Interference No. 105,188
Short v. Punnonnen
According to one embodiment, plasmid DNA is introduced
into a simian COS cell line . . . by known methods of
transfection . . . and allowed to replicate and express cDNA
inserts. The transfectants expressing B7-1 antigen are
5 depleted with an anti-B7-1 monoclonal antibody . . . and
anti-murine IgG and IgM coated immunomagnetic beads.
Transfectants expressing human B7-2 antigen can be
positively selected by reacting the transfectants with the
fusion proteins CTLA4Ig and CD28Ig, followed by panning with
10 anti-human Ig antibody coated plates. Although human
CTLA4Ig and CD28Ig fusion protiens were used in the examples
described herein, given the cross-species reactivity between
B7-1 and, for example murine B7-1, it can be expected that
other fusion proteins reactive with another cross-reactive
15 species could be used. After panning, episomal DNA is
recovered from the panned cells and transformed into a
competent bacterial host . . . . Plasmid DNA is
subsequently reintroduced into COS cells and the cycle of
expression and panning repeated at least two times. After
20 the final cycle, plasmid DNA is prepared from individual
colonies, transfected into COS cells and analyzed for
expression of novel B lymphocyte antigens by indirect
immunofluorescence with, for example, CTLA4Ig and CD28Ig.
25 Example 4 (Exh. 2040, B. Cloning Procedure, p. 72, ll. 6-32) and
Example 6 (Exh. 2040, B. Cloning Procedure, p. 80, ll. 8-34)
explain how the recursive screening steps of the procedures
described in the Freeman PCT select polynucleotide sequences
which encode novel human or murine B7-2 variants having markedly
30 enhanced or inhibited immune responses to a vaccine vector.
Focusing on the detailed teachings of the Freeman PCT, we
find that one method described in this primary reference for
obtaining an immunomodulatory polynucleotide that encodes a B7-2
antigen which has an altered modulatory effect on an immune
35 response induced by a genetic vaccine vector as compared to the
-12-
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