We propose to conjugate paclitaxel to a single-chain antibody that specifically binds to the extr... more We propose to conjugate paclitaxel to a single-chain antibody that specifically binds to the extracellular domain of the EGFR. The scFv-paclitaxel conjugate will be able to localize to the tumor rapidly, penetrate deeper, be eliminated quickly, and induce fewer side effects for the patient, thereby increasing the efficacy of the scFvpaclitaxel conjugate. An additional benefit with the scFv-paclitaxel conjugate is the fact that taxol is a radiosensitizing drug, therefore radiation treatment can be used following the administration of the scFvpaclitaxel conjugate. The EGFR overexpressing breast cancer cell line, MDA-MB-468 will be used in this proposal to show "proof of concept". Cell proliferation assays will be used to examine the cytotoxicity of the scFv-paclitaxel conjugate as compared to paclitaxel, scFv or the combination of the two agents. Cell death will be examined using two different apoptosis assays, annexin V-FITC and TUNEL. Since taxol is a radiosensitizing drug, radiation will be included in the cell proliferation and apoptosis assays. Clonogenic assays will be done to determine the radiosensitizing ability of the scFv-paclitaxel conjugate as compare to each agent alone. 14. SUBJECT TERMS 15. NUMBER OF PAGES Single-chain antibody, taxol, breast cancer, conjugate 12 16. PRICE CODE 17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19. SECURITY CLASSIFICATION 20. LIMITATION OF ABSTRACT OF REPORT OF THIS PAGE
Analogs of marine alkaloid, makaluvamine, bearing substituted benzyl and substituted phenethyl si... more Analogs of marine alkaloid, makaluvamine, bearing substituted benzyl and substituted phenethyl side chains have been synthesized and their antiproliferative activities have been evaluated. 4-Methyl, 4-chloro, and 4-fluoro substituted benzyl analogs possessed pronounced antiproliferative effects on the breast cancer cell line, MCF-7 at IC(50) values of 2.3 microM, 1.8 microM, and 2.8 microM, respectively. 4-Methyl, 4-chloro, and 3,4-methylenedioxy derivatives showed the best activity against MCF-7 among the phenethyl analogs with IC(50) values of 2.3 microM, 2.8 microM, and 2.4muM, respectively. In general, methoxy substitutions resulted in slight loss in activity in both benzyl and phenethyl series. Benzyl, 4-fluorobenzyl, 3,4-dimethoxyphenethyl, and 3,4-methylenedioxyphenethyl analogs were tested by NCI in their 60 cell lines in vitro human cancer cell screen. All four compounds showed excellent inhibition against several tested cancer cell lines. Benzyl and 4-fluorobenzyl analogs were relatively more active than 3,4-dimethoxy phenethyl and 3,4-methylenedioxy phenethyl analogs. In NCI assays, the best LogGI(50) values were shown by the fluorobenzyl analog against the renal cancer cell line RXF-393 (<-8.0M) and dimethoxy phenethyl analog against the CNS cancer cell line, SF-268 (<-8.0M). The best LogLC(50) value was shown by the fluorobenzyl analog against the breast cancer cell line MCF-7 (-6.01 M).
Epidermal growth factor receptor (EGFr) is overexpressed in a majority of head and neck squamous ... more Epidermal growth factor receptor (EGFr) is overexpressed in a majority of head and neck squamous cell carcinomas, and this overexpression is associated with a poor prognosis. Therefore, EGFr has become the target of investigations aimed at disabling the receptor to determine whether this process leads to improved tumor kill with conventional treatment. C225 is an anti-EGFr monoclonal antibody that inhibits receptor activity by blocking the ligand binding site. A panel of human head and neck squamous cell carcinoma cell lines was used to study the combination of C225 and radiation. It was determined that the combination of C225 (5 microgram/mL) delivered simultaneously with radiation (3 Gy) resulted in a greater decrement in cellular proliferation than either treatment alone. This reduction in proliferation correlated with reduced EGFr tyrosine phosphorylation and a reduction in phosphorylated signal transducer and activator of transcription-3 (STAT-3) protein (known to protect cells from apoptosis). Also, the decrement in proliferation correlated with increased apoptotic events, thereby indirectly linking C225/radiation-induced regulation of STAT-3 protein to apoptosis. This preclinical work serves as important support for the ongoing clinical investigation of C225 and radiotherapy for patients with head and neck carcinomas. The initial results of these clinical studies have been promising.
