Abstract 1235: Impact of anti-CD3 and tumor-target binder affinities on in-vitro potency, in-vivo efficacy, and cytokine release

Abstract Background: First generation T cell bispecifics (TCBs) were developed by utilizing high CD3 and tumor target binder affinities to induce a potent response. Instrumentalizing this, TCBs have clinically proven to elicit efficacious target cell killing and anti-tumor efficacy, but they encountered hurdles related to cytokine release. Recent studies suggested that reducing CD3 binder affinity could lead to an efficient tumor killing with lower cytokine levels. However, the answer to whether cytokine release is mainly dependent on CD3 binder affinity or a multifactorial phenomenon remains ambiguous. Here, we aim to characterize the efficacy-safety relationship of different TCBs through investigating the interplay between CD3 binder affinity and the affinity of the tumor target-specific binder. For that purpose, a series of TCBs with varying CD3 affinities, and two target binders of high and low affinities were generated in the 2:1 TCB format, resulting in a bivalent binding to the tumor antigen and a monovalent one to CD3, which offers a unique opportunity to enhance the activity of TCBs. Methods: Firstly, 2:1 TCBs comprising either the high or low affinity target binder and varying CD3 binder affinities were co-cultured in-vitro with human peripheral blood mononuclear cells (PBMCs), and human tumor cell lines, in order to assess T cell mediated cytotoxicity and cytokine release at different time points. Additionally, the impact on in-vivo efficacy, cytokine release, and pharmacokinetics was assessed in humanized mice implanted with a patient-derived xenograft model. Results: Based on the dose-response in the cytotoxicity assay, the CD3 binders were classified into high, intermediate, and low levels of affinity. As expected, we observed that the in-vitro T cell cytotoxicity, in-vivo tumor growth inhibition (TGI), and cytokine release levels were maximal with the highest affine CD3 binder. Simultaneously, we have observed a decrease in cytotoxicity response with decreasing CD3 affinity compared to the high affinity binder. However, this was accompanied by an atypical decrease in cytokine release. Particularly, the TCB comprising the intermediate CD3 affinity binder illustrated a comparable in-vitro cytotoxicity and in-vivo TGI to the high affinity binder with a marked decrease in cytokine release [4x reduction in-vitro and 6x reduction in-vivo]. Upon testing the TCBs comprising the low affinity target binder with the same CD3 binders, we observed an additional decrease in cytokine release with the decreasing CD3 affinity. In conclusion, we designed and generated TCBs with varying affinities in both the CD3 and target binding arms. Reducing cytokine release while maintaining adequate efficacy is feasible through CD3 binder affinity attenuation; however, the target binder affinity should be taken into consideration when interpreting our findings and designing new molecules. Citation Format: Omar Abdelmotaleb, Anneliese Schneider, Thomas Hofer, Johannes Sam, Martin Lechmann, Inja Waldhauer, Ali Bransi, Miro Eigenmann, Anne Freimoser-Grundschober, Christian Gassner, Alex Odermatt, Peter Brünker, Sara Colombetti, Christian Klein. Impact of anti-CD3 and tumor-target binder affinities on in-vitro potency, in-vivo efficacy, and cytokine release [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1235.