Multiparatopic antibodies induce targeted downregulation of programmed death-ligand 1.

Programmed death-ligand 1 (PD-L1) drives inhibition of antigen-specific T cell responses through engagement of its receptor programmed death-1 (PD-1) on activated T cells. Overexpression of these immune checkpoint proteins in the tumor microenvironment has motivated the design of targeted antibodies that disrupt this interaction. Despite clinical success of these antibodies, response rates remain low, necessitating novel approaches to enhance performance. Here, we report the development of antibody fusion proteins that block immune checkpoint pathways through a distinct mechanism targeting molecular trafficking. By engaging multiple receptor epitopes on PD-L1, our engineered multiparatopic antibodies induce rapid clustering, internalization, and degradation in an epitope- and topology-dependent manner. The complementary mechanisms of ligand blockade and receptor downregulation led to more durable immune cell activation and dramatically reduced PD-L1 availability in mouse tumors. Collectively, these multiparatopic antibodies offer mechanistic insight into immune checkpoint protein trafficking and how it may be manipulated to reprogram immune outcomes.

DGKα/ζ inhibition lowers the TCR affinity threshold and potentiates antitumor immunity.

Diacylglycerol kinases (DGKs) attenuate diacylglycerol (DAG) signaling by converting DAG to phosphatidic acid, thereby suppressing pathways downstream of T cell receptor signaling. Using a dual DGKα/ζ inhibitor (DGKi), tumor-specific CD8 T cells with different affinities (TRP1high and TRP1low), and altered peptide ligands, we demonstrate that inhibition of DGKα/ζ can lower the signaling threshold for T cell priming. TRP1high and TRP1low CD8 T cells produced more effector cytokines in the presence of cognate antigen and DGKi. Effector TRP1high- and TRP1low-mediated cytolysis of tumor cells with low antigen load required antigen recognition, was mediated by interferon-γ, and augmented by DGKi. Adoptive T cell transfer into mice bearing pancreatic or melanoma tumors synergized with single-agent DGKi or DGKi and antiprogrammed cell death protein 1 (PD-1), with increased expansion of low-affinity T cells and increased cytokine production observed in tumors of treated mice. Collectively, our findings highlight DGKα/ζ as therapeutic targets for augmenting tumor-specific CD8 T cell function.

Blockade of innate inflammatory cytokines TNFα, IL-1ß, or IL-6 overcomes virotherapy-induced cancer equilibrium to promote tumor regression.

Cancer therapeutics can lead to immune equilibrium in which the immune response controls tumor cell expansion without fully eliminating the cancer. The factors involved in this equilibrium remain incompletely understood, especially those that would antagonize the anti-tumor immune response and lead to tumor outgrowth. We previously demonstrated that continuous treatment with a non-replicating herpes simplex virus 1 expressing interleukin (IL)-12 induces a state of cancer immune equilibrium highly dependent on interferon-γ. We profiled the IL-12 virotherapy-induced immune equilibrium in murine melanoma, identifying blockade of innate inflammatory cytokines, tumor necrosis factor alpha (TNFα), IL-1ß, or IL-6 as possible synergistic interventions. Antibody depletions of each of these cytokines enhanced survival in mice treated with IL-12 virotherapy and helped to overcome equilibrium in some tumors. Single-cell RNA-sequencing demonstrated that blockade of inflammatory cytokines resulted in downregulation of overlapping inflammatory pathways in macrophages, shifting immune equilibrium towards tumor clearance, and raising the possibility that TNFα blockade could synergize with existing cancer immunotherapies.

IFNγ is a central node of cancer immune equilibrium.

Tumors in immune equilibrium are held in balance between outgrowth and destruction by the immune system. The equilibrium phase defines the duration of clinical remission and stable disease, and escape from equilibrium remains a major clinical problem. Using a non-replicating HSV-1 vector expressing interleukin-12 (d106S-IL12), we developed a mouse model of therapy-induced immune equilibrium, a phenomenon previously seen only in humans. This immune equilibrium was centrally reliant on interferon-γ (IFNγ). CD8+ T cell direct recognition of MHC class I, perforin/granzyme-mediated cytotoxicity, and extrinsic death receptor signaling such as Fas/FasL were all individually dispensable for equilibrium. IFNγ was critically important and played redundant roles in host and tumor cells such that IFNγ sensing in either compartment was sufficient for immune equilibrium. We propose that these redundant mechanisms of action are integrated by IFNγ to protect from oncogenic or chronic viral threats and establish IFNγ as a central node in therapy-induced immune equilibrium.

