Novel CD2-Targeting Trispecific Immune Effector Cell Engager (TRIEC) Coupled with CD19 CAR-T Overcomes Resistant B-Cell Lymphoma

Background: Loss or down-regulation of CD19 on B-cell lymphomas is a well described resistance mechanism to CD19-directed chimeric antigen receptor (CAR) T-cell therapies. Loss of CD58, ligand for CD2 on T-cells, has recently been identified as another mechanism of resistance to CD19-directed CAR-T cells. CD2 is a costimulatory receptor on T-cells, present in the immune synapse and important for T-cell activation and adhesion to antigen-presenting cells. We report here findings of a novel CD19-directed CAR design incorporating a secreted trispecific immune effector cell engager (TRIEC) consisting of three single chain variable fragments (scfv) targeting CD79b, CD2, and CD3 connected by glycine(4)serine linkers.

Methods: Tumor models of resistant malignant B-cell lines were made through modulation or knockout of CD19 expression, with or without concomitant knockout of CD58 in Jeko-1 (Mantle Cell Lymphoma), Nalm6 (Acute Lymphoblastic Leukemia), and Raji (Burkitt lymphoma) cell lines. Primary human T-cells were transduced with a lentivirus encoding for second generation CD19 CAR and TRIEC molecule, CD19 CAR alone, or CD19 CAR with null TRIEC absent CD2 binding (CD2 scfv replaced with EGFR scfv).

Results: CD19-directed second generation CAR T cells demonstrate reduced cytotoxicity and lower levels of cytotoxic cytokines when exposed to CD19low, CD19low/CD58KO, and CD19KO B-cell tumor lines compared to CD19/CD58 unmodified parental B-cell lines in vitro, and progressive decrement in tumor clearance of CD19low, CD19low/CD58KO, and CD19KO B-cell tumors in vivo. TRIEC molecules show specific binding of CD79b, CD2 (TRIEC only), EGFR (null TRIEC only) and CD3 antigen targets as assessed by anti-glycine(4)serine linker antibody. Untransduced T-cells co-cultured with Jeko-1 cells demonstrate increased cytolysis in the presence of TRIEC-containing supernatant vs null supernatant (p<0.0001). CD19 CAR, CD19 CAR + null TRIEC, and CD19 CAR + TRIEC constructs demonstrate equivalent activation and proliferation in the absence of tumor. CD19 CAR/TRIEC constructs demonstrate superior in vitro cytotoxicity against parental (p=.004), CD19low/58KO (p=.0011), and CD19KO (p=.0002) Jeko-1 and Raji 19low/58KO (p<.0001) cell lines compared to CD19 CAR and CD19 CAR/null TRIEC constructs.

Conclusion: Harnessing CD2 signaling through incorporation of a first-in-class secreted trispecific CD79b/CD2/CD3 TRIEC enhances killing of CAR-resistant lymphoma models. This is the first use of a cell-secreted trispecific CD2/CD3 T-cell engaging molecule (TRIEC). CD19 CARs secreting TRIECs are effective against CD19 low/negative and CD58KO B-cell lines. Incorporation of CD2 binding into the TRIEC molecule increases cytotoxicity without eliciting nonspecific activation or unstimulated proliferation of CAR-T cells in the absence of tumor. In vivo studies are ongoing to evaluate CAR + TRIEC efficacy in NOD-Scid-common gamma chain knockout (NSG) mice engrafted with CAR-resistant B-cell tumors. Incorporation of CD2 signaling into CAR-T cells represents an attractive addition to circumvent tumor-intrinsic resistance mechanisms to current CD19-directed CAR-T cell therapies.