Fig 2: PSMA and CD137 expression measured by IHC. A, Primary human prostate cancer tumor co-stained for PSMA (green) and CD137 (brown). Arrows indicate patches of CD137-positive cells. B, PSMA positivity across a selection of human multitumor tissue microarrays. Shown are percent of cells in a core showing any level of PSMA positivity by IHC as quantified by image analysis. Where duplicate cores have been stained and scored, results are presented as the average. C, Density of CD137-positive cells per mm2 of viable tissue. Cells with any level of CD137 expression as determined by image analysis of IHC staining on sample cores are shown. Where duplicate cores have been stained and scored, results are presented as the average. D, PSMA expression in sites of primary and metastatic human prostate cancer are shown—any level of PSMA expression in a given cell is counted as positive. The median value for each anatomical site is indicated. Boxes represent inner quartiles. Each dot represents a sample from an individual patient. E, CD137-positive cell density in regions determined by image analysis to be tumor. F, The density of CD137-positive cells in the tumor-adjacent nontumor stromal tissue.

Fig 3: CB307 activity in combination with immune checkpoint inhibitors. A, Schematic of a 2D primary cell-based experimental system assessing CB307 combination with immune checkpoint inhibitors. SEB is used to induce CD137 expression on primary T cells. B, IL2 secretion following drug treatment in the presence of PSMA-expressing tumor cells. Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001. C, Isobologram of CB307-pembrolizumab additivity as assessed in 2D cocultures. D, Comparison of IL2 production between PD-L1–expressing versus PD-L1 CRISPR gene deleted nonexpressing tumor cells. In both cases, PSMA expression is similar. E, Schematic of a 3D tumor spheroid experimental system, whereby tumor cells fluoresce red whilst immune cells appear black. F, Single-agent versus combination drug activity measured as reduction in tumor cell count and (G) increasing CD8+ T-cell count after 10 days in culture. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001. H, Changing red fluorescent tumor spheroid and surrounding black immune cell “cloud” over time.

Fig 4: CB307 design and induced T-cell phenotypes. A, Illustration of CB307 format: three VH domains separated by peptide linkers. B, PSMA expression on cell lines. DU145-PSMA is isogenic to DU145-Parent other than for constitutive stable PSMA expression. C, Schematic of a Jurkat-based CD137 agonism reporter system whereby CB307 confers the clustering status of PSMA upon CD137 to generate a CD137 agonist signal. CD137 signaling is measured by luciferase expression from an NFκB-based reporter gene. D, Comparison of the reporter gene signal in the presence (DU145-PSMA) and absence (DU145-parent) of PSMA expression. E, Schematic of a primary cell CD137 activity assay whereby PBMCs stimulated by plate-bound αCD3 are co-incubated with tumor cells in the presence of CB307, leading to (F) PSMA-dependent secretion of IFNγ, (G) PSMA-dependent production of granzyme B, and (H) PSMA-dependent production of Bcl.xL. I, PSMA-dependent proliferation indicated by Ki67 expression. J, CB307 pharmacodynamic effects on NK cells. Statistical significance of CB307 treated versus untreated outcomes with PSMA-expressing tumor cells is indicated: **, P < 0.01; ****, P < 0.0001.

Fig 5: PD-L1, 4-1BB, CD3, and CD8 tumor expression in MC38 tumor-bearing h4-1BB KI or WT mice that received 89Zr-mPD-L1xh4-1BB or 89Zr-hPD-L1xh4-1BB. (A) Overview of experimental groups and binding specificity of mPD-L1xh4-1BB and hPD-L1xh4-1BB in h4-1BB KI and WT mice. (B) Quantification of PD-L1 expression (C) Quantification of CD3 expression (D) Quantification of CD8 expression (E) Quantification of 4-1BB expression. Representative IHC images are depicted horizontally next to the quantification graphs. The quantifications are expressed as a percentage of DAB-positive regions in tumor tissue. Tumor expression levels of the PD-L1 and 4-1BB binding group at 200 µg 4 days post-injection are presented as yellow dots and 7 days post-injection as yellow diamonds. Tumor expression levels at day 4 post-injection in the PD-L1 binding group are presented as red dots and the 4-1BB binding group as blue dots, both are dosed at 200 µg. In the non-specific binding group, tumor expression levels of the 30 µg dose at 7 days post-injection and 200 µg at 4 days post-injection are presented. DAB: diaminobenzidine; h: human; IHC: immunohistochemistry; m: murine; ns: not significant; *: P ≤ 0.05; **: P ≤ 0.01.