[Cancer Research 60, 4175-4178, August 1, 2000]
© 2000 American Association for Cancer Research
Experimental Therapeutics |
One-Step Single-Chain Fv Recombinant Antibody-based Purification of gp96 for Vaccine Development1
Danièle Arnold-Schild,
Christian Kleist,
Martin Welschof2,
Gerhard Opelz,
Hans-Georg Rammensee,
Hansjörg Schild3 and
Peter Terness
Institute for Cell Biology, Department of Immunology, University of Tübingen, D-72076 Tübingen, Germany [D. A-S., H-G. R., H. S.], and the Institute for Immunology, Department of Transplantation Immunology, University of Heidelberg, D-69120 Heidelberg, Germany [C. K., M. W., G. O., P. T.]
 |
ABSTRACT
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Heat shock proteins such as gp96 (grp94) isolated from tumor or
infected cells are able to induce specific cytotoxic T-cell responses
and protective immunity. To facilitate rapid and efficient isolation,
we generated gp96-specific recombinant single-chain Fv (scFv)
antibodies from a semisynthetic phage display library. When immobilized
on Sepharose beads, these antibodies allow a high-yield, one-step
purification of native gp96 molecules from both mouse and human tumor
cell lysates. gp96 molecules eluted from these affinity columns under
mild conditions are still capable of generating antigen-specific CTL
responses in mice. Thus, scFv-purified gp96 is still associated with
peptides; however, in contrast to conventionally purified gp96,
scFv-isolated gp96 is free of contaminating material such as mitogenic
concanavalin A and proteolytic cathepsins. With the help of these
high-yield antibody columns, it is now possible to rapidly isolate
immunogenic gp96-peptide complexes from small amounts of tumor material
to a purity that allows their use in cancer immunotherapy protocols.
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INTRODUCTION
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HSPs4
provide an immunogenic context to peptides that associate with them
inside tumor or infected cells. These peptides are acquired by HSPs in
the cytosol (e.g., for HSP70) or in the ER (for gp96). If
such HSP-peptide complexes are injected into mice, potent immune
responses can be induced. These responses are specific for
HSP-associated peptides, as demonstrated by the induction of specific
CTL responses against tumor cells, minor H and viral antigens (reviewed
in Refs. 1
and 2
). Importantly, HSP
immunization of mice also leads to protective immunity against tumor
challenge. The ER-resident HSP gp96 is most efficient in this regard
(3)
.
What makes HSPs, especially gp96, so effective in vivo is
not completely understood. Recent experiments, however, showed that
APCs are equipped with a receptor specific for HSPs, resulting in the
efficient uptake of HSPs (4
, 5)
and the presentation of
the associated peptides to T cells, as postulated previously
(6)
. On the basis of these features, HSPs can be used for
cancer immunotherapy in mice (7)
. Furthermore, HSPs appear
to be ideal candidates for patient individual immunotherapy protocols.
However, this very promising application will require the rapid
purification of HSP molecules from a limited amount of sample material
at a very high purity. Highly specific mAbs against the HSPs,
especially against gp96 molecules, would be the ideal tool. However,
the existing antibodies are of limited suitability because they react
poorly with native molecules. This behavior might be a consequence of
the intracellular localization of gp96. The anti-gp96 antibodies,
however, may react with the native gp96 in the ER of the respective
B-cell hybridoma. This interaction can be expected to interfere with
normal gp96 function and the secretion of antibodies.
To circumvent this problem inherent to conserved ER proteins, we have
generated recombinant antibodies specific for gp96 molecules from a
semisynthetic phage display library (8)
. The selection of
the phages was deliberately performed with native gp96 molecules; thus,
the resulting recombinant antibodies were able to efficiently recognize
native gp96 molecules even after immobilization on Sepharose beads.
These antibodies will be a precious tool for the rapid purification of
intact gp96-peptide complexes from small quantities of tumor material
to be used in autologous cancer immunotherapy protocols.
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MATERIALS AND METHODS
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Mice and Antibodies.
C57BL/6 mice obtained from Charles River WIGA (Sulzfeld,
Germany) and BALB.B mice obtained from Harlan Winkelmann (Borchen,
Germany) were maintained in the animal facilities at the Institute for
Cell Biology (University of Tübingen, Tübingen, Germany).
mAbs to gp96 (anti-Grp94, SPA-850, and clone 9G10) and immunoglobulin
heavy chain-binding protein (SPA-827 and clone 10C3) were obtained from
StressGen Biotechnologies Corp. (Victoria, British Columbia, Canada).
