An Allosteric Anti-tryptase Antibody for the Treatment of Mast Cell-Mediated Severe Asthma.

Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.

Homeostatic functions of monocytes and interstitial lung macrophages are regulated via collagen domain-binding receptor LAIR1.

Myeloid cells encounter stromal cells and their matrix determinants on a continual basis during their residence in any given organ. Here, we examined the impact of the collagen receptor LAIR1 on myeloid cell homeostasis and function. LAIR1 was highly expressed in the myeloid lineage and enriched in non-classical monocytes. Proteomic definition of the LAIR1 interactome identified stromal factor Colec12 as a high-affinity LAIR1 ligand. Proteomic profiling of LAIR1 signaling triggered by Collagen1 and Colec12 highlighted pathways associated with survival, proliferation, and differentiation. Lair1-/- mice had reduced frequencies of Ly6C- monocytes, which were associated with altered proliferation and apoptosis of non-classical monocytes from bone marrow and altered heterogeneity of interstitial macrophages in lung. Myeloid-specific LAIR1 deficiency promoted metastatic growth in a melanoma model and LAIR1 expression associated with improved clinical outcomes in human metastatic melanoma. Thus, monocytes and macrophages rely on LAIR1 sensing of stromal determinants for fitness and function, with relevance in homeostasis and disease.

Anti-α-synuclein c-terminal antibodies block PFF uptake and accumulation of phospho-synuclein in preclinical models of Parkinson's disease.

Parkinson's disease (PD), a neurodegenerative disease affecting dopaminergic (DA) neurons, is characterized by decline of motor function and cognition. Dopaminergic cell loss is associated with accumulation of toxic alpha synuclein aggregates. As DA neuron death occurs late in the disease, therapeutics that block the spread of alpha synuclein may offer functional benefit and delay disease progression. To test this hypothesis, we generated antibodies to the C terminal region of synuclein with high nanomolar affinity and characterized them in in vitro and in vivo models of spread. Interestingly, we found that only antibodies with high affinity to the distal most portion of the C-terminus robustly reduced uptake of alpha synuclein preformed fibrils (PFF) and accumulation of phospho (S129) alpha synuclein in cell culture. Additionally, the antibody treatment blocked the spread of phospho (S129) alpha synuclein associated-pathology in a mouse model of synucleinopathy. Blockade of neuronal PFF uptake by different antibodies was more predictive of in vivo activity than their binding potency to monomeric or oligomeric forms of alpha synuclein. These data demonstrate that antibodies directed to the C-terminus of the alpha synuclein have differential effects on target engagement and efficacy. Furthermore, our data provides additional support for the development of alpha synuclein antibodies as a therapeutic strategy for PD patients.

Development of an ultra-sensitive human IL-33 biomarker assay for age-related macular degeneration and asthma drug development.

BACKGROUND: Over the past decade, human Interleukin 33 (hIL-33) has emerged as a key contributor to the pathogenesis of numerous inflammatory diseases. Despite the existence of several commercial hIL-33 assays spanning multiple platform technologies, their ability to provide accurate hIL-33 concentration measurements and to differentiate between active (reduced) and inactive (oxidized) hIL-33 in various matrices remains uncertain. This is especially true for lower sample volumes, matrices with low hIL-33 concentrations, and matrices with elevated levels of soluble Interleukin 1 Receptor-Like 1 (sST2), an inactive form of ST2 that competes with membrane bound ST2 for hIL-33 binding. RESULTS: We tested the performance of several commercially available hIL-33 detection assays in various human matrices and found that most of these assays lacked the sensitivity to accurately detect reduced hIL-33 at biologically relevant levels (sub-to-low pg/mL), especially in the presence of human sST2 (hsST2), and/or lacked sufficient target specificity. To address this, we developed and validated a sensitive and specific enzyme-linked immunosorbent assay (ELISA) capable of detecting reduced and total hIL-33 levels even in the presence of high concentrations of sST2. By incorporating the immuno-polymerase chain reaction (iPCR) platform, we further increased the sensitivity of this assay for the reduced form of hIL-33 by ~ 52-fold. Using this hIL-33 iPCR assay, we detected hIL-33 in postmortem human vitreous humor (VH) samples from donors with age-related macular degeneration (AMD) and found significantly increased hIL-33 levels when compared to control individuals. No statistically significant difference was observed in aqueous humor (AH) from AMD donors nor in plasma and nasosorption fluid (NF) from asthma patients compared to control individuals. CONCLUSIONS: Unlike existing commercial hIL-33 assays, our hIL-33 bioassays are highly sensitive and specific and can accurately quantify hIL-33 in various human clinical matrices, including those with high levels of hsST2. Our results provide a proof of concept of the utility of these assays in clinical trials targeting the hIL-33/hST2 pathway.

