In PD-1?/CTLA-4+ cells, MGD019 blockade of CTLA-4 ligand binding is normally reduced compared to that mediated by anti-CTLA-4 mAbs, while in TIL-like dual-expressing cells, the potency of CTLA-4 blockade can increase by 2 orders of magnitude (Figure?2), in agreement with an avidity-driven effect.36 Consistent DDR1-IN-1 with this observation, dual checkpoint blockade by MGD019 in primary T?cell models provided an enhanced effect in a subset of donors selected for poor response to PD-1 blockade PD-1 Blockade Studies Patients whole blood samples (per time point/per patient) were incubated with commercial, APC-labeled, MGD019-competing anti-PD-1 mAb (clone J105, eBioscience, San Diego, USA). analysis of a 35-healthy adult tissue TMA revealed no expression of PD-1 or CTLA-4, with the exception of lymphoid organs (thymus, tonsils, and lymph nodes) and rare occurrences in the stroma of colon and pancreas (data not DDR1-IN-1 shown). Notably, PD-1 and CTLA-4 expression in normal lymphoid tissues was observed in distinct, spatially separated cell populations, in contrast to the pattern of co-expression observed in TILs (Figure?1B). Digital quantitation confirmed a higher proportion of PD-1/CTLA-4 double-positive cells in ovarian (Figure?1B), breast, lung, colon, and rectal cancer specimens relative to those observed in normal lymphoid tissues (Figures 1C and S1). Flow cytometry studies comparing circulating T?cells from healthy donors and TILs from patients with various cancers confirmed cell surface protein expression (Figures 1D and 1E). On average, 14.6% of TILs expressed both PD-1 and CTLA-4, Kcnmb1 51.8% expressed PD-1 alone, 2.8% expressed CTLA-4 alone, and 30.8% did not express either protein. This observation is in line with a prior report of a high occurrence of PD-1 and CTLA-4 expression on TILs.29 Interestingly, a small but detectable fraction (<0.25%) of circulating T?cells from tumor patients co-expressed PD-1 and CTLA-4, while no circulating double-positive cells were detected in healthy donors (Figures 1D and 1E). These data indicate that cells co-expressing PD-1 and CTLA-4 are prevalent in the TME but virtually absent in healthy tissues. This observation further implies that targeting dual PD-1-/CTLA-4-expressing cells may provide an opportunity for selective checkpoint blockade in the TME, while relatively reducing effects in normal tissues. Open in a separate window Figure?1 Cells Co-expressing PD-1 and CTLA-4 Are More Prevalent in the Tumor Microenvironment (A) RNA hybridization of PD-1 and CTLA-4 probes in ovarian cancer tumor cores (N?= 21) analyzed using RNAscope and quantified with HALO software. Each square represents an individual core, with red and blue circles representing the indicated frequency of PD-1 and CTLA-4 expression, respectively. The first square shows PD-1 and CTLA-4 expression in a non-malignant ovary sample. (B) RNA hybridization of PD-1 (red) and CTLA-4 (blue) probes visualized by RNAscope in representative tumor microarray core or healthy tonsil samples. (C) Fraction of cells co-expressing PD-1 and CTLA-4 RNA detected by ISH in lymphoid organs from healthy donors (N?= 7) or tumor samples from randomly selected patients (N?= 12).?Means and standard deviations (SDs) are shown. (D) Peripheral blood mononuclear cells DDR1-IN-1 (PBMCs) from healthy donors (N?= 8) and PBMCs (N?= 27) or dissociated tumor cells (DTCs) (N?= 7) from patients with various cancers were stained for PD-1 and CTLA-4 expression and analyzed by flow cytometry. Box and whiskers plots depict the minimum, first quartile, median, third quartile, and maximum. Gated on viable CD45+/CD3+ cells. (E) Representative fluorescence-activated cell sorting (FACS) images from (D) gated on viable T?cells. See also Figure?S1. Engineering and Characterization of MGD019, a PD-1 x CTLA-4 Bispecific Molecule Featuring Complete Blockade of PD-1 and Variable Inhibition of CTLA-4 To build DDR1-IN-1 a molecule capable of stringent, uniform blockade of PD-1 and conditional blockade of CTLA-4, we selected a high-affinity, clinically validated anti-PD-1 mAb30,31 and an anti-CTLA-4 mAb with ligand-blocking properties similar to that of ipilimumab (see Method Details) as a precursor for the PD-1 and CTLA-4 arms, respectively. A PD-1 x CTLA-4 bispecific molecule was constructed on the DART platform32.