Fig 2: PD-L1 blockade reinforces anti-HLA-G Nb-CAR-?dT to eliminate PD-L1-overexpressing immune escape variants. A) Schematic diagram of anti-HLA-G CAR construct. HLA-G-targeted Nb#1 and Nb#2 were ligated with CD8a hinge/TM, followed by a 4-1BB and a CD3? intracellular domains, which was driven by EF-1a promoter using lentiviral vector. B) Generation of immune escape variants following anti-HLA-G CAR-?dT challenge in vivo. Briefly, NSG mice (n = 5) were orthotopically implanted with luciferase-expressing MDA-MB-231 cells. After 7 d, the mice were infused with anti-HLA-G Nb-CAR-?dT cells (1 × 107) via tail vein injection weekly for 8 weeks. The mice were sacrificed at 63 days after the first challenge of Nb-CAR-?dT, and then residual tumors were harvested. Subsequently, the isolated cells were re-challenged with anti-HLA-G Nb-CAR-?dT at an E:T ratio of 3:1 for 72 h. C–E) Three immune escape variants generated from individual mice showed consistently upregulated PD-L1 levels. C) Bioluminescent signals were detected using IVIS after the MDA-MB-231 tumor-bearing mice were treated with or without HLA-G-targeted Nb-CAR-?dT cells. D) Bioluminescence of the residual clones (231-R1, -R2, -R3) was measured using IVIS. Parental MDA-MB-231 and MDA-MB-231-luc cells were used as the control (left). Expression levels of ICPs were determined through flow cytometry (right). E) PD-L1 expression in these clones was also confirmed by immunoblotting. F) Combination with atezolizumab enhanced anti-HLA-G Nb-CAR-?dT-induced cytotoxicity against the immune escape variants. Parental MDA-MB-231, 231-R1, -R2, and -R3 cells were pretreated with or without 10 µg mL-1 atezolizumab for 15 min and followed by coculture with or without parental or anti-HLA-G CAR-?dT cells for 48 h at an E:T ratio of 3:1. Subsequently, we determined specific lysis through a LIVE/DEAD cell-mediated cytotoxicity assay. G-I) PD-L1 blockade reverses the sensitivity of 231-R3 tumor cells to anti-HLA-G CAR-?dT challenge in vivo. G) NSG mice (n = 5) were orthotopically implanted with 231-R3 cells. After 10 d, the mice were injected with or without atezolizumab (20 mg kg-1) combined with parental or anti-HLA-G CAR-?dT weekly for 4 weeks through the tail vein. H,I) Bioluminescence of the 231-R3 tumors was measured (H,I), and J) survival rates were recorded. These results suggested that PD-L1 is upregulated in persistent tumor cells after HLA-G CAR-?dT challenge, and the blockade of PD-L1 restored their sensitivity to HLA-G CAR-?dT cells. Results are representative of at least three independent experiments. Data represent the mean ± SD, n = 4; *p < 0.05; **p < 0.01; ***p < 0.001; and #, not detectable based on paired Student's t-tests. For tumor growth and survival rate comparisons, the Kaplan–Meier method and log-rank test were performed.

Fig 3: Expression of PD-L1 in NSCLC and TNBC cells interferes with their sensitivity to HLA-G targeted CAR-?dT cells. A,B) HLA-G and PD-L1 were frequently expressed in the same lung (n = 24) and TNBC (n = 30) tumor lesions. HLA-G and PD-L1 expressions were determined by 2-plexed IHC staining using specific antibodies (left). Scale bar = 20 µm of. HLA-G and PD-L1 expression levels in tumor lesions and normal adjacent tissues were shown as a tissue cytometry dot-plot (middle panel) and the quantified data are displayed (right panel). C,D) Expression levels of HLA-G and PD-L1 in lung cancer and breast cancer cell lines. C) A549 and H1975; D) MCF-7, T47D and MDA-MB-231 cells were harvested to evaluate the levels of HLA-G and PD-L1 by immunoblotting (left panels) and flow cytometry (right panels). E) PD-L1 blockade re-enforced cytotoxicity induced by Nb-CAR-?dT cells in HLA-Ghigh/PD-L1high tumor cells. MDA-MB-231 and H1975 cells were pretreated with or without 10 µg mL-1 atezolizumab for 15 min and then cocultured with parental or Nb-CAR-?dT cells (E:T = 3:1) for 48 h. F) PD-L1 antagonized HLA-G targeted CAR-?dT-induced cytolysis. Mock or PD-L1-overexpressed A549 cells were treated with or without 10 µg mL-1 atezolizumab for 15 min and then co-incubated with parental or Nb-CAR-?dT cells (E:T = 3:1) for 48 h. Specific lysis was performed by the LIVE/DEAD cell-mediated cytotoxicity assay using flow cytometry analysis. These results suggested that elevated PD-L1 levels on tumor cells may be a risk of immune escape from Nb-CAR-?dT therapy in solid tumors. Results are representative of at least three independent experiments. Data represent the mean ± SD, n = 3–4, *p < 0.05; **p < 0.01; and ***p < 0.001 based on paired Student's or Student's t-tests.

