- Email: [email protected]
151
278
Abstract / Cytokine 63 (2013) 243–314
with greatly improved efficacy in pre-clinical tumor models relative to IL-2. The polymer conjugation strategy was designed to improve efficacy and safety by optimizing localization to the tumor, by enhancing the PK profile through reduced Cmax and increased exposure, and by altering the receptor binding profile to enhance tumor killing, without any changes to the amino-acid sequence. We present in vitro and in vivo data demonstrating the achievement of these objectives, and discuss how this approach may be generalized to enhance other cytokine-based therapies. http://dx.doi.org/10.1016/j.cyto.2013.06.151
149 Cell crowding results in accumulation of interferon-stimulated genes independently of STAT1 Iryna Kolosenko a, Elin Edsbäcker a, Mårten Fryknäs b, Per Johnsson a, Dan Grandér a, Katja Pokrovskaja Tamm a, Stig Lindera a, Angelo De Milito a, a Department of Oncology– Pathology, Karolinska Institutet, Stockholm, Sweden, b Division of Clinical Pharmacology, Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden Personalized anti-cancer therapy requires a set of markers predicting therapy response and clinical outcome. Recently, interferon-related DNA damage signature (IRDS) has been correlated to chemo- and radiotherapy resistance in patients and experimental systems. We demonstrated that multicellular spheroids from HCT116 cell line characterized by chemotherapy–resistant phenotype also activate interferon-stimulated genes (ISGs) with a genetic signature similar to IRDS. Our data showed that cell crowding was responsible for driving the ISGs’ expression in this system. We demonstrated that culturing cell lines of different origin as monolayers for prolonged periods of time results in gradual accumulation of interferon (IFN)-related genes STAT1, IRF9, STAT2, OAS1, IFITM1 and IFI27. Further, interferon-stimulated response element (ISRE) reporter was induced under these conditions suggesting a common mechanism for these genes’ induction. Interestingly, conditioned media from confluent cells also conferred ISRE activation and upregulation of protein levels of IRF9 implying involvement of a yet unknown secreted paracrine factor. In order to understand the mechanism of ISGs’ induction upon cell crowding, we manipulated the levels of STAT1, IRF9 and STAT2, all members of the ISGF3 complex known to drive the transcription in response to type I IFNs. Knocking down STAT1 did not influence gradual increase of the expression of IFN-regulated genes and IRF9 accumulation was not impeded in a STAT1-negative cell line. On the contrary, manipulating IRF9 levels affected the expression of ISGs, including STAT1, suggesting that IRF9 and not STAT1 is the driver of ISGs in this system. Interestingly, knocking down STAT2 ablated induction of ISGs indicating that IRF9 and STAT2 might constitute an alternative complex capable of binding to ISRE of the target genes. Taken together, our data demonstrate that cell density is accompanied by activation of the ISGs that belong to the IRDS driven by IRF9 and STAT2 and mediated by a paracrine factor.
PEGylated cytokines depended on molecular weight of the attached PEGs and was 30%–80% of the activity of unmodified proteins. Fluorescent spectra and CD spectra for PEG-proteins and their native analogs were nearly superimposed across the nearto far-UV spectrum, suggesting that pegylation had no significant effect on the secondary or tertiary conformation of the cytokines. It was shown that the trypsin resistance and thermal stability were significantly improved in PEGylated proteins. The native proteins aggregated rapidly in 100–200 min, whereas the PEG-proteins showed no aggregation more for 24 h. The results of preclinical studies have demonstrated that PEGylated proteins have significantly better pharmacological properties than their unmodified analogs. The systemic clearance of PEGylated cytokines decreased 20–30-fold and the serum half-life increased 12–16-fold compared to unconjugated proteins. Conclusions: The two PEG-cytokine conjugates were selected for further studies in clinical trials. http://dx.doi.org/10.1016/j.cyto.2013.06.153