Standard Purification of gp96.
gp96 was purified as described previously (9)
.
Selection of gp96-specific scFv Ab-expressing Phages.
A human semisynthetic phage display library (8)
was
used for selection of scFv Abs directed against mgp96. For the
construction of the library, synthetic CDR3 loops comprising random
sequences of 4–12 amino acids were introduced into 50 variable heavy
chain germ-line genes (10)
. Subsequently, the heavy chain
repertoire was combined in the phagemid vector pHEN1 (11)
with the unmutated rearranged V
3 variable light chain
gene IGLV3S1/DPL16 (12
, 13) . After preparation of
scFv-expressing phage particles in Escherichia coli TG1,
5 x 1012 (colony-forming units)
phages suspended in MPBS were introduced for panning into Nunc Maxisorb
immunotubes (Nunc, Roskilde, Denmark) coated with 80 µg mgp96.
Unspecifically adsorbed phages were removed by intensive washings.
Specific phages were eluted with 100 mM
triethylamine (pH 12), neutralized, expanded, and reused for further
selections up to a total number of four rounds. Binding specificity was
verified by phage ELISA. Briefly, microtiter plates were coated with
gp96 (0.5 µg/well) and blocked with 2% MPBS, and phage particles
(1010 colony-forming units) were added. Chicken
anti-gp96 polyclonal serum (1:500) served as a positive control, and
PBS served as a negative control. The system was developed by adding
rabbit anti-M13 phage Ab (Stratagene), PO-conjugated goat
antirabbit immunoglobulin and substrate (3,3',
5,5'-tetramethyl-benzidine). Absorbance at 620 nm was measured. Single
colonies (n = 77) were generated after the
fourth selection round, and Ab diversity was examined by digestion of
plasmid DNA with the restriction enzyme BstNI. Three
restriction patterns were identified, and their nucleotide sequences
were determined (377 DNA sequencer; Applied Biosystems Inc., Foster
City, CA). The sequences of the scFv Abs reported in this study have
been deposited in the European Molecular Biology Laboratory Nucleotide
Sequence Database (accession numbers AJ252274, AJ252275, and
AJ2552276).
Production of Soluble gp96-specific scFv Abs.
The gene segments of three gp96-specific scFv Abs (B10C, G12D, and
H11B) were obtained via NcoI-NotI digestion,
ligated into the plasmid pHOG21 (14)
, and expressed in
E. coli XL1-Blue (Stratagene). The soluble fraction of the
periplasmic extract and the culture supernatant were combined,
concentrated, and dialyzed against 50 mM Tris-HCl
and 1 M NaCl (pH 7.0).
Chromatographic Purification of Soluble scFv Abs.
All scFv Abs contain a poly-His tag that allows their purification by
immobilized metal affinity chromatography. Samples were loaded onto a
Chelating Sepharose Fast Flow column (Amersham Pharmacia Biotech,
Uppsala, Sweden) previously charged with Ni2+ and
equilibrated with dialyzing buffer. After extensive washing the bound
material was eluted with 250 mM imidazole and dialyzed
against PBS. Its purity was tested by PAGE.
Western and Dot Blot Analysis of Soluble scFv Abs.
mgp96 was separated by SDS-PAGE under reducing (100 mM
ß-mercaptoethanol + 2% SDS) conditions and transferred to
nitrocellulose. For dot blot analysis, 500 ng of mgp96 were immobilized
on nitrocellulose membrane under native (PBS), denaturing (2% SDS), or
reducing (100 mM ß-mercaptoethanol + 2% SDS)
conditions. Western and dot blot membranes were blocked with 2% MPBS.
gp96 was detected with rat IgG anti-Grp94 (SPA-850; StressGen
Biotechnologies Corp.) and PO-conjugated rabbit antirat IgG (DAKO,
Glostrup, Denmark) or with our scFv Abs. All scFv Abs carry a c-myc
tag, allowing labeling with mouse IgG antihuman c-myc (Genosys, The
Woodlands, TX) and PO-conjugated rabbit-anti-mouse Ab (DAKO). Detection
of soluble scFv Abs by ELISA was performed as described for phages. The
binding of scFv Abs was tested at various dilutions (1:1 to 1:8192).
Immunoprecipitations.
