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Here, we explore whether PEGylation of antibodies can modulate their biodistribution to the eye, an organ once thought to be immune privileged but has recently been shown to be accessible to IV-administered large molecules, such as antibodies. We chose to PEGylate an anti-MerTK antibody, a target with known potential for ocular toxicity, to minimize biodistribution to retinal pigment epithelial cells (RPEs) in the eye by increasing the hydrodynamic volume of the antibody. We used site-specific conjugation to an engineered cysteine on anti-MerTK antibody to chemically attach 40-kDa branched or linear PEG polymers. Despite reduced binding to MerTK on cells, site-specifically PEGylated anti-MerTK retained similar potency in inhibiting MerTK-mediated macrophage efferocytosis of apoptotic cells. Importantly, we found that PEGylation of anti-MerTK significantly reduced MerTK receptor occupancy in RPE cells in both naïve mice and MC-38 tumor-bearing mice, with the branched PEG exhibiting a greater effect than linear PEG. Furthermore, similar to unconjugated anti-MerTK, PEGylated anti-MerTK antibody triggered type I IFN response and exhibited antitumor effect in syngeneic mouse tumor studies. Our results demonstrate the potential of PEGylation to control ocular biodistribution of antibodies.

Immunotherapy with checkpoint inhibitors has proved to be highly effective, with durable responses in a subset of patients. Given their encouraging clinical activity, checkpoint inhibitors are increasingly being tested in clinical trials in combination with chemotherapy. In many instances, there is little understanding of how chemotherapy might influence the quality of the immune response generated by checkpoint inhibitors. In this study, we evaluated the impact of chemotherapy alone or in combination with anti–PD-L1 in a responsive syngeneic tumor model. Although multiple classes of chemotherapy treatment reduced immune cell numbers and activity in peripheral tissues, chemotherapy did not antagonize but in many cases augmented the antitumor activity mediated by anti–PD-L1. This dichotomy between the detrimental effects in peripheral tissues and enhanced antitumor activity was largely explained by the reduced dependence on incoming cells for antitumor efficacy in already established tumors. The effects of the various chemotherapies were also agent specific, and synergy with anti–PD-L1 was achieved by different mechanisms that ultimately helped establish a new threshold for response. These results rationalize the combination of chemotherapy with immunotherapy and suggest that, despite the negative systemic effects of chemotherapy, effective combinations can be obtained through distinct mechanisms acting within the tumor.

During cytotoxic T cell activation, lymphocyte function-associated antigen-1 (LFA-1) engages its ligands on antigen-presenting cells (APCs) or target cells to enhance T cell priming or lytic activity. Inhibiting LFA-1 dampens T cell-dependent symptoms in inflammation, autoimmune diseases, and graft-versus-host disease. However, the therapeutic potential of augmenting LFA-1 function is less explored. Here, we show that genetic deletion or inhibition of mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) enhances LFA-1 activation on CD8 T cells and improves their adherence to APCs or LFA-1 ligand. In addition, loss of Map4k4 increases CD8 T cell priming, which culminates in enhanced antigen-dependent activation, proliferation, cytokine production, and cytotoxic activity, resulting in impaired tumor growth and improved response to viral infection. LFA-1 inhibition reverses these phenotypes. The ERM (ezrin, radixin, and moesin) proteins reportedly regulate T cell-APC conjugation, but the molecular regulator and effector of ERM proteins in T cells have not been defined. In this study, we demonstrate that the ERM proteins serve as mediators between MAP4K4 and LFA-1. Last, systematic analyses of many organs revealed that inducible whole-body deletion of Map4k4 in adult animals is tolerated under homeostatic conditions. Our results uncover MAP4K4 as a potential target to augment antitumor and antiviral immunity.