1. Semin Radiat Oncol. 2002 Jul;12(3 Suppl 2):11-20. Epidermal growth factor receptor as a therap... more 1. Semin Radiat Oncol. 2002 Jul;12(3 Suppl 2):11-20. Epidermal growth factor receptor as a therapeutic target in head and neck cancer. Bonner JA, De Los Santos J, Waksal HW, Needle MN, Trummel HQ, Raisch KP. University of Alabama at Birmingham, 35294-3300, USA. ...
Epidermal growth factor receptor (EGFr) is overexpressed in a majority of head and neck squamous ... more Epidermal growth factor receptor (EGFr) is overexpressed in a majority of head and neck squamous cell carcinomas, and this overexpression is associated with a poor prognosis. Therefore, EGFr has become the target of investigations aimed at disabling the receptor to determine whether this process leads to improved tumor kill with conventional treatment. C225 is an anti-EGFr monoclonal antibody that inhibits receptor activity by blocking the ligand binding site. A panel of human head and neck squamous cell carcinoma cell lines was used to study the combination of C225 and radiation. It was determined that the combination of C225 (5 microgram/mL) delivered simultaneously with radiation (3 Gy) resulted in a greater decrement in cellular proliferation than either treatment alone. This reduction in proliferation correlated with reduced EGFr tyrosine phosphorylation and a reduction in phosphorylated signal transducer and activator of transcription-3 (STAT-3) protein (known to protect cells...
The first ten amino acids of hexokinase II are import for binding to the MOM. Mutating the fifth ... more The first ten amino acids of hexokinase II are import for binding to the MOM. Mutating the fifth amino acid abolished mitochondrial binding. Determining the binding site is important for identifying novel compounds that can disrupt HKII binding to VDAC.
During the screening of human lymphoblastoid cells as suitable hosts for retrovirus transmission ... more During the screening of human lymphoblastoid cells as suitable hosts for retrovirus transmission studies, the Epstein-Barr virus (EBV)-negative, B-lymphoblastoid cell line DG-75 was found to be chronically infected with a heretofore unrecognized retrovirus. Two DG-75 sublines obtained from different sources (designated UW and KAR) were found to produce constitutively particles identified as retroviral by electron microscopy and reverse transcriptase activity. The ultrastructure, morphogenesis, and density in sucrose of the particles were typical of C-type retroviruses. Immunoblot analysis of the DG-75(UW) retrovirus proteins showed antigenic similarity to Moloney murine leukemia virus. A third DG-75 subline in early passage, designated HAD, was free of retrovirus. The DG-75(UW) retrovirus was infectious and produced progeny virions that could be passaged to uninfected cells. We have thus demonstrated that DG-75 cells, which have been used extensively in studies of the biological eff...
The goal of this study was to design paclitaxel (PTX)-monoclonal antibody (mAb) prodrug conjugate... more The goal of this study was to design paclitaxel (PTX)-monoclonal antibody (mAb) prodrug conjugates (PTXMAbs) with the ability to deliver therapeutically significant doses of the drug to the tumor while avoiding the previously observed solubility limitations of conjugates with PTX:mAb molar ratios of >3. New PTX conjugates were synthesized using the discrete poly(ethylene glycol) (dPEG) as linkers. These compounds, PTX-L-Lys[(dPEG12)(3)-dPEG4]-dPEG6-NHS (9a and 9b, for L = GL or SX, respectively), were then conjugated to the antiepidermal growth factor receptor mAb, C225 at increasing PTX:C225 ratios, producing completely soluble conjugates. Unlike the earlier PTXMAbs, buffered solutions of these conjugates remained homogeneous for extended periods of time. Fluorescence-activated cell sorting (FACS) analysis indicated preserved immunogenicity of the conjugates at all four substitution ratios, while cytotoxicity studies in MDA-MB-468 breast cancer cells indicated preservation of dr...
