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*
The Wistar Institute, Philadelphia, PA 19104;
University of Pennsylvania, Philadelphia, PA 19104; and
SmithKline Beecham, King of Prussia, PA 19406
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have used the Ab (Fab) phage display approach to identify melanoma-associated protein Ags as candidate vaccines for melanoma patients. A Fab library was established from the B cells of a melanoma patient in complete remission after vaccination with allogeneic tumor cells (7). From this library, a Fab (A-18) was isolated that bound not only to melanoma tissues but also to cells derived from various normal tissues (7). Because of its expression by normal tissues, the A-18 Ag was not identified. During the course of those studies, another combinatorial Fab (A10-5) was isolated, which is described in detail in this study.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Table I
lists the cell lines used in this study. The
EBV-transformed B cell line RS, established from the lymphocytes of a
healthy donor as described (8), was grown in RPMI 1640
medium supplemented with 10% FBS. Melanocyte cell lines FM1024, -2404,
and -2408; fibroblast cell lines FF1065 and FF2207; and melanoma cell
lines WM1158, -1552C, -35, and -793 were obtained from Drs. Meenhard
Herlyn and Elliot Levine (The Wistar Institute, Philadelphia, PA).
These cell lines were propagated in MCDB 153 medium supplemented with
2% FBS. Melanoma cell lines Mel-2, -3, -4, and -B used for vaccination
of the patient have been described (9) and were maintained
in RPMI 1640 medium supplemented with 10% FBS. Melanoma cell line
DM196 was provided by Dr. Timothy Darrow (Duke University, Durham, NC)
and was maintained in MCDB 153/10% FBS medium. Melanoma cell line
A375, colorectal carcinoma cell line SW1116, breast carcinoma cell
lines MCF-7 and MDA231, and glioma cell line U87 MG were purchased from
American Type Culture Collection (Manassas, VA). These cell lines were
maintained in MCDB 153/2% FBS medium, with the exception of breast and
glioma cell lines, which were maintained in RPMI 1640/10% FBS medium.
African green monkey kidney COS-7 cells were maintained in DMEM/10%
FBS (Lifetech, Rockville, MD).
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The phage-Fab library derived from the PBMC of melanoma patient GL has been described (7). This patient demonstrated serum Abs that bound specifically to melanoma cell lines, but not to colorectal carcinoma cell lines (7). Briefly, total RNA was isolated from 108 PBