90
Combinatorial Phage Antibody Libraries
of donor serum taken concomitantly with
bone marrow samples indicated that these
patients possessed measurable antibody
titers against a plethora of different viruses.
The libraries were then independently
panned against antigens from the various
pathogens to enrich for antigen-binding
clones. Recombinant Fabs against HIV-
1,
RSV,
herpes
simplex
virus
(HSV)
1 and 2, varicella zoster virus (VZV),
cytomegalovirus (CMV), and rubella were
isolated from a single library, attesting
to the antibody diversity captured during
library construction. Essentially, existing
antibody responses demonstrable at the
serological level could be cloned and
characterized at the molecular level.
Some of these Fab fragments were
obtained by panning against lysates of
virus-infected cells, rather than with re-
combinant or puri±ed viral antigens. As
NNE
NE
Protein
D
Directed antigen capture
Mixture e.g. viral lysate
Human anti-D (NE)
Capture with antibody
against Protein D (NNE)
Pan human library
Protein
D
Nonneutralizing
epitope
(NNE)
Neutralizing
epitope
(NE)
Protein
B
Protein
C
Protein
A
Fig. 6
Antigen capture prior to library
panning. Library panning may be
focused on a neutralizing epitope of a
given antigen by Frst capturing the
antigen, using a mouse antibody or a
recombinant ±ab that recognizes a
nonneutralizing epitope. The method is
advantageous when recombinant or
puriFed forms of an antigen are not
available.
a consequence, these experiments often
generated antibodies against abundant,
highly immunogenic antigens that do not
elicit neutralizing antibodies in a natu-
ral immune response. To increase the
probability of selecting neutralizing anti-
bodies when puri±ed antigen preparations
are not available, a simple antigen-capture
technique was developed. In this proce-
dure, antigens known to contain important
neutralizing epitopes (NE) are captured
(typically from a lysate of virus-infected
cells) onto a solid surface by an anti-
body speci±c for a nonneutralizing epitope
(NNE) on the same antigen. Unbound ma-
terial is then washed away, and panning is
performed against the remaining captured
antigen. In this way, only Fabs recog-
nizing epitopes of the captured antigen
(other than that occupied by the capturing
antibody) are selected. Figure 6 gives an
example taken from a capture-panning of
the HSV glycoprotein D.
Two recombinant antibodies with excep-
tional neutralizing properties have been
isolated against RSV and HSV. The RSV-
speci±c antibody was derived from a library
prepared from a HIV-1-positive donor with
high serum antibody titer to recombinant
RSV surface glycoprotein FG. Panning the
library against FG yielded the antibody
RSV 19, which was shown to potently neu-
tralize a diverse collection of ±eld isolates.
RSV 19 was later evaluated as an Fab for
therapeutic ef±cacy in a murine model
of RSV infection, and when administered
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