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Methods: List of Antibodies, reagents and cell lines Table S1: Distribution of EphA3 protein in human cancers assessed by IHC. Table S2: Kinetic properties of mouse IIIA4 and chimeric IIIA4 for binding to EphA3 Figure S1: Expression patterns of EphA3 in human cancer tissue samples and cell lines. Figure S2: EphA3 is not expressed in normal adult mouse tissues and cell lines. Figure S3: Tumour targeting characteristics of the chIIIA4 antibody. Figure S4: chIIIA4 accumulation into tumour xenografts and association with [FITC] lectin-stained tumour vessels. Figure S5: The EphA3+ stromal tumour infiltrate contains a BM-derived cell population with mesenchymal stromal cell surface phenotype. Figure S6: Bone-marrow origin of EphA3+ tumour stromal cells. Figure S7: EphA3+ MSC's are not exposed to tumour endogenous ephrins; their activation with pre-clustered chIIIA4 triggers cell contraction and apoptosis. Figure S8: cIIIA4 antibody treatment-effects in PCa tumours.

(1) Influenza viruses constantly change and evade prior immune responses, forcing seasonal re-vaccinations with updated vaccines. Current FDA-approved vaccine manufacturing technologies are too slow and/or expensive to quickly adapt to mid-season changes in the virus or to the emergence of pandemic strains. Therefore, cost-effective vaccine technologies that can quickly adapt to newly emerged strains are desirable. (2) The filamentous fungal host

EphA3 is expressed in solid tumors and leukemias and is an attractive target for the therapy. We have generated a panel of Humaneered® antibodies to the ligand-binding domain using a Fab epitope-focused library that has the same specificity as monoclonal antibody mIIIA4. A high-affinity antibody was selected that competes with the mIIIA4 antibody for binding to EphA3 and has an improved affinity of ∼1 nM. In order to generate an antibody with potent cell-killing activity the variable regions were assembled with human IgG1k constant regions and expressed in a Chinese hamster ovary (CHO) cell line deficient in fucosyl transferase. Non-fucosylated antibodies have been reported to have enhanced binding affinity for the IgG receptor CD16a (FcγRIIIa). The affinity of the antibody for recombinant CD16a was enhanced approximately 10-fold. This resulted in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity against EphA3-expressing leukemic cells, providing a potent antibody for the evaluation as a therapeutic agent.

A Biacore T100 optical biosensor was used to characterize the binding kinetics of a panel of antigen binding fragments (Fabs) directed against the PcrV protein from Pseudomonas aeruginosa. PcrV protein forms part of the type III secretion system complex of this opportunistic pathogen. We demonstrate that the biosensor response data for each Fab collected from three different surface densities of the antigen could be fit globally to a simple 1:1 interaction model. Importantly, we found that the Fabs with the slowest dissociation rate provided the best protection in cell cytotoxicity studies. To further characterize the Fab interactions, binding data were automatically acquired at different temperatures and under different buffer conditions. The comprehensive characterization of these Fabs shows how Biacore T100 can be used to complement protein therapeutic discovery programs from basic research to the selection of therapeutic candidates.

Lipopolysaccharide (LPS) induces expression of tumor necrosis factor alpha (TNFalpha) and other pro-inflammatory cytokines in macrophages. Following its induction, TNFalpha gene transcription is rapidly attenuated, in part due to the accumulation of NF-kappaB p50 homodimers that bind to three kappaB sites in the TNFalpha promoter. Here we have investigated the inhibitory role of BCL-3, an IkappaB-like protein that interacts exclusively with p50 and p52 homodimers. BCL-3 was induced by LPS with delayed kinetics and was associated with p50 in the nucleus. Forced expression of BCL-3 suppressed LPS-induced transcription from the TNFalpha promoter and inhibited two artificial promoters composed of TNFalphakappaB sites that preferentially bind p50 dimers. BCL-3-mediated repression was reversed by trichostatin A and was enhanced by overexpression of HDAC-1, indicating that transcriptional attenuation involves recruitment of histone deacetylase. Analysis of macrophages from p50 and BCL-3 knock-out mice revealed that both transcription factors negatively regulate TNFalpha expression and that BCL-3 inhibits IL-1alpha and IL-1beta. In contrast, induction of the anti-inflammatory cytokine IL-10 was reduced in BCL-3 null macrophages. BCL-3 was not required for the production of p50 homodimers but BCL-3 expression was severely diminished in p50-deficient cells. Together, these findings indicate that p50 and BCL-3 function as anti-inflammatory regulators in macrophages by attenuating transcription of pro-inflammatory cytokines and activating IL-10 expression.

