88
Combinatorial Phage Antibody Libraries
major viral infections. In summary, there
is a signiFcant body of evidence under-
lining the proven and potential role of
antibodies in prevention and therapy of
viral infection. Sections 4.1.1 and 4.1.2 de-
scribe the application of phage antibody
libraries in the generation of recombi-
nant ±abs against clinically important
pathogens and tumor-associated antigens.
4.1.1
Antibodies against Viruses and
Prions
We have extensive experience in recover-
ing and characterizing antibodies against
viruses (particularly HIV-1) and prion pro-
teins and will therefore present some
of these antibodies as examples in this
section. One of the Frst human phage
display antibody libraries taken from an
immune source was prepared using bone
marrow from an individual who had been
infected for several years with HIV-1 but
was asymptomatic. As would be expected,
the patient showed strong serological reac-
tivity against the HIV-1 surface glycopro-
tein gp120. By panning the library phage
against recombinant gp120 immobilized
on ELISA wells, it was possible to isolate a
large number of recombinant ±abs react-
ing with the panning antigen, but not with
wells coated with bovine serum albumin.
DNA sequences from the variable do-
mains of these 33 positive clones revealed
that 10 clones possessed unique heavy
chains and 20 clones possessed unique
light chains. The sequencing data revealed
a phenomenon termed
chain promiscuity
;
that is, heavy chains that were identical,
or very closely related, were seen pair-
ing with light chains of very a different
sequence. This observation made it clear
that the heavy- and light-chain pairings
found in library-generated ±abs are not
necessarily those encountered
in vivo
.A
l
l
the speciFc ±abs were subsequently shown
to compete with soluble CD4 for binding
to gp120. This accords with the Fnding
that more than 50% of the gp120 reactivity
present in the donor serum may be in-
hibited by CD4. Moreover, a cocktail of 3
±abs was able to inhibit more than 50%
of gp120 serum reactivity from a selection
of seropositive donors. These observations
indicate that despite chain promiscuity,
antibodies generated from combinatorial
libraries are relevant to and reflective of
the
in vivo
response.
Ultimately, the more interesting ±ab an-
tibodies are those that are not only able
to neutralize viruses
in vitro
but protect
against infection
in vivo
. To determine
their ability to inhibit HIV-1 infection,
±abs were assessed in p24 ELISA and
syncytia assays. Most ±ab clones showed
little or no neutralizing activity, but one
group of closely sequence-related ±abs
was able to neutralize 50% of the virus
in both assays at a concentration of ap-
proximately 1
µ
gmL
1
.Oneofthese±ab
clones (termed
b
12) has been linked to ±c
to generate whole IgG1 (see Section 3). In
this form, it has been shown to be the most
effective anti-HIV-1 antibody produced to
date, combining potent neutralizing ac-
tivity with broad strain cross-reactivity
against a diverse panel of virus primary
isolates. SigniFcantly, passive adminis-
tration of IgG1 b12 to macaques has
been shown to completely protect against
vaginal challenge of the simian-HIV hy-
brid (SHIV
162P4
). In this experiment, the
serum titer of IgG1 b12 required for ster-
ilizing immunity was roughly 100 times
greater than the 90% neutralization titer
of the challenge virus. The X-ray crystal
structure of whole IgG1 b12 has now been
determined and is the Frst structure to
be determined of an entire human IgG1
molecule (±ig. 5). The long CDR H3 loop is
predicted to be important for its anti-HIV-1
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