Cells (107) were metabolically labeled in the
presence of [35S]methionine (150 µCi) in 10
ml of methionine-free RPMI 1640 for 16 h. After lysis of
the cells in PBS containing 0.5% NP40 or in hypotonic buffer [30
mM NaHCO3 (pH 7.1); both lysis
buffers supplemented with iodoacetamide and phenylmethylsulfonyl
fluoride], the lysates were preadsorbed overnight with protein
G-Sepharose (Amersham Pharmacia Biotech). Specific mAbs or scFv
anti-gp96 Abs and anti-c-myc Ab were added to the cleared lysates at 10
µg/ml for 90 min before the complexes were isolated with protein
G-Sepharose. In some cases, the lysates were precleared with BSA
coupled to Sepharose beads, and the precipitations were performed
directly with BSA-Sepharose as a control or with scFv anti-gp96 coupled
to Sepharose beads. The immunocomplexes were separated by SDS-PAGE.
Fixed gels were dried and exposed to a phosphorimager screen.
Purification of gp96 Using scFv Abs Coupled to Sepharose Beads.
Five mg of scFv anti-gp96 or BSA were coupled to 0.5 mg of
CNBr-activated Sepharose (Pharmacia). IGELa2 cell pellets (1 ml) were
homogenized as described previously (9)
. The homogenates
were ultracentrifuged for 60 min at 100,000 x g, and the supernatants were applied to the control
BSA-Sepharose column followed by the scFv anti-gp96 column. After
extensive washing with PBS containing 0.5% NaCl, gp96 was eluted with
100 mM sodium acetate (pH 4.5) containing 0.15
M NaCl, 100 mM
diethanolamine (pH 10.5) containing 0.15 M NaCl,
or PBS (pH 7.4) containing 1.3 M NaCl. The
fractions were tested in SDS-PAGE and Western blot using mAb SPA-850
(StressGen Biotechnologies Corp.). The absorbance at 280 nm was
measured to determine the approximate concentration of gp96 by using an
extinction coefficient of 1.0.
Immunization of Mice and Generation of CTLs.
The 8–10-week-old C57BL/6 mice were immunized with 30 µg of
gp96 purified from IGELa2 cells using the standard method or the scFv
anti-gp96 method in 300 µl of PBS or with 80 µl of Sepharose beads
(beads volume) coupled to BSA or to scFv complexed to gp96 in 300 µl
of PBS. CTLs were generated as described previously (9)
.
Cell Culture and CTL Assays.
The mouse IGELa2 and the human C1R cell lines were obtained from
American Type Culture Collection (Manassas, VA) and cultured in RPMI
1640 containing 10% FCS supplemented with L-glutamine (0.3
mg/ml), penicillin/streptomycin (100 units/ml), and 2-ME (2
µl/ml). CTL lines were generated, and CTL assays were performed as
described previously (9)
using Con A blasts of spleen
cells as target cells.
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RESULTS AND DISCUSSION
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Generation of Recombinant scFv anti-gp96 Abs.
A semisynthetic phage display library generated from 50 variable human
heavy chain germ-line genes with randomly varied CDR3 loops and one
human V3 variable light chain gene was used
(8)
. Specific phages were obtained by four consecutive
selection rounds performed in tubes coated with mgp96. A continuous
increase of binding to mgp96 as tested by phage ELISA was noted after
each round (binding activity after the fourth round: selected
phages = 1.406 and negative control = 0.065 A620 nm). Among 77 single phage
colonies, three Ab clones with different CDR3 regions (B10C, G12D, and
H11B) were identified by sequence analyses (Fig. 1
). All belonged to the VH1 gene family. As
expected, the light chains of the Abs were identical. After recloning
into the pHOG21 plasmid (14)
and expression in E.
coli, the Abs showed a good binding activity in ELISA (clone
B10C = 1.297, G12D = 0.651,
H11B = 0.557, positive control = 0.773,
and negative control = 0.003
A620 nm). Dot blot analysis of
purified Abs on nitrocellulose membranes coated with reduced,
denatured, or native mgp96 revealed binding only to the native protein
(Fig. 2
).
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Fig. 1. Sequence analysis of the recombinant scFv anti-gp96 Abs.
Amino acid sequences of the VH and VL chains
are shown. The VH gene segments of the B10C, G12D, and H11B
Ab clones belong to the VH1 gene family and are derived
from the germ-line gene DP-3. The VL gene
segment sequences are identical in all clones and match the germ-line
V3 gene segment IGLV3S1/DPL16.
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Fig. 2. Binding activity of the recombinant scFv anti-gp96 Abs.