Eph receptor tyrosine kinases are critical for cell–cell communication during normal and oncogenic tissue patterning and tumor growth. Somatic mutation profiles of several cancer genomes suggest EphA3 as a tumor suppressor, but its oncogenic expression pattern and role in tumorigenesis remain largely undefined. Here, we report unexpected EphA3 overexpression within the microenvironment of a range of human cancers and mouse tumor xenografts where its activation inhibits tumor growth. EphA3 is found on mouse bone marrow–derived cells with mesenchymal and myeloid phenotypes, and activation of EphA3+/CD90+/Sca1+ mesenchymal/stromal cells with an EphA3 agonist leads to cell contraction, cell–cell segregation, and apoptosis. Treatment of mice with an agonistic α-EphA3 antibody inhibits tumor growth by severely disrupting the integrity and function of newly formed tumor stroma and microvasculature. Our data define EphA3 as a novel target for selective ablation of the tumor microenvironment and demonstrate the potential of EphA3 agonists for anticancer therapy. Cancer Res; 74(16); 4470–81. ©2014 AACR.

The transcription factor CCAAT/enhancer binding protein β (C/EBPβ, or NF-IL6) is expressed in macrophages, where it participates in lipopolysaccharide (LPS)-mediated induction of proinflammatory cytokine genes such as interleukin-6 (IL-6) and IL-1β. We have identified activities in conditioned medium from a macrophage tumor cell line that regulates the expression, localization, and transcriptional activity of C/EBPβ. One factor was shown to be tumor necrosis factor- (TNF-), which increased C/EBPβ expression by a posttranscriptional mechanism. A second activity, designated autocrine macrophage factor (AMF), elicited a change in C/EBPβ localization from a punctate nuclear staining pattern to diffuse nuclear distribution. The punctate form of C/EBPβ correlated with increased susceptibility of this protein to cleavage by an endogenous protease during nuclear extract preparation. Conditioned medium stimulated the ability of C/EBPβ to transactivate a reporter gene and activated the expression of two cytokine genes that are putative targets of C/EBPβ. These observations suggest that diffuse distribution of C/EBPβ in the nucleus corresponds to an activated form of this protein. AMF activity could not be mimicked by an extensive set of recombinant cytokines and growth factors and therefore may represent a novel extracellular factor.

C/EBPα, -β, and -δ are members of the CCAAT/enhancer binding protein family of transcriptional regulators. All three of these factors are expressed by bone marrow-derived macrophages, with the DNA binding activity of C/EBPβ and -δ increased by treatment with LPS while that of C/EBPα is decreased. We have ectopically expressed each C/EBP protein in P388 lymphoblasts. The expression of any of these transcription factors is sufficient to confer the LPS-inducible expression of IL-6 and monocyte chemoattractant protein-1 to lymphoblasts, which normally lack C/EBP factors and do not display LPS induction of proinflammatory cytokines. Thus, the activities of C/EBPα, -β, and -δ are redundant in regard to the expression of IL-6 and monocyte chemoattractant protein-1. Since C/EBPβ-deficient mice have been reported to be largely normal in their expression of proinflammatory cytokines, it is likely that the lack of C/EBPβ is compensated for by the induction of C/EBPδ upon LPS treatment.

Granulocyte-macrophage–colony-stimulating factor (GM-CSF) hypersensitivity is a hallmark of juvenile myelomonocytic leukemia (JMML) but has not been systematically shown in the related human disease chronic myelomonocytic leukemia (CMML). We find that primary CMML samples demonstrate GM-CSF–dependent hypersensitivity by hematopoietic colony formation assays and phospho-STAT5 (pSTAT5) flow cytometry compared with healthy donors. Among CMML patients, the pSTAT5 hypersensitive response positively correlated with high-risk disease, peripheral leukocytes, monocytes, and signaling-associated mutations. When compared with IL-3 and G-CSF, GM-CSF hypersensitivity was cytokine specific and thus a possible target for intervention in CMML. To explore this possibility, we treated primary CMML cells with KB003, a novel monoclonal anti–GM-CSF antibody, and JAK2 inhibitors. We found that an elevated proportion of immature GM-CSF receptor-α(R) subunit-expressing cells were present in the bone marrow myeloid compartment of CMML. In survival assays, we found that myeloid and monocytic progenitors were sensitive to GM-CSF signal inhibition. Our data indicate that a committed myeloid precursor expressing CD38 may represent the progenitor population with enhanced GM-CSF dependence in CMML, consistent with results in JMML. These preclinical data indicate that GM-CSF signaling inhibitors merit further investigation in CMML and that GM-CSFR expression on myeloid progenitors may be a biomarker for this therapy.

