Cancer Immune Therapy Edited by G. Stuhler and P. Walden ...

17.7 Isolation of New and Improved Antibody Fragments as Targeting Moieties
lar, because advances in this field are dependent not only on the identification of new
targets on cancer cells, but also on the development of new and very specific targeting
moieties such as antibody fragments (scFvs). Phage-display technology enables one
now to select such molecules against unique targets, especially when hybridoma technology
fails to produce antibodies against an antigen or when non-immunogenic or
conserved targets between species are being used. Alternatives to phage display for
making fully human antibodies are technologies developed using transgenic mice (xenomice).
These transgenic mice have been engineered to lack the native murine immune
repertoire and instead harbor most of the human immune system V genes in
the germline [168, 169]. Injection of these ªhumanizedº animals with a foreign antigen
or hapten effectively evokes an immune response and a human-like antibody is
produced in the B cells. The antibody genes can be recovered from B cells either by
PCR and library selection or by fusion into a monoclonal cell line by classic hybridoma
technology. Several examples of recombinant Fv±immunotoxins that were constructed
from scFvs isolated by phage display have already been reported [113, 114,
170]and are being considered for use in clinical trials.
The phage-display approach has been used to isolate a scFv that binds with high affinity
to a mutant form of the EGF receptor in which a deletion of a portion of the extracellular
domain of the receptor generates a tumor-specific [113]. Another novel target
for cancer therapy could be cancer-specific peptides presented on human leukocyte
antigen (HLA) molecules on the surface of tumor cells. To accomplish this, it will be
necessary to isolate antibodies that recognize tumor-specific peptides associated with
class I MHC molecules on tumor cells. As a first step in this direction, a recombinant
immunotoxin has been constructed using an antibody that was isolated by phage display
and that binds specifically to peptide/MHC complexes found on virally infected
cells [170±172]. This recombinant immunotoxin was cytotoxic only to cells specifically
expressing hemagglutinin peptide HA255±262 in complex with H-2K k (mouse class I
MHC) and was not cytotoxic to cells that express other peptides associated with H-2K k
nor to cells not expressing H-2K k . These studies indicate that, if antibodies that recognize
tumor-specific peptides in the context of class I MHC molecules can be developed,
they should be very useful agents for targeted cancer immunotherapy. Recently,
the isolation of a human antibody directed against a peptide encoded by the melanoma-associated
antigen MAGE-A1 presented by HLA-A1 molecules it was reported
[173, 174]. A large phage Fab antibody repertoire was selected on a recombinant version
of the complex. One of the selected phage antibodies shows binding to HLA-A1
complexed with the MAGE-A1 peptide, but does not show binding to HLA-A1 complexed
with a peptide encoded by gene MAGE-A3 and differing from the MAGE-A1
peptide by only three residues. Phages carrying this recombinant antibody bind to
HLA-A1 + cells only after in vitro loading with MAGE-A1 peptide. It remains now to see
if such human anti-MHC/peptide complexes may prove useful for monitoring the cell
surface expression of these complexes and, eventually, as a targeting reagent for the
specific killing of tumor cells expressing tumor peptide/MHC complexes. The isolation
of such rare antibodies against unique tumor targets is a proof for the powerful
abilities of antibody phage-display technology for the development of new generations
of targeting molecules for cancer therapy and diagnosis.
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