151 CXCR3 deficient mice are resistant to herpes simplex virus type 1 infection associated with a drop in viral titer in the ependymal region of the brain Chandra Kroll a, Min Zheng b, Daniel J.J. Carr a,b, a Departments of Microbiology, Immunology, Oklahoma City, OK, United States, b Departments of Ophthalmology OUHSC, Oklahoma City, OK, United States Herpes simplex virus type 1 (HSV-1) continues to be the leading cause of sporadic frank encephalitis and associated neurological immunopathology despite antiviral therapy. Microglia, resident immune cells of central nervous tissue, are crucial for viral surveillance during HSV-1 infection. Moreover, the chemokine CXCL10 is highly expressed during HSV-1 encephalitis and its receptor, CXCR3, is expressed by microglia. Thus, signaling by CXCR3 is a candidate for activating microglia and directing them to the site of infection. Therefore, we hypothesized that CXCR3 plays a critical role in resistance to HSV-1 encephalitis by recruiting and activating microglia. Using a corneal infection model, HSV-1-infected CXCR3 deficient mice (CXCR3 KO) were found to be more resistant to HSV-1 infection compared to C56BL/6 (WT) mice based on infectious virus recovered from the ependymal region of the brain. These results are consistent with the enhanced cumulative survival of CXCR3 KO mice. There were no differences in resident microglial cell numbers recovered from brain and the ependymal region of infected WT and CXCR3 KO mice. Also, in investigating the T cell populations that infiltrate this region following ocular HSV-1 infection, we found no differences when comparing WT with CXCR3 KO mice. We did find however, that infected CXCR3 KO mice express an increase in MHC class II activation expressed by microglia proximal to the ependyma. Studies are ongoing to assess the functional aspects of microglial cells within infected tissue to prevent viral load from progressing throughout brain cortex.
http://dx.doi.org/10.1016/j.cyto.2013.06.152
http://dx.doi.org/10.1016/j.cyto.2013.06.154
150 Physico-chemical properties, pharmacokinetics and pharmacodynamics of pegylated cytokines
152 STAT4 negatively regulate hepatocyte survival during auto-immune liver inflammation
D. Korzhavin a, E. Rudenko b, E. Morozova b, T. Chernovskaya b, a Lomonosov Moscow University of Fine Chemical Technology, Russia, b Biopharmaceutical company ‘‘BIOCAD”, Russia
Pawan Kumar, Kong Chen, Xuejun Zhao, Derek Pociask, Jay K. Kolls, Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA 15224, United States
Introduction: Covalent attachment of poly (ethylene glycol) (PEG) to therapeutic proteins can be used to enhance of pharmacological effectiveness of drug. In comparison with unmodified analogs, PEGylated proteins have better physical and thermal stability, greater protection against proteolytic cleavage, reduced immunogenicity and toxicity and improved pharmacokinetic (PK) and pharmacodynamic (PD) profiles. Methods: Physicochemical properties of PEGylated proteins were analyzed in comparison with unmodified proteins by SDS–PAGE, SEC-HPLC, RP-HPLC, mass spectrometry, peptide mapping, circular dichroism, fluorescence spectroscopy and in vitro specific activity. Preclinical studies were conducted with animals to determine toxicities, safety and PK/PD characteristics of new PEG- conjugates. Results: The series of new conjugates of human recombinant cytokines (IFN, G-CSF) were prepared with PEGs of different structure and lengths specifically attached to the N-terminal amino group of the proteins. The methods of single step chromatographic purification to produce mono PEGylated proteins were developed. The functional activity of the
Autoimmune hepatitis is a condition where immune cells target and destroy liver cells. To date, molecular mechanisms of inflammation mediated fibrosis and necrotic changes in liver cells are poorly understood. STAT4 is expressed in immune cells as well structural cells such as hepatocytes. However, the role of STAT4 in autoimmune hepatitis is ill-defined. Therefore, we investigated the role of STAT4 signaling in Concanavalin A (ConA)-induced auto-immune hepatitis model. Our data indicate STAT4 -/- mice but not WT, IL-12p35 -/- or IL-12p40 -/- mice were resistant to ConA-induced hepatitis as measured by liver pathology and the serum alanine aminotransferase (ALT) level. Histopathological analysis of liver sections showed significant necrotic lesion in WT, IL-12p35 -/- and IL-12p40 -/- but not in STAT4 -/- mice, indicating a IL-12-independent role of STAT4. Furthermore, no significant difference in serum ALT and necrotic lesions were observed when WT or STAT4 -/- spleenocytes were adoptive transfer into lymphocyte deficient Rag2gc -/- mice, suggesting that protection in STAT4 -/- is independent of lymphocyte STAT4. Interestingly, serum IFNg level were significantly reduced in both IL-12p35 -/- and STAT4 -/- mice, and injection of
