Introduction

Chimeric antigen receptor (CAR)-T cells targeting CD19 have revolutionized the treatment of human B cell malignancies. However, post-treatment loss of CD19 has emerged as an adaptive mechanism of resistance. Comparative trials at the University of Pennsylvania are investigating the activity of canine CAR-T cells targeting either CD19 or CD20 in canine B cell lymphoma patients. Similarly, expansion of CD20-negative B cells was observed in dogs treated with anti-CD20 CAR-T cells. Bispecific CAR-T cells that simultaneously target two antigens to mitigate antigen escape have encouraging efficacy profiles in human patients. Thus, we designed a dual-targeting CAR specific for canine B cell antigens.

Methods

A second-generation canine CAR construct targeting both CD19 and CD20 was cloned into the MSGV1 backbone. Chimeric retrovirus encoding RD114 and VSV-G envelope proteins and the CAR construct was made using GP2-293 cells. Jurkat NFAT-GFP reporter cells and primary T cells from dogs diagnosed with high grade B cell lymphoma were retrovirally transduced. Antigen specific reactivity was assessed against cell lines with variable CD19 and CD20 expression patterns.

Results

The CD19-CD20 CAR was efficiently transduced, with stable expression in both Jurkat and primary canine T cells. Jurkat reporters revealed dual-specific CAR signaling after co-culture with CD19+ and/or CD20+ target cells. Primary canine CAR-T cells expanded in vitro and exhibited CAR-specific proliferation and cytotoxicity against CD19+ CD20+ target cells.

Conclusion

Preclinical manufacture of functional, bispecific anti-CD19 anti-CD20 canine CAR-T cells is feasible. A clinical trial recruiting dogs diagnosed with B cell cancer will assess safety and efficacy in vivo.

Funding Information

Shuman Translational Research Fellowship (MJA); NCI K08CA252619 (MJA); PetCo Love (NJM).