# Characterization of a Potential Therapeutic Anti-Canine PD-1 Single Domain Antibody Produced in Yeast

## TL;DR

Researchers developed a low-cost, yeast-produced antibody targeting canine PD-1, a potential treatment for dog cancer.

## Contribution

A yeast-expressible single-domain antibody against canine PD-1 was developed and characterized for veterinary immunotherapy.

## Key findings

- Clone STX-1b5 binds canine PD-1 with nanomolar affinity and blocks PDL-1 interaction.
- STX-1b5 is efficiently produced in a yeast-based recombinant system.
- In silico modeling supports STX-1b5's interaction with key PD-1 residues.

## Abstract

Cancer is the leading cause of death in dogs, with one in three dogs expected to die of cancer. Although promising in the treatment of many human cancers, monoclonal antibodies (MAb) as immunotherapeutics are costly and exhibit poor transferability between species, inhibiting their use in veterinary clinics. Nanobodies are single-domain antibodies (SDAb) that share the same antigen specificity as Mab but have added benefits, including high modularity, allowing use in a variety of protein expression systems that facilitate desirable recombinant modifications to the primary protein sequence. We describe the generation, discovery, production, and functional characterization of an SDAb against canine PD-1 (caPD1) using alpaca immunization and phage display library antigen selection. We further show the amenability of our SDAb to production by a yeast expression system, illustrating a potential path for the advancement of functional, low-cost immunotherapeutics in veterinary medicine.

A single domain antibody (SDAb) targeting canine PD-1 was developed as a potential immunotherapeutic for canine cancer. An alpaca was immunized with canine PD-1 protein, and a phage-display library was constructed using mRNA isolated from peripheral lymphocytes. Screening of the library yielded multiple SDAb candidates capable of nanomolar binding to canine PD-1. Among these, clone STX-1b5 demonstrated high expression in a yeast-based recombinant system and was selected for further characterization. Binding and competition assays using ELISA confirmed its ability to bind canine PD-1 and block PDL-1 interaction. In silico structural modeling supported the interaction of STX-1b5 with key PD-1 residues implicated in ligand binding. These findings support the feasibility of using SDAbs and cost-effective yeast expression systems to generate immunotherapeutics for veterinary use, with STX-1b5 representing a promising lead candidate for future clinical development.