Purified scFv Abs were tested for binding to mgp96 immobilized on a
nitrocellulose membrane under (1) reducing (SDS + ß-mercaptoethanol), (2) denaturing (SDS), and
(3) native (PBS) conditions. The membrane was developed
with mouse IgG antihuman c-myc mAb, polyclonal PO-conjugated rabbit
antimouse IgG Ab, and substrate. The rat IgG anti-gp96 (SPA-850) mAb
served as a positive control, and a scFv anti-estradiol Ab served as a
negative control. All recombinant scFv Abs bound only the native
antigen.
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scFv Abs Precipitate Native gp96 Molecules.
Our recombinant scFv gp96-specific Abs recognize their antigen in a
native conformation if tested in an ELISA. The commercially available
mAb against gp96 (SPA850) does this poorly, limiting its use for
affinity purification of gp96. To examine the ability of the
recombinant scFv Abs to interact with native gp96 molecules, we
performed immunoprecipitation experiments after lysing the radiolabeled
cells in a hypotonic buffer that allows the proteins to stay in their
native conformation. As expected, only a weak band corresponding to the
size of gp96 was precipitated using SPA-850 (Fig. 3
, Lane SPA-850) or SPA-847, using SPA-850 or SPA-847
antibodies specific for immunoglobulin heavy chain-binding protein and
cross-reacting to gp96 (Fig. 3
, Lane SPA-847). In contrast,
gp96 molecules were nicely precipitated using the scFv anti-gp96 Abs
(Fig. 3
, Lane H11B). Thus, our recombinant Abs recognize
native gp96 molecules much better than the mAb does. The clones B10C
and G12D showed equal results in precipitating native gp96 molecules
(data not shown).
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Fig. 3. Precipitation of native gp96 from mouse tumor cell
lysates. Hypotonic lysates of radiolabeled IGELa2 cells were incubated
with the indicated Abs, followed by protein G-Sepharose precipitation
and SDS-PAGE. The control lane shows precipitation with protein
G-Sepharose alone.
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Chromatographic Purification of gp96 with scFv Anti-gp96 Sepharose
Beads.
To isolate gp96 by a one-step chromatographic purification, we coupled
the scFv anti-gp96 Ab H11B to Sepharose beads. BSA coupled to Sepharose
beads was used as a precolumn to adsorb unspecific material. Precleared
hypotonic lysates of the radiolabeled mouse cell line IGELa2 were
incubated directly with the scFv-Sepharose beads. The strong
radiolabeled band of the protein isolated with H11B-Sepharose beads
that migrated close to the 94 kDa marker band demonstrated that
the immobilized recombinant scFv H11B Ab is able to recognize gp96
molecules (Fig. 4
). This band is absent when BSA-Sepharose beads are used for
immunoprecipitation (Fig. 4
). For use in a clinically applicable gp96
purification protocol, the recombinant scFv Abs should also recognize
native human gp96 molecules. To test whether our recombinant anti-gp96
clones are able to do so, immunoprecipitation experiments were repeated
using hypotonic lysates of human C1R cells. In contrast to immobilized
BSA (Fig. 4
), immobilized scFv H11B Ab was able to precipitate human
gp96 molecules (Fig. 4
). The differences in intensity between the mouse
and human gp96 bands in Fig. 4
are due to higher expression levels of
gp96 in IGELa2 cells than in C1R
cells.5
Immobilized B10C or G12D scFv Abs are able to precipitate murine and
human gp96 molecules as well.
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Fig. 4. Chromatographic purification of native gp96 molecules.
Hypotonic lysates of radiolabeled mouse IGELa2 and human C1R cells were
incubated with anti-gp96 scFv Abs or BSA as a negative control
immobilized on Sepharose beads. Immuncomplexes were separated by
SDS-PAGE.
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scFv-purified gp96 Activates Specific CTLs in Vivo.
Because native gp96 molecules are recognized by the immobilized scFv
Abs, we tested whether they are still associated with antigenic
peptides and able to induce specific immune responses. The recombinant
scFv column was loaded with hypotonic lysates of IGELa2 cells (of
BALB/c origin; expressing H2d), and the
gp96/scFv-Sepharose beads were injected into C57BL/6 mice.