Assess interaction of pazopanib, an oral antivascular endothelial growth factor inhibitor, with r... more Assess interaction of pazopanib, an oral antivascular endothelial growth factor inhibitor, with radiation in tumor xenograft models. Flank xenografts in female athymic nude mice of human lung cancer cell line, A549, and head and neck cancer cell line, UM-SCC-6, were allowed to grow to ∼5×5 mm. Groups were then treated with pazopanib and/or escalating doses of radiation and tumor measurements over time compared with untreated tumor-bearing controls. Pazopanib (100 mg/kg) began 7 days before radiation and continued for 28 days. Daily radiation was 0.5, 1, 2, or 3 Gy ×5 days. Tumors in the A549 control group reached >4× the original size by day 36 postradiation. All treatment groups had less robust tumor growth (p<0.05) and the group receiving pazopanib+3 Gy radiation/day had tumor regression to less than baseline. In the UM-SCC-6-tumor-bearing animals, tumors in all treatment groups had less robust growth than untreated controls after day 23 post-treatment. The combination of pa...
The human B-lymphoblastoid cell line, designated JHK-3, with pre-B-cell characteristics, chronica... more The human B-lymphoblastoid cell line, designated JHK-3, with pre-B-cell characteristics, chronically produces two viruses, Epstein-Barr virus (EBV) and JHK virus, an apparently novel retrovirus. The JHK-3 cells are much more productive of extracellular EBV than the high-producer marmoset line B95-8. The extracellular virus of the JHK-3 EBV strain is relatively fragile, more broadly dispersed in an ultracentrifuged sucrose gradient than the B95-8 EBV and more susceptible to disruption by combined treatment with urea and dithiothreitol. By restriction fragment length polymorphism analysis, the JHK-3 EBV strain resembles the EBV strain FF-41. The JHK-3 cells also produce an incompletely characterized, relatively fragile, enveloped, icosahedral RNA virus that contains Mn++-dependent reverse transcriptase. JHK virions measure 85 nm in ultrathin sections, much smaller than other Retroviridae. The JHK virus exhibits a distinctive morphogenesis, most nearly resembling C-type retroviruses. The JHK-3 cell line provides a human cell model for investigating virus/virus interactions and their pathogenetic effects on host cells which chronically and simultaneously produce DNA and RNA viruses.
We propose to conjugate paclitaxel to a single-chain antibody that specifically binds to the extr... more We propose to conjugate paclitaxel to a single-chain antibody that specifically binds to the extracellular domain of the EGFR. The scFv-paclitaxel conjugate will be able to localize to the tumor rapidly, penetrate deeper, be eliminated quickly, and induce fewer side effects for the patient, thereby increasing the efficacy of the scFvpaclitaxel conjugate. An additional benefit with the scFv-paclitaxel conjugate is the fact that taxol is a radiosensitizing drug, therefore radiation treatment can be used following the administration of the scFvpaclitaxel conjugate. The EGFR overexpressing breast cancer cell line, MDA-MB-468 will be used in this proposal to show "proof of concept". Cell proliferation assays will be used to examine the cytotoxicity of the scFv-paclitaxel conjugate as compared to paclitaxel, scFv or the combination of the two agents. Cell death will be examined using two different apoptosis assays, annexin V-FITC and TUNEL. Since taxol is a radiosensitizing drug, radiation will be included in the cell proliferation and apoptosis assays. Clonogenic assays will be done to determine the radiosensitizing ability of the scFv-paclitaxel conjugate as compare to each agent alone. 14. SUBJECT TERMS 15. NUMBER OF PAGES Single-chain antibody, taxol, breast cancer, conjugate 12 16. PRICE CODE 17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19. SECURITY CLASSIFICATION 20. LIMITATION OF ABSTRACT OF REPORT OF THIS PAGE
Analogs of marine alkaloid, makaluvamine, bearing substituted benzyl and substituted phenethyl si... more Analogs of marine alkaloid, makaluvamine, bearing substituted benzyl and substituted phenethyl side chains have been synthesized and their antiproliferative activities have been evaluated. 4-Methyl, 4-chloro, and 4-fluoro substituted benzyl analogs possessed pronounced antiproliferative effects on the breast cancer cell line, MCF-7 at IC(50) values of 2.3 microM, 1.8 microM, and 2.8 microM, respectively. 4-Methyl, 4-chloro, and 3,4-methylenedioxy derivatives showed the best activity against MCF-7 among the phenethyl analogs with IC(50) values of 2.3 microM, 2.8 microM, and 2.4muM, respectively. In general, methoxy substitutions resulted in slight loss in activity in both benzyl and phenethyl series. Benzyl, 4-fluorobenzyl, 3,4-dimethoxyphenethyl, and 3,4-methylenedioxyphenethyl analogs were tested by NCI in their 60 cell lines in vitro human cancer cell screen. All four compounds showed excellent inhibition against several tested cancer cell lines. Benzyl and 4-fluorobenzyl analogs were relatively more active than 3,4-dimethoxy phenethyl and 3,4-methylenedioxy phenethyl analogs. In NCI assays, the best LogGI(50) values were shown by the fluorobenzyl analog against the renal cancer cell line RXF-393 (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;-8.0M) and dimethoxy phenethyl analog against the CNS cancer cell line, SF-268 (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;-8.0M). The best LogLC(50) value was shown by the fluorobenzyl analog against the breast cancer cell line MCF-7 (-6.01 M).
