Fig 2: PD-1 and GITR receptor copy number, expression and anti-PD-1-GITR-L cross-reactivity on normal human and cynomolgus monkey tissues by IHC, and Hexagonal conformation model of GITR-L-GITR complex.(A and B) GITR and PD-1 receptor copy number on PHA activated (t = 48 hrs) human (A) and cynomolgus monkey (B) PBMCs (n = 8 donors per group). Data are presented as mean values +/- SEM. Statistical significance was calculated with two-way ANOVA with Tukey’s correction for multiple comparisons (statistic refer to GITR vs PD-1 expression). (C) Representative human and Cyno PD-1 and GITR expression by IHC (representative staining of n = 2 tissues) on tonsil, lymph node, spleen, and stomach tissues. FFPE tissue samples were used for PD-1 and GITR (Human TMA: ZPL2 and ZPL3, and Cyno: I09447 and I08762) staining. Scale bars indicate 50 µm (PD-1) and 60 µm (GITR) (original magnification 40x). (D) Representative anti-huPD-1-huGITR-L bispecific binding on normal human and cynomolgus monkey tissues using human to human pre-complex conjugated IHC staining method. Scale bars indicate 200 µm (original magnification 20x, representative staining of n = 2 tissues). (E) GITR-L trimeric units are shown in light blue, and GITR monomeric units from different GITR trimers are shown in pink and orange. Source data

Fig 3: Expression of PD-1 and GITR in human T cells and in vitro characterization of anti-huPD-1-huGITR-L bispecific fusion protein.a–d, Percentage of PD-1 and GITR double-positive T cells following anti-CD3/CD28 activation of human CD4+ (a) and CD8+ T cells (b) (n = 3 donors, data presented as mean ± s.e.m.), and correlation of GITR and PD-1 mRNA-seq expression in live cells (c) (n = 21 tumors) and T cells (d) (n = 18 tumors) sorted from HNSCC tumor samples. Statistical significance was calculated with nonlinear regression. Live and T cells, P = 0.00001 and P = 0.067, respectively. e, Representative CD8, PD-1, GITR and FoxP3 multiplex immunofluorescence imaging (excluding PanCK, CD3 and DAPI) and spatial distribution cell density heatmaps (cells mm–2) (CD8, PD-1 and GITR) of FFPE sections of primary HNSCC (excluded compartment) and matching lymph node metastases (LN mets) (20×). Selected areas of GITR+PD-1+CD8+ T cells are indicated in white boxes. Experiment is representative of n = 3 tumor samples. f,g, Schematic diagram of anti-PD-1–GITR-L bispecific (f) and an example of a 2D class average based on negative-stain TEM of the whole molecule (g). h,i, Binding of anti-huPD-1-huGITR-L to huPD-1- (h) and GITR-transfected (i) HEK293S cells (n = 2 technical cell culture replicates within a single experiment). j,k, Binding of anti-huPD-1-huGITR-L to human CD4+CD45RA–CCR7+ central memory T cells (TCM) (j) and CD4+CD45RA–CCR7– effector memory T cells (TEM) (k) (n = 2 donors). l,m, GITR-NF?B (l) and PD-1/PD-L1 NFAT signaling (m) on HEK293-NF?B-huGITR+ and Jurkat-NFAT-huPD-1+ (with CHO-K-PD-L1+ cells) reporter cells following threefold titration of the anti-huPD-1-huGITR-L bispecific (n = 2 technical cell culture replicates within a single experiment). MFI, median fluorescence intensity. RLU, relative luminescence units.Source data

Fig 4: The anti-muPD-1-muGITR-L bispecific induces dose-dependent growth inhibition and peripheral target engagement, T cell activation and proliferation in CT26 and EMT6 tumor-bearing mice.a,b, Growth inhibition of CT26 and EMT6 cells in syngeneic mice by anti-muPD-muGITR-L. Titration of the bispecific at indicated doses was administered IV following one dose in CT26 (a) and EMT6 (b), respectively. Each point on the curve represents mean tumor volume for each group (n = 10 mice). c,d, Serum concentration versus time profile following IV administration of the bispecific at the indicated doses in CT26 (c) and EMT6 (d) (n = 4 mice). e–j, Flow cytometry analysis of blood lymphocytes after treatment with anti-muPD-1-muGITR-L. Blood samples were collected from CT26 and EMT6 tumor-bearing mice at the indicated times following either treatment with isotype control or titration of anti-muPD-muGITR-L administered IV following one dose, at the indicated doses. CD4+ T cells were assessed for percentage of GITR (CT26 (e) and EMT6 (g)) and PD-1 (CT26 (f) and EMT6 (h)) expression. Percentage target saturation was standardized to 0% at t = 0. TIGIT+, CD62–CD44+ TCM and Ki67+ are shown as a percentage of CD4+ while Ki67+ is shown as a percentage of CD8+ T cells in the blood (CT26 (i) and EMT6 (j)). Results for five animals per group from a single experiment were averaged, and standard deviations are shown, Statistical significance was calculated using two-way ANOVA with Tukey’s correction for multiple comparisons (statistics refer to anti-PD-1–GITR-L bispecific (25.8, 8.6 or 12.9, 2.9 or 6.5 mg kg–1) versus isotype control). LLOQ, lower level of quantitation.Source data

Fig 5: Human PD-1 and GITR expression on TILs by mRNA-seq and IHC and LC-MS of anti-huPD-1-huGITR-Lbispecific.(A) Gene mRNA expression of GITR and PD-1 across different tumor types (UCSF-IPI, n = 50 tumors). For box-and-whiskers plots, boxes represent 25th and 75th percentiles, center lines indicate median values and whiskers represent minimum and maximum values. (B) Representative human PD-1 and GITR expression by IHC on colon, pancreatic and breast cancer tissues. Frozen tissue microarray samples were used for PD-1 and GITR staining. Scale bars indicate 50 µm (original magnification 40x, representative staining of n = 5 tissues). (C) Percentage of GITR+PD-1+ cells of total CD8+ T cells within inflamed, excluded and desert compartments of HNSCC including the tumor, stroma, and tumor-proximal lymph node aggregates (n = 8 samples) (D) LC-MS of reduced and de-glycosylated anti-huPD-1-huGITR-L bispecific (hIgG1-LALA) with theoretical and experimental masses of light (LC) and heavy chain (HC).