Anti-T Cell Receptor Vβ Chain-Specific CAR-iNKT Cells As a Precise Strategy to Treat Mature T Cell Lymphomas

Background Most mature T cell malignancies including those caused by Human T cell Leukemia virus type 1 (HTLV-1) have a poor prognosis. To address the pressing clinical need for new therapeutic approaches, we developed chimeric antigen receptors (CAR) which target a group of subunits of the T cell receptor (TCR) β chain that are constitutively, stably and clonally expressed by mature T cell malignancies. As expression of each TCRVβ subunit is restricted to <9% of healthy T cells, this approach would preserve >90% of the healthy T cell repertoire, while the use of invariant natural killer T (iNKT) cells as effectors would allow delivery of an allogeneic 'off-the-shelf treatment’ without need for TCR deletion.

Methods We generated second-generation CD8α leader peptide-VL-linker-VH-CD8α hinge-CD8αTM-CD28-CD3ζ CAR lentiviral constructs targeting TCRVβ1, 2 and 9. CARs were transduced into anti-CD3/CD28-activated bulk peripheral blood mononuclear cells (for CAR-T effectors) or iNKT cells purified using 6B11 mAb-coated magnetic beads. CAR effectors were expanded by culturing in the presence of 150 U/ml IL-15. CAR targets were in vitro-expanded primary T cells which had been magnetically purified on the basis of TCRVβ expression or JRT3 T3.5 T acute lymphoblastic leukemia (T-ALL) cells transduced to express a TCRβ chain of interest. Cytotoxicity of CAR-transduced effectors was assessed by quantifying the frequency of 7AAD+ CFSE-labelled targets after 4-24h culture in the presence or absence of CAR-T or CAR-iNKT effectors. The frequency of pentamer-stained T cells and the expression of cytokines, granzyme, perforin, and HTLV-1 Tax were quantified by flow cytometry. We established a xenograft mouse model of T cell lymphoma by subcutaneous injection of 5x10⁶ TCRVβ2-transduced JRT3 T3.5 cells with matrigel into the flank of NSG mice (n=5-7). Mice were either left untreated or were treated with 1x10⁶ anti-Vβ or anti-CD19 CAR-iNKT intravenously when palpable tumors became evident (day 18). Tumor size and body weight were measured weekly until the experiment was terminated (day 48) and the excised tumors were weighed.

Results As a proof-of-concept, we developed CARs specific for TCRVβ1, 2 and 9. We found that anti-Vβ CAR-T cells efficiently and selectively killed normal T cells and JRT3 T3.5 targets that expressed the cognate TCRVβ while secreting tumoricidal cytokines and leaving >90% of the physiological TCR repertoire intact. Using iNKT cells as CAR effectors, we demonstrated that ligation of the anti-Vβ CAR induced degranulation (CD107a mobilization), secretion of IFN-γ and TNF-α, and enhanced expression of perforin and granzyme by iNKT effectors. Anti-Vβ CAR-iNKT could kill in vitro-expanded primary T cells, JRT3 T3.5 T-ALL targets expressing the cognate TCRVβ and ex-vivo Adult T cell leukemia (ATL) cells (n=2 patients) which express the corresponding TCRVβ subunit, with minimal off-target killing of T cells which express other TCRVβ subunits. CAR-iNKT cytotoxicity, but not CAR-T cytotoxicity was significantly enhanced when challenged with CD1d-expressing targets that had been pulsed with the iNKT cell-selective antigen α-Galactosylceramide. Importantly, targeting individual TCRVβ subunits in T cells in the blood of HLA-A*0201 HTLV-1 carriers (n=3) spared influenza virus and HTLV-1-specific CTL as assessed by HLA-A*0201/peptide pentamer staining and did not activate viral gene expression in HTLV-1-infected T cells as assessed by Tax staining. Finally, anti-Vβ CAR-iNKT but not anti-CD19 CAR-iNKT significantly (p<0.01) reduced tumor size in a subcutaneous model of T cell lymphoma. Tumor-bearing mice that received iNKT effectors showed no signs of graft-versus-host disease.

Conclusion We provide proof-of-concept of effective and selective anti-TCRVβ CAR-T and -iNKT cell therapy for TCRβ chain-expressing T cell malignancies with CAR-iNKT being suitable for allogeneic, off-the-shelf immunotherapy.

Ponnusamy:Arovella Therapeutics: Current equity holder in publicly-traded company. Cook:Abbvie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Karadimitris:Arovella Therapeutics: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties, Research Funding.