Targeted inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) can induce regression of tumors bearing activating mutations in the Ras pathway but rarely leads to tumor eradication. Although combining MEK inhibition with T-cell-directed immunotherapy might lead to more durable efficacy, T cell responses are themselves at least partially dependent on MEK activity. We show here that MEK inhibition did profoundly block naive CD8(+) T cell priming in tumor-bearing mice, but actually increased the number of effector-phenotype antigen-specific CD8(+) T cells within the tumor. MEK inhibition protected tumor-infiltrating CD8(+) T cells from death driven by chronic TCR stimulation while sparing cytotoxic activity. Combining MEK inhibition with anti-programmed death-ligand 1 (PD-L1) resulted in synergistic and durable tumor regression even where either agent alone was only modestly effective. Thus, despite the central importance of the MAP kinase pathway in some aspects of T cell function, MEK-targeted agents can be compatible with T-cell-dependent immunotherapy.

Summary Clearance of apoptotic cells by macrophages prevents excessive inflammation and supports immune tolerance. Here, we examined the effect of blocking apoptotic cell clearance on anti-tumor immune response. We generated an antibody that selectively inhibited efferocytosis by phagocytic receptor MerTK. Blockade of MerTK resulted in accumulation of apoptotic cells within tumors and triggered a type I interferon response. Treatment of tumor-bearing mice with anti-MerTK antibody stimulated T cell activation and synergized with anti-PD-1 or anti-PD-L1 therapy. The anti-tumor effect induced by anti-MerTK treatment was lost in Stinggt/gt mice, but not in Cgas−/− mice. Abolishing cGAMP production in Cgas−/− tumor cells, depletion of extracellular ATP, or inactivation of the ATP-gated P2X7R channel also compromised the effects of MerTK blockade. Mechanistically, extracellular ATP acted via P2X7R to enhance the transport of extracellular cGAMP into macrophages and subsequent STING activation. Thus, MerTK blockade increases tumor immunogenicity and potentiates anti-tumor immunity, which has implications for cancer immunotherapy.

Autophagy is a cell survival process essential for the regulation of immune responses to infections. However, the role of T cell autophagy in anti-tumor immunity is less clear. Here, we demonstrate a cell-autonomous role for autophagy in the regulation of CD8

Cell migration is a stepwise process that coordinates multiple molecular machineries. Using in vitro angiogenesis screens with short interfering RNA and chemical inhibitors, we define here a MAP4K4–moesin–talin–β1-integrin molecular pathway that promotes efficient plasma membrane retraction during endothelial cell migration. Loss of MAP4K4 decreased membrane dynamics, slowed endothelial cell migration, and impaired angiogenesis in vitro and in vivo. In migrating endothelial cells, MAP4K4 phosphorylates moesin in retracting membranes at sites of focal adhesion disassembly. Epistasis analyses indicated that moesin functions downstream of MAP4K4 to inactivate integrin by competing with talin for binding to β1-integrin intracellular domain. Consequently, loss of moesin (encoded by the MSN gene) or MAP4K4 reduced adhesion disassembly rate in endothelial cells. Additionally, α5β1-integrin blockade reversed the membrane retraction defects associated with loss of Map4k4 in vitro and in vivo. Our study uncovers a novel aspect of endothelial cell migration. Finally, loss of MAP4K4 function suppressed pathological angiogenesis in disease models, identifying MAP4K4 as a potential therapeutic target. A new MAP4K4–moesin–talin–β1-integrin pathway regulating endothelial cell motility was discovered through chemical and siRNA screens; loss of Map4k4 or inhibition of MAP4K4 kinase activity altered the sprout morphology of endothelial cells during angiogenesis by blocking moesin phosphorylation, which regulates the disassembly of focal adhesions, demonstrating that this pathway is involved in both normal and pathological angiogenesis. Cell migration is crucial in important biological processes such as embryogenesis, inflammation and angiogenesis, but many aspects of the cell motility machinery remain to be defined at the molecular level. Here Weilan Ye and colleagues identify a previously unknown pathway regulating endothelial cell motility. Using chemical and RNA screens, they found that a selective inhibitor of MAP4K4 altered the sprout morphology of endothelial cells during angiogenesis. The inhibitor caused endothelial cells to accumulate long, thin subcellular protrusions, suggesting a failure to retract these protrusions. The authors show that MAP4K4 phosphorylates moesin, which regulates the disassembly of focal adhesions by inactivating β1-integrin, and they demonstrate that this MAP4K4–moesin–talin–β1-integrin pathway plays a role in both normal and pathological angiogenesis.

Molecularly targeted drug therapies have revolutionized cancer treatment; however, resistance remains a major limitation to their overall efficacy. Epithelial-to-mesenchymal transition (EMT) has been linked to acquired resistance to tyrosine kinase inhibitors (TKI), independent of mutational resistance mechanisms. AXL is a receptor tyrosine kinase associated with EMT that has been implicated in drug resistance and has emerged as a candidate therapeutic target. Across 643 human cancer cell lines that were analyzed, elevated AXL was strongly associated with a mesenchymal phenotype, particularly in triple-negative breast cancer and non–small cell lung cancer. In an unbiased screen of small-molecule inhibitors of cancer-relevant processes, we discovered that AXL inhibition was specifically synergistic with antimitotic agents in killing cancer cells that had undergone EMT and demonstrated associated TKI resistance. However, we did not find that AXL inhibition alone could overcome acquired resistance to EGFR TKIs in the EMT setting, as previously reported. These findings reveal a novel cotreatment strategy for tumors displaying mesenchymal features that otherwise render them treatment refractory. Cancer Res; 74(20); 5878–90. ©2014 AACR.

Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is a serine/threonine kinase implicated in the regulation of many biological processes. A fragment-based lead discovery approach was used to generate potent and selective MAP4K4 inhibitors. The fragment hit pursued in this article had excellent ligand efficiency (LE), an important attribute for subsequent successful optimization into drug-like lead compounds. The optimization efforts eventually led us to focus on the pyridopyrimidine series, from which 6-(2-fluoropyridin-4-yl)pyrido[3,2-d]pyrimidin-4-amine (29) was identified. This compound had low nanomolar potency, excellent kinase selectivity, and good in vivo exposure, and demonstrated in vivo pharmacodynamic effects in a human tumor xenograft model.

BACKGROUND: Normal "wear and tear" of bones during weight bearing activity creates microdamage that triggers bone to heal itself.OBJECTIVE: A controlled laboratory study was carried out to determine the effect of lithotripsy on bone apposition and resorption in osteopenic, hind-limb suspended, aged rats compared to age-matched controls allowed normal weight bearing (cage) activity. METHODS: First, we tested the feasibility of using a clinical lithotripsy device, designed for treatment of kidney stones, to create microdamage in bone. In a second step, we tested the hypothesis that microdamage induced through lithotripsy treatment increases bone apposition in aged rats. In a third step, we exposed osteopenic, aged rats to lithotripsy to evaluate the effectiveness of lithotripsy in counteracting bone loss due to simulated disuse. RESULTS: Both in aged, weight bearing as well as aged, osteopenic rats, we showed that lithotripsy effectively increases the area of bone apposition along the periosteal and endosteal surfaces. While new bone apposition concentrates in areas of lithotripsy treatment in aged bone of weight bearing rats, new bone apposition extends beyond the immediate treatment site (to the contralateral limb) of osteopenic animals. Furthermore, bone resorption decreases in osteopenic (hindlimb suspended) and aged rat femora treated with lithotripsy, compared to baseline and hindlimb suspended controls. This decrease in resorption is not observed in the contralateral limb of osteopenic animals.CONCLUSION: Taken as a whole, lithotripsy may offer a viable treatment method for disuse osteopenia or osteoporosis, particularly for aging individuals or for those who are limited in their weight bearing activities.

Purpose: Immunodeficient mice transplanted with subcutaneous tumors (xenograft or allograft) are widely used as a model of preclinical activity for the discovery and development of anticancer drug candidates. Despite their widespread use, there is a widely held view that these models provide minimal predictive value for discerning clinically active versus inactive agents. To improve the predictive nature of these models, we have carried out a retrospective population pharmacokinetic–pharmacodynamic (PK–PD) analysis of relevant xenograft/allograft efficacy data for eight agents (molecularly targeted and cytotoxic) with known clinical outcome. Experimental Design: PK–PD modeling was carried out to first characterize the relationship between drug concentration and antitumor activity for each agent in dose-ranging xenograft or allograft experiments. Next, simulations of tumor growth inhibition (TGI) in xenografts/allografts at clinically relevant doses and schedules were carried out by replacing the murine pharmacokinetics, which were used to build the PK–PD model with human pharmacokinetics obtained from literature to account for species differences in pharmacokinetics. Results: A significant correlation (r = 0.91, P = 0.0008) was observed between simulated xenograft/allograft TGI driven by human pharmacokinetics and clinical response but not when TGI observed at maximum tolerated doses in mice was correlated with clinical response (r = 0.36, P = 0.34). Conclusions: On the basis of these analyses, agents that led to greater than 60% TGI in preclinical models, at clinically relevant exposures, are more likely to lead to responses in the clinic. A proposed strategy for the use of murine subcutaneous models for compound selection in anticancer drug discovery is discussed. Clin Cancer Res; 18(14); 3846–55. ©2012 AACR.

CYP Oxidoreductase (Por) is the essential electron donor for all CYP enzymes and is responsible for the activation of CYP. The Taconic Hepatic CYP Reductase Null (HRN) mouse model possesses a targeted mutation that results in liver-specific deletion of the Por gene thereby resulting in a disruption of CYP metabolism in the liver. The objectives of these studies were to further characterize the HRN mouse using probe drugs metabolized by CYP. In addition, tumor exposure in xenograft tumor bearing HRN immune-compromised (nude) mice was also determined. In HRN mice following intravenous (iv) administration of midazolam, clearance (CL) was reduced by ∼ 80% compared to wild-type mice (WT). After oral administration, the AUC of midazolam was increased by ∼ 20-fold in HRN mice compared to WT mice; this greater effect suggests that hepatic first pass plays a role in the oral CL of midazolam. A 50% and an 80% decrease in CL were also observed in HRN mice following iv administration of docetaxel and theophylline, respectively, compared to WT mice. In addition, a 2- to 3-fold increase in tumor concentrations of G4222, a tool compound, were observed in tumor bearing HRN nude mice compared to tumor bearing nude WT mice. The observations from these experiments demonstrate that, for compounds that are extensively metabolized by hepatic CYP, the HRN mouse model could potentially be valuable for evaluating in vivo efficacy of tool compounds in drug discovery where high hepatic CL and low exposure may prevent in vivo evaluation of a new chemical entity.

There are concerns regarding increased cancer incidence in patients treated with ezetimibe, an inhibitor of the absorption of dietary cholesterol. Here we tested the hypothesis that ezetimibe will accelerate mammary tumorigenesis in rats. The drug was administered at a dose of 1 ppm in an AIN-93G diet that contained 0.3% cholesterol. This experimental diet and control diets that contained either no additions or cholesterol or ezetimibe only, were fed to groups of 30 Sprague-Dawley rats 3 days after they were treated with 50 mg/kg methylnitrosourea (MNU). All rats were euthanized 22 weeks after MNU administration. Tumor multiplicity was significantly smaller in rats fed cholesterol than those fed no cholesterol (1.84 +/- 0.42 vs. 3.86 +/- 0.86 respectively, P0.05), but was significantly greater in the cholesterol/ezetimibe group than the group fed only cholesterol (3.48 +/- 0.59 vs. 1.84 +/- 0.42 respectively, P0.04). The average weight of tumors/rat was also significantly larger in the cholesterol/ezetimibe group than those fed cholesterol alone (5.67 +/- 1.15 vs. 2.56 +/- 0.71 respectively, P0.04). As expected, ezetimibe prevented the cholesterol raising effect of the dietary cholesterol. These results show that ezetimibe reverses the inhibitory effect of dietary cholesterol on the development of rat mammary tumors.