Enhancing CAR T Cell Anti-Tumor Efficacy through Secreted Single Chain Variable Fragment (scFv) Immune Checkpoint Blockade
Authors: Rafiq Sarwish , Jackson Hollie J. , Yeku Oladapo , Purdon Terence J , van Leeuwen Dayenne G. , Curran Kevin J. , Ahmed Ryan N , Cullen Grace D , Yan Su , Wang Pei , Xiang Jingyi , Liu Cheng , Brentjens Renier J. 
Published: April 2021

Abstract
T cell therapies have had valuable clinical responses in patients with cancer. Chimeric antigen receptor (CAR) T cells can be genetically engineered to recognize tumor cells and CAR T cell therapy has shown impressive results in the setting of B cell acute lymphoblastic leukemia but has been less effective in treating other types of hematologic and solid tumors. The inhibitory tumor microenvironment (TME), including expression of ligands that bind inhibitory receptors on T cells, e.g. programmed death receptor 1 (PD-1), can dampen CAR T cell responses. Separately, immune checkpoint blockade therapy involving the disruption of PD-1 and programmed death receptor ligand1 (PD-L1) interaction allows for re-activation of tumor-infiltrating lymphocytes (TIL) to have anti-tumor function. This approach has shown clinical responses in a range of malignancies, but has been less efficacious in poorly immunogenic tumors.
To prevent PD-1-mediated dampening of CAR T cell function, we have co-modified CAR T cells to secrete PD-1 blocking single chain variable fragments (scFv). We first designed mouse constructs with which we could investigate the scFv-secreting CAR T cells in the context of a syngeneic immune-competent intact TME. CAR constructs were engineered directed against either human CD19 or MUC-16 (ecto) with mouse signaling domains and a anti-mouse PD-1 scFv. Mouse T cells transduced with these constructs expressed the CAR on the surface and secreted detectable amounts of scFv that bound to mouse PD-1. The scFv-secreting CAR T cells were cytotoxic and produced IFN-g when co-cultured with PD-L1 expressing tumors in vitro . We utilized a syngeneic mouse model to study scFv secreting CAR T cells in a model with an intact TME. In tumor-bearing mice treated with CAR T cells, scFv-secreting CAR T cells enhanced survival as compared to second generation CAR T cells. The survival benefit achieved with scFv-secreting CAR T cells was comparable to that achieved with systemic infusion of PD-1 blocking antibody, but with localized delivery of PD-1 blockade. Mice treated with scFv-secreting CAR T cells had detectable scFv in vivo in the TME. Lastly, long term surviving mice had detectable CAR T cells in the bone marrow by PCR, demonstrating persistence and suggesting an immunological memory.
We next aimed to translate PD-1 blocking scFv CAR T cells to a clinically relevant human model utilizing a novel anti-human PD-1 blocking scFv. CAR constructs were engineered with recognition domains directed against human CD19 or MUC-16 (ecto) and human signaling domains. Human T cells modified with the CAR constructs express the CAR on the surface and secrete detectable amounts of PD-1 blocking scFv. The scFv binds to human PD-1 and scFv-secreting CAR T cells are cytotoxic to PD-L1 expressing tumors. Expression of PD-1-blocking scFv enhances CAR T cell function against PD-L1 expressing tumors in xenograft models of hematological and solid tumors by enhancing survival in tumor-bearing mice as compared to second generation CAR T cells. Furthermore, scFv-secreting CAR T cells exhibit in vivo bystander T cell enhancement of function, suggesting scFv-secreting CAR T cells can reactivate endogenous TILs in the TME.
These data support the novel concept that localized delivery of scFv by CAR T cells can successfully block PD-1 binding to PD-L1 and work in an autocrine manner to prevent dampening of CAR T cell responses as well as a paracrine manner to activate endogenous tumor infiltrating lymphocytes to enhance the overall anti-tumor efficacy of CAR T cell therapy.

Disclosures
Curran: Juno Therapeutics: Research Funding; Novartis: Consultancy. Yan: Eureka Therapeutics Inc: Employment. Wang: Eureka Therapeutics Inc.: Employment, Equity Ownership. Xiang: Eureka Therapeutics Inc.: Employment. Liu: Eureka Therpeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.