Development of Novel Chimeric Antigen Receptors Targeting the Gamma-Delta (γδ) T-Cell Receptor
Authors: Cutmore Lauren C , Lam Norris , Amatya Christina , Weissler Katherine A , Natrakul Danielle A , Kochenderfer James N 
Published: December 2024

γδ T-cell receptors (TCRs) are made up of γ and δ chains that are each composed of a constant region and a variable region. Several aggressive malignancies express γδ TCRs including hepatosplenic T-cell lymphoma and some cases of T-cell acute lymphoblastic leukemia. γδ T cells might also play a role in autoimmune diseases. New treatments are needed for γδ T-cell malignancies and autoimmune diseases.

Chimeric antigen receptors (CARs) are artificial proteins containing an antigen-recognition domain and cell signaling domains. We designed a series of CARs targeting the γδ TCR. To conduct experiments, we transduced primary human T cells with γ-retroviruses encoding CARs. We assessed CAR single-chain variable fragments (scFv), hinge+transmembrane (HTM) domains, and costimulatory domains.

To select the optimal scFv, we cocultured CAR-expressing T cells with target cells and performed interferonγ (IFNγ) enzyme-linked immunosorbent assays (ELISA) on culture supernatants. We selected CARs that caused the maximum antigen-specific T-cell activation as defined by the greatest fold-increase in IFNγ release of CAR T cells cocultured with γδ TCR-expressing target cells versus CAR T cells cultured alone. γδ TCR-expressing cell lines used were Loucy, MOLT13, and BE13. There was greater antigen-specific IFNγ release with T cells expressing a scFv designated MGDL than an scFv designated gdL. The MGDL-28Z CAR has the MDGL scFv, CD28 HTM, CD28 costimulatory domain, and a CD3ζ activation domain. The gdL-28Z CAR is identical except it has the gdL scFv. Ratios of IFNγ release of CAR T cells cocultured with γδ TCR-expressing target cells versus T cells cultured alone were higher for MGDL-28Z versus gdL-28Z when T cells were cocultured with Loucy (P=0.016) and MOLT13 (P=0.014). Statistics by 2-tailed ratio paired t test; n=4. We demonstrated that all CARs with MGDL or gdL scFvs recognized a γδ TCR constant region shared by all γδ TCRs.

We next assessed how the HTM domains affected antigen-specific IFNγ release. We compared a CAR designated MGDL-CD828Z to MGDL-28Z. These CARs are identical except MGDL-CD828Z has a CD8α HTM while MGDL-28Z has a CD28 HTM. The ratios of IFNγ release of CAR T cells cocultured with γδ TCR-expressing target cells versus T cells cultured alone were higher for MGDL-28Z versus MGDL-CD828Z when the CAR T cells were cultured with Loucy (P=0.018), MOLT13 (P=0.023), or BE13 (P=0.001). Statistics by 2-tailed ratio paired t test; n=7. In addition, interleukin-2 release was higher for T cells expressing MGDL-28Z versus T cells expressing MGDL-CD828Z when T cells were cocultured with Loucy (P=0.034), MOLT13 (P=0.006), or BE13 (P=0.026). Statistics by 2-tailed paired t test; n=7.

We assessed in vitro proliferation of T cells expressing CD28-containing CARs versus a 4-1BB-containing CAR, MGDL-CD8BBZ. Higher numbers of T cells expressing MGDL-CD828Z versus MGDL-CD8BBZ (P=0.030) accumulated after 4 days of coculture with γδ TCR-expressing target cells. Also, higher numbers of T cells expressing MGDL-28Z versus MGDL-CD8BBZ (P=0.033) accumulated after 4 days of coculture with γδ TCR-expressing target cells. Statistics were two-tailed paired t tests; n=3.

During in vitro cytotoxicity assays, T cells expressing MGDL-28Z, MGDL-CD828Z, and MGDL-CD8BBZ exhibited similar levels of cytotoxicity against Loucy and MOLT13.

To assess in vivo anti-tumor activity of CAR T cells, nod-scid common γ-chain knockout (NSG) mice were injected intravenously (i.v.) with either MOLT13 or BE13 cells. MOLT13 and BE13 expressed luciferase for bioluminescence imaging.

MGDL-28Z-expressing T cells eliminated established MOLT13 cells in a dose-dependent manner. Twenty-seven days after i.v. CAR T-cell infusion, the mean MOLT13 burden measured by bioluminescence was lower for mice treated with 8x106 versus 0.5x106 CAR T cells (P=0.039; 2-tailed unpaired t test).

BE13 burdens were established in mice, and the mice received an i.v. infusion of MGDL-28Z-expressing T cells or were untreated. There was longer survival for BE13-bearing mice receiving a dose of 5x106 T cells/mouse versus untreated mice (P=0.003) and mice receiving 2x106 T cells/mouse (P=0.002; log rank test; n=5 mice per group).

In summary, we developed CARs that recognized the constant region of the γδ TCR in vitro and eliminated malignancy from mice. These CARs could be used for clinical treatment of γδ TCR-expressing malignancies and possibly some autoimmune diseases.