Abstract / Cytokine 63 (2013) 243–314
with greatly improved efficacy in pre-clinical tumor models relative to IL-2. The polymer conjugation strategy was designed to improve efficacy and safety by optimizing localization to the tumor, by enhancing the PK profile through reduced Cmax and increased exposure, and by altering the receptor binding profile to enhance tumor killing, without any changes to the amino-acid sequence. We present in vitro and in vivo data demonstrating the achievement of these objectives, and discuss how this approach may be generalized to enhance other cytokine-based therapies. http://dx.doi.org/10.1016/j.cyto.2013.06.151
149 Cell crowding results in accumulation of interferon-stimulated genes independently of STAT1 Iryna Kolosenko a, Elin Edsbäcker a, Mårten Fryknäs b, Per Johnsson a, Dan Grandér a, Katja Pokrovskaja Tamm a, Stig Lindera a, Angelo De Milito a, a Department of Oncology– Pathology, Karolinska Institutet, Stockholm, Sweden, b Division of Clinical Pharmacology, Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden Personalized anti-cancer therapy requires a set of markers predicting therapy response and clinical outcome. Recently, interferon-related DNA damage signature (IRDS) has been correlated to chemo- and radiotherapy resistance in patients and experimental systems. We demonstrated that multicellular spheroids from HCT116 cell line characterized by chemotherapy–resistant phenotype also activate interferon-stimulated genes (ISGs) with a genetic signature similar to IRDS. Our data showed that cell crowding was responsible for driving the ISGs’ expression in this system. We demonstrated that culturing cell lines of different origin as monolayers for prolonged periods of time results in gradual accumulation of interferon (IFN)-related genes STAT1, IRF9, STAT2, OAS1, IFITM1 and IFI27. Further, interferon-stimulated response element (ISRE) reporter was induced under these conditions suggesting a common mechanism for these genes’ induction. Interestingly, conditioned media from confluent cells also conferred ISRE activation and upregulation of protein levels of IRF9 implying involvement of a yet unknown secreted paracrine factor. In order to understand the mechanism of ISGs’ induction upon cell crowding, we manipulated the levels of STAT1, IRF9 and STAT2, all members of the ISGF3 complex known to drive the transcription in response to type I IFNs. Knocking down STAT1 did not influence gradual increase of the expression of IFN-regulated genes and IRF9 accumulation was not impeded in a STAT1-negative cell line. On the contrary, manipulating IRF9 levels affected the expression of ISGs, including STAT1, suggesting that IRF9 and not STAT1 is the driver of ISGs in this system. Interestingly, knocking down STAT2 ablated induction of ISGs indicating that IRF9 and STAT2 might constitute an alternative complex capable of binding to ISRE of the target genes. Taken together, our data demonstrate that cell density is accompanied by activation of the ISGs that belong to the IRDS driven by IRF9 and STAT2 and mediated by a paracrine factor.
PEGylated cytokines depended on molecular weight of the attached PEGs and was 30%–80% of the activity of unmodified proteins. Fluorescent spectra and CD spectra for PEG-proteins and their native analogs were nearly superimposed across the nearto far-UV spectrum, suggesting that pegylation had no significant effect on the secondary or tertiary conformation of the cytokines. It was shown that the trypsin resistance and thermal stability were significantly improved in PEGylated proteins. The native proteins aggregated rapidly in 100–200 min, whereas the PEG-proteins showed no aggregation more for 24 h. The results of preclinical studies have demonstrated that PEGylated proteins have significantly better pharmacological properties than their unmodified analogs. The systemic clearance of PEGylated cytokines decreased 20–30-fold and the serum half-life increased 12–16-fold compared to unconjugated proteins. Conclusions: The two PEG-cytokine conjugates were selected for further studies in clinical trials. http://dx.doi.org/10.1016/j.cyto.2013.06.153
