D1.379 - AllergoOncology: Generation of Equinized Anti-PD-L1 mAbs with Specific Equine Constant Regions by MOE-PCR Ligation-Independent Cloning (LIC)

Horses, dogs, and humans naturally develop both allergies and cancer; however, effective immunotherapies, particularly checkpoint inhibitors, remain largely unavailable for veterinary applications. Immunotherapy has revolutionized human cancer treatment, with monoclonal antibodies (mAbs) targeting immune checkpoints such as PD-1 and PD-L1 demonstrating significant clinical success. Our previous work successfully generated a chimeric human-canine anti-PD-L1 antibody using the variable regions of Atezolizumab, enabling species-specific checkpoint inhibition in dogs. Building on these findings, we aimed to develop equinized anti-PD-L1 mAbs by incorporating equine IgG constant regions using MOE-PCR, a ligation-independent cloning (LIC) system, to facilitate the adaptation of checkpoint blockade therapies for equine oncology.

Multiple sequence alignment (MSA) and phylogenetic analysis were performed using EMBL-EBI’s Multiple Sequence Alignment Job Dispatcher with the Clustal Omega program to assess PD-L1 homology across species. The binding efficiency of biotin-labeled human anti-PD-1 (Pembrolizumab, Nivolumab, Cemiplimab) and anti-PD-L1 (Atezolizumab, Avelumab, Durvalumab) mAbs was evaluated by flow cytometry on equine peripheral blood mononuclear cells (PBMCs) and the equine malignant melanoma cell line MelDuWI. We employed MOE-PCR, a LIC-based system, to generate equinized anti-PD-L1 mAbs, using a pVitro1 cassette designed for rapid recombination of human variable heavy and light chain sequences with equine IgG constant regions, retrieved from the International Immunogenetics Information System (IMGT).

Multiple sequence alignment and phylogenetic analysis confirmed the high conservation of PD-L1 across species, with 80.07% identity between equine and human PD-L1 and 84.38% identity between equine and canine PD-L1. Flow cytometry analysis demonstrated strong cross-reactivity of Atezolizumab with equine PBMCs and the equine malignant melanoma cell line MelDuWI, supporting its potential as a parental sequence for equinized mAb development. Using MOE-PCR LIC, we successfully inserted human variable regions into an equine IgG framework, generating equinized anti-PD-L1 mAbs for potential application in equine oncology.

Expanding upon our previous development of a chimeric human-canine anti-PD-L1 antibody, this study presents a novel approach for generating equine-specific checkpoint inhibitors. These equinized anti-PD-L1 mAbs offer a promising step toward implementing immune checkpoint blockade in equine oncology, addressing a critical gap in veterinary cancer immunotherapy.