cIAP1/2 Antagonism Induces Antigen-Specific T Cell-Dependent Immunity.

Checkpoint blockade immunotherapy has failed in pancreatic cancer and other poorly responsive tumor types in part due to inadequate T cell priming. Naive T cells can receive costimulation not only via CD28 but also through TNF superfamily receptors that signal via NF-κB. Antagonists of the ubiquitin ligases cellular inhibitor of apoptosis protein (cIAP)1/2, also called second mitochondria-derived activator of caspases (SMAC) mimetics, induce degradation of cIAP1/2 proteins, allowing for the accumulation of NIK and constitutive, ligand-independent activation of alternate NF-κB signaling that mimics costimulation in T cells. In tumor cells, cIAP1/2 antagonists can increase TNF production and TNF-mediated apoptosis; however, pancreatic cancer cells are resistant to cytokine-mediated apoptosis, even in the presence of cIAP1/2 antagonism. Dendritic cell activation is enhanced by cIAP1/2 antagonism in vitro, and intratumoral dendritic cells show higher expression of MHC class II in tumors from cIAP1/2 antagonism-treated mice. In this study, we use in vivo mouse models of syngeneic pancreatic cancer that generate endogenous T cell responses ranging from moderate to poor. Across multiple models, cIAP1/2 antagonism has pleiotropic beneficial effects on antitumor immunity, including direct effects on tumor-specific T cells leading to overall increased activation, increased control of tumor growth in vivo, synergy with multiple immunotherapy modalities, and immunologic memory. In contrast to checkpoint blockade, cIAP1/2 antagonism does not increase intratumoral T cell frequencies. Furthermore, we confirm our previous findings that even poorly immunogenic tumors with a paucity of T cells can experience T cell-dependent antitumor immunity, and we provide transcriptional clues into how these rare T cells coordinate downstream immune responses.

Ornithine aminotransferase supports polyamine synthesis in pancreatic cancer.

There is a need to develop effective therapies for pancreatic ductal adenocarcinoma (PDA), a highly lethal malignancy with increasing incidence1 and poor prognosis2. Although targeting tumour metabolism has been the focus of intense investigation for more than a decade, tumour metabolic plasticity and high risk of toxicity have limited this anticancer strategy3,4. Here we use genetic and pharmacological approaches in human and mouse in vitro and in vivo models to show that PDA has a distinct dependence on de novo ornithine synthesis from glutamine. We find that this process, which is mediated through ornithine aminotransferase (OAT), supports polyamine synthesis and is required for tumour growth. This directional OAT activity is usually largely restricted to infancy and contrasts with the reliance of most adult normal tissues and other cancer types on arginine-derived ornithine for polyamine synthesis5,6. This dependency associates with arginine depletion in the PDA tumour microenvironment and is driven by mutant KRAS. Activated KRAS induces the expression of OAT and polyamine synthesis enzymes, leading to alterations in the transcriptome and open chromatin landscape in PDA tumour cells. The distinct dependence of PDA, but not normal tissue, on OAT-mediated de novo ornithine synthesis provides an attractive therapeutic window for treating patients with pancreatic cancer with minimal toxicity.

A split, conditionally active mimetic of IL-2 reduces the toxicity of systemic cytokine therapy.

The therapeutic potential of recombinant cytokines has been limited by the severe side effects of systemic administration. We describe a strategy to reduce the dose-limiting toxicities of monomeric cytokines by designing two components that require colocalization for activity and that can be independently targeted to restrict activity to cells expressing two surface markers. We demonstrate the approach with a previously designed mimetic of cytokines interleukin-2 and interleukin-15-Neoleukin-2/15 (Neo-2/15)-both for trans-activating immune cells surrounding targeted tumor cells and for cis-activating directly targeted immune cells. In trans-activation mode, tumor antigen targeting of the two components enhanced antitumor activity and attenuated toxicity compared with systemic treatment in syngeneic mouse melanoma models. In cis-activation mode, immune cell targeting of the two components selectively expanded CD8+ T cells in a syngeneic mouse melanoma model and promoted chimeric antigen receptor T cell activation in a lymphoma xenograft model, enhancing antitumor efficacy in both cases.

Engineering T cell memory for antitumor immunity.

Cancer therapy with the T cell growth factor interleukin (IL)-2 is limited by low response rates and toxicity. Multiple protein engineering strategies have attempted to improve IL-2 therapy, typically through enhanced IL-2 receptor (IL-2R) binding. Intriguingly, Mo et al. show that an IL-2R partial agonist may dramatically improve IL-2 responses by altering T cell differentiation.

cIAP1/2 antagonism eliminates MHC class I-negative tumors through T cell-dependent reprogramming of mononuclear phagocytes.

Loss of major histocompatibility complex (MHC) class I and interferon-γ (IFN-γ) sensing are major causes of primary and acquired resistance to checkpoint blockade immunotherapy. Thus, additional treatment options are needed for tumors that lose expression of MHC class I. The cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/2) regulate classical and alternative nuclear factor κB (NF-κB) signaling. Induction of noncanonical NF-κB signaling with cIAP1/2 antagonists mimics costimulatory signaling, augmenting antitumor immunity. We show that induction of noncanonical NF-κB signaling induces T cell-dependent immune responses, even in ß2-microglobulin (ß2M)-deficient tumors, demonstrating that direct CD8 T cell recognition of tumor cell-expressed MHC class I is not required. Instead, T cell-produced lymphotoxin reprograms both mouse and human macrophages to be tumoricidal. In wild-type mice, but not mice incapable of antigen-specific T cell responses, cIAP1/2 antagonism reduces tumor burden by increasing phagocytosis of live tumor cells. Efficacy is augmented by combination with CD47 blockade. Thus, activation of noncanonical NF-κB stimulates a T cell-macrophage axis that curtails growth of tumors that are resistant to checkpoint blockade because of loss of MHC class I or IFN-γ sensing. These findings provide a potential mechanism for controlling checkpoint blockade refractory tumors.

Immune receptor inhibition through enforced phosphatase recruitment.

Antibodies that antagonize extracellular receptor-ligand interactions are used as therapeutic agents for many diseases to inhibit signalling by cell-surface receptors1. 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 CD452,3, 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 SIRPα 'don't eat me' signal with a SIRPα-CD45 RIPR molecule potentiates antibody-dependent cellular phagocytosis beyond that of SIRPα blockade alone. RIPR represents a general strategy for direct attenuation of signalling by kinase-activated cell-surface receptors.