Abstract 1885: Selection of an optimal anti-PD-L1 single domain antibody format for the vectorization into oncolytic vaccinia virus and the generation of bispecific immunomodulators
Authors: Marchand Jean-Baptiste , Pintado Elodie , Dunlop Marshall , Remy Christelle , Kleinpeter Patricia , Shön Shirley , Laetitia Fend , Brandely Renée , Suhner Delphine , Winter Eline , Silvestre Nathalie , Huguet Claire , Fitzgerald Peter , Quéméneur Eric 
Published: April 2023

Abstract
Background: Single domain antibodies (sdAbs) isolated after immunization of camelids are particularly attractive formats for their high modularity and small size allowing a better diffusion into tumors. However, the short in vivo half-life of sdAbs, related to the lack of a Fc domain, limits their clinical application. By replicating specifically into tumor cells, the oncolytic vaccinia virus (VACV) is an optimal vector to deliver and maintain high intra-tumoral concentrations of therapeutic sdAb. Moreover, sdAb targeting immunological targets, such as PD-L1, may synergize the anti-tumoral activity of VACV. Randox and Transgene report the selection and characterization of a sdAb targeting the human PD-L1 and the design of optimal formats, including bispecific anti-PD-L1-CD40 ligand, for vectorization into VACV.
Methods: Alpacas were immunized with human PD-L1 protein and sdAb coding sequences were isolated by PCR. Anti-PD-L1 sdAb binders were selected by phage display and sdAb blockers of PD-L1/PD-1 interaction were identified by ELISA. The ability of the selected sdAb to disrupt the PD-L1/PD-1 interaction was verified on transformed and primary cells. To fine-tune an optimal anti-PD-L1, several sdAb formats were designed and vectorized into VACV. The sdAb format exhibiting the best PD-L1/PD-1 blocking activity was selected by the screening of culture supernatants of several VACV-sdAb infected tumor cells. Finally, anti-PD-L1 sdAb-CD40L fusions were designed to generate a strong CD40 agonist active only in a PD-L1 positive environment.
Results: SdAb clone 1A1 exhibited the best PD-L1/PD-1 blocking activity which remained unchanged after extensive humanization (latterly becoming named GS542). GS542 was vectorized in VACV as monomeric single chain homodimer, and fused to Fc domain or antibody hinge domain to foster dimerization together with full length IgG1 avelumab as anti-PD-L1 benchmark. All constructs were expressed by infected tumor cells. The single chain homodimer displayed the best PD-L1/PD-1 blocking activity, superior to that of avelumab. Moreover, GS542-CD40L fusions were designed to take advantage of the natural trimerization of CD40L to increase the avidity for PD-L1 while clustering CD40L at the surface of PD-L1+ cells to trans-activate the CD40 pathway. Evaluation of these GS542-CD40L fusions showed strong CD40 agonist activity depending on the presence of PD-L1+ cells making these constructs safer CD40 agonists.
Conclusion: An anti-PD-L1 sdAb with a strong blocking activity was selected, humanized and evaluated under different VACV-vectorized formats. The single chain homodimeric sdAb expressed by VACV was identified as the best PD-L1/PD-1 blocking format. Furthermore, bispecific anti-PD-L1 sdAb-CD40L fusions that exhibited strong CD40 agonist activity was charactered within a PD-L1+ environment.
Citation Format: Jean-Baptiste Marchand, Elodie Pintado, Marshall Dunlop, Christelle Remy, Patricia Kleinpeter, Shirley Shön, Fend Laetitia, Renée Brandely, Delphine Suhner, Eline Winter, Nathalie Silvestre, Claire Huguet, Peter Fitzgerald, Eric Quéméneur. Selection of an optimal anti-PD-L1 single domain antibody format for the vectorization into oncolytic vaccinia virus and the generation of bispecific immunomodulators [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 1885.