Relieving Restriction in Resting CD8+ T Cells Increases Gene Transfer and Improves Tumor Killing after Transduction with CD8-Targeted CD19CAR Fusosome
Authors: Ott Mariliis , Aamer Hadega , Humes Daryl , Burch Reuben , Proano Christina , Dolinski Brian , Vedenova Diana , Benson Ashley , White Ryan , Tareen Semih , Green Jesse , Elpek Kutlu G , van Hoeven Neal , Fry Terry J 
Published: December 2024

While chimeric antigen receptor (CAR) T cells have proven to be effective for the treatment of hematological cancers, currently approved autologous CAR T therapies rely on individual manufacturing runs involving apheresis and cellular expansion prior to lymphodepletion and infusion. We have been developing an off-the-shelf therapy to generate CD19CAR T cells by in vivo delivery of targeted lentiviral vectors which would avoid these drawbacks. One advantage of in vivo delivery of a CD8-targeted CD19CAR fusosome is its potential to transduce and generate CD19CAR T cells from resting CD8+ T cell subsets that maintain plasticity and expansion potential beyond that of ex vivo manufactured CAR T cells. However, resting T cells express several cellular restriction factors that render them difficult to transduce. Restriction factors have been studied extensively in CD4+ T cells, but their role in CD8+ T cells is less defined. While we found that CD8-targeted fusosome transduces multiple phenotypic subsets of resting CD8+ T cells, including naïve, effector, and effector memory cells, we hypothesized that overcoming restriction in these cells could lead to greater gene transfer and generation of CD19CAR T cells.
Here, we evaluated the role of key restriction factors in resting CD8+ T cells by performing CRISPR/Cas9 knockout studies. We identified IFITM1 and SAMHD1 as critical factors that act through distinct mechanisms to block efficient transduction of resting CD8+ T cells by CD8-targeted fusosome. As part of an effort to increase transduction efficiency, we identified temsirolimus and IL-7 as clinically relevant pharmaceutical agents that lift restriction by IFITM1 and SAMHD1, respectively, in resting CD8+ T cells. Pre-treatment of resting CD8+ T cells with temsirolimus and IL-7 alone or in combination prior to transduction by CD8-targeted CD19CAR fusosome resulted in significantly increased gene transfer and surface expression of CD19CAR (At 10 SupT1 IU/CD8, the increase in percent transduction was on average 2.4-fold with IL-7, 10.3-fold with temsirolimus, or 25.0-fold with the combination, using cells from 6 donors).
While IL-7 supports T cell expansion and function apart from regulating SAMHD1 activity, temsirolimus is an immunosuppressive molecule that inhibits mTOR and therefore can interfere with cytotoxic function, expansion, and survival of T cells. We tested the functional effect of temsirolimus on resting CD8+ T cells prior to transduction and exposure to NALM6 tumor cells. Pre-treatment with temsirolimus reduced CAR-driven cytotoxic ability and expansion of transduced resting CD8+ T cells compared to untreated cells from two donors.
To identify a novel pharmaceutical agent that could relieve IFITM1-mediated restriction in CD8+ T cells without negatively affecting cytotoxic function, we performed a high-throughput screen of clinically-approved small molecules for their ability to increase transduction in resting T cells. Amphotericin B was identified as an agent that led to IFITM1 degradation in resting CD8+ T cells and increased their transduction by fusosome (At 5 SupT1 IU/T cell, percent transduction was increased by an average of 5.7-fold using cells from 4 donors). In addition, pre-treatment of resting CD8+ T cells with amphotericin B prior to transduction by CD8-targeted CD19CAR fusosome led to an increased ability to control NALM6 tumor cell growth over a serial challenge assay without a delay in cytotoxicity or expansion.
Avoiding lymphodepletion and the complexities of autologous CAR T manufacturing makes in vivo delivery of a CD8-targeted CD19CAR fusosome an attractive option for the treatment of B cell malignancies and other B cell-mediated diseases. The findings reported here further our understanding of resting CD8+ T cell transduction, informing the potential use of pharmaceutical interventions or vector modifications to overcome restriction and increase the potency of a CD8-targeted fusosome.