Abstract 5294: Conditionally active CD28xVISTA bispecific antibodies induce myeloid-driven tumor-specific T-cell co-stimulation for improved cancer immunotherapy

Abstract Introduction: Tumor-specific recruitment of co-stimulatory bispecific antibodies (bsAbs) has emerged as a promising therapeutic strategy. Here, we investigated pH-selective CD28xVISTA bsAbs to act selectively within the acidic tumor microenvironment (TME). Our CD28xVISTA bsAbs are designed for tripartite “trans-activation” of CD28 in the TME, aiming for enhanced T-cell-mediated cancer cell killing while minimizing systemic T-cell activation and Cytokine Release Syndrome (CRS) risk. Experimental Procedures: We evaluated various CD28xVISTA bsAbs, focusing on a prototype 1+2 format with monovalent CD28 binding, bivalent VISTA binding and Fc receptor interaction null mutations (BS2). BS2 was tested for T-cell trans-activation using Jurkat-IL-2-luciferase reporter cells in the presence of HEK293 cells expressing membrane bound OKT3-scFv and CHO cells expressing human VISTA. BS2’s potency was further evaluated in an xCelligence-based human T-cell killing assay that dynamically monitors growth of LNCaP prostate cancer cells co-cultured with VISTA+ Kasumi-3 cells, alone or in combination with a CD3xPSMA bispecific T-cell engager. T-cell activation and proliferation were also measured using flow cytometry with CD3, CD4, CD8 and CD25 markers. Additionally, we modified BS2 to introduce pH-selective VISTA binding, limiting its activity to the TME, and tested this version in a syngeneic mouse model in combination with anti-murine PD-1 (anti-mPD-1). Finally, cytokine release from human PBMCs, co-cultured with human umbilical vein endothelial cells (HUVECs) and treated with the pH-selective BS2 (or TGN1412 as a positive control), was measured using a bead-based multiplex immune assay. Results: Our data show that the combination of anti-mPD-1 and our prototype CD28xVISTA bsAb (BS2) with pH-selective VISTA binding significantly inhibited tumor growth in vivo. In vitro, CD28xVISTA co-stimulation in trans by BS2 potentiated the activity of a CD3xPSMA bispecific T-cell engager in our human T-cell killing assay. Cytokine release assessments demonstrated negligible induction of inflammatory cytokines, indicating a favorable safety profile for this antibody. Conclusion: Our study demonstrates the feasibility of cis and trans CD28 co-stimulation using a CD28xVISTA bsAb (BS2). This approach, which bypasses the need for tumor-associated antigens (TAAs) required by other CD28xTAA bispecifics in development, suggests a potentially safer alternative for T-cell engagement and stimulation. Moreover, our novel myeloid-directed TME-selective co-stimulation strategy with a CD28xVISTA bsAb may broaden the potential for T-cell engagement approaches in solid tumors by enabling rational combinations with existing CD3xTAA T-cell engagers without competing for the same target. Citation Format: Thomas Thisted, Zhi-Gang Jiang, Zuzana Biesova, Adejumoke Onumajuru, Yuliya Kleschenko, Kanam Malhotra, Vikas Saxena, Arnab Mukherjee, F. Donelson Smith, Edward H. van der Horst. Conditionally active CD28xVISTA bispecific antibodies induce myeloid-driven tumor-specific T-cell co-stimulation for improved cancer immunotherapy [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 5294.