Abstract 4436: A novel antibody-enabled dual precision targeted protein stabilization (TPS2) that augments anti-tumor immune response by targeting CBL-B inhibitor to exhausted T cells while blocking checkpoint molecule, PD-1
Authors: Lim Joanne , Skaletskaya Anna , Permpoon Uttapol , Kim Yeonjoon , Saini Shikha , Takrouri Khuloud , Ribkovskaia Zinaida , Bhar Palash , Fishkin Nathan , Choi Dong-Ki , Park Ji Hyun , Palacino James , Park Peter U. 
Published: April 2023

Abstract
It is well-established that T cells play a key role in the success of cancer immunotherapies. The goals of T cell-targeting therapies have been to restore the effector function of tumor-specific T cells that are either dysfunctional due to the immunosuppressive mechanisms in the tumor microenvironment, the lack of co-stimulatory signals, or negative regulation by expression of checkpoint molecules such as PD-1. E3 ligase Casitas B-Lineage Lymphoma Proto-Oncogene B (CBL-B) has recently gained attention as a master regulator of multiple immune-activation mechanisms. Inhibition of CBL-B enables the activation of antigen-specific T cells, and has been shown in syngeneic mouse tumor models to be efficacious as a cancer immunotherapy agent. CBL-B inhibition has also been shown to enhance and prolong the effects of anti-PD-1 antibody (Ab) therapy. However, genetic knockout of CBL-B in mice led to spontaneous autoimmunity characterized by auto-antibody production and infiltration of activated T cells and B cells into multiple organs that could result in tissue damage. In order to mitigate safety issues that could result from systemic CBL-B inhibition, we developed novel antibody-drug conjugates (ADCs) that target CBL-B inhibitors (CBL-Bi) to T cells via binding to PD-1. Using PD-1-expressing Jurkat NFAT reporter cells and mixed lymphocyte reaction assay, we demonstrated that our TPS2 approach enhanced the activation of T cells in vitro compared to treatment with anti-PD-1 Ab alone. Evaluation of pathways downstream of TCR activation in primary exhausted T cells showed increased Notch1, phospho-PLCγ2, and ZAP-70 accumulation following anti-PD-1/CBL-Bi treatment and is correlated with IFNγ induction. When autologous mature dendritic cells were co-cultured with dissociated tumor cells from melanoma patients, the highest magnitude of tumor-infiltrating lymphocyte activation was observed using delivery of CBL-Bi by anti-PD-1 ADC, compared to CBL-Bi alone, anti-PD-1 Ab alone, or combination treatment. The potential of anti-PD-1/CBL-Bi ADC for activation effector T cell function is also evident by increased intratumoral transcript levels of Granzyme B and Perforin following treatment in a humanized mouse model. In conclusion, we validated that the biological activity of CBL-Bi is retained after conjugation with an anti-PD-1 antibody. The use of anti-PD-1 Ab for developing the ADC not only targets the CBL-Bi payload to exhausted T cells but also blocks negative regulation of T cells via PD-1. Together, anti-PD-1/CBL-Bi ADC enhanced T cell activation in vitro as well as in vivo. Evaluation of safety and efficacy in animal tumor models is ongoing, to support further development of the TPS2 approach.
Citation Format: Joanne Lim, Anna Skaletskaya, Uttapol Permpoon, Yeonjoon Kim, Shikha Saini, Khuloud Takrouri, Zinaida Ribkovskaia, Palash Bhar, Nathan Fishkin, Dong-Ki Choi, Ji Hyun Park, James Palacino, Peter U. Park. A novel antibody-enabled dual precision targeted protein stabilization (TPS2) that augments anti-tumor immune response by targeting CBL-B inhibitor to exhausted T cells while blocking checkpoint molecule, PD-1. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4436.