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McGuinness and Arced 641
in G418. Thus, the LNCX-GPI-CD80 viral vector was transduced using lipofectin
into Cos cells, which were subsequently selected for resistance to neomycin.
Surviving colonies were cloned using sterile glass cylinders and expanded in
culture. The expression of CD80 was determined by flow cytometric analysis using
an anti-CD80 mAb (Becton Dickinson) or CTLA4Ig. 35
Expression and purification of costimulatory conjugates
Transfected Cos cells (Cos-hB71FC) were grown in 18X T-175 flasks. When
confluent, the cells were removed by scraping and pelleted by centrifugation. The
pelleted cells were lysed in 50 mM Tris-HCl, pH 8.0, 50 mM octyl-B glucoside, 5
mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1% aprotinin at
4°C, overnight. The lysate was clarified by ultracentrifuge at 93,000g at 4°C for 1
hour. The clarified lysate was passed through a protein G column (Pierce),
crosslinked to anti-human B7 mAb using disuccinimidyl suberate (DSS). The
column was washed with 40 mL of 50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1%
Triton X-100, then 20 mL 20 mM triethylamine, pH 10.5, plus 1% octyl-B-
glucoside, followed by 10 mL of 50 mM Tris-HCl, pH 8.0, 1% octyl-B-glucoside.
The protein was eluted using 50 mM glycine-HCl, pH 3.0, 200 mM NaCl, 1%
octyl-B-glucoside as 1 mL fractions into tubes with 50 uL of 1 M Tris-HCl, pH 9.0.
The appropriate fractions were pooled and then dialyzed against PBS for 24 hours
with buffer changes every 8 hours. 80-83
tested directly.
The purified chimeric molecule was then
Testing incorporation of costimulatory conjugates into cell membranes
Either cell lines or de novo tumor cells were collected and washed three times
with phosphate-buffered saline (PBS) and 1% fetal bovine serum (FBS). The cells
were then resuspended in PBS and 1% FBS. Purified GPI-linked B71 and the target
cells were incubated at 37°C on a rocking table at 5 rpm for 2-4 hours. The cells
were then washed 3X with PBS and 1% FBS, then tested by flow cytometry for
CD80 expression using an anti-human CD80 mAb (clone L307.4 from Becton
Dickinson). This test was also repeated using cells that were irradiated with 32 cGy
to determine how long the cells would continue to express CD80.
RESULTS
Target specific costimulatory conjugates
Based on published DNA sequence data and the work of Chan et al., 70
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have generated murine CD80(mB7-l)/GM-CSF chimeric cDNAs. This was
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