BSA-Sepharose beads incubated with the cell lysate were used as a
negative control. Ten days after immunization, recipient splenocytes
were stimulated with irradiated BALB.B spleen cells (of the
H2b haplotype). Because mice of BALB origin
differ from C57BL/6 mice at multiple minor H genes and because gp96 is
able to induce cross-priming (9
, 15)
, BALB.B-reactive CTLs
should have been induced in the recipients. As shown in Fig. 5B
, mice immunized with the gp96/scFv-Sepharose bead complexes
generated CTLs able to recognize minor H antigens expressed on BALB.B
blasts. In the control experiment, no CTL activity was detected (Fig. 5A
). These results demonstrate that affinity-purified gp96
is associated with antigenic peptides and able to induce a specific
immune response. To test the usefulness of affinity-purified gp96
molecules for clinical applications, we tried to elute gp96 molecules
from the Sepharose beads while retaining their immunogenicity. Only the
diethanolamine elution buffer with a pH of 10.5 or a buffer containing
a high salt concentration (1.3 M NaCl)
successfully released the gp96 molecules, as detected by dot blot
analysis (data not shown). A buffer with a pH of 4.5 was unable to
elute gp96 from the H11B column. To test whether the eluted gp96
molecules are still immunogenic, we repeated cross-priming experiments
by immunizing C57BL/6 mice with IGELa2 gp96 eluted from the
gp96/scFv-Sepharose beads. gp96 molecules eluted from the scFv
H11B-Sepharose column using the high salt buffer (1.3
M NaCl) were indeed able to generate
BALB.B-specific CTLs (Fig. 5F
). The affinity-purified gp96
molecules were as efficient in generating a CTL response as the gp96
isolated from the same mouse IGELa2 cells using the standard
purification protocol (Fig. 5D
). Both protocols resulted in
the purification of comparable amounts of gp96 from a 1-ml cell pellet
(about 200 µg; data not shown). However, no CTL response was observed
in the mouse immunized with gp96 eluted from the scFv column using the
pH 10.5 buffer (Fig. 5E
), suggesting a loss of peptides, and
no CTL response was observed in untreated C57BL/6 mice (Fig. 5C
).
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Fig. 5. Activation of CTL by scFv-purified gp96 molecules. C57BL/6
mice immunized i.p. with PBS, A; gp96 bound to anti-gp96
scFv-Sepharose, B; untreated, C;
gp96 molecules purified according to standard protocols,
D; pH 10.5-eluted gp96 molecules, E; 1.3
M NaCl-eluted gp96 molecules, F. After 9
days, splenocytes were stimulated with irradiated BALB.B spleen cells
for 5 days, and CTL activity was tested on C57BL/6 (•) and BALB.B
( ) ConA blasts.
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In summary, we have selected the antigen-binding domains of
gp96-specific Abs from a semisynthetic immunoglobulin gene library. The
resulting scFv Abs have been used for the development of a fast,
one-step chromatographic purification method for the isolation of gp96
molecules. Like gp96 molecules isolated by the conventional procedure,
these are still able to induce specific immune responses. The
latter, however, requires ConA-Sepharose column purification, resulting
in contamination of gp96 with ConA due to bleeding from the column.
Because ConA is a T-cell mitogen, it might harmfully interfere with the
vaccinee’s immune system. In addition, proteases such as cathepsin E
have been shown to contaminate conventionally purified gp96
preparations (16)
. All of the potential risks due to gp96
contaminants can be avoided by using affinity purification with the
recombinant scFv Abs. In addition, the anti-gp96 scFv Abs speed up the
purification process and allow the processing of small tumor samples.
These features render our recombinant anti-gp96 scFv Abs an ideal tool
for the purification of gp96 to be used in the treatment of human
cancer and infectious diseases.
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ACKNOWLEDGMENTS
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We thank A. Nissim and G. Winter (MRC Center, Cambridge,
United Kingdom) for the semisynthetic Ab gene library. The excellent
technical assistance of Martina Finger, Claudia Schmid, and Sylvia
Bayertz is gratefully acknowledged. We thank Olga Kouprianova for
helpful discussions and Ulrich Christ and Joannis Mytilineos for
sequencing the scFv Abs.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by Deutsche Forschungsgemeinschaft
Leibnizprogram Grant Ra369/4-1 (to H-G. R.), Deutsche
Forschungsgemeinschaft Sonderforschungsbereich Grant 510, C1 (to
H. S.), and European Union Biomed Grant 95-1627. D. A-S. and C. K.
contributed equally to this work. 
2 Present address: BASF-LYNX Bioscience AG, Im
Neuenheimer Feld 519, D-69120 Heidelberg, Germany.
