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-GAL EPITOPE
The use of antibody engineering to manipulate and reshape
antibodies has led to the production of antibodies with the ability to alter
the course of immune responses by functioning to bind and inactivate molecules,
including glycoproteins, at both intracellular and extracellular levels. Single
chain antibodies have been demonstrated to have a variety of clinical
applications at the therapeutic level. In the xenograft model, the rejection of
pig organs transplanted into humans is mediated by xenoantibodies that react
with the -gal epitope. Human patients exposed to pig cells
mount a xenoantibody response that is encoded by IgVH genes derived
from the IGHV3-11 germline progenitor. We have recently used the colony filter
hybridization technique to demonstrate a significant increase in the frequency
of expression of genes with a specific VDJ gene configuration in IgM and IgG
cDNA libraries prepared from the peripheral blood of patients exposed to pig
cells. The restricted nature of this response strongly suggests that a specific
and targeted manipulation designed to alter this xenoantibody response has the
potential to prolong xenograft survival. We therefore cloned the IgHV3-11 gene
encoding this antibody along with a light chain variable region into a phagemid
vector (pHEN2) using an overlap extension PCR technique. The introduction of an
amber mutation between the antibody genes and gIII allows soluble scFv
fragments to be produced directly using the technique of induction with IPTG in
a nonsuppressor E.coli strain. The soluble scFv fragments were purified and
screened for binding to the -gal epitope in an ELISA assay using
bovine thyroglobulin and mouse laminin as antigenic targets. We constructed two
clones producing single chain antibodies encoded by IGHV3-11 germline
progenitors. Both clones demonstrated strong reactivity for the -gal epitope expressed on
both bovine thryroglobulin and mouse laminin. The reactivity of human single
chain xenoantibodies to single stranded DNA and human thyroglobulin in an ELISA
assay was used to determine whether human xenoreactive antibodies are
polyreactive or monospecific and to address whether these clones demonstrated
autoantibody activity. One clone demonstrated reactivity with the -gal
epitope in the absence of autoantibody activity and one clone demonstrated both
autoantibody and -gal reactivity. DNA sequencing will be used to
address the role of specific amino acid substitutions in altering the
specificity of the xenoantibody response.