Granulocyte macrophage colony-stimulating factor (GM-CSF) promotes infarct expansion and inappropriate collagen synthesis in a myocardial infarction (MI). This study was designed to determine if treatment with anti-GM-CSF will inhibit macrophage migration, preserve function, and limit left ventricular (LV) remodeling in the rat coronary artery ligation model. Treatment with a monoclonal antibody to GM-CSF (5 mg/kg) was initiated 24 hours before coronary artery ligation and continued every 3 days for 3 weeks. Left coronary arteries of rats were ligated, animals were recovered, and cardiac function was evaluated 3 weeks postligation. Tissue samples were processed for histochemistry. Anti-GM-CSF treatment increased LV ejection fraction (37 ± 3% vs 47 ± 5%) and decreased LV end systolic diameter (0.75 ± 0.12 vs 0.59 ± 0.05 cm) with no changes in LV systolic pressure (109 ± 4 vs 104 ± 5 mm Hg), LV end diastolic pressure (22 ± 4 vs 21 ± 2 mm Hg), LV end diastolic diameter (0.96 ± 0.04 vs 0.92 ± 0.05 cm), or the time constant of LV relaxation tau (25.4 ± +2.4 vs 22.7 ± 1.4 milliseconds) (P < 0.05). Significantly lower numbers of tissue macrophages and significant reductions in infarct size were found in the myocardium of antibody-treated animals (81 ± 21.24 vs 195 ± 31.7 positive cells per 0.105 mm, compared with controls. These findings suggest that inhibition of macrophage migration may be beneficial in the treatment of heart failure after MI.

DNA photolyases catalyze the light-dependent repair of pyrimidine dimers in DNA. We have utilized chemical modification and site-directed mutagenesis to probe the interactions involved in substrate recognition by the yeast photolyase Phr1. Lys517 was protected from reductive methylation in the presence of substrate, but not in its absence, and the specific and nonspecific association constants for substrate binding by Phr1 (Lys517-->Ala) were decreased 10-fold. These results establish a role for Lys517 in substrate binding. Mutations at Arg507, Lys463, and Trp387 reduced both the overall affinity for substrate and substrate discrimination. Sites of altered interactions in ES complexes were identified by methylation and ethylation interference techniques. Interaction with the base immediately 3' to the dimer was altered in the Phr1(Lys517-->Ala). DNA complex, whereas interactions with the phosphate and base immediately 5' to the dimer were reduced when Phr1(Arg507-->Ala) bound substrate. Multiple interactions 5' and 3' to the dimer were perturbed in complexes containing Phr1(Trp387-->Ala) or Phr1(Lys463-->Ala). In addition the quantum yield for dimer photolysis by Phr1(Trp387-->Ala) was reduced 3-fold. The locations of these mutations establish that a portion of the DNA binding domain is comprised of residues in the highly conserved carboxyl-terminal half of the enzyme.

Photolyases contain two chromophores, flavin plus either methenyltetrahydrofolate (MTHF) or 8-OH-5-deazaflavin (HDF). Amino acid sequence comparison reveals that all photolyases sequenced to date have extensive sequence homology in the carboxyl-terminal half; in the amino-terminal region the folate and deazaflavin class enzymes are more homologous to other members of the same class. This modular arrangement of sequence homologies suggests that the amino-terminal half of photolyase is involved in MTHF or HDF binding whereas the carboxyl-terminal half carries the flavin binding site. In this study we attempted to identify such structural domains of yeast photolyase by partial proteolysis and gene fusion techniques. Partial digestion with chymotrypsin yielded an amino-terminal 34-kDa fragment containing tightly bound MTHF and a carboxyl-terminal 20-kDa polypeptide which lacked chromophore or DNA binding activity. However, a fusion protein carrying the carboxyl-terminal 275 amino acids of yeast photolyase bound specifically to FAD but not to MTHF or DNA. We conclude that the amino-terminal half of yeast photolyase constitutes the folate binding domain and that the carboxyl-terminal half carries the flavin binding site.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause acute lung injury and mortality through the delivery of exotoxins by the type III secretion system (TTSS). PcrV is an important structural protein of the TTSS. An engineered human antibody Fab fragment that binds to the P. aeruginosa PcrV protein with high affinity has been identified and has potent in vitro neutralization activity against the TTSS. The instillation of a single dose of Fab into the lungs of mice provided protection against lethal pulmonary challenge of P. aeruginosa and led to a substantial reduction of viable bacterial counts in the lungs. These results demonstrate that blocking of the TTSS by a Fab lacking antibody Fc-mediated effector functions can be sufficient for the effective clearance of pulmonary P. aeruginosa infection.

The CCAAT/enhancer-binding proteins (C/EBPs) are basic leucine zipper transcription factors that play important roles in regulating cell growth and differentiation. C/EBP proteins form leucine zipper-mediated homodimers but are also capable of heterodimerizing with other C/EBPs in vitro. Here we show that C/EBPbeta occurs predominantly as a heterodimer that displays rapid mobility in gel shift assays. Biochemical fractionation and antibody supershift assays demonstrate that the C/EBPbeta heterodimeric partner is C/EBPgamma (Ig/EBP), a C/EBP protein that has been implicated as an inhibitor of other family members. Although most cell types express C/EBPbeta.C/EBPgamma heterodimers, macrophages contain a C/EBPbeta partner that is serologically distinct from C/EBPgamma. We found that C/EBPgamma blocked the ability of C/EBPbeta and C/EBPgamma to activate a reporter gene in L cell fibroblasts but did not inhibit a chimeric C/EBPbeta protein containing the GCN4 leucine zipper. Repression by C/EBPgamma occurs at the level of transactivation and requires heterodimerization with the C/EBP partner. C/EBPgamma was an ineffective repressor in HepG2 hepatoma cells despite forming C/EBP heterodimers, and C/EBPalpha was not effectively inhibited in either L or HepG2 cells. Our findings demonstrate that C/EBPgamma modulates C/EBP activity in a cell- and isoform-specific manner.

Although several novel agents are currently in clinical trials for eosinophilic disorders, none has demonstrated efficacy in reducing blood and tissue eosinophilia in all subjects. Additional approaches are clearly needed.We sought to explore the potential of the human eosinophil surface receptor epidermal growth factor-like module containing mucin-like hormone receptor 1 (EMR1) as a therapeutic target for eosinophilic disorders.EMR1 expression was assessed in blood and bone marrow specimens from eosinophilic and healthy subjects, cell lines, CD34(+) cells differentiated in vitro, and tissue biopsy specimens by using flow cytometry, quantitative PCR, and immunostaining. Eosinophil targeting by a novel, humanized, afucosylated anti-EMR1 IgG1 was evaluated in vitro by using a natural killer cell-mediated killing assay and in vivo in cynomolgus monkeys.Analysis of blood and bone marrow cells from healthy and eosinophilic donors and in vitro-differentiated CD34(+) cells confirmed restriction of human EMR1 surface and mRNA expression to mature eosinophils. Tissue eosinophils also expressed EMR1. Although EMR1 was highly expressed on eosinophils from all subjects, surface expression was negatively correlated with absolute eosinophil counts (r = -0.46, P.001), and soluble plasma levels correlated positively with absolute eosinophil counts (r = 0.69, P.001), suggesting modulation of EMR1 in vivo. Nevertheless, afucosylated anti-EMR1 mAb dramatically enhanced natural killer cell-mediated killing of eosinophils from healthy and eosinophilic donors and induced a rapid and sustained depletion of eosinophils in monkeys.EMR1 expression is restricted to mature blood and tissue eosinophils. Targeting of eosinophils with afucosylated anti-EMR1 antibody shows promise as a treatment for eosinophilic disorders.

