Blockade of epidermal growth factor receptor (EGFR) signaling with specific inhibitors of the EGFR tyrosine kinase retards cellular proliferation and arrests the growth of tumor xenografts. AG1478, an inhibitor of the EGFR tyrosine kinase, is used in laboratory studies; however, its therapeutic potential has not been elucidated. Therefore, we evaluated an aqueous form of AG1478 for its antitumor activity in mice bearing human xenografts expressing the WT EGFR or a naturally occurring ligand-independent truncation of the EGFR [delta2-7 (de2-7) EGFR or EGFRvIII]. Parenteral administration of soluble AG1478 blocked phosphorylation of the EGFR at the tumor site and inhibited the growth of A431 xenografts that overexpress the WT EGFR and glioma xenografts expressing the de2-7 EGFR. Strikingly, even subtherapeutic doses of AG1478 significantly enhanced the efficacy of cytotoxic drugs, with the combination of AG1478 and temozolomide displaying synergistic antitumor activity against human glioma xenografts. AG1478 was also examined in combination with mAb 806, an anti-EGFR antibody that was raised against the de2-7 EGFR but unexpectedly also binds a subset of the EGFR expressed in cells exhibiting amplification of the EGFR gene. The combination of AG1478 and mAb 806 displayed additive, and in some cases synergistic, antitumor activity against tumor xenografts overexpressing the EGFR. Here, we demonstrate that different classes of inhibitors to the EGFR can have synergistic antitumor activity in vivo. These results establish the antitumor efficacy of the EGFR inhibitor AG1478 and provide a rationale for its clinical evaluation in combination with both chemotherapy and other EGFR therapeutics.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 a-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability , Protein Sci 2:1472-1481). To further probe the structurefunction relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of a-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.

Mice defective in both granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colonystimulating factor (GM-CSF) have severely impaired neutrophil production and function, yet these mice respond to acute pathogen challenge with a significant neutrophil response. We have recently reported the development of an in vitro system to detect granulopoietic cytokines secreted from cells isolated from G-CSF, GM-CSF double knockout mice.The conditioned media produced by these cells after stimulation with lipopolysaccharide or Candida albicans supports the production and differentiation of granulocytes (ie, the conditioned media contains neutrophil promoting activity [NPA]). We now show that the NPA in the G-CSF ؊/؊ / GM-CSF ؊/؊ conditioned media requires interleukin-6 (IL6), is abolished by soluble gp130, and can be specifically immunodepleted by an anti-IL6R antibody. NPA effects on bone marrow cells are also mimicked by Hyper-IL6, and the soluble IL6R is present in NPA. These results show that the IL6/sIL6R complex is the major effector of NPA. NPA production by mice defective for both G-CSF and GM-CSF uncovers an alternative pathway to granulocyte production, which is activated after exposure to pathogens. IntroductionNeutrophils and monocytes play a pivotal role in host defenses against bacterial and fungal infections. The main modulators of granulocyte production and activation are the classic hemopoietic cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF). 1-4 Indeed, several studies have shown that mice defective in both G-CSF and GM-CSF have profound chronic neutropenia, granulocyte, and macrophage progenitor cell deficiency, impaired neutrophil mobilization, impaired reproductive capacity, perturbed neonatal granulopoiesis, and amyloidosis. [5][6][7] However, acute challenge of G-CSF Ϫ/Ϫ and GM-CSF Ϫ/Ϫ mice with pathogens, such as Candida albicans, results in elevated numbers of monocyte and granulocyte progenitor cells in the bone marrow and in a significant peripheral neutrophilia, 8 suggesting that there must be alternative regulators of neutrophil production in response to acute inflammatory challenges.We have reported recently that conditioned media (CM) from mouse embryo fibroblasts (MEFs) derived from compound G-CSF Ϫ/Ϫ /GM-CSF Ϫ/Ϫ animals exposed to lipopolysaccharide (LPS) or heat-inactivated C albicans stimulate a mixed population of myeloid progenitor cells to produce mature neutrophils in vitro. 9 Thus, cytokines other than G-and GM-CSF, produced by MEFs in response to LPS challenge, can stimulate neutrophil production and function. We have ruled out the contribution of some cytokines, including interleukin-3 (IL3), SCF, MIP1␣, and RANTES, 9 to this neutrophil-promoting activity (NPA) Surprisingly, we uncovered a critical role for M-CSF in the production of NPA; however, M-CSF itself is not sufficient for the generation of neutrophils in vitro from myeloid progenitor cells of compound mutant G-CSF Ϫ/Ϫ, GM-CSF Ϫ/Ϫ mice....