Epidermal growth factor receptor (EGFr) is overexpressed in a majority of head and neck squamous ... more Epidermal growth factor receptor (EGFr) is overexpressed in a majority of head and neck squamous cell carcinomas, and this overexpression is associated with a poor prognosis. Therefore, EGFr has become the target of investigations aimed at disabling the receptor to determine whether this process leads to improved tumor kill with conventional treatment. C225 is an anti-EGFr monoclonal antibody that inhibits receptor activity by blocking the ligand binding site. A panel of human head and neck squamous cell carcinoma cell lines was used to study the combination of C225 and radiation. It was determined that the combination of C225 (5 microgram/mL) delivered simultaneously with radiation (3 Gy) resulted in a greater decrement in cellular proliferation than either treatment alone. This reduction in proliferation correlated with reduced EGFr tyrosine phosphorylation and a reduction in phosphorylated signal transducer and activator of transcription-3 (STAT-3) protein (known to protect cells from apoptosis). Also, the decrement in proliferation correlated with increased apoptotic events, thereby indirectly linking C225/radiation-induced regulation of STAT-3 protein to apoptosis. This preclinical work serves as important support for the ongoing clinical investigation of C225 and radiotherapy for patients with head and neck carcinomas. The initial results of these clinical studies have been promising.
1. Semin Radiat Oncol. 2002 Jul;12(3 Suppl 2):11-20. Epidermal growth factor receptor as a therap... more 1. Semin Radiat Oncol. 2002 Jul;12(3 Suppl 2):11-20. Epidermal growth factor receptor as a therapeutic target in head and neck cancer. Bonner JA, De Los Santos J, Waksal HW, Needle MN, Trummel HQ, Raisch KP. University of Alabama at Birmingham, 35294-3300, USA. ...
Epidermal growth factor receptor (EGFr) is overexpressed in a majority of head and neck squamous ... more Epidermal growth factor receptor (EGFr) is overexpressed in a majority of head and neck squamous cell carcinomas, and this overexpression is associated with a poor prognosis. Therefore, EGFr has become the target of investigations aimed at disabling the receptor to determine whether this process leads to improved tumor kill with conventional treatment. C225 is an anti-EGFr monoclonal antibody that inhibits receptor activity by blocking the ligand binding site. A panel of human head and neck squamous cell carcinoma cell lines was used to study the combination of C225 and radiation. It was determined that the combination of C225 (5 microgram/mL) delivered simultaneously with radiation (3 Gy) resulted in a greater decrement in cellular proliferation than either treatment alone. This reduction in proliferation correlated with reduced EGFr tyrosine phosphorylation and a reduction in phosphorylated signal transducer and activator of transcription-3 (STAT-3) protein (known to protect cells...
The first ten amino acids of hexokinase II are import for binding to the MOM. Mutating the fifth ... more The first ten amino acids of hexokinase II are import for binding to the MOM. Mutating the fifth amino acid abolished mitochondrial binding. Determining the binding site is important for identifying novel compounds that can disrupt HKII binding to VDAC.