C/EBPalpha, beta, and delta are all expressed by bone marrow-derived macrophages. Ectopic expression of any of these transcription factors is sufficient to confer lipopolysaccharide (LPS)-inducible expression of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) to a B lymphoblast cell line, which normally lacks C/EBP factors and does not display LPS induction of proinflammatory cytokines. Thus, the activities of C/EBPalpha, beta, and delta are redundant in regard to expression of IL-6 and MCP-1. Surprisingly, the bZIP region of C/EBPbeta, which lacks any previously described activation domains, can also confer LPS-inducible expression of IL-6 and MCP-1 in stable transfectants. Transient transfections reveal that the bZIP regions of C/EBPbeta, C/EBPdelta, and, to a lesser extent, C/EBPalpha can activate the IL-6 promoter and augment its induction by LPS. Furthermore, the transdominant inhibitor, LIP, can activate expression from the IL-6 promoter. The ability of the C/EBPbeta bZIP region to activate the IL-6 promoter in transient transfections is completely dependent upon an intact NF-kappaB-binding site, supporting a model where the bZIP protein primarily functions to augment the activity of NF-kappaB. Replacement of the leucine zipper of C/EBPbeta with that of GCN4 yields a chimeric protein that can dimerize and specifically bind to a C/EBP consensus sequence, but shows a markedly reduced ability to activate IL-6 and MCP-1 expression. These results implicate the leucine zipper domain in some function other than dimerization with known C/EBP family members, and suggest that C/EBP redundancy in regulating cytokine expression may result from their highly related bZIP regions.

The rat CYP2D5 P-450 gene is activated in the liver during postnatal development. We previously showed that liver-specific transcription of the CYP2D5 gene is dictated by a proximal promoter element, termed 2D5, that is composed of a binding site for Sp1 or a related factor, and an adjacent cryptic C/EBP (CCAAT/enhancer-binding protein) site. Despite the fact that both C/EBP alpha and C/EBP beta are expressed abundantly in liver, only C/EBP beta is capable of stimulating the 2D5 promoter in HepG2 hepatocarcinoma cells. In addition, activation of the 2D5 promoter by C/EBP beta is completely dependent on the presence of the Sp1 site. Domain switch experiments reveal that C/EBP beta proteins containing either the leucine zipper or the activation domain of C/EBP alpha are unable to stimulate the 2D5 promoter yet are fully capable of transactivating an artificial promoter bearing a high-affinity C/EBP site. Thus, the leucine zipper and the activation domain of C/EBP beta are absolutely required to support transactivation of the 2D5 promoter. Using Drosophila cells that lack endogenous Sp1 activity, we show that the serine/threonine- and glutamine-rich activation domains A and B of Sp1 are required for efficient cooperatively with C/EBP beta. Furthermore, analysis of c/ebp beta-deficient mice shows that mutant animals are defective in expression of a murine CYP2D5 homolog in hepatic cells, confirming the selective ability of C/EBP beta to activate this liver-specific P-450 gene in vivo. Our findings illustrate that two members of a transcription factor family can achieve distinct target gene specificities through differential interactions with a cooperating Sp1 protein.

Two members of the C/EBP family of basic region-leucine zipper proteins enriched in the liver, C/EBP (C/EBP alpha) and CRP2 (C/EBP beta), were previously shown to transactivate the albumin promoter in a cell type-dependent manner. These proteins function efficiently in HepG2 hepatoma cells, but inefficiently in HeLa (epithelial) and L (fibroblastic) cells. Here we have investigated the mechanism for cell-specific control of CRP2 activity. We show that CRP2 contains a negative regulatory region composed of two elements, RD1 and RD2. Deletions of RD2 relieve the inhibition of CRP2 activity in L cells, but do not affect CRP2 function in HepG2 cells. These deletions also increase the DNA binding activity of CRP2 approximately 3-fold, suggesting that RD2-mediated repression of DNA binding activity is responsible for CRP2 inhibition in L cells. The adjacent RD1 element functions independently of RD2 and modulates the CRP2 activation domain, which we show to be composed of three subdomains that are conserved within the C/EBP protein family. RD1 does not affect cell type specificity, but inhibits the transactivation potential of GAL4-CRP2 hybrid proteins by 50-fold. These findings suggest that CRP2 assumes a tightly folded conformation in which the DNA binding and activation domains are masked by interactions with the regulatory domain and that to function efficiently in HepG2 cells the protein must undergo an activation step. We propose that relief of inhibition conferred by the regulatory domains also accounts for CRP2 activation in response to extracellular signals.

