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Title: Recombinant Proteome Expression and Antibody Selection

Authors:
;
Publication Date:
Research Org.:
CYTODESIGN, INC
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
899263
Report Number(s):
DOE-ER84277-FINAL TECH REPORT
DOE Contract Number:
FG02-05ER84277
Type / Phase:
SBIR
Resource Type:
Technical Report
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; proteome, expression, high-throughput, antibody, interaction, mapping, genome, optimization

Citation Formats

Robert F Balint, and Xiaoli Cheng. Recombinant Proteome Expression and Antibody Selection. United States: N. p., 2007. Web.
Robert F Balint, & Xiaoli Cheng. Recombinant Proteome Expression and Antibody Selection. United States.
Robert F Balint, and Xiaoli Cheng. Tue . "Recombinant Proteome Expression and Antibody Selection". United States. doi:.
@article{osti_899263,
title = {Recombinant Proteome Expression and Antibody Selection},
author = {Robert F Balint and Xiaoli Cheng},
abstractNote = {},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Tue Feb 13 00:00:00 EST 2007},
month = {Tue Feb 13 00:00:00 EST 2007}
}

Technical Report:
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  • The general objective of this research is to combine genetically derived molecular constructs reactive with tenascin, with appropriate radionuclides and labeling methods in order to generate more effective diagnostic and therapeutic reagents for oncologic nuclear medicine. Tenascin, a polymorphic extracellular matrix glycoprotein, is of interest because of its high expression on glioma, melanoma, as well as prostate and breast carcinoma. Recently, we have also documented high levels of tenascin in lymphomas, particularly those of higher grade, making the potential clinical impact of tenascin-specific radiodiagnostics and therapeutics even greater. An essential feature of our work plan is the ability to exploitmore » our extensive clinical experience in order to design second-generation constructs with properties which could improve clinical efficacy. To date, we have treated over 150 brain tumor patients with 131I-labeled murine 81C6, an antibody which binds specifically to the alternatively spliced fibronectin type III repeats CD of the tenascin molecule. During the current grant period, we have made several observations which form the basis for our proposed specific aims. First, tissue distribution and catabolism experiments in animal models have demonstrated enhanced stability for a chimeric construct composed of murine variable regions and human IgG2 constant domains. Furthermore, pharmacokinetic studies in patients with 131I-labeled chimeric 81C6 have shown significantly longer retention in glioma tumor resection cavities compared with its murine parent. Second, we have initiated the first clinical trial of an endoradiotherapeutic labeled with the 7.2-hr -particle emitter 211At. Twelve glioma patients have received 211At-labeled chimeric 81C6 directly into their brain tumor resection cavity, and very encouraging results have been obtained. Now that the feasibility of human studies with 211At, has been demonstrated, the development and evaluation of anti-tenascin constructs with optimized properties for use in tandem with short half life radionuclides such as 211At ( as well as 1.8-hr 18F for PET imaging) is warranted. Our specific aims are: 1) to construct a bivalent, anti-tenascin molecule containing murine 81C6 variable regions and the human IgG2 hinge region. Both the CH2 domain deletion construct (CH2) and F(ab’)2 will be investigated; 2) to construct a single-chain Fv dimer or multimer with adequate stability, affinity and immunoreactivity for use in tandem with 211At for therapy and 18F for imaging; 3) to generate higher affinity scFv constructs reactive with the alternatively spliced fibronectin type III repeats CD of the tenascin molecule via phage display technology and site-directed mutagenesis; 4) to label promising anti-tenascin constructs with radioiodine, 211At, and 18F and evaluate their potential as radiodiagnostic and radiotherapeutic agents. The proposed studies include: characterization of affinity and immunoreactivity after labeling; evaluation of tissue distribution and projected dosimetry in normal mice, and athymic rodents with subcutaneous, intracranial and neoplastic meningitis xenografts; investigation of the nature of low and high molecular weight labeled catabolites generated in mice; and assessment of cytotoxicity in vitro and in vivo models of human glioma, and possibly, other tenascin expressing tumors; and 5) to investigate strategies for labeling scFv monomers and dimers which will minimize retention of the radiohalogen in the kidneys through the use of negatively charged templates.« less
  • A mutant of human butyryicholinesterase, C117H, was expressed in CHO Ki cells. This Cl 17H mutant had the ability to hydrolyze organophosphates including the nerve agents sarin and VX, the pesticide paraoxon, and the drug echothiopate. The rate of paraoxon hydrolysis was accelerated 40,000 fold over the rate by wild-type butyrylcholinesterase. A second project to shorten the butyrylcholinesterase protein found that 3 types of deletion were compatible with fully active enzyme: deletion of 40 amino acids from the C-terminus, deletion of 3 carbohydrate attachment sites, and deletion of the interchain disulfide bond at Cys 371. However, deletion of 8 ormore » 59 amino acids from the N-terminus. or deletion of intrachain disulfide bonds yielded inactive enzyme. A third project to understand one of the common genetic variants in the American population, the D7OC mutant of butyryicholinesterase, studied the steady-state kinetic parameters of mutants D7OG, Q119Y, A277W, C283D and wild-type. It was concluded that Asp 70 was the major component of the peripheral anionic site of butyryicholinesterase.« less