CD28 CAR T Cells for the Treatment of T Cell Malignancies

While synthetic immunotherapy has been introduced into the standard of care treatment of patients with B-lineage malignancies, patients with T-lineage malignancies have not gained benefit from this novel approach, yet. The main reason is the challenging choice of target antigen in T-lineage malignancies such as pediatric T cell acute lymphoblastic leukemia (T-ALL). As activating mutations of CD28 have already been described in T cell malignancies, we set out to quantify surface expression of CD28 and other costimulatory molecules on primary pediatric T-ALL samples and explore the utility of this functionally relevant molecule as a novel target antigen. Bone marrow (BM) samples of pediatric T-ALL patients (n=54) at time of diagnosis and healthy control individuals (n=14) were collected. We quantified surface expression of CD28 and 25 additional co-stimulatory or co-inhibitory molecules by flow cytometry after gating on T cell precursors (living singlets, CD45 dim/SSC-A low and CD7 +). Subsequently, we generated a set of 20 CD28 directed second-generation chimeric antigen receptors (CARs) based on five different monoclonal antibodies with variation in hinge domain and light chain / heavy chain chronology. We analyzed specific CAR activity against CD28 expressing T-ALL cell lines after retroviral transduction into primary human T cells. Additional CD28 knockout (KO) by CRISPR/Cas9 could prevent T cell fratricide. CAR constructs were subjected to in vivo testing in a T-ALL NSG mouse model with transplantation of 7.5e4 CCRF-CEM cells transduced with firefly luciferase on day 0 and transfer of 2.5e6 CD28 KO T cells on day 3 harboring either of both CD28 CAR molecules. We used CD7 CAR T cells as positive control, CD19 CAR T cells and CD28 KO T cells without CAR as negative controls. We observed significant upregulation of CD28 on T-ALL leukemia when compared to healthy BM donors (Figure 1A, mean 68.8% vs. 3.7%, p=0.0002). We confirmed upregulation of CD28 in published T-ALL RNA-expression data sets. Interestingly, other co-stimulatory molecules such as CD127 were upregulated, too (mean 50.0% vs. 14.6%, p=0.0001), while co-inhibitory molecules such as CD160, TIGIT and TIM-3 were strongly downregulated (>10fold and p