"Switch" Transmembrane Immunomodulatory Proteins (TIPs) Consisting of High-Affinity PD-1 Extracellular Domains (PD-1 vIgDs) and Costimulatory Intracellular Domains Potently Enhance the Activity of TCR-Engineered T Cells

Background: T cell receptor (TCR)-engineered T cell therapies hold great promise as personalized, adoptive anticancer treatments, but clinical experience to date has demonstrated generally only modest efficacy, attributed in large part to unfavorable factors in the tumor microenvironment. This includes the presence of receptors, such as PD-L1, that can inhibit T cell responses, and/or insufficient engineered T cell longevity. Common strategies to address such limitations have involved attempts to provide additional costimulatory signals to T cells. However, addition of costimulatory signals alone may not be sufficient to overcome PD-L1-mediated inhibition. We have utilized our variant immunoglobulin domain (vIgD) platform, based on the directed evolution of immunoglobulin superfamily (IgSF) members, to develop PD-1 domains with higher affinity for PD-L1 and then substituted costimulatory intracellular signaling domains for the native PD-1 inhibitory intracellular region with the hypothesis that PD-L1 engagement of these Transmembrane Immune-modulatory Proteins (TIPs) would result in costimulation rather than inhibitory signaling. Such "Switch" TIPs that consist of a checkpoint-inhibitory extracellular PD-1 vIgD and intracellular costimulatory domains may therefore improve the activity of TCR-engineered T cells by providing costimulation while preventing inhibitory signaling through native PD-1.

Methods: Variant PD-1 extracellular domains (PD-1 vIgDs) were generated using random mutagenesis and FACS-based selection of yeast displayed proteins selecting for increased binding to PD-L1. These variants were then fused to intracellular signaling domains from CD28, ICOS and CD137 either singly or together in combinations. These constructs were expressed via lentivirus in primary human T cells along with a TCR recognizing an HPV16 E6 peptide (E6 TCR). Surface expression was confirmed by flow cytometry, and activity was assessed against HPV+ tumor cell lines via proliferation, cytokine production (IFNg, TNFa and IL-2) and cytotoxicity.

Results: TIPs including PD-1 vIgDs selected for increased PD-L1 binding and various combinations of intracellular signaling domains potently enhanced the activity of E6 TCR-engineered T cells, including killing of HPV+ target cells (Figure 1a) as well as target-driven proliferation and cytokine production (Figure 1b). Activity was consistently superior to TIPs consisting of only a PD-1 extracellular vIgD, or TIPs which included a wild-type extracellular PD-1 domain. Importantly, TIP activity was abrogated in the presence of an anti-PD-L1 antibody, demonstrating PD-L1 dependence of the costimulatory activity.

Conclusions: "Switch" TIPs consisting of high-affinity PD-1 vIgD extracellular domains and costimulatory intracellular signaling domains potently augment the antitumor activity of TCR-engineered T cells as judged by proliferation, cytokine production and cytotoxicity. Ongoing studies continue to explore this and analogous strategies with other IgSF-based vIgDs and/or costimulatory domains and will hopefully significantly enhance the clinical efficacy of engineered T cells in both solid and hematological malignancies.

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