Fibroblast Activation Protein Cleaves and Inactivates Fibroblast Growth Factor 21.

FGF21 is a stress-induced hormone with potent anti-obesity, insulin-sensitizing, and hepatoprotective properties. Although proteolytic cleavage of recombinant human FGF21 in preclinical species has been observed previously, the regulation of endogenously produced FGF21 is not well understood. Here we identify fibroblast activation protein (FAP) as the enzyme that cleaves and inactivates human FGF21. A selective chemical inhibitor, immunodepletion, or genetic deletion of Fap stabilized recombinant human FGF21 in serum. In addition, administration of a selective FAP inhibitor acutely increased circulating intact FGF21 levels in cynomolgus monkeys. On the basis of our findings, we propose selective FAP inhibition as a potential therapeutic approach to increase endogenous FGF21 activity for the treatment of obesity, type 2 diabetes, non-alcoholic steatohepatitis, and related metabolic disorders.

TGFß2 and TGFß3 isoforms drive fibrotic disease pathogenesis.

Transforming growth factor-ß (TGFß) is a key driver of fibrogenesis. Three TGFß isoforms (TGFß1, TGFß2, and TGFß3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFß2 and TGFß3 have not been well characterized. Here, we show that the latent forms of TGFß2 and TGFß3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFß1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFß2 and TGFß3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFß isoform-selective antibodies demonstrated that TGFß2 and TGFß3 are independently involved in mouse fibrosis models in vivo, and selective TGFß2 and TGFß3 inhibition does not lead to the increased inflammation observed with pan-TGFß isoform inhibition. A cocrystal structure of a TGFß2-anti-TGFß2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFß2 and/or TGFß3 while sparing TGFß1 may alleviate fibrosis without toxicity concerns associated with pan-TGFß blockade.

Immune repertoire mining for rapid affinity optimization of mouse monoclonal antibodies.

Traditional hybridoma and B cell cloning antibody discovery platforms have inherent limits in immune repertoire sampling depth. One consequence is that monoclonal antibody (mAb) leads often lack the necessary affinity for therapeutic applications, thus requiring labor-intensive and time-consuming affinity in vitro engineering optimization steps. Here, we show that high-affinity variants of mouse-derived mAbs can be rapidly obtained by testing of somatic sequence variants obtained by deep sequencing of antibody variable regions in immune repertories from immunized mice, even with a relatively sparse sampling of sequence variants from large sequence datasets. Affinity improvements can be achieved for mAbs with a wide range of affinities. The optimized antibody variants derived from immune repertoire mining have no detectable in vitro off-target binding and have in vivo clearance comparable to the parental mAbs, essential properties in therapeutic antibody leads. As generation of antibody variants in vitro is replaced by mining of variants generated in vivo, the procedure can be applied to rapidly identify affinity-optimized mAb variants.

Structural basis of the broadly neutralizing anti-interferon-α antibody rontalizumab.

Interferons-alpha (IFN-α) are the expressed gene products comprising thirteen type I interferons with protein pairwise sequence similarities in the 77-96% range. Three other widely expressed human type I interferons, IFN-ß, IFN-κ and IFN-ω have sequences 29-33%, 29-32% and 56-60% similar to the IFN-αs, respectively. Type I interferons act on immune cells by producing subtly different immune-modulatory effects upon binding to the extracellular domains of a heterodimeric cell-surface receptor composed of IFNAR1 and IFNAR2, most notably anti-viral effects. IFN-α has been used to treat infection by hepatitis-virus type C (HCV) and a correlation between hyperactivity of IFN-α-induced signaling and systemic lupus erythematosis (SLE), or lupus, has been noted. Anti-IFN-α antibodies including rontalizumab have been under clinical study for the treatment of lupus. To better understand the rontalizumab mechanism of action and specificity, we determined the X-ray crystal structure of the Fab fragment of rontalizumab bound to human IFN-α2 at 3Å resolution and find substantial overlap of the antibody and IFNA2 epitopes on IFN-α2.

Fibroblast growth factor 19 increases metabolic rate and reverses dietary and leptin-deficient diabetes.

