Fig 1: Characterization of the Nb-CAR.BiTE construct. A) Schematic representation of mRNA and lentiviral HLA-G Nb-CAR, with or without combined secretable PD-L1/CD3e-targeted Nb-BiTE constructs. The bi-epitopic Nb-CAR comprises two tandem extracellular HLA-G Nbs fused with a CD8a hinge/TM, followed by a modified 4-1BB cytosolic domain (4-1BB/Tyk) and a modified CD3? intracellular domain (CD3?/ITAM) linked to a self-cleavage peptide P2A to separate the secretable bivalent Nb-BiTE, which includes two PD-L1 Nbs linked to a CD3? Nb. B) The blocking activity of HLA-G Nbs. The blockade activity of HLA-G Nb clones #1 and #2 or HLA-G mAb 87G to LILRB1/HLA-G (upper panels) and KIR2DL4/HLA-G (bottom panel) were determined by competitive ELISA. C) HLA-G Nb moiety capable to bind to all HLA-G isoforms. The binding activity of HLA-G Nb clones and it derived bi-epitopic Nb moiety to recombinant HLA-G-ß2M (corresponding to HLA-G G1 and G5), HLA-G a1a3 domains (G2 and G6), HLA-G a1a2 domains (G4) and HLA-G a1 domain (G5) were examined by ELISA-based binding assay. D) PD-L1 blockade Nbs. The activity of PD-L1 Nb clones #1 and #2 was determined by PD-L1/PD-1 blockade bioassay. E) Biofunction and affinity of CD3e Nb. PBMCs (1 × 106 cells) were treated with/ without 100 ng CD3e Nb or OKT3. The enriched ?dT cells from PBMCs were supplemented with 1000 IL mL-1 IL-2 and then treated with or without 100 ng CD3e Nb or OKT3. After 6 d, the images were taken (upper panel), Scale bar = 20 µm, and the cell numbers were counted and normalized to the CD3+ cell populations (bottom panel). Evaluation of the novel CAR signaling cassette. F) Bi-epitopic HLA-G Nb-CARs were engineered using 4-1BB-CD3? (BB-3z) or modified signaling cassettes (IFNAR/BB-3z or BBTyk-3z). G) BB-3z-, IFNAR/BB-3z-, or BBTyk-3z-based HLA-G Nb-CAR lentiviral particles (MOI = 3) were transduced into ?dT cells, and after three days, the expression of Nb-CAR was determined by flow cytometry. H,I) Parental and BB-3z-, IFNAR/BB-3z-, or BBTyk-3z-Nb-CAR-?dT cells were cocultured with MDA-MB-231 cells at an E:T ratio of 3:1. H) After 2 h, the phosphorylation status of STAT2 and Syk/ZAP70 in the CAR-?dT cells was determined; and I) after 24 h, the induced cytotoxicity was determined using a LIVE/DEAD cell-mediated cytotoxicity assay (upper left panel); CD107a expression in CAR-?dT cells was detected by flow cytometry using specific antibodies (bottom left panel); and their supernatants were harvested to detect the contents of granzyme B, IFN-?, TNF-a, and IL-17A by ELISA. These data demonstrated that the elements for engineering the Nb-CAR.BiTE construct were functional and may be superior than that of the conventional CAR design. Results are representative of at least three independent experiments. Data represent the mean ± SD, n = 3; *p < 0.05; **p < 0.01; and ***p < 0.001 based on paired Student's t-test.