Fig 2: Evaluation of the toxicological, PK and PD characteristics of HER2-XPAT protein and unmasked HER2-uTCE in NHPs.HER2-XPAT protein was administered via short i.v. infusions (∼1–3 h), whereas the unmasked counterpart, due to its short half-life, was administered via 48-h continuous i.v. infusion to provide prolonged systemic exposure. a, A dose-escalation study with a single dose of HER2-XPAT protein administered to each cynomolgus monkey. *At doses below 21 mg kg−1, a HER2-XPAT prototype was used. b, Dose de-escalation study of uTCE in NHPs administered as a 48-h continuous infusion due to its short half-life. c, Plasma concentrations in NHPs following administration of a single i.v. dose of HER2-XPAT protein (42 mg kg−1, n = 2 NHPs; 25 mg kg−1, n = 4 NHPs; 6 mg kg−1, n = 12 NHPs; 2 mg kg−1, n = 12 NHPs) or a 48-h continuous infusion of uTCE (0.2 mg kg−1 d−1, n = 1 NHP; 0.1 mg kg−1 d−1, n = 1 NHP; 0.06 mg kg−1 d−1, n = 1 NHP). Data represent mean plasma concentration (±s.d. when n > 3 NHP treated) for the NHPs treated at each dose within one single experiment. d,e, Activation of circulating CD4+ T cells (d) and CD8+ T cells (e) 24 h after drug administration (HER2-XPAT protein, n = 16 NHPs; HER2-uTCE, n = 5 NHPs). f–h, Highest plasma levels of TNF-α (f), IL-6 (g) and IFN-γ (h) measured 0–24 h following drug administration (HER2-XPAT protein, n = 23 NHPs; HER2-uTCE, n = 8 NHPs). Note that the normal ranges for cytokine levels in NHP are as follows: IL-6 0.3–1.2 pg ml−1, TNF-α 1–10 pg ml−1, IFN-γ 1.1–8.0 pg ml−1 (ref. 44). Data in d–f represent a single sample from each NHP receiving the test material within each single experiment. TNF, tumor necrosis factor; IFN, interferon.Source data

Fig 3: Binding and in vitro activity of HER2-XPAT protein, its partially unmasked metabolites and unmasked HER2-TCE.a, Binding affinities to human HER2 and CD3 at 37 °C by surface plasmon resonance. Data are KD (n = 12 technical replicates of a single experiment for HER2-XPAT protein and HER2(1x-N); n = 8 for HER2(1x-C) and uTCE). Surface plasmon resonance sensorgrams for these data are provided in Supplementary Figs. 3–10. b–d, In vitro tumor cytotoxicity of HER2-XPAT protein and its metabolites following a 48-h incubation with co-cultures of huPBMCs and the high HER2-expressing human tumor cell lines (1:1 effector–target ratio) SKOV3 (b), BT-474 (c) or the medium-low HER2-expressing MCF7 cell line (d). e, In vitro cytotoxicity of HER2-XPAT protein and its metabolites against BT-474 cells co-cultured with huPBMCs (1:1 effector–target ratio). f, Impact of the protease-cleavable linker on in vitro cytotoxicity versus BT-474 cells co-cultured with huPBMCs. g,h, CD69-positive T cells (g) and IL-2 secretion (h) following 72-h incubation of huPBMC/SKOV3 co-cultures with HER2-XPAT protein or its unmasked form (uTCE). i, Target-dependent T-cell activation with HER2-XPAT protein and its metabolites. CD3-expressing Jurkat reporter T cells were incubated with BT-474 cells at a 5:1 effector–target ratio for 6 h, followed by quantification of NFAT-induced luciferase activity and measured in relative luminescence units (RLUs). Mean data for n = 2 technical replicates within one single experiment (b–i). Extended Data Table 1 provides a summary of EC50 values for the different forms of the HER2-XPAT proteins in the cytotoxicity and reporter T-cell activation assays.Source data

Fig 4: Incorporation of CD96 endodomain in 4D5-z CAR-T cells impairs their cytotoxicity against HER2+ tumor cells in vivo. (A) Schematic diagram of the tumor model using SK-BR-3 cells in NSG mice and treatment with non-CAR-T or CAR-T cells. (B) Selected images of tumor bioluminescence (BLI) at indicated time points (days) prior to and post T cell infusion. (C) Average radiance of tumor burden in mice at indicated time points post infusion of non-CAR-T, 4D5-z or 4D5-96z CAR-T cells. (D) Ratio of tumor-free and tumor-bearing mice of all mice examined at 14 days post T cell infusion. Data in (B,C) are representative of 2 independent experiments. Data in (C) are based on 5 or more mice analyzed with each symbol representing one mouse and red horizontal bars indicating the mean. Data in (D) are pooled from mice examined in 2 independent experiments; Kruskal-Wallis test (C) or Fisher exact test (D), *, p < 0.05; **, p < 0.005.

Fig 5: Cytotoxic capacity against ErbB2-positive 3D tumor spheroids. (A) ErbB2 surface expression on RH30 cells was confirmed by flow cytometry. (B) Expression of the anti-ErbB2-targeted CAR on NK-92/5.28.z cells was confirmed by flow cytometry. (C) Exemplary coculture images of NK-92 or NK-92/5.28.z cells added to established 3D RH30GFP/luc+ tumor spheroids on day 4 are shown. Imaging was performed on days 4, 5, 6, 8 and 10. (D) The areas with green fluorescent signals were quantified using Fiji. Data of three independent experiments are shown as mean ± SD. Differences were analyzed with a one-way ANOVA using the Bonferroni method and were considered significant for p< 0.05 (*), p< 0.01 (**) or ns.