Preclinical Development of a T-Cell ALL CAR Demonstrates That Differences in CAR Membrane Distribution May Impact Efficacy
Authors: Shalabi Haneen , Qin Haiying , Wanhainen Kelsey , Smith Jillian , Orentas Rimas , Shah Nirali N , Maude Shannon L , Tasian Sarah K , Teachey David T , Khan Javed , Fry Terry J 
Published: October 2019

Background: Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is an uncommon childhood leukemia that has been associated with very poor clinical outcomes in some studies. ETP-ALL cells arrest at a more immature differentiation stage than other T-lymphoblasts, and are hypothesized to retain multi-lineage differentiation potential, which may contribute to chemoresistance with standard lymphoid-directed therapy. Based on the recent clinical success of chimeric antigen receptor (CAR)-modified T-cells in children with B-ALL, we sought to identify potential surface protein targets on ETP lymphoblasts using differential gene expression analysis combined with a bioinformatic algorithm to predict surface expression.

Methods: Cell-surface targets on ETP-ALL were predicted by identifying overexpressed transcripts based on gene expression and a bioinformatic algorithm to predict surface expression. Using several gene expression platforms and reference databases, (Oncogenomics website-Pediatric Oncology Branch, NCI, Gene Expression Omnibus, Gene Ontology, Human Protein References Database) ETP-ALL samples were compared to peripheral blood mononuclear cell (PBMC) controls on an individual transcript basis. A list of the top 25 transcripts was generated based on cell surface proteins, and the resultant list ordered by the degree of difference from PBMC controls. We next used human leukemia cells from six established ETP-ALL patient-derived xenograft (PDX) models using flow cytometry to evaluate for cell surface expression of proteins encoded by the overexpressed transcripts. Additionally, since CD7 and CD33 expression on ETP-ALL patient samples is universal with minimal normal tissue distribution, we developed two new second-generation anti-CD7 or anti-CD33 CAR constructs using a 41-BB/CD3ζ backbone.

Results: Multiple gene transcripts encoding cell surface proteins potentially amenable to CAR T-cell targeting were overexpressed in ETP-ALL cells in comparison to PBMC controls. Many of these proteins are involved in cell signaling, cell adhesion, and metastasis, and thus potentially important for leukemic cell survival. TSPAN7 (also known as TALLA-1) was the strongest differentially expressed transcript. Despite identification of several transcripts, we did not detect increased surface expression of multiple antigens that were identified as top 25 transcripts, including TALLA-1, MCAM, EPHB6, or TSLPR. Interestingly, TALLA-1 was expressed on the more mature T-cell ALL lines, JURKAT and HPB-AU, suggesting that the surface expression of TALLA protein may be developmentally regulated. Although a new target could not be identified, given the universal expression of CD7 and CD33 on ETP-ALL, we proceeded with development of CARs targeting these antigens. CD33 CAR T-cells had excellent in vitro activity in human AML cell line MOLM-14 with minimal anti-leukemia activity in six tested ETP-ALL PDX models, perhaps due to their lower CD33 expression. We next tested T-cells transduced with a bicistronic CD7-redirected CAR with a truncated EGFR (EGFRt) to facilitate measurement of transduction efficiency and to provide a CAR deletion method. Despite high EGFRt surface expression in transduced T-cells, these CD7 CAR T-cells did not demonstrate in vitro activity against ETP-ALL or mature T-ALL samples despite high CD7 surface expression on all leukemia cell lines. We postulated that abnormal CAR distribution within the T-cell itself could be a potential factor in the observed lack of CD7 CAR T-cell activity. Using fluorescent-labeling to assess CAR surface membrane distribution, we detected high intracellular expression of the CD7 CAR, and noted that it did not traffic to the cell surface.

Conclusions: We applied multimodal techniques to evaluate for cell surface expression on ETP-ALL that could serve as a target for immunotherapy. Although novel targets could not be identified, we were able to design an active anti-CD33 CAR. Further studies are in progress to evaluate what degree of antigen expression is needed to be amenable to targeted therapy. Additionally, ongoing studies are assessing whether optimization of CAR design can enhance cell surface trafficking and thereby potentially improve the anti-leukemia efficacy of CD7 CAR T-cells.

Disclosures

Orentas: Lentigen Technology, Inc.: Employment. Maude:Novartis: Consultancy. Teachey:Novartis: Research Funding.