During the screening of human lymphoblastoid cells as suitable hosts for retrovirus transmission ... more During the screening of human lymphoblastoid cells as suitable hosts for retrovirus transmission studies, the Epstein-Barr virus (EBV)-negative, B-lymphoblastoid cell line DG-75 was found to be chronically infected with a heretofore unrecognized retrovirus. Two DG-75 sublines obtained from different sources (designated UW and KAR) were found to produce constitutively particles identified as retroviral by electron microscopy and reverse transcriptase activity. The ultrastructure, morphogenesis, and density in sucrose of the particles were typical of C-type retroviruses. Immunoblot analysis of the DG-75(UW) retrovirus proteins showed antigenic similarity to Moloney murine leukemia virus. A third DG-75 subline in early passage, designated HAD, was free of retrovirus. The DG-75(UW) retrovirus was infectious and produced progeny virions that could be passaged to uninfected cells. We have thus demonstrated that DG-75 cells, which have been used extensively in studies of the biological eff...
The goal of this study was to design paclitaxel (PTX)-monoclonal antibody (mAb) prodrug conjugate... more The goal of this study was to design paclitaxel (PTX)-monoclonal antibody (mAb) prodrug conjugates (PTXMAbs) with the ability to deliver therapeutically significant doses of the drug to the tumor while avoiding the previously observed solubility limitations of conjugates with PTX:mAb molar ratios of >3. New PTX conjugates were synthesized using the discrete poly(ethylene glycol) (dPEG) as linkers. These compounds, PTX-L-Lys[(dPEG12)(3)-dPEG4]-dPEG6-NHS (9a and 9b, for L = GL or SX, respectively), were then conjugated to the antiepidermal growth factor receptor mAb, C225 at increasing PTX:C225 ratios, producing completely soluble conjugates. Unlike the earlier PTXMAbs, buffered solutions of these conjugates remained homogeneous for extended periods of time. Fluorescence-activated cell sorting (FACS) analysis indicated preserved immunogenicity of the conjugates at all four substitution ratios, while cytotoxicity studies in MDA-MB-468 breast cancer cells indicated preservation of dr...
Assess interaction of pazopanib, an oral antivascular endothelial growth factor inhibitor, with r... more Assess interaction of pazopanib, an oral antivascular endothelial growth factor inhibitor, with radiation in tumor xenograft models. Flank xenografts in female athymic nude mice of human lung cancer cell line, A549, and head and neck cancer cell line, UM-SCC-6, were allowed to grow to ∼5×5 mm. Groups were then treated with pazopanib and/or escalating doses of radiation and tumor measurements over time compared with untreated tumor-bearing controls. Pazopanib (100 mg/kg) began 7 days before radiation and continued for 28 days. Daily radiation was 0.5, 1, 2, or 3 Gy ×5 days. Tumors in the A549 control group reached >4× the original size by day 36 postradiation. All treatment groups had less robust tumor growth (p<0.05) and the group receiving pazopanib+3 Gy radiation/day had tumor regression to less than baseline. In the UM-SCC-6-tumor-bearing animals, tumors in all treatment groups had less robust growth than untreated controls after day 23 post-treatment. The combination of pa...
The human B-lymphoblastoid cell line, designated JHK-3, with pre-B-cell characteristics, chronica... more The human B-lymphoblastoid cell line, designated JHK-3, with pre-B-cell characteristics, chronically produces two viruses, Epstein-Barr virus (EBV) and JHK virus, an apparently novel retrovirus. The JHK-3 cells are much more productive of extracellular EBV than the high-producer marmoset line B95-8. The extracellular virus of the JHK-3 EBV strain is relatively fragile, more broadly dispersed in an ultracentrifuged sucrose gradient than the B95-8 EBV and more susceptible to disruption by combined treatment with urea and dithiothreitol. By restriction fragment length polymorphism analysis, the JHK-3 EBV strain resembles the EBV strain FF-41. The JHK-3 cells also produce an incompletely characterized, relatively fragile, enveloped, icosahedral RNA virus that contains Mn++-dependent reverse transcriptase. JHK virions measure 85 nm in ultrathin sections, much smaller than other Retroviridae. The JHK virus exhibits a distinctive morphogenesis, most nearly resembling C-type retroviruses. The JHK-3 cell line provides a human cell model for investigating virus/virus interactions and their pathogenetic effects on host cells which chronically and simultaneously produce DNA and RNA viruses.
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