Abstract 3793 Introduction: Chronic Myelomonocytic Leukemia (CMML) is a genetically diverse hematologic malignancy characterized by cytopenias with or without leukocytosis, marrow dysplasia, monocytosis, splenomegaly, and a propensity to transform to Acute Myeloid Leukemia (AML). It is among the most aggressive chronic myeloid malignancies with a three year overall survival approximating twenty percent. The current treatment armamentarium is generally ineffective and mostly derived from studies that focused on Myelodysplastic Syndromes (MDS). As has been well defined in Juvenile Myelomonocytic Leukemia (JMML), we previously reported that primary CMML bone marrow mononuclear cells exhibit GM-CSF-dependent pSTAT5 hypersensitivity. To determine whether this signaling pathway is a viable therapeutic target, we present the preclinical characterization of KB003 activity on primary CMML bone marrow mononuclear cells. Methods: KB003 was engineered and provided by KaloBios Pharmaceuticals. The ability of KB003 to neutralize GM-CSF was tested in two ways using doses that ranged from .01 to 10,000 ng/ml of KB003. First, 10 ng/ml of GM-CSF was used to induce IL-8 production by the U937 cell line as measured by ELISA. Second, the GM-CSF-dependent cell line, MO7e, was cultured in the absence and in the presence of 0.1, 1, and 10 ng/ml of GM-CSF with or without KB003. Apoptosis and viability was assessed by annexin and 4′,6-diamidino-2-phenylindole (DAPI) staining by flow cytometry. To determine anti-GM-CSF sensitivity in CMML, primary cryoperserved bone marrow samples from 10 patients were reconstituted in prewarmed StemSpan H3000 with 10% FBS and microcultured in a 96 well plate with increasing doses of GM-CSF and KB003. After a 48-hour incubation period, cells were stained with myeloid-specific antibodies (CD34, CD38, CD14, CD33) and measured by multi-color flow cytometry. DAPI was used to measure cell viability. MethoCult hematopoietic progenitor assays were used to quantify the expansion and differentiation of CMML mononuclear cells in the presence of GM-CSF and escalating concentrations of KB003 in those samples with sufficient cell number for analysis (n=7). Results: KB003 effectively neutralized GM-CSF-induced IL-8 secretion in U937 cells with an IC50 of 48.2 ng/ml, as shown in Figure 1a. A dose of 0.1, 1, and 10 ng/ml of GM-CSF was used to protect MO7e cells from apoptosis and a survival benefit was achieved at each dose tested (Fig 1b). In this assay, KB003 neutralized 0.1 ng/ml of GM-CSF, but was unable to neutralize GM-CSF at higher doses (10 ng/ml). Using CMML samples, doses ranging from 10 to 100 μg/ml of KB003 were tested in apoptosis and in colony formation assays. Using the gating strategy shown in Figure 2a, the percentage of mature (CD33+/CD14+) and immature (CD33+/CD14-) myeloid subpopulations within the viable gate after KB003 treatment was decreased (Figure 2b). As expected, KB003 had no effect on CD3+ T-cells in the mixed culture populations. In addition to myeloid subpopulations identified by CD14, the CD33+/CD38+ cells were also more sensitive than CD33+/CD38- or CD38+/CD34+ inhibition by KB003 (Figure 2c, p Conclusion: GM-CSF neutralization using KB003 suppresses CMML progenitor survival in vitro. These preclinical data suggest that KB003 and other GM-CSF signaling axis inhibitors merit further investigation in CMML. A more committed myeloid precursor expressing CD38 may represent the progenitor population with enhanced GM-CSF dependence in CMML. This is consistent with the results showing CD38 to be a marker of GM-CSF-dependent pSTAT5 sensitivity in JMML and may prove to be predictive biomarker for anti-GM-CSF therapy in CMML. Disclosures: No relevant conflicts of interest to declare.

