Abstract 3179: Design and activity of 2nd generation, dual-targeted CAR T cell factories against ccRCC
Authors: Wang Yufei , Suarez Eloah Rabello , Chang Matthew , Jennings Rebecca , Signoretti Sabina , Zhu Quan , Marasco Wayne 
Published: August 2019

Abstract
Clear cell renal cell carcinoma (ccRCC) is the major type of RCC which is among the 10 most common cancers in both men and women. Chimeric Antigen Receptor (CAR) T cells have proven to be a powerful, clinically translatable immunotherapy for hematologic malignancies. However, these results have not been translatable to solid tumors due to inefficient homing of CAR T cells, the suppressive tumor microenvironment, and on-target off-tumor toxicities resulted from the sharing of CAR T targeting epitopes on healthy tissues. To combat the suppressive microenvironment, immune checkpoint blockade has shown promising effect on antitumor response by restoring the local antitumor immunity. CAR T cell factories were designed to empower CAR T cells through the secretion of human anti-immune checkpoint inhibitor monoclonal antibodies (mAbs) locally at the tumor site. Our results show a dramatic improvement in CAR T killing of ccRCC in vitro and in vivoby reversing CAR T cell and tumor infiltrating lymphocyte (TIL) exhaustion.
CAIX is an ideal target for ccRCC therapy and used as a CAR target for the first clinical trial. However, it led to adverse side effects (ADEs) due to CAIX expression on the bile duct. Therefore, it is crucial to develop a CAR with elevated efficacy and safety (limited on-target off-tumor effect). To achieve that, the 2nd targeting scFv was introduced in the CAR T cell factory together with anti-CAIX scFv to enable the CAR to target two unique antigens simultaneously. By IHC staining of ccRCC patient samples, we found that target B is an ideal target to be utilized as the 2nd target since it is highly expressed on ccRCC and co-expressed with CAIX.
Our 27 billion-member human scFv-phage display library was panned against the antigen expressing skrc-59 ccRCC cells to identify novel scFvs. Their binding kinetics (Kon/Koff) were then measured and scFvs with desirable kinetics were evaluated for their ability to bind with antigen expressing cells. Ideal candidates were cloned into vectors where anti-target B and anti-CAIX scFvs were combined in different permutations by changing the order of the two targeting scFvs with various linkers connected to a costimulatory domain (CD28, 41BB) and an activating domain (CD3). Primary T cells isolated from PBMCs were transduced to express dual CAR and were tested against different cell lines in vitro. For further evaluation in vivo, a humanized orthotopic ccRCC mouse model was established by injecting luciferized ccRCC cells under the kidney capsule of NSG-SGM3 mice with a reconstituted human immune system.
In summary, utilizing a dual CAR T discovery platform, we generated a series of CARs with different scFvs, linkers, and hinges. By combining the best dual CAR with immune checkpoint blockade as payload, we propose that lead 2nd generation CAR T cell factory candidates will be discovered that should mitigate against ADEs on normal tissues and hold great promise for the treatment of ccRCC.
Citation Format: Yufei Wang, Eloah Rabello Suarez, Matthew Chang, Rebecca Jennings, Sabina Signoretti, Quan Zhu, Wayne Marasco. Design and activity of 2nd generation, dual-targeted CAR T cell factories against ccRCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3179.