Hormonal control of metabolic rate can be important in regulating the imbalance between energy intake and expenditure that underlies the development of obesity. In mice fed a high-fat diet, human fibroblast growth factor 19 (FGF19) increased metabolic rate [1.53 +/- 0.06 liters O(2)/h.kg(0.75) (vehicle) vs. 1.93 +/- 0.05 liters O(2)/h.kg(0.75) (FGF19); P < 0.001] and decreased respiratory quotient [0.82 +/- 0.01 (vehicle) vs. 0.80 +/- 0.01 (FGF19); P < 0.05]. In contrast to the vehicle-treated mice that gained weight (0.14 +/- 0.05 g/mouse.d), FGF19-treated mice lost weight (-0.13 +/- 0.03 g/mouse.d; P < 0.001) without a significant change in food intake. Furthermore, in addition to a reduction in weight gain, treatment with FGF19 prevented or reversed the diabetes that develops in mice made obese by genetic ablation of brown adipose tissue or genetic absence of leptin. To explore the mechanisms underlying the FGF19-mediated increase in metabolic rate, we profiled the FGF19-induced gene expression changes in the liver and brown fat. In brown adipose tissue, chronic exposure to FGF19 led to a gene expression profile that is consistent with activation of this tissue. We also found that FGF19 acutely increased liver expression of the leptin receptor (1.8-fold; P < 0.05) and decreased the expression of acetyl coenzyme A carboxylase 2 (0.6-fold; P < 0.05). The gene expression changes were consistent with the experimentally determined increase in fat oxidation and decrease in liver triglycerides. Thus, FGF19 is able to increase metabolic rate concurrently with an increase in fatty acid oxidation.

Critical role of activation induced cytidine deaminase in experimental autoimmune encephalomyelitis.

Multiple Sclerosis (MS) is a neurodegenerative autoimmune disorder caused by chronic inflammation and demyelination within the central nervous system (CNS). Clinical studies in MS patients have demonstrated efficacy with B cell targeted therapies such as anti-CD20. However, the exact role that B cells play in the disease process is unclear. Activation Induced cytidine deaminase (AID) is an essential enzyme for the processes of antibody affinity maturation and isotype switching. To evaluate the impact of affinity maturation and isotype switching, we have interrogated the effect of AID-deficiency in an animal model of MS. Here, we show that the severity of experimental autoimmune encephalomyelitis (EAE) induced by the extracellular domain of human myelin oligodendrocyte glycoprotein (MOG1-125) is significantly reduced in Aicda deficient mice, which, unlike wild-type mice, lack serum IgG to myelin associated antigens. MOG specific T cell responses are comparable between wild-type and Aicda knockout mice suggesting an active role for antigen experienced B cells. Thus affinity maturation and/or class switching are critical processes in the pathogenesis of EAE.

Development and preclinical characterization of a humanized antibody targeting CXCL12.

PURPOSE: Our goal was to develop a potent humanized antibody against mouse/human CXCL12. This report summarized its in vitro and in vivo activities. EXPERIMENTAL DESIGN: Cell surface binding and cell migration assays were used to select neutralizing hamster antibodies, followed by testing in several animal models. Monoclonal antibody (mAb) 30D8 was selected for humanization based on its in vitro and in vivo activities. RESULTS: 30D8, a hamster antibody against mouse and human CXCL12α, CXCL12ß, and CXCL12γ, was shown to dose-dependently block CXCL12α binding to CXCR4 and CXCR7, and CXCL12α-induced Jurkat cell migration in vitro. Inhibition of primary tumor growth and/or metastasis was observed in several models. 30D8 alone significantly ameliorated arthritis in a mouse collagen-induced arthritis model (CIA). Combination with a TNF-α antagonist was additive. In addition, 30D8 inhibited 50% of laser-induced choroidal neovascularization (CNV) in mice. Humanized 30D8 (hu30D8) showed similar in vitro and in vivo activities as the parental hamster antibody. A crystal structure of the hu30D8 Fab/CXCL12α complex in combination with mutational analysis revealed a "hot spot" around residues Asn(44)/Asn(45) of CXCL12α and part of the RFFESH region required for CXCL12α binding to CXCR4 and CXCR7. Finally, hu30D8 exhibited fast clearance in cynomolgus monkeys but not in rats. CONCLUSION: CXCL12 is an attractive target for treatment of cancer and inflammation-related diseases; hu30D8 is suitable for testing this hypothesis in humans.

Pegylated kunitz domain inhibitor suppresses hepsin-mediated invasive tumor growth and metastasis.

The transmembrane serine protease hepsin is one of the most highly upregulated genes in prostate cancer. Here, we investigated its tumor-promoting activity by use of a mouse orthotopic prostate cancer model. First, we compared the tumor growth of low hepsin-expressing LnCaP-17 cells with hepsin-overexpressing LnCaP-34 cells. After implantation of cells into the left anterior prostate lobe, LnCaP-34 tumors not only grew faster based on increased serum prostate-specific antigen levels but also metastasized to local lymph nodes and, most remarkably, invaded the contralateral side of the prostate at a rate of 100% compared with only 18% for LnCaP-17 tumors. The increased tumor growth was not due to nonspecific gene expression changes and was not predicted from the unaltered in vitro growth and invasion of LnCaP-34 cells. A likely explanation is that the in vivo effects of hepsin were mediated by specific hepsin substrates present in the tumor stroma. In a second study, mice bearing LnCaP-34 tumors were treated with a PEGylated form of Kunitz domain-1, a potent hepsin active site inhibitor derived from hepatocyte growth factor activator inhibitor-1 (K(i)(app) 0.30 +/- 0.02 nmol/L). Treatment of established tumors with PEGylated Kunitz domain-1 decreased contralateral prostate invasion (46% weight reduction) and lymph node metastasis (50% inhibition). Moreover, serum prostate-specific antigen level remained reduced during the entire treatment period, reaching a maximal reduction of 76% after 5 weeks of dosing. The findings show that hepsin promotes invasive prostate tumor growth and metastasis and suggest that active site-directed hepsin inhibition could be effective in prostate cancer therapy.

Mice expressing a humanized form of VEGF-A may provide insights into the safety and efficacy of anti-VEGF antibodies.

VEGF-A is important in tumor angiogenesis, and a humanized anti-VEGF-A monoclonal antibody (bevacizumab) has been approved by the FDA as a treatment for metastatic colorectal and nonsquamous, non-small-cell lung cancer in combination with chemotherapy. However, contributions of both tumor- and stromal-cell derived VEGF-A to vascularization of human tumors grown in immunodeficient mice hindered direct comparison between the pharmacological effects of anti-VEGF antibodies with different abilities to block host VEGF. Therefore, by gene replacement technology, we engineered mice to express a humanized form of VEGF-A (hum-X VEGF) that is recognized by many anti-VEGF antibodies and has biochemical and biological properties comparable with WT mouse and human VEGF-A. The hum-X VEGF mouse model was then used to compare the activity and safety of a panel of VEGF Mabs with different affinities for VEGF-A. Although in vitro studies clearly showed a correlation between binding affinity and potency at blocking endothelial cell proliferation stimulated by VEGF, in vivo experiments failed to document any consistent correlation between antibody affinity and the ability to inhibit tumor growth and angiogenesis in most animal models. However, higher-affinity antibodies were more likely to result in glomerulosclerosis during long-term treatment.

Cutting edge: novel human dendritic cell- and monocyte-attracting chemokine-like protein identified by fold recognition methods.

Chemokines play an important role in the immune system by regulating cell trafficking in homeostasis and inflammation. In this study, we report the identification and characterization of a novel cytokine-like protein, DMC (dendritic cell and monocyte chemokine-like protein), which attracts dendritic cells and monocytes. The key to the identification of this putative new chemokine was the application of threading techniques to its uncharacterized sequence. Based on our studies, DMC is predicted to have an IL-8-like chemokine fold and to be structurally and functionally related to CXCL8 and CXCL14. Consistent with our predictions, DMC induces migration of monocytes and immature dendritic cells. Expression studies show that DMC is constitutively expressed in lung, suggesting a potential role for DMC in recruiting monocytes and dendritic cells from blood into lung parenchyma.

Tissue expression, protease specificity, and Kunitz domain functions of hepatocyte growth factor activator inhibitor-1B (HAI-1B), a new splice variant of HAI-1.

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is an integral membrane protein expressed on epithelial cells and contains two extracellular Kunitz domains (N-terminal KD1 and C-terminal KD2) known to inhibit trypsin-like serine proteases. In tumorigenesis and tissue regeneration, HAI-1 regulates the hepatocyte growth factor (HGF)/c-Met pathway by inhibiting the activity of HGF activator (HGFA) and matriptase, two serine proteases that convert pro-HGF into its biologically active form. By screening a placental cDNA library, we discovered a new splice variant of HAI-1 designated HAI-1B that contains an extra 16 amino acids adjacent to the C terminus of KD1. To investigate possible consequences on Kunitz domain function, a soluble form of HAI-1B (sHAI-1B) comprising the entire extracellular domain was produced. First, we found that sHAI-1B displayed remarkable enzyme specificity by potently inhibiting only HGFA (IC50 = 30.5 nm), matriptase (IC50 = 16.5 nm), and trypsin (IC50 = 2.4 nm) among 16 serine proteases examined, including plasminogen activators (urokinase- and tissue-type plasminogen activators), coagulation enzymes thrombin, factors VIIa, Xa, XIa, and XIIa, and activated protein C. Relatively weak inhibition was found for plasmin (IC50 = 399 nm) and plasma kallikrein (IC50 = 686 nm). Second, the functions of the KD1 and KD2 domains in sHAI-1B were investigated using P1 residue-directed mutagenesis to show that inhibition of HGFA, matriptase, trypsin, and plasmin was due to KD1 and not KD2. Furthermore, analysis by reverse transcription-PCR demonstrated that HAI-1B and HAI-1 were co-expressed in normal tissues and various epithelial-derived cancer cell lines. Both isoforms were up-regulated in eight examined ovarian carcinoma specimens, three of which had higher levels of HAI-1B RNA than of HAI-1 RNA. Therefore, previously demonstrated roles of HAI-1 in various physiological and pathological processes likely involve both HAI-1B and HAI-1.