C/EBP-related proteins 2 and 3 (CRP2 and CRP3) are differentially expressed by P388 lymphoblasts and their derivative P388D1(IL1) macrophages. We have ectopically expressed CRP2, the predominant CRP in macrophages, in P388 lymphoblasts. The expression of CRP2 is sufficient to confer the lipopolysaccharide (LPS)-inducible expression of interleukin 6 and monocyte chemoattractant protein 1 to lymphoblasts, which normally do not display LPS induction of inflammatory cytokines. Consistent with these findings, the expression of CRP2 antisense RNA blocks the LPS induction of IL-6 expression in P388D1(IL1) macrophages. This work clearly establishes the essential role of CRP2 in the induction of cytokine genes by LPS. Additionally, these data add MCP-1 to the list of cytokines showing an involvement of CRP2 in their expression.

Abstract 1713 Background: Chronic Myelomonocytic Leukemia (CMML) and Juvenile Myelomonocytic leukemia (JMML) are classified as MDS/MPN in the WHO classification system. Despite sharing clinical and histological features, CMML is characterized by a heterogeneous collection of molecular lesions while JMML is defined by well-established molecular aberrations clustered along the RAS pathway leading directly to GM-CSF hypersensitivity; a pathognomonic characteristic of JMML. Here we test whether a molecular signature for GM-CSF hypersensitivity in JMML, determined by the pSTAT5 activation assay, is also present in CMML and whether this signature clusters within a specific CMML subgroup. Methods: Cryopreserved bone marrow aspirates from 24 patients with newly diagnosed or relapsed CMML were obtained from the Moffitt Cancer Center Tissue Repository. Cells were thawed and rested in Stem Span H3000 with 10% FBS for 2 hours and then either starved for one hour in serum-free media, serum free group (n=12), or rested in Stem Span for an additional hour, serum group (n=12), prior to stimulation with G-CSF, IL-3, or GM-CSF for 15 minutes and then fixed and permeabilized with formaldehyde and methanol, as previously described. Samples were stained with an anti-pSTAT5(Y-694) antibody and analyzed by flow cytometry (Kotecha, Cancer Cell. 2009). Cells stained with isotype-control antibody were used to establish the threshold for basal STAT5 phosphorylation. Because STAT5 was constitutively phosphorylated in serum, and to a lesser extent in serum-free conditions, inducible cytokine activation was defined as the percentage of pSTAT5 positive cells above untreated samples in both CMML and healthy controls. A retrospective chart review was performed to obtain clinical variables including age, sex, WHO classification, Dusseldorf scoring system, MD Anderson scoring system, WBC, peripheral monocyte count, blast percentage, anemia, platelet count, splenomegaly, and metaphase cytogenetics. Results: The percentage of pSTAT5 responsive cells after G-CSF stimulation with doses up to 10 ng/ml was similar in cases and normal BM controls (p=0.14), whereas, a statistically significant increase in the percentage of inducible pSTAT5 positive cells was observed with GM-CSF 0.1 ng/ml (p=0.04), GM-CSF 1 ng/ml (p=0.02), and GM-CSF 10 ng/ml (p=0.01) in CMML BM cells compared to healthy donor BM cells, as shown in Figure 1. Using one standard deviation below the mean as a cut point, only 5 patients failed to show GM-CSF hypersensitivity in the serum (n=3) and serum-free groups (n=2), respectively. IL-3 and GM-CSF play similar roles in hematopoietic growth through the activation of JAK2/STAT5 and share a common beta-chain required for signaling. Signaling mediated by GM-CSF and IL3 converge to activate RAS and other downstream intermediates that regulate DNA synthesis, cell-cycle progression and suppression of apoptosis. The concentration of IL3 required to induce STAT5 phosphorylation was 10-fold greater than GM-CSF in CMML cells, but the percentage of cells responsive to IL3 was greater in CMML cases compared to controls at 10 ng/ml (p=0.02). Analysis of the percentage of GM-CSF hypersensitive cells and clinical parameters revealed no associations with age at onset, WHO classification, Dusseldorf scoring system, MD Anderson scoring system, blast percentage, anemia, platelet count, splenomegaly, or karyotype. The percentage of pSTAT5 positive cells with GM-CSF 0.1 ng/ml positively correlated with the total leukocyte (p=0.03) and total monocyte (p=0.02) count indicating that the JAK2/STAT5 signaling response is indicative of disease burden. Conclusions: Based on the threshold for cytokine stimulation and percentage of cells that display pSTAT5 induction, CMML appears to preferentially utilize GM-CSF for survival and/or expansion. Although RAS mutations were not assessed, CMML cells were preferentially sensitive to GM-CSF in newly diagnosed cases independent of cytogenetic abnormalities suggesting that JMML and CMML share biological features of GM-CSF hypersensitivity. Disclosures: Padron: KaloBios Pharmaceuticals, Inc.: Research Funding. Bebbington:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership. Baer:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership.

