Glycopeptide-Specific Chimeric Antigen Receptor Targeting of T Cell Leukemia

Background

Adoptive transfer of T cells redirected with chimeric antigen receptors (CARs) has shown tremendous efficacy in targeting CD19-expressing leukemic cells; however, these CAR T cells also deplete normal CD19-expressing B cells. Although B cell lymphopenia can be overcome by gamma-globulin supplementation, other forms of on-target, off-site toxicity emphasizes the need for cancer cell-specific epitopes in CAR T cell therapy. To date, T cell leukemia has not been targeted due to possible fratricide because of shared antigens expressed on leukemic T cells and CAR T cells. Cellular transformation often produces altered glycosylation patterns, making cancer-specific glycoantigens attractive targets for CAR development. Here we demonstrate the successful eradication of the Jurkat T cell leukemia with Tn-specific CAR T cells.

Methods

The Jurkat E6.1 T cell leukemia line contains a frameshift mutation in the T synthase chaperone protein Cosmc that prevents core 1 structure formation on O-linked protein glycosylation, resulting in the presentation of Tn antigen on many glycoproteins. Here we describe the development of a CAR using a scFv sequence derived from the 5E5 mAb, which was developed against the Tn antigen of Mucin-1 (Muc1). A CAR was constructed by linking the 5E5 scFv to CD8alpha hinge and transmembrane region to the signaling endodomain comprised of 4-1BB and CD3zeta. The resultant Tn specific CAR was expressed in a lentiviral vector.

Results

In preliminary experiments Tn-CAR T cells had cytotoxicity to a variety of primary ovarian, breast and pancreatic tumor cells. Specific killing of Jurkat T leukemic cells was demonstrated by lysis of wild type Jurkat while the CAR T cells failed to kill Jurakat after overexpression of Cosmc. In specific, expression of full-length Cosmc expression in the Jurkat E6.1 cell line abolished staining by the 5E5 mAb, restoredT synthase activity, and protected the cells from lysis by 5E5-CAR T cells. Human T cells expressing the 5E5-CAR demonstrate rapid clearance of established Jurkat tumors in NSG mouse xenograft models, as assessed by serial bioluminescence imaging and prolonged survival compared to mice expressing a control CD19-specific CAR T cells (P<0.005).

Conclusions

These results demonstrate that the 5E5-CAR is specific for the Tn antigen and provides a potent cancer-specific immunotherapy for the targeting of non-dispensable tissues such as T cell leukemias.