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Background and Aims Proteomics holds promise for individualizing cancer treatment. We analyzed to what extent the proteomic landscape of human colorectal cancer (CRC) is maintained in established CRC cell lines and the utility of proteomics for predicting therapeutic responses. Methods Proteomic and transcriptomic analyses were performed on 44 CRC cell lines, compared against primary CRCs (n=95) and normal tissues (n=60), and integrated with genomic and drug sensitivity data. Results Cell lines mirrored the proteomic aberrations of primary tumors, in particular for intrinsic programs. Tumor relationships of protein expression with DNA copy number aberrations and signatures of post-transcriptional regulation were recapitulated in cell lines. The five proteomic subtypes previously identified in tumors were represented among cell lines. Nonetheless, systematic differences between cell line and tumor proteomes were apparent, attributable to stroma, extrinsic signaling and growth conditions. Contribution of tumor stroma obscured signatures of DNA mismatch repair identified in cell lines with a hypermutation phenotype. Global proteomic data showed improved utility for predicting both known drug-target relationships and overall drug sensitivity as compared to genomic or transcriptomic measurements. Inhibition of targetable proteins associated with drug responses further identified corresponding synergistic or antagonistic drug combinations. Our data provide evidence for CRC proteomic subtype-specific drug responses. Conclusions Proteomes of established CRC cell line are representative of primary tumors. Proteomic data tend to exhibit improved prediction of drug sensitivity as compared to genomic and transcriptomic profiles. Our integrative proteogenomic analysis highlights the potential of proteome profiling to inform personalized cancer medicine.

The high affinity interleukin-6 (IL-6) signaling complex consists of IL-6 and two membrane-associated receptor components: a low affinity but specific IL-6 receptor and the affinity converter/signal transducing protein gp130. Monomeric (IL-6 M ) and dimeric (IL-6 D ) forms of Escherichia coli-derived human IL-6 and the extracellular ("soluble") portions of the IL-6 receptor (sIL-6R) and gp130 have been purified in order to investigate the effect of IL-6 dimerization on binding to the receptor complex. Although IL-6 D has a higher binding affinity for immobilized sIL-6R, as determined by biosensor analysis employing surface plasmon resonance detection, IL-6 M is more potent than IL-6 D in a STAT3 phosphorylation assay. The difference in potency is significantly less pronounced when measured in the murine 7TD1 hybridoma growth factor assay and the human hepatoma HepG2 bioassay due to time-dependent dissociation at 37°C of IL-6 dimers into active monomers. The increased binding affinity of IL-6 D appears to be due to its ability to cross-link two sIL-6R molecules on the biosensor surface. Studies of the IL-6 ternary complex formation demonstrated that the reduced biological potency of IL-6 D resulted from a decreased ability of the IL-6 D ⅐(sIL-6R) 2 complex to couple with the soluble portion of gp130. These data imply that IL-6-induced dimerization of sIL-6R is not the driving force in promoting formation of the hexameric (IL-6⅐IL-6R⅐gp130) 2 complex. A model is presented whereby the trimeric complex of IL-6R, gp130, and IL-6 M forms before the functional hexamer. Due to its increased affinity for the IL-6R but its decreased ability to couple with gp130, we suggest that a stable IL-6 dimer may be an efficient IL-6 antagonist.

The interaction of recombinant human interleukin-6 (IL-6) with the soluble extracellular form of its receptor (sIL-6R) has been characterized by the application of expressions developed for quantitative affinity chromatography to results obtained with a biosensor based on surface plasmon resonance detection. First, the interaction of sIL-6R with IL-6 covalently attached to the biosensor-chip was characterized from the dependence of the surface plasmon resonance response upon the concentration of receptor injected into the biosensor. A binding constant for the interaction between sIL-6R and IL-6 was then determined from the biosensor response observed for mixtures of IL-6 and receptor--a procedure that is shown to provide unequivocal characterization of the competing reaction, irrespective of the model used to describe the biphasic interaction between partitioning receptor and immobilized IL-6. A binding constant of 5 x 10(7) M-1 has been obtained for the interaction of sIL-6R with two equivalent and independent sites on an essentially dimeric IL-6 preparation produced using the pUC vector system, and also for the interaction of sIL-6R with a monomeric IL-6 preparation that was univalent in its interaction with receptor.

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