Combinatorial Phage Antibody Libraries
93
have given insight into the nature of the
antibody response within tumors. For ex-
ample, phage display libraries have been
prepared from tumor-in±ltrating B-cells
revealing an oligoclonal antibody response
against the intracellular autoantigen actin
that becomes translocated to the cell sur-
face upon apoptosis. Another group, using
a similar approach, provides evidence for
a bias toward intracellular antigens in the
local antitumor immune response in a col-
orectal patient. Antibodies have also been
selected against a number of gene prod-
ucts frequently found to be important in
oncogenesis. An antibody library prepared
from a colorectal cancer patient was used
to isolate antibodies against the tumor
suppressor p53. In another study, an anti-
body was selected against a stage-speci±c
melanoma-associated antigen, and the an-
tibody was used to identify a novel target,
a 23 kDa glycoprotein, p23.
4.1.3
Intracellular Antibodies or
‘‘Intrabodies’’
A relatively recent application of antibodies
has been their use within the cell to mod-
ulate cellular function. Certain antibodies
are able to bind to their target in the rel-
atively oxidizing environment within the
cytoplasm, and they can either act by inac-
tivating a deleterious biochemical pathway
or by initiating or enhancing a desirable
one. In some cases, existing speci±cities
can be engineered as scFvs speci±cally for
intracellular expression, for example, by
r
emo
v
ingC
y
sr
e
s
idu
e
sandth
ens
e
l
e
c
t
-
ing highly stable and functional variants
using phage display. A number of ex-
periments have demonstrated the ability
of intrabodies to combat HIV-1
in vitro
.
Potential therapeutic applications of intra-
bodies hinge upon the potential of gene
therapy, an area in which there has been
a growing foundation of basic science
and progress.
4.2
Antibodies in the Absence of Immunization
Isolating antibodies from an immune
source
has
the
advantage
that
the
molecules will already have been selected
by the host. However, there are many
molecules of importance (e.g. self or
highly conserved antigens) against which
no high-af±nity antibodies will be pro-
duced in the normal human host. Libraries
prepared from ‘‘nonimmunized’’ or unim-
munized sources provide the possibility
of isolating these speci±cities. Moreover,
given that libraries can be made approx-
imating to the size of an animal’s entire
antibody repertoire, antibodies to a vast
number of different antigens could be-
come accessible from a single source.
4.2.1
Naive Libraries
Two main types of nonimmunized li-
braries have been described. In the ±rst,
‘‘naive’’ antibody repertoires have been
prepared using V genes from the IgD
and IgM antibody classes. These antibody
gene pools are less biased by the immune
history of the host than those encoding
IgG. Antibodies against a range of differ-
ent molecules, including haptens and a
number of self antigens, have been se-
lected from such libraries. The antibodies
have been shown to be highly speci±c,
and, in some cases, of high (nM) af±nity.
More generally, however, they are of much
lower af±nity (1
×
10
5
to 1
×
10
6
M
1
)
than those typically obtained from IgG li-
braries. In addition, from libraries of about
10
8
members, often only a small number
of different clones of the desired speci-
±city are selected. One of the most dif±cult
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