Pharmacodynamic measures within tumors expose differential activity of PD(L)-1 antibody therapeutics.

Macromolecules such as monoclonal antibodies (mAbs) are likely to experience poor tumor penetration because of their large size, and thus low drug exposure of target cells within a tumor could contribute to suboptimal responses. Given the challenge of inadequate quantitative tools to assess mAb activity within tumors, we hypothesized that measurement of accessible target levels in tumors could elucidate the pharmacologic activity of a mAb and could be used to compare the activity of different mAbs. Using positron emission tomography (PET), we measured the pharmacodynamics of immune checkpoint protein programmed-death ligand 1 (PD-L1) to evaluate pharmacologic effects of mAbs targeting PD-L1 and its receptor programmed cell death protein 1 (PD-1). For PD-L1 quantification, we first developed a small peptide-based fluorine-18-labeled PET imaging agent, [18F]DK222, which provided high-contrast images in preclinical models. We then quantified accessible PD-L1 levels in the tumor bed during treatment with anti-PD-1 and anti-PD-L1 mAbs. Applying mixed-effects models to these data, we found subtle differences in the pharmacodynamic effects of two anti-PD-1 mAbs (nivolumab and pembrolizumab). In contrast, we observed starkly divergent target engagement with anti-PD-L1 mAbs (atezolizumab, avelumab, and durvalumab) that were administered at equivalent doses, correlating with differential effects on tumor growth. Thus, we show that measuring PD-L1 pharmacodynamics informs mechanistic understanding of therapeutic mAbs targeting PD-L1 and PD-1. These findings demonstrate the value of quantifying target pharmacodynamics to elucidate the pharmacologic activity of mAbs, independent of mAb biophysical properties and inclusive of all physiological variables, which are highly heterogeneous within and across tumors and patients.

Structure-Guided Molecular Engineering of a Vascular Endothelial Growth Factor Antagonist to Treat Retinal Diseases.

BACKGROUND: Ocular neovascularization is a hallmark of retinal diseases including neovascular age-related macular degeneration and diabetic retinopathy, two leading causes of blindness in adults. Neovascularization is driven by the interaction of soluble vascular endothelial growth factor (VEGF) ligands with transmembrane VEGF receptors (VEGFR), and inhibition of the VEGF pathway has shown tremendous clinical promise. However, anti-VEGF therapies require invasive intravitreal injections at frequent intervals and high doses, and many patients show incomplete responses to current drugs due to the lack of sustained VEGF signaling suppression. METHODS: We synthesized insights from structural biology with molecular engineering technologies to engineer an anti-VEGF antagonist protein. Starting from the clinically approved decoy receptor protein aflibercept, we strategically designed a yeast-displayed mutagenic library of variants and isolated clones with superior VEGF affinity compared to the clinical drug. Our lead engineered protein was expressed in the choroidal space of rat eyes via nonviral gene delivery. RESULTS: Using a structure-informed directed evolution approach, we identified multiple promising anti-VEGF antagonist proteins with improved target affinity. Improvements were primarily mediated through reduction in dissociation rate, and structurally significant convergent sequence mutations were identified. Nonviral gene transfer of our engineered antagonist protein demonstrated robust and durable expression in the choroid of treated rats one month post-injection. CONCLUSIONS: We engineered a novel anti-VEGF protein as a new weapon against retinal diseases and demonstrated safe and noninvasive ocular delivery in rats. Furthermore, our structure-guided design approach presents a general strategy for discovery of targeted protein drugs for a vast array of applications.

Structural Basis for Signaling Through Shared Common γ Chain Cytokines.

The common γ chain (γc) family of hematopoietic cytokines consists of six distinct four α-helix bundle soluble ligands that signal through receptors which include the shared γc subunit to coordinate a wide range of physiological processes, in particular, those related to innate and adaptive immune function. Since the first crystallographic structure of a γc family cytokine/receptor signaling complex (the active Interleukin-2 [IL-2] quaternary complex) was determined in 2005 [1], tremendous progress has been made in the structural characterization of this protein family, transforming our understanding of the molecular mechanisms underlying immune activity. Although many conserved features of γc family cytokine complex architecture have emerged, distinguishing details have been observed for individual cytokine complexes that rationalize their unique functional properties. Much work remains to be done in the molecular characterization of γc family signaling, particularly with regard to intracellular activation events, and looking forward, new technologies in structural biophysics will offer further insight into the biology of cytokine signaling to inform the design of targeted therapeutics for treatment of immune-linked diseases such as cancer, infection, and autoimmune disorders.