3 To whom requests for reprints should be
addressed, at Institute for Cell Biology, Department of Immunology,
University of Tübingen, Auf der Morgenstelle 15, D-72076
Tübingen, Germany. E-mail: hansjoerg.schild{at}uni-tuebingen.de
4 The abbreviations used are: HSP, heat shock
protein; minor H, minor histocompatibility; VH and
VL, variable region of the heavy and the light chain,
respectively; CDR, complementarity-determining region; ConA,
concanavalin A; ER, endoplasmic reticulum; mAb, monoclonal antibody;
Ab, antibody; MPBS, PBS with 2% skimmed milk powder; mgp96; mouse
gp96; PO, peroxidase; scFv, single-chain Fv.
5 D. Arnold-Schild and H. Schild, unpublished
observation.
Received 2/25/00.
Accepted 6/ 2/00.
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REFERENCES
|
|---|
-
Srivastava P. K., Menoret A., Basu S., Binder R. J., McQuade K. L. Heat shock proteins come of age: primitive functions acquire new roles in an adaptive world. Immunity, 8: 657-665, 1998.[Medline]
-
Schild H., Arnold-Schild D., Lammert E., Rammensee H. G. Stress proteins and immunity mediated by cytotoxic T lymphocytes. Curr. Opin. Immunol., 11: 109-113, 1999.[Medline]
-
Udono H., Srivastava P. K. Comparison of tumor-specific immunogenicities of stress-induced proteins gp96, hsp90, and hsp70. J. Immunol., 152: 5398-5403, 1994.[Abstract]
-
Arnold-Schild D., Hanau D., Spehner D., Schmid C., Rammensee H. G., de la Salle H., Schild H. Cutting edge: receptor-mediated endocytosis of heat shock proteins by professional antigen-presenting cells. J. Immunol., 162: 3757-3760, 1999.[Abstract/Free Full Text]
-
Wassenberg J. J., Dezfulian C., Nicchitta C. V. Receptor mediated and fluid phase pathways for internalization of the ER Hsp90 chaperone GRP94 in murine macrophages. J. Cell Sci., 112: 2167-2175, 1999.[Abstract]
-
Srivastava P. K., Udono H., Blachere N. E., Li Z. Heat shock proteins transfer peptides during antigen processing and CTL priming. Immunogenetics, 39: 93-98, 1994.[Medline]
-
Tamura Y., Peng P., Liu K., Daou M., Srivastava P. K. Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations. Science (Washington DC), 278: 117-120, 1997.[Abstract/Free Full Text]
-
Nissim A., Hoogenboom H. R., Tomlinson I. M., Flynn G., Midgley C., Lane D., Winter G. Antibody fragments from a "single pot" phage display library as immunochemical reagents. EMBO J., 13: 692-698, 1994.[Medline]
-
Arnold D., Faath S., Rammensee H., Schild H. Cross-priming of minor histocompatibility antigen-specific cytotoxic T cells upon immunization with the heat shock protein gp96. J. Exp. Med., 182: 885-889, 1995.[Abstract/Free Full Text]
-
Tomlinson I. M., Walter G., Marks J. D., Llewelyn M. B., Winter G. The repertoire of human germline VH sequences reveals about fifty groups of VH segments with different hypervariable loops. J. Mol. Biol., 227: 776-798, 1992.[Medline]
-
Hoogenboom H. R., Griffiths A. D., Johnson K. S., Chiswell D. J., Hudson P., Winter G. Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res., 19: 4133-4137, 1991.[Abstract/Free Full Text]
-
Frippiat J. P., Chuchana P., Bernard F., Buluwela L., Lefranc G., Lefranc M. P. First genomic sequence of a human Ig variable gene belonging to subgroup III. Nucleic Acids Res., 18: 7134 1990.[Free Full Text]
-
Williams S. C., Winter G. Cloning and sequencing of human immunoglobulin V gene segments. Eur. J. Immunol., 23: 1456-1461, 1993.[Medline]
-
Kipriyanov S. M., Little M. Affinity purification of tagged recombinant proteins using immobilized single chain Fv fragments. Anal. Biochem., 244: 189-191, 1997.[Medline]
-
Arnold D., Wahl C., Faath S., Rammensee H. G., Schild H. Influences of transporter associated with antigen processing (TAP) on the repertoire of peptides associated with the endoplasmic reticulum-resident stress protein gp96. J. Exp. Med., 186: 461-466, 1997.[Abstract/Free Full Text]
-
Arnold D., Keilholz W., Schild H., Dumrese T., Stevanovic S., Rammensee H. G. Substrate specificity of cathepsins D and E determined by N-terminal and C-terminal sequencing of peptide pools. Eur. J. Biochem., 249: 171-179, 1997.[Medline]
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ABSTRACT
INTRODUCTION
