Fig 2: ILT3 × CD3 bispecific T cell engager antibody for MM(A) Overall survival of patients with de novo MM based on ILT3 expression. Patients with an ILT3 expression above the median gene expression present a statistically significant worse survival. Survival analysis was performed using the R package Survival with log rank test and hazard ratio statistical tests and the MMRF cohort, as we previously described.14(B) T cells isolated from healthy donors were activated with CD3/CD28 beads and co-cultured with U266 MM cells expressing an empty vector (controls) or ILT3 knockout (KO) U266 MM cells for 48 h at a target:effector 1:2 ratio. CFSE-positive T cells were counted by flow cytometry. ∗∗p ≤ 0.01. Data represent the results of three independent biological experiments. Statistical analyses were performed using Student’s t test.(C) Schematic representation of the bivalent anti-ILT3 × CD3 bispecific antibody that we developed. T cell engager was designed on a human IgG1 backbone. The bivalent tandem anti-ILT3 Fabs are positioned on the Knob Fc, and the anti-CD3 scFv is positioned on the Hole Fc. The anti-ILT3 Fab targeting arm contains humanized variable heavy (vH) and variable light (vL) sequences that were selected from an antibody campaign against the extracellular domain of human ILT3. Data represent the results of three independent biological experiments.(D) Killing assays of 3 RFP-labeled MM cell lines (U266, H929, and MM1.S cells). Cells are co-cultured with T cells (target:effector [T:E] ratio 1:5) and treated with ILT3 × CD3 antibody or control antibody (anti-KLH). A caspase cleavage domain coupled to a green DNA-binding fluorescent label is released upon cleavage by activated caspase 3/7 and quantified.(E) We generated a BCMA-KO U266 cell line and determined the killing ability of the ILT3 × CD3 antibody in co-culture with healthy T cells. The cell line was RFP labeled as described for (D).(F) T cells isolated from healthy donors were co-cultured with primary samples from patients who relapsed post-BCMA-CAR T cell therapy. The ILT3 × CD3 antibody was added to the culture, and T cell activation was measured by CD25 expression with flow cytometry. Percentage of CD3+CD25+ T cells was compared between the group treated with the ILT3 × CD3 antibody and the group treated with the control antibody (anti-KLH). Untreated cells and T cells alone served as additional controls. ∗∗p ≤ 0.01. Statistical analyses were performed using Student’s t test.(G) In vivo treatment with ILT3 × CD3 antibody (1 mpk). NSG mice were injected with MM U266 cells and healthy T cells from healthy volunteers and treated with 4 weekly doses of ILT3 × CD3 antibody or control IgG. Lambda chain concentration in mice serum by ELISA at multiple time points served as measurement of the tumor burden. ∗p ≤ 0.05, ∗∗p ≤ 0.01. Data represent the results of two technical replicates. Statistical analyses were performed using Student’s t test.(H) Overall mice survival from the experiment described in (G). ∗p ≤ 0.05. Statistical analyses were performed using log rank (Mantel-Cox) test.(I) NSG mice were injected with BCMA-KO MM U266 cells and healthy T cells from healthy volunteers and treated with 4 weekly doses of ILT3 × CD3 antibody (1 mpk) or control IgG. Lambda chain concentration in mice serum by ELISA at multiple time points served as measurement of tumor burden. ∗p ≤ 0.05, ∗∗p ≤ 0.01. Data represent the results of two technical replicates. Statistical analyses were performed using Student’s t test.(J) Overall mice survival from the experiment described in (I). ∗p ≤ 0.05. Statistical analyses were performed using log rank (Mantel-Cox) test.Data in (D)–(G) and (I) represent means ± SD.

Fig 3: Validation step in primary patient samples(A) Target expression levels normalized by the loading control (VCP) based on western blot validation in 31 MM patient samples.(B) Frequency of target expression in primary MM patient samples by western blot.(C) Representative western blot (WB) data of 11 top candidate targets in MM primary patient samples. Numbers refer to patient samples used. VCP serves as loading control. Complete set of data in Figure S2A.(D) Target expression in purified healthy hematopoietic stem cells by flow cytometry. Data representing the results of three independent biological experiments in primary healthy CD34+CD38−CD45RA−CD90+CD49f+ HSCs by flow cytometry. Gating strategy is shown in Figure S2C. Data represent means ± SD.(E) Target expression in freshly purified and activated T cells by flow cytometry. T cell activation is confirmed by measuring CD25 and HLADR upon treatment with CD3/CD28 beads at days 0, 4, and 5 (see Figure S2D). Data represent the results of three independent biological experiments. Data represent means ± SD.(F) Schematic representation of the stepwise pipeline utilized for target discovery and relative numbers of candidates obtained per step.

