autologous blood and marrow transplantation - Blog Science ...

642 Chapter 13: Immunotherapy
accomplished by first isolating murine CD80 cDNA from total RNA of
lipopolysaccharide (LPS)-stimulated splenocytes by reverse transcription
polymerase chain reaction (RT-PCR) amplification using oligonucleotide primers
spanning either the entire open reading frame (ORF) of CD80 or the sequence just
distal to the signal peptide (termed B7-1/MP for mature peptide). The 5' primers
were tailed with a Hindlll restriction enzyme sequence while the 3' primers were
extended with an overhanging linker encoding the Gly4SerGly4SerGly4-
SerGly4Ser amino acid sequence to serve as a hinge for the chimeric molecule. The
murine GM-CSF cDNA was isolated from RNA of LPS-induced WEHI cells using
a 5' primer encoding both GM-CSF sequence and the extended nucleotide
sequence coding for the overhanging linker noted above as well as a 3' primer
complementary to the 3' end of the GM-CSF mRNA and extended with sequence
encoding a Hindlll restriction enzyme site.
The amplified CD80 and GM-CSF cDNAs with their overhanging linkers were
gel-purified and allowed to anneal to form a single molecule followed by amplification
using the murine CD80 5' primers and the GM-CSF 3' primers. The
CD80/GM-CSF chimeric cDNA was then cloned into prokaryotic and eukaryotic
expression vectors. A schematic representation of the strategy used to create this
chimeric cDNA is shown in Fig. 2. Figure 3 shows the PCR amplicons for CD80
full-length and mature peptide, the secreted portion of GM-CSF, and the chimeric
products after the reannealing PCR amplification of products.
After the cloning of this target specific costimulatory cDNA into the LNCX
retrovirus, Cos cells were transfected and clones selected for neomycin resistance.
While multiple clones were shown to express intact chimeric mRNA, it was not
possible to demonstrate sufficient levels of chimeric protein either by using
bioassays for GM-CSF activity or by Western blot analysis using antibodies
directed to CD80. Subsequent experiments have now shown that this particular
protein is unstable (possibly due to the linker used) and thus would not accumulate
to levels allowing us to isolate sufficient amounts for biological studies. We
therefore also began to explore an alternative approach.
Costimulatory conjugates for generic use
Another approach for the transfer and expression of proteins on cell surfaces
takes advantage of modifying proteins so that they can be directly incorporated into
membranes. One such modification is the glycosylphosphatidylinositol (GPI)
linkage, which anchors certain classes of proteins to cell membranes. 84,85
Proteins
that are GPI-anchored are synthesized and then enzymatically processed such that
a signal sequence (as yet not precisely defined) is recognized, resulting in the
enzymatic addition of the GPI linkage to the protein. The GPI linkage then allows
the protein to be inserted into the bilipid membrane of a cell and expressed on the