title: Refined generation of chimeric antigen receptor T cells by dasatinib and urolithin A
Creators: name: Braun, Angela
Publisher: name: UNSPECIFIED
Year: 2024

description: In recent years, genetic modification of T lymphocytes has revolutionized the treatment of certain types of hematopoietic cancer. Equipping T lymphocytes with chimeric antigen receptors (CARs) directed towards tumor-associated antigens (TAAs) has shown great clinical success in tumor cell elimination. However, there are still limitations to CAR T cell therapy that hinder its broader application and clinical efficacy. These limitations include the laborious manufacturing process and the quality of the resulting CAR T cells in terms of their efficacy and persistence. During manufacturing, the transduction of T cells, most often using lentiviral vectors (LVs), comprises extensive isolation and activation of T cells to ensure safe and efficient gene delivery. To reduce the risk of unwanted transduction and allow transduction of non-activated T cells, T cell-targeted LVs have been developed. Among these, CD3-LV is of potential relevance since the CD3 receptor in complex with the T cell receptor (TCR) is exclusively expressed on T cells and CD3-LV has been shown to activate and transduce non-activated T cells. However, CD3-LV-induced T cell activation increases CD3:TCR complex downregulation, eventually dampening gene transfer rates and preventing its clinical application. 
Regarding the quality of the CAR T cell product, CAR T cell activity is often hampered by limited engraftment and persistence caused by differentiation and exhaustion of the CAR T cells. Therefore, CAR T cells with the favorable T stem cell memory (TSCM) phenotype, have shown to be superior over conventional CAR T cells, as they greatly expand in vivo and have prolonged antitumor response. So far, attempts to increase the frequency of CAR TSCM cells have been restricted to introducing additional steps to the manufacturing process, such as cell sorting, enrichment, and reprogramming. 

To address the two limitations explained above, the small molecules dasatinib and urolithin A (UA) were used to help refine the generation of CAR T cells. Dasatinib is a tyrosine kinase inhibitor (TKI) known to inhibit phosphorylation by the T cell specific tyrosine kinase Lck and thereby preventing T cell activation. UA on the other hand, has been shown to induce mitophagy which is associated with immunomodulatory effects such as an enhanced antitumor efficacy.

Incubation of activated peripheral blood mononuclear cells (PBMCs) with dasatinib prior to transduction with CD3-LV(GFP) significantly increased transduction efficiency by 3- to 10-fold in a time- and dose-dependent manner. The maximal enhancing effect was achieved when 50 nM dasatinib was applied for five hours during transduction, resulting in over 60% transduced cells. The transduction enhancing effect of dasatinib, did not impair viability or cell proliferation and was further increased when combined with the transduction enhancer vectofusin-1 (VF-1). Importantly, dasatinib also increased the transduction of cytokine-only stimulated PBMCs and non activated PBMCs in whole blood. In an in vivo approach, treatment with dasatinib did not interfere with the selectivity of CD3-LV, and one mouse treated with dasatinib prior to CD3-LV administration showed a higher transduction level and vector genome integration compared to untreated mice. While dasatinib increased transduction efficiency of CD3 LV in all these setups, it had no effect on transduction using CD4-, CD8- or VSV LV. The inhibition of endocytosis or other tyrosine kinases did not enhance transduction by CD3-LV. Instead, preventing CD3-LV-induced T cell activation with dasatinib increased CD3 receptor availability and improved CD3-LV binding to the cells, most likely being the cause for dasatinib’s enhancing effect. When CD19.CAR was delivered instead of the gfp reporter gene, dasatinib had the same effect on enhancing CD3-LV transduction. The presence of dasatinib during transduction with CD3-LV resulted in CAR T cell levels similar to those achieved with VSV-LV. These CAR T cells were able to efficiently kill tumor cells while expressing lower levels of exhaustion markers and exhibiting a slightly more naïve phenotype. While tumor cell killing was not affected by dasatinib, the secretion of pro-inflammatory cytokines during killing was reduced for CAR T cells generated in presence of dasatinib, regardless of whether they were transduced with CD3- or VSV-LV. Thus, dasatinib not only reduced CD3-LV-induced stimulation, resulting in increased CD3 LV-mediated gene transfer, but also reduced transduction-induced stimulation by LVs in general. 
The second part of this thesis focused on the effect of UA on T cells and the generation of CAR T cells. Cultivation of T cells directly after transduction with LVs in medium containing 25 µM UA resulted in a 10-fold expansion of TSCM cells which among CAR-expressing T cells resulted in 50% TSCM cells. This shift in phenotype is most likely attributed to the induction of mitophagy. Cultivation in UA containing medium had no effect on the transduction efficiency or CAR expression. The high proportion of CAR TSCM cells did not impair killing efficacy of neither CD19.CAR nor CEA.CAR T cells. Accordingly, the transduction enhancing effect of dasatinib with CD3-LV was combined with the expansion of CEA.CAR TSCM cells by UA, which allowed the selective transduction of 40% of the T cells, of which 30% showed to have the TSCM phenotype, resulting in a high proportion of functional CAR TSCM cells. 

This work represents the first description of dasatinib as transduction enhancer for CD3-LV thus identifying a new class of transduction enhancers. Dasatinib is a member of a here firstly described class of transduction enhancers that do not act by facilitating vector-cell interaction outside the cell, but instead increase the availability of the target receptor through intracellular mechanisms, in this case Lck inhibition. Thereby, dasatinib prevents CD3:TCR downregulation, increases the binding of CD3 LV to the cell and by fusion of more CD3-LV particles with the cell, helps evade possible intrinsic restriction factors (RFs). 
In addition, a simple protocol for the expansion of CAR TSCM cells was developed in this work. The cultivation of cells in UA containing medium significantly increases the amount of CAR TSCM cells and provides a simplified method to enrich TSCM cells compared to methods described so far. This demonstrates the ability of UA to reprogram human T cells towards the TSCM phenotype by inducing mitophagy, thereby enhancing T cell fitness and providing a valuable source for future tumor therapies.
Taken together, the results obtained in this work demonstrate the potency of small molecules to further refine CAR T cell generation by representing a viable approach to facilitate gene delivery and generate a favorable CAR T cell phenotype, helping to improve therapeutic outcomes.