Fig 4: Nb-CAR.BiTE-?dT cells are effective to treat tumor cells with HLA-G and/or PD-L1 expression in vitro. A,B) Nb-CAR.BiTE-?dT cells effectively ablated HLA-G+ and/or PD-L1-overexpressed tumor cells. Parental, Nb-CAR, or Nb-CAR.BiTE-?dT cells were cocultured with 1 × 105 MDA-MB-231 or H1975 cells A) mock or PD-L1-stably overexpressing A549 cells B) at an E:T ratio of 1:1–10:1 for 72 h. Induced cytotoxicity was measured by LIVE/DEAD cell-mediated cytotoxicity assay C) Nb-CAR.BiTE-?dT cells have potent cytotoxicity against the PD-L1-overexpressing immune escape variants. We cocultured 1 × 105 parental MDA-MB-231, 231-R1, -R2 or -R3 cells with Parental, Nb-CAR, or Nb-CAR.BiTE-?dT cells at an E:T ratio of 3:1 for 72 h. Specific lysis was determined using LIVE/DEAD cell-mediated cytotoxicity assay. D) ?dT effector cells retain their natural anti-tumor effect on tumor cells with low levels of CAR or BiTE antigen expression. HLA-G and PD-L1 levels in H1975 and MDA-MB-231 cells were detected through flow cytometry following HLA-G or PD-L1 stable knockdown (left panel). HLA-G- or PD-L1-knockdown H1975 and MDA-MB-231 cells (1 × 105) were co-incubated with parental, Nb-CAR-expressing, or Nb-CAR.BiTE-expressing ?dT cells at an E:T ratio of 1:1, 2:1, 3:1, and 6:1 for 72 h. Subsequently, specific lysis of target cells was determined by a LIVE/DEAD cell-mediated cytotoxicity assay. E,F) PD-L1 on tumor cells determines the sensitivity of secreted Nb-BiTE, and both HLA-G and PD-L1 expression affect the cytolysis induced by ?dT cells. E) Schematic illustration of non-contact coculture system for Nb-CAR.BiTE-?dT cells, which release Nb-BiTE to engage ?dT cells against H1975 cells. F) H1975 cells were transfected with or without siHLA-G and/or siPD-L1 for 48 h, then seeded in the bottom wells (1 × 105 cells per well), and then cocultured with parental or Nb-CAR-?dT cells (E:T = 1:1) with or without Nb-CAR.BiTE-?dT cells in the top transwell inserts (5 × 105 cells per insert) for 72 h. Subsequently, the specific lysis of target cells was examined using a LIVE/DEAD cell-mediated cytotoxicity assay. These results suggested that Nb-CAR.BiTE-?dT cells are effective to against HLA-G and/or PD-L1 positive tumor cells even antigens are low expressed. Results are representative of at least three independent experiments. Data represent the mean ± SD, n = 4, *,#,X p < 0.05; **,##,XX p < 0.01; ***,###,XX X p < 0.001; stars represent significant differences between parental and Nb-CAR.BiTE-?dT; pound signs represent significant differences between parental and Nb-CAR-?dT; and double daggers represent significant differences between Nb-CAR-?dT and Nb-CAR.BiTE-?dT based on paired Student's t-tests.

Fig 5: 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.