation Format:
Aiko Matsuda, Isabella M. Stathas, Irina V. Balyasnikova, Leo I. Gordon, Adam Y. Lin. Bispecific antibody dose optimization in a syngeneic lymphoma model and development of bispecific nanoshells (BINS) for enhanced immune targeting [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr A032.