Fig 4: Gene-function validation in hypoxic PH mice.C57BL/6 mice exposed to hypoxia (10% O2) for 4 weeks exhibited higher RVSP (a) and RVHI (b) compared to that of mice in normoxia (21% O2) (n = 10 for per group, ***p < 0.001; Student’s t test. Data were presented as mean ± SD). c Representative images of hematoxylin and eosin (H&E) staining of lung tissues from mice under normoxic or hypoxic condition. Scale bars, 20 μm. d Assessment of pulmonary vascular remodeling by determination of the ratio of the media cross-sectional area to the total vessel cross-sectional area in each group (n = 10 for per group, ***p < 0.001; Student’s t test. Data were presented as mean ± SD). e The expression of Prf1, Spon2, Tbx21, Cd28 and Cd3e in lung tissues of the mice under normoxic or hypoxic condition at mRNA levels (n = 7–8 for per group, *p < 0.05, **p < 0.01; Student’s t test. Data were presented as mean ± SD). f–i Representative images of immunoblottings and the quantification of the expression of Cd28 (g), Spon2 (h) and Tbx21 (i) in lung tissues of the control mice and hypoxic PH mice at protein levels (n = 8 for per group, **p < 0.01, ***p < 0.001; Student’s t test. Data were presented as mean ± SD). j Immunoblottings for the expression of Cd28, Tbx21 and Spon2 in RAW264.7 monocytes/macrophages in response to CoCl2 (150 μmol/L) or vehicle for 24 h. k Immunoblottings for the expression of CD28, TBX21 and SPON2 in THP-1 monocytes in response to CoCl2 (150 μmol/L) or vehicle for 24 h. l Quantification of the expression of Cd28 (CD28), Tbx21 (TBX21) and Spon2 (SPON2) in RAW264.7 and THP-1 cells (n = 4 for per group, *p < 0.05, **p < 0.01; Student’s t test. Data were presented as mean ± SD). m Representative images of immunoblottings of Jurkat Clone E6-1 cells transfected with CD28 siRNA (siCD28) or control siRNA (siControl). n Viability of human PASMCs co-cultured with or without CD28-silencing or control Jurkat cells for 48 h (n = 5 for per group, *p < 0.05, **p < 0.01; one-way ANOVA, Tukey’s post-hoc test. Data were presented as mean ± SD). PH pulmonary hypertension, RVSP right ventricular systolic pressure, RVHI right ventricular hypertrophy index, PASMCs pulmonary arterial smooth muscle cells. Prf1 Perforin 1, Spon2, Spondin 2, Tbx21 t-box transcription factor 21.

Fig 5: Cytolytic activities of T cells expressing CARs with varied CD3-derived endodomains. a Schematic diagram of CARs constructed using varied scFvs and endodomains. b Human T cells isolated from PBMCs were infected with recombinant lentiviruses for indicated CARs, and the percentages of CAR-expressing cells were measured through FCM assay for the cotranslated GFP. H28Z refers to T cells expressing a CAR composed of a HER2 scFv, a CD28-derived costimulatory domain and an ICD from CD3 ζ chain, whereas others represent CARs containing an ICD from the δ (D), ε (E) or γ (G) chain of CD3, respectively. UTD, untransduced cells. c CAR-T cells were cocultured with PC-9 cells modified to express HER2 at varied E/T ratios for 16 h, and the percentages of cell lysis were calculated and plotted. d CAR-T cells were co-cultured with Dil-labeled PC-9 cells or those modified to express HER2 (E:T = 2:1) for 6 h, and cells were observed via fluorescence microscopy. Bar, 100 μm. e CAR-T cells and malignant cells were cocultured for 6 h (E:T = 1:1). FCM assay was used to evaluate cell degranulation marked by membrane CD107a levels and to measure the release of cytotoxic granules containing perforin and granzyme B. f HER2-targeted CAR-T cells were stimulated by incubation with PC-9 cells overexpressing HER2 for indicated times. Cells were then harvested for Western blot analysis. The intensities of the protein bands were quantified by the ImageJ software, and the ratios of phosphorylated to total protein levels were plotted. g Patient-derived organoids (PDOs) of HER2-positive ovarian cancer were established, followed by coculture with 3 × 105 CAR-T cells. The infiltration of CAR-T cells was imaged (red) 48 h later, and neoplastic cells undergoing apoptosis were visualized using a dye-conjugated substrate for active cleaved caspase 3/7 (green). Scale bars, 20 μm. Data are representative images and expressed as the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001; ns, non-significant