Abstract 6372: In situ detection of various activated immune checkpoints via next-generation proximity ligation assays in tumor tissue
Authors: Edén Desirée , Ieong Ka I Au , Cook Naomi , Raykova Doroteya , Wicher Agata Zieba 
Published: April 2023

Abstract
Immune checkpoints (ICs), which are inhibitory signaling pathways that can down-modulate the immune responses of T cells, are pivotal in peripheral tissues and for maintaining immune self-tolerance. Among the many molecularly defined IC proteins, some of the most studied ones are CTLA-4 and its binding partners important for early T-cell co-inhibition, along with the PD-1/PD-L1 axis proteins that carry out late co-inhibitory signals. Notably, in addition to antigen presenting and other immune cells, many tumors also express CTLA-4 and PD-L1, which facilitate tumor evasion from the immune system. The inhibition of these IC proteins has revolutionized the field of cancer therapy, but its efficiency has been limited to a poorly defined subset of patients. Patient outcomes are likely to have a stronger correlation to high levels of PD-1/PD-L1 interaction and other hallmarks of pathway activation, such as PD-1 phosphorylation and recruitment of SHP-2, than to the overexpression of a single IC protein alone. To improve the predictive value of tissue staining, we created an immuno-oncology line of ultrasensitive kits based on proximity ligation, which detect the activation of multiple ICs. Naveni™ PD1/PD-L1 is a tool for direct visualization of the interaction between PD-1 and PD-L1, and has been verified in various FFPE tumor tissues. Naveni™ pY PD1 sensitively detects PD1 phosphorylation, which is the first step in the PD-1/PD-L1 inhibitory pathway activation. To study the subsequent recruitment of SHP-2 to the activated PD-1 receptor, which is crucial in immune cells but dispensable when the pathway is activated between two tumor cells, one can use the upcoming Naveni™ PD1/SHP-2 kit. Furthermore, we are developing assays to observe the competing interactions of CTLA-4 and CD28 with their binding partners CD80 and CD86, the interplay of which is pivotal for the initiation of early co-inhibition. All kits can be used on consecutive tissue sections to obtain a comprehensive picture of tumor IC pathway activation, which may not always correspond to CTLA-4, PD-1 or PD-L1 overexpression. While these kits have a chromogenic readout applicable to brightfield microscopy, we also have preliminary data on a PD-1/PD-L1 assay with a fluorescent readout thanks to which it can also be multiplexed. The multiplex feature generates an immune profile which may help improve immunotherapeutic strategies. The Naveni™ assays can be applied in basic research to elucidate the interplay of IC axes and downstream molecules, in pre-clinical and clinical research to compare stainings with the existing IHC assays and evaluate the potential prognostic value of interaction detection, and in pharma, aiding the development of new drugs or bispecific antibodies.
Citation Format: Desirée Edén, Ka I Au Ieong, Naomi Cook, Doroteya Raykova, Agata Zieba Wicher. In situ detection of various activated immune checkpoints via next-generation proximity ligation assays in tumor tissue. [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 6372.