autologous blood and marrow transplantation - Blog Science ...

644 Chapter 13: Immunotherapy
Figure 3
cell's surface. The possibility therefore exists for generating chimeric cDNAs
composed of sequence encoding a desired protein joined to sequence encoding GPI
linkage signals, thus providing the means to produce and purify GPI-linked
receptors, ligands, or other signaling molecules.
To test this possibility, we cloned the cDNA sequence encoding the external
domain of the human and murine CD80 receptor along with the cDNA sequence
encoding the GPI linkage signals from the CD16 antigen as shown in Fig. 4. 77,79
The PCR products generated using this strategy are shown in Fig. 5. The chimeric
cDNA was then cloned into the LNCX retrovirus and used to transduce Cos cells.
The transduced Cos cells were selected for resistance to neomycin, and clones were
isolated, expanded, and tested for surface expression of CD80. Several producer
lines were isolated that expressed high levels of GPI-linked CD80 (Fig. 6).
Purification of the GPI-linked CD80 receptor was accomplished by affinity
chromatography using either an anti-CD80 monoclonal antibody or CTLA4Ig
directly coupled to Sepharose beads. Purified human GPI-CD80 was then tested for
its ability to properly incorporate into membranes of various cell lines. Figure 7
shows the efficient incorporation of purified GPI-CD80 molecules into the
membranes of a Ewing's sarcoma cell line our laboratory generated from a patient.
Figure 8 shows the incorporation of human GPI-CD80 into the membranes of a
human acute myelogenous leukemia cell line, M07e. The incorporation of this