Fernandes et al. showed that tonic intracellular PD-1 signaling inhibits T cell activation and is unimpeded by PD-1, PD-L1, and/or PD-L2 blockade. A PD-1/CD45 bispecific diabody recruited the cis CD45 intracellular phosphatase domain to reduce PD-1 phosphorylation and amplify T cell activation better than PD-1 axis-blocking antibodies. In mouse tumor models, an anti-mouse PD-1 scFv linked to an anti-mouse CD45 nanobody reduced growth of small cell lung cancer and a colon adenocarcinoma, decreased exhausted T cells, and increased CD8+ T effector memory cells. Phosphatase recruitment to CD28, CTLA-4, or SIRPα also attenuated signaling.
Contributed by Paula Hochman
ABSTRACT: Antibodies that antagonize extracellular receptor-ligand interactions are used as therapeutic agents for many diseases to inhibit signalling by cell-surface receptors(1). However, this approach does not directly prevent intracellular signalling, such as through tonic or sustained signalling after ligand engagement. Here we present an alternative approach for attenuating cell-surface receptor signalling, termed receptor inhibition by phosphatase recruitment (RIPR). This approach compels cis-ligation of cell-surface receptors containing ITAM, ITIM or ITSM tyrosine phosphorylation motifs to the promiscuous cell-surface phosphatase CD45(2,3), which results in the direct intracellular dephosphorylation of tyrosine residues on the receptor target. As an example, we found that tonic signalling by the programmed cell death-1 receptor (PD-1) results in residual suppression of T cell activation, but is not inhibited by ligand-antagonist antibodies. We engineered a PD-1 molecule, which we denote RIPR-PD1, that induces cross-linking of PD-1 to CD45 and inhibits both tonic and ligand-activated signalling. RIPR-PD1 demonstrated enhanced inhibition of checkpoint blockade compared with ligand blocking by anti-PD1 antibodies, and increased therapeutic efficacy over anti-PD1 in mouse tumour models. We also show that the RIPR strategy extends to other immune-receptor targets that contain activating or inhibitory ITIM, ITSM or ITAM motifs; for example, inhibition of the macrophage SIRP_ 'don't eat me' signal with a SIRP_-CD45 RIPR molecule potentiates antibody-dependent cellular phagocytosis beyond that of SIRP_ blockade alone. RIPR represents a general strategy for direct attenuation of signalling by kinase-activated cell-surface receptors.
Author Info: (1) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. Department of Structural Biology, Stanford University School of Medi
Author Info: (1) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA. (2) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA. (3) Department of Pediatrics, Stanford University, Stanford, CA, USA. Department of Radiation Oncology, Stanford University, Stanford, CA, USA. (4) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA. (5) Department of Medicine, Division of Gastroenterology, Massachusetts General Hospital, Boston, MA, USA. (6) Department of Medicine, Division of Hematology, Stanford University School of Medicine, Stanford, CA, USA. (7) Department of Medicine, Division of Hematology, Stanford University School of Medicine, Stanford, CA, USA. (8) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA. (9) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA. (10) Department of Pediatrics, Stanford University, Stanford, CA, USA. Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA. (11) Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA. (12) Department of Pediatrics, Stanford University, Stanford, CA, USA. Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA. Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA. (13) Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA, USA. (14) Department of Medicine, Harvard Medical School, Boston, MA, USA. (15) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA. (16) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA. (17) Department of Pediatrics, Stanford University, Stanford, CA, USA. Department of Genetics, Stanford University, Stanford, CA, USA. (18) Department of Medicine, Division of Gastroenterology, Massachusetts General Hospital, Boston, MA, USA. Department of Medicine, Harvard Medical School, Boston, MA, USA. (19) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. kcgarcia@stanford.edu. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA. kcgarcia@stanford.edu. Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA. kcgarcia@stanford.edu.