151 CXCR3 deficient mice are resistant to herpes simplex virus type 1 infection associated with a drop in viral titer in the ependymal region of the brain Chandra Kroll a, Min Zheng b, Daniel J.J. Carr a,b, a Departments of Microbiology, Immunology, Oklahoma City, OK, United States, b Departments of Ophthalmology OUHSC, Oklahoma City, OK, United States Herpes simplex virus type 1 (HSV-1) continues to be the leading cause of sporadic frank encephalitis and associated neurological immunopathology despite antiviral therapy. Microglia, resident immune cells of central nervous tissue, are crucial for viral surveillance during HSV-1 infection. Moreover, the chemokine CXCL10 is highly expressed during HSV-1 encephalitis and its receptor, CXCR3, is expressed by microglia. Thus, signaling by CXCR3 is a candidate for activating microglia and directing them to the site of infection. Therefore, we hypothesized that CXCR3 plays a critical role in resistance to HSV-1 encephalitis by recruiting and activating microglia. Using a corneal infection model, HSV-1-infected CXCR3 deficient mice (CXCR3 KO) were found to be more resistant to HSV-1 infection compared to C56BL/6 (WT) mice based on infectious virus recovered from the ependymal region of the brain. These results are consistent with the enhanced cumulative survival of CXCR3 KO mice. There were no differences in resident microglial cell numbers recovered from brain and the ependymal region of infected WT and CXCR3 KO mice. Also, in investigating the T cell populations that infiltrate this region following ocular HSV-1 infection, we found no differences when comparing WT with CXCR3 KO mice. We did find however, that infected CXCR3 KO mice express an increase in MHC class II activation expressed by microglia proximal to the ependyma. Studies are ongoing to assess the functional aspects of microglial cells within infected tissue to prevent viral load from progressing throughout brain cortex.
http://dx.doi.org/10.1016/j.cyto.2013.06.152
http://dx.doi.org/10.1016/j.cyto.2013.06.154
150 Physico-chemical properties, pharmacokinetics and pharmacodynamics of pegylated cytokines
152 STAT4 negatively regulate hepatocyte survival during auto-immune liver inflammation
D. Korzhavin a, E. Rudenko b, E. Morozova b, T. Chernovskaya b, a Lomonosov Moscow University of Fine Chemical Technology, Russia, b Biopharmaceutical company ‘‘BIOCAD”, Russia
Pawan Kumar, Kong Chen, Xuejun Zhao, Derek Pociask, Jay K. Kolls, Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA 15224, United States
Introduction: Covalent attachment of poly (ethylene glycol) (PEG) to therapeutic proteins can be used to enhance of pharmacological effectiveness of drug. In comparison with unmodified analogs, PEGylated proteins have better physical and thermal stability, greater protection against proteolytic cleavage, reduced immunogenicity and toxicity and improved pharmacokinetic (PK) and pharmacodynamic (PD) profiles. Methods: Physicochemical properties of PEGylated proteins were analyzed in comparison with unmodified proteins by SDS–PAGE, SEC-HPLC, RP-HPLC, mass spectrometry, peptide mapping, circular dichroism, fluorescence spectroscopy and in vitro specific activity. Preclinical studies were conducted with animals to determine toxicities, safety and PK/PD characteristics of new PEG- conjugates. Results: The series of new conjugates of human recombinant cytokines (IFN, G-CSF) were prepared with PEGs of different structure and lengths specifically attached to the N-terminal amino group of the proteins. The methods of single step chromatographic purification to produce mono PEGylated proteins were developed. The functional activity of the
Autoimmune hepatitis is a condition where immune cells target and destroy liver cells. To date, molecular mechanisms of inflammation mediated fibrosis and necrotic changes in liver cells are poorly understood. STAT4 is expressed in immune cells as well structural cells such as hepatocytes. However, the role of STAT4 in autoimmune hepatitis is ill-defined. Therefore, we investigated the role of STAT4 signaling in Concanavalin A (ConA)-induced auto-immune hepatitis model. Our data indicate STAT4 -/- mice but not WT, IL-12p35 -/- or IL-12p40 -/- mice were resistant to ConA-induced hepatitis as measured by liver pathology and the serum alanine aminotransferase (ALT) level. Histopathological analysis of liver sections showed significant necrotic lesion in WT, IL-12p35 -/- and IL-12p40 -/- but not in STAT4 -/- mice, indicating a IL-12-independent role of STAT4. Furthermore, no significant difference in serum ALT and necrotic lesions were observed when WT or STAT4 -/- spleenocytes were adoptive transfer into lymphocyte deficient Rag2gc -/- mice, suggesting that protection in STAT4 -/- is independent of lymphocyte STAT4. Interestingly, serum IFNg level were significantly reduced in both IL-12p35 -/- and STAT4 -/- mice, and injection of