Development of chimeric antigen receptors targeting T-cell malignancies using two structurally different anti-CD5 antigen binding domains in NK and CRISPR-edited T cell lines

Abstract

Relapsed T-cell malignancies have poor outcomes when treated with chemotherapy, but survival after allogeneic bone marrow transplantation (BMT) approaches 50%. A limitation to BMT is the difficulty of achieving remission prior to transplant. Chimeric antigen receptor (CAR) T-cell therapy has shown successes in B-cell malignancies. This approach is difficult to adapt for the treatment of T-cell disease due to lack of a T-lymphoblast specific antigen and the fratricide of CAR T cells that occurs with T-cell antigen targeting. To circumvent this problem two approaches were investigated. First, a natural killer (NK) cell line, which does not express CD5, was used for CAR expression. Second, CRISPR-Cas9 genome editing technology was used to knockout CD5 expression in CD5-positive Jurkat T cells and in primary T cells, allowing for the use of CD5-negative T cells for CAR expression. Two structurally distinct anti-CD5 sequences were also tested, i) a traditional immunoglobulin-based single chain variable fragment (scFv) and ii) a lamprey-derived variable lymphocyte receptor (VLR), which we previously showed can be used for CAR-based recognition. Our results show i) both CARs yield comparable T-cell activation and NK cell-based cytotoxicity when targeting CD5-positive cells, ii) CD5-edited CAR-modified Jurkat T cells have reduced self-activation compared to that of CD5-positive CAR-modified T cells, iii) CD5-edited CAR-modified Jurkat T cells have increased activation in the presence of CD5-positive target cells compared to that of CD5-positive CAR-modified T cells, and iv) although modest effects were seen, a mouse model using the CAR-expressing NK cell line showed the scFv-CAR was superior to the VLR-CAR in delaying disease progression.

CAR-Expressing Natural Killer Cells for Cancer Retargeting

“…Approaches directed against this antigen face the same difficulties as CD7-directed approaches since CD5 is also a pan T-cell marker. Two studies showed that NK-92 expressing CD5-directed CARs were efficient in vitro against CD5-positive cell lines and primary tumour cells, and developed anti-tumour activity in vivo [33, 34]. Additionally, the use of anti-CD3 CAR-equipped NK-92 against primary peripheral T-cell lymphoma cells and T-cell leukaemia cell lines was investigated showing cytotoxic activity in vitro and against Jurkat cells in a xenogeneic mouse model [35].…”

Targeting T cell malignancies using CAR-based immunotherapy: challenges and potential solutions

“…Various approaches have been used to overcome these challenges, including CRISPR-Cas9 genome editing to remove the antigen from the CAR T cells [45][46][47], Tet-OFF expression system to limit fratricide during ex vivo expansion [48], protein expression blocker (PEBL) to retain the antigen in the ER/Golgi to prevent cell surface expression [49,50], or using CAR-modified natural killer cells instead of T cells [47,[51][52][53][54]. Additionally, to date, four targets have been investigated as targets for CAR T cell therapy for the treatment of T cell malignancies with limited to no expression in the normal population of T cells, CD30, CD37, TRBC1, and CD1a [55][56][57][58].…”

“…As a result, fratricide was observed only transiently, allowing the CD5-CAR T cells to expand. These cells had significant Genome editing of target antigen [45][46][47] Targeting antigens with limited expression on T cells (e.g., CD30, CD37, TRBC1, CD1a) [55][56][57][58] Tet-OFF expression system [48] Protein expression blockers (PEBLs) [49] Using NK cells or NK-92 cells [47,[51][52][53][54]60] T cell aplasia Targeting antigens with limited expression on T cells (e.g., CD30, CD37, TRBC1, CD1a) [55][56][57][58] mRNA electroporation…”

“…Allogeneic CAR T cells with TRAC locus editing [46,61] Using NK cells or NK-92 cells [47,[51][52][53][54]60] Using γδ T cells in vitro cytotoxicity against two T-ALL cell lines and primary T-ALL cells and delayed leukemia progression in two different CD5-positive T-ALL models [59]. Based on these results, CD5-CAR T cells with a CD28 costimulatory domain are being tested in patients with relapsed or refractory T cell disease (MAGENTA trial, NCT03081910).…”

Cellular therapy: Immune‐related complications

“…Allogeneic CD7 directed CAR‐T that have been modified by CRISPR/Cas9 to not express CD7 are in development for T‐cell malignancies . In addition, CAR‐T directed at CD2, CD5, and CD38 are under development for T‐cell cancers, including T‐ALL . CD123 and CD33 directed CAR‐T approaches are being explored in acute myelogenous leukemia (AML) .…”