Introduction

Chimeric antigen receptor (CAR) T cells have shown remarkable efficiencies in hematological malignancies however, their effectiveness in solid tumors is disappointing. One contributing factor might be the negative influence of checkpoints on CART cells in the tumor microenvironment. Engagement of programmed cell death protein-1 (PD-1) on T cells by PD-L1 inhibits effector function. We hypothesized that a combination of PD-1:PD-L1 inhibition with CART cells could increase their effectiveness in solid tumors. Inhibitory canine scFv antibody fragments will be employed in next generation CART cell design to generate CART that constitutively secrete anti-PD1 to increase efficiency in solid tumors.

Methods

Canine (c)PD-1-specific scFvs isolated from a fully canine scFv phage display library were assessed in an ELISA-based assay for their ability to inhibit cPD-1/cPD-L1 interaction. Inhibitory clones were reformatted as full-length monoclonal antibodies and their affinity for cPD-1 was analyzed by surface plasmon resonance, and their ability to detect cPD-1 in FFPE specimens was determined by IHC.

Results

Three anti-PD1 scFv clones strongly inhibited cPD-1/cPD-L1 interactions. Full-length antibodies were generated for two clones which showed single digit and subnanomolar binding affinities for cPD-1. One clone detected cPD-1 expression in FFPE lymph node specimens. Preliminary data suggests that this clone can also enhance canine effector T cell function following mitogen activation in vitro.

Conclusion

We have identified 2 anti-cPD-1 clones that have potential for use either as a mAb or as scFv secreted from next generation CART cells to enhance effector T cell function in solid tumors.

Funding Information

V Foundation for Cancer Research, NCI/NIH/SBIR Contract 75N91018C000042, JSPS KAKENHI Grant Number 21J01155