Abstract 2897 EphA3 is a receptor tyrosine kinase expressed at high level on the surface of some solid tumors and certain leukemias. KB004 is a high-affinity non-fucosylated, recombinant antibody developed by KaloBios derived from a mouse monoclonal anti-EphA3 antibody using Antibody Humaneering™ Technology. The antibody binds EphA3 from human and cynomolgus macaque with equivalent affinity (610 pM for human and 570 pM for cynomolgus as determined by surface plasmon resonance (spr) analysis) making the latter a relevant toxicology species. KB004 has a direct pro-apoptotic effect on EphA3-positive cells and a induces potent antibody-dependent cellular cytotoxicity (ADCC) activity mediated by CD16-expressing effector cells. The non-fucosylated antibody has enhanced affinity for both human and cynomolgus CD16a. The affinity of KB004 for cynomolgus CD16a (7.2 nM, determined by spr analysis) was comparable to the affinity for the high-affinity variant of CD16a from humans (6.6 nM), indicating that KB004 has similar potential for ADCC in cynomolgus macaques. We first demonstrated cell-surface expression of EphA3 by flow cytometry in approximately 50% of primary human leukemia cells. Here we extend the analysis to multiple myeloma (MM) in which 3/5 patient samples show EphA3 expression. EphA3 expression was confirmed in a panel of chronic myeloid leukemia (CML) samples [n=14] and in acute myeloid leukemia (AML) [n=40] in which EphA3+ cells were identified in each French American British (FAB) classification sub-type except M0, for which only two samples were studied. CD123-positive leukemia stem cells (CD34+ CD38− CD123+) also displayed surface expression of EphA3. KB004 induced apoptosis in EphA3+ primary cells from each of the diseases analyzed, including imatinib-sensitive and resistant CML, with no activity in EphA3-negative specimens, including normal CD34+ bone marrow cells. KB004 inhibited myeloid leukemia colony formation (CFU-L) from primary AML samples in methylcellulose colony assays without affecting normal hematopoietic colony recovery and also showed dose-dependent inhibition of long-term culture-initiating cells (LTC-IC) from AML stem cells. EphA3 expression was not detected on primary human CD34+ bone marrow cells from healthy individuals. These data support the development of an anti-EphA3 antibody for the treatment of hematologic malignancies. The selective activity against leukemic stem cells supports a novel strategy for therapeutic targeting of leukemia-initiating cells. Disclosures: No relevant conflicts of interest to declare.

Macrophages are a major source of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), which are expressed during conditions of inflammation, infection, or injury. We identified an activity secreted by a macrophage tumor cell line that negatively regulates bacterial lipopolysaccharide (LPS)-induced expression of TNF-alpha. This activity, termed TNF-alpha-inhibiting factor (TIF), suppressed the induction of TNF-alpha expression in macrophages, whereas induction of three other proinflammatory cytokines (interleukin-1beta [IL-1beta], IL-6, and monocyte chemoattractant protein 1) was accelerated or enhanced. A similar or identical inhibitory activity was secreted by IC-21 macrophages following LPS stimulation. Inhibition of TNF-alpha expression by macrophage conditioned medium was associated with selective induction of the NF-kappaB p50 subunit. Hyperinduction of p50 occurred with delayed kinetics in LPS-stimulated macrophages but not in fibroblasts. Overexpression of p50 blocked LPS-induced transcription from a TNF-alpha promoter reporter construct, showing that this transcription factor is an inhibitor of the TNF-alpha gene. Repression of the TNF-alpha promoter by TIF required a distal region that includes three NF-kappaB binding sites with preferential affinity for p50 homodimers. Thus, the selective repression of the TNF-alpha promoter by TIF may be explained by the specific binding of inhibitory p50 homodimers. We propose that TIF serves as a negative autocrine signal to attenuate TNF-alpha expression in activated macrophages. TIF is distinct from the known TNF-alpha-inhibiting factors IL-4, IL-10, and transforming growth factor beta and may represent a novel cytokine.