Abstract 7242: Immunostimulatory gene therapy co-expressing immune activators TMZ-CD40L and 4-1BBL with a PD-L1-targeting Affibody molecule to reactivate T cell immunity

Abstract

Abstract Immunotherapy has been used to make great strides in helping cancer patients, and new approaches to reactivate patient immunity are constantly being developed. We are developing immunostimulatory gene therapy using adenoviruses as gene vehicles to inflame the tumor microenvironment and drive anti-tumor immunity. One consequence of immune activation is PD-L1 upregulation, so to optimize our treatment, we are using a novel gene therapy vehicle expressing both immune activators and a PD-L1-blocking Affibody molecule. Affibody molecules are tri-helical proteins engineered to bind specific target molecules with high affinity. These molecules have successfully been used to image cancer cells in the body and can block target proteins. Affibody molecules are advantageous over other affinity molecules due to their small size, averaging about 6 kDa, and their high stability. The small size makes an Affibody molecule an ideal candidate for use in viral vectors, where genetic space is limited. In the present study, we tested the biological activity of a PD-L1 targeted Affibody molecule expressed from a viral vehicle compared to the synthetic protein, using a Bio-IC in vitro luciferase assay. Further, the capacity of the PD-L1-binding Affibody molecule to prevent inhibitory signaling was evaluated in an ovarian cancer/immune cell co-culture assay. Our results demonstrate that cell lines infected with viral gene vehicles successfully express and release the Affibody molecule as demonstrated with PCR and ELISA. Furthermore, the supernatants containing the Affibody molecule and synthetic Affibody molecule inhibited the activity of PD-L1 in the BioIC assay. Next, an adenovirus vector containing trimerized-CD40L and 4-1BBL (LOAd703), known to upregulate PD-L1 in stimulated dendritic cells, was modified to co-express the anti-PD-L1 Affibody molecule (LOAd724). Gene expression was confirmed using PCR, flow cytometry (CD40L, 4-1BBL) and ELISA (anti-PD-L1 Affibody molecule). The tumor/immune cell co-culture is underway. In summary, Affibody molecules can be expressed from adenoviral gene vehicles alone or in combination with immunostimulatory genes. Further studies are warranted to understand the role of local PD-L1 blockade in the tumor microenvironment following LOAd724 therapy. Citation Format: Luke Adam White, Iris Vaask, Jonas Härdin, Emma Eriksson, Anna Sandegren, Fredrik Y. Frejd, Tanja Lövgren, Angelica Loskog. Immunostimulatory gene therapy co-expressing immune activators TMZ-CD40L and 4-1BBL with a PD-L1-targeting Affibody molecule to reactivate T cell immunity [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 7242.