APX005M, a CD40 agonist antibody with unique epitope specificity and Fc receptor binding profile for optimal therapeutic application.

Targeting CD40 with agonist antibodies is a promising approach to cancer immunotherapy. CD40 acts as a master regulator of immunity by mobilizing multiple arms of the immune system to initiate highly effective CD8 + T-cell-mediated responses against foreign pathogens and tumors. The clinical development of CD40 agonist antibodies requires careful optimization of the antibody to maximize therapeutic efficacy while minimizing adverse effects. Both epitope specificity and isotype are critical for CD40 agonist antibody mechanism of action and potency. We developed a novel antibody, APX005M, which binds with high affinity to the CD40 ligand-binding site on CD40 and is optimized for selective interaction with Fcγ receptors to enhance agonistic potency while limiting less desirable Fc-effector functions like antibody-dependent cellular cytotoxicity of CD40-expressing immune cells. APX005M is a highly potent inducer of innate and adaptive immune effector responses and represents a promising CD40 agonist antibody for induction of an effective anti-tumor immune response with a favorable safety profile.

Cytosolic Receptor Melanoma Differentiation-Associated Protein 5 Mediates Preconditioning-Induced Neuroprotection Against Cerebral Ischemic Injury.

BACKGROUND AND PURPOSE: Preconditioning with poly-l-lysine and carboxymethylcellulose (ICLC) provides robust neuroprotection from cerebral ischemia in a mouse stroke model. However, the receptor that mediates neuroprotection is unknown. As a synthetic double-stranded RNA, poly-ICLC may bind endosomal Toll-like receptor 3 or one of the cytosolic retinoic acid-inducible gene-I-like receptor family members, retinoic acid-inducible gene-I, or melanoma differentiation-associated protein 5. Activation of these receptors culminates in type I interferons (IFN-α/ß) induction-a response required for poly-ICLC-induced neuroprotection. In this study, we investigate the receptor required for poly-ICLC-induced neuroprotection. METHODS: Toll-like receptor 3, melanoma differentiation-associated protein 5-, and IFN-promoter stimulator 1-deficient mice were treated with poly-ICLC 24 hours before middle cerebral artery occlusion. Infarct volume was measured 24 hours after stroke to identify the receptor signaling pathways involved in protection. IFN-α/ß induction was measured in plasma samples collected 6 hours after poly-ICLC treatment. IFN-ß-deficient mice were used to test the requirement of IFN-ß for poly-ICLC-induced neuroprotection. Mice were treated with recombinant IFN-α-A to test the role of IFN-α as a potential mediator of neuroprotection. RESULTS: Poly-ICLC induction of both neuroprotection and systemic IFN-α/ß requires the cytosolic receptor melanoma differentiation-associated protein 5 and the adapter molecule IFN-promoter stimulator 1, whereas it is independent of Toll-like receptor 3. IFN-ß is not required for poly-ICLC-induced neuroprotection. IFN-α treatment protects against stroke. CONCLUSIONS: Poly-ICLC preconditioning is mediated by melanoma differentiation-associated protein 5 and its adaptor molecule IFN-promoter stimulator 1. This is the first evidence that a cytosolic receptor can mediate neuroprotection, providing a new target for the development of therapeutic agents to protect the brain from ischemic injury.

Steps to translate preconditioning from basic research to the clinic.

Efforts to treat cardiovascular and cerebrovascular diseases often focus on the mitigation of ischemia-reperfusion (I/R) injury. Many treatments or "preconditioners" are known to provide substantial protection against the I/R injury when administered prior to the event. Brief periods of ischemia itself have been validated as a means to achieve neuroprotection in many experimental disease settings, in multiple organ systems, and in multiple species suggesting a common pathway leading to tolerance. In addition, pharmacological agents that act as potent preconditioners have been described. Experimental induction of neuroprotection using these various preconditioning paradigms has provided a unique window into the brain's endogenous protective mechanisms. Moreover, preconditioning agents themselves hold significant promise as clinical-stage therapies for prevention of I/R injury. The aim of this article is to explore several key steps involved in the preclinical validation of preconditioning agents prior to the conduct of clinical studies in humans. Drug development is difficult, expensive and relies on multi-factorial analysis of data from diverse disciplines. Importantly, there is no single path for the preclinical development of a novel therapeutic and no proven strategy to ensure success in clinical translation. Rather, the conduct of a diverse array of robust preclinical studies reduces the risk of clinical failure by varying degrees depending upon the relevance of preclinical models and drug pharmacology to humans. A strong sense of urgency and high tolerance of failure are often required to achieve success in the development of novel treatment paradigms for complex human conditions.

Poly-ICLC preconditioning protects the blood-brain barrier against ischemic injury in vitro through type I interferon signaling.

Preconditioning with a low dose of harmful stimulus prior to injury induces tolerance to a subsequent ischemic challenge resulting in neuroprotection against stroke. Experimental models of preconditioning primarily focus on neurons as the cellular target of cerebral protection, while less attention has been paid to the cerebrovascular compartment, whose role in the pathogenesis of ischemic brain injury is crucial. We have shown that preconditioning with polyinosinic polycytidylic acid (poly-ICLC) protects against cerebral ischemic damage. To delineate the mechanism of poly-ICLC protection, we investigated whether poly-ICLC preconditioning preserves the function of the blood-brain barrier (BBB) in response to ischemic injury. Using an in vitro BBB model, we found that poly-ICLC treatment prior to exposure to oxygen-glucose deprivation maintained the paracellular and transcellular transport across the endothelium and attenuated the drop in transendothelial electric resistance. We found that poly-ICLC treatment induced interferon (IFN) ß mRNA expression in astrocytes and microglia and that type I IFN signaling in brain microvascular endothelial cells was required for protection. Importantly, this implicates a potential mechanism underlying neuroprotection in our in vivo experimental stroke model, where type I IFN signaling is required for poly-ICLC-induced neuroprotection against ischemic injury. In conclusion, we are the first to show that preconditioning with poly-ICLC attenuates ischemia-induced BBB dysfunction. This mechanism is likely an important feature of poly-ICLC-mediated neuroprotection and highlights the therapeutic potential of targeting BBB signaling pathways to protect the brain against stroke.

TLR9 bone marrow chimeric mice define a role for cerebral TNF in neuroprotection induced by CpG preconditioning.

Systemic preconditioning with the TLR9 ligand CpG induces neuroprotection against brain ischemic injury through a tumor necrosis factor (TNF)-dependent mechanism. It is unclear how systemic administration of CpG engages the brain to induce the protective phenotype. To address this, we created TLR9-deficient reciprocal bone marrow chimeric mice lacking TLR9 on either hematopoietic cells or radiation-resistant cells of nonhematopoietic origin. We report that wild-type mice reconstituted with TLR9-deficient hematopoietic cells failed to show neuroprotection after systemic CpG preconditioning. Further, while hematopoietic expression of TLR9 is required for CpG-induced neuroprotection it is not sufficient to restore protection to TLR9-deficient mice that are reconstituted with hematopoietic cells bearing TLR9. To determine whether the absence of protection was associated with TNF, we examined TNF levels in the systemic circulation and the brain. We found that although TNF is required for CpG preconditioning, systemic TNF levels did not correlate with the protective phenotype. However, induction of cerebral TNF mRNA required expression of TLR9 on both hematopoietic and nonhematopoietic cells and correlated with neuroprotection. In accordance with these results, we show the therapeutic potential of intranasal CpG preconditioning, which induces brain TNF mRNA and robust neuroprotection with no concomitant increase in systemic levels of TNF.

Poly-IC preconditioning protects against cerebral and renal ischemia-reperfusion injury.

Preconditioning induces ischemic tolerance, which confers robust protection against ischemic damage. We show marked protection with polyinosinic polycytidylic acid (poly-IC) preconditioning in three models of murine ischemia-reperfusion injury. Poly-IC preconditioning induced protection against ischemia modeled in vitro in brain cortical cells and in vivo in models of brain ischemia and renal ischemia. Further, unlike other Toll-like receptor (TLR) ligands, which generally induce significant inflammatory responses, poly-IC elicits only modest systemic inflammation. Results show that poly-IC is a new powerful prophylactic treatment that offers promise as a clinical therapeutic strategy to minimize damage in patient populations at risk of ischemic injury.

Differential maturation of murine bone-marrow derived dendritic cells with lipopolysaccharide and tumor necrosis factor-alpha.

Dendritic cells (DCs) play a key role in the interface between the innate and acquired immune systems. In response to both exogenous as well as endogenous signals, DCs undergo a programmed maturation to become an efficient, antigen-presenting cell. Yet little is known regarding the differential responses by endogenous versus exogenous stimuli on DC maturation. In the present report, we have compared the phenotypic, functional, and genome-wide expression responses associated with maturation by bone marrow derived DCs to either an endogenous danger signal, tumor necrosis factor-alpha (TNF-alpha), or a microbial product, bacterial lipopolysaccharide (LPS). Examination of the cell surface expression of DCs as well as cytokine production demonstrated that patterns of DC maturation varied dramatically depending upon the stimulus. Whereas LPS was highly effective in terms of inducing phenotypic and functional maturation, TNF-alpha exposure produced a phenotypically distinct DC. Gene expression patterns in DCs 6 and 24 h after LPS and TNF-alpha exposure revealed that these activation signals produce fundamentally different genomic responses. Supervised analysis revealed that the expression of 929 probe sets discriminated among the treatment groups, and the patterns of gene expression in TNF-alpha stimulated DCs were more similar to unstimulated cells at both 6 and 24 h post-stimulation than to LPS-stimulated cells at the same time points. These findings reveal that DCs are capable of a varying phenotypic response to different antigens and endogenous signals.

Functional modification of dendritic cells with recombinant adenovirus encoding interleukin 10 for the treatment of sepsis.

Control of dendritic cell (DC) function is critical for strategies to modulate innate and acquired immune responses. We examined whether transduction of murine DCs with adenoviral vectors (Adv) expressing interleukin (IL)-10 could alter their phenotype and T cell stimulatory function. Murine bone marrow-derived DCs were transduced with AdV encoding human IL-10 or green fluorescent protein (GFP). Whereas transduction of immature DCs with AdV/GFP resulted in dose-dependent maturation, DCs transduced with Adv/IL-10 maintained an immature state with low major histocompatibility complex (MHC) class II, CD86, and IL-12 expression. The Adv/IL-10 transduced DCs were phenotypically unique, characterized by suppression of IL-12 expression, failure to stimulate Th1 or Th2 cytokine responses, and retained capacity to endocytose antigen. Importantly, Adv/IL-10-transduced DCs were biologically active in vivo, in that administration of these DCs into mice before a generalized peritonitis significantly improved survival. We conclude that Adv/IL-10 transduction of DCs provides an efficient means to modulate DC function. The capacity to modify DCs by adenoviral expression of IL-10 may provide a novel ex vivo or in vivo approach to mitigate acute and chronic inflammatory diseases like sepsis.

Influence of recombinant adenovirus on liver injury in endotoxicosis and its modulation by IL-10 expression.

Adenovirus-based gene therapy offers a unique opportunity to target gene expression to the liver by systemic delivery. However, systemic administration of a first generation adenoviral construct elicits an inflammatory response leading to TNF-alpha-dependent liver injury. The aim of this study was to evaluate whether the systemic administration of recombinant adenovirus exacerbates a subsequent TNF-alpha-dependent liver injury induced by D-galactosamine and lipopolysaccharide. Surprisingly, low-dose adenovirus administration (10(5) particles) protects, while high-dose adenovirus (10(10) particles) is associated with an exaggerated hepatic inflammatory response from a subsequent D-galactosamine and lipopolysaccharide challenge. This exacerbation is TNF-alpha dependent, since treatment with a TNF inhibitor fully protects against the liver injury. Moreover, intravenous administration of an adenoviral construct expressing the anti-inflammatory protein interleukin-10 reduces TNF-alpha appearance and attenuates the increased hepatocyte injury. Taken together, this report demonstrates potential additive effects of TNF-alpha responses induced by adenovirus and other inflammatory signals, and suggests that the response can be mitigated by relative adenovirus particle dose or by inhibitors, such as TNF-binding protein or interleukin 10.

Characterization of the systemic loss of dendritic cells in murine lymph nodes during polymicrobial sepsis.

Dendritic cells (DCs) play a key role in critical illness and are depleted in spleens from septic patients and mice. To date, few studies have characterized the systemic effect of sepsis on DC populations in lymphoid tissues. We analyzed the phenotype of DCs and Th cells present in the local (mesenteric) and distant (inguinal and popliteal) lymph nodes of mice with induced polymicrobial sepsis (cecal ligation and puncture). Flow cytometry and immunohistochemical staining demonstrated that there was a significant local (mesenteric nodes) and partial systemic (inguinal, but not popliteal nodes) loss of DCs from lymph nodes in septic mice, and that this process was associated with increased apoptosis. This sepsis-induced loss of DCs occurred after CD3(+)CD4(+) T cell activation and loss in the lymph nodes, and the loss of DCs was not preceded by any sustained increase in their maturation status. In addition, there was no preferential loss of either mature/activated (MHCII(high)/CD86(high)) or immature (MHCII(low)/CD86(low)) DCs during sepsis. However, there was a preferential loss of CD8(+) DCs in the local and distant lymph nodes. The loss of DCs in lymphoid tissue, particularly CD8(+) lymphoid-derived DCs, may contribute to the alterations in acquired immune status that frequently accompany sepsis.

Aberrant signaling in the TNFalpha/TNF receptor 1 pathway of the NZM2410 lupus-prone mouse.

The purpose of this study was to evaluate the ability to induce TNFalpha-dependent apoptosis in vivo in predisease lupus-prone NZM2410 and derived B6.NZM congenic mouse strains. An endotoxicosis model that utilizes LPS and d-galactosamine to induce mortality by TNFalpha/TNFR1-dependent hepatocyte apoptosis was used to assess TNFalpha production, apoptotic signaling, and effects on the production of IL-6 and IL-10. NZM2410 was found to be resistant to endotoxicosis and to produce significantly less TNFalpha-induced IL-6 and IL-10. At low doses of LPS, partial resistance was associated with the Tnfa(w) allele. At higher doses of LPS, partial resistance cosegregated with lupus-susceptibility loci and functionally mapped downstream of caspase 3. Additional partial resistance in NZM2410 was also found upstream of FADD. These results demonstrate the existence of multiple defects in the TNFalpha/TNFR1 signaling pathway in the NZM2410 mouse and their relevance to lupus pathogenesis is discussed.

Increased survival in sepsis by in vivo adenovirus-induced expression of IL-10 in dendritic cells.

The dendritic cell (DC) is the most potent APC of the immune system, capable of stimulating naive T cells to proliferate and differentiate into effector T cells. Recombinant adenovirus (Adv) readily transduces DCs in vitro allowing directed delivery of transgenes that modify DC function and immune responses. In this study we demonstrate that footpad injection of a recombinant Adv readily targets transduction of myeloid and lymphoid DCs in the draining popliteal lymph node, but not in other lymphoid organs. Popliteal DCs transduced with an empty recombinant Adv undergo maturation, as determined by high MHC class II and CD86 expression. However, transduction with vectors expressing human IL-10 limit DC maturation and associated T cell activation in the draining lymph node. The extent of IL-10 expression is dose dependent; transduction with low particle numbers (10(5)) yields only local expression, while transduction with higher particle numbers (10(7) and 10(10)) leads additionally to IL-10 appearance in the circulation. Furthermore, local DC expression of human IL-10 following in vivo transduction with low particle numbers (10(5)) significantly improves survival following cecal ligation and puncture, suggesting that compartmental modulation of DC function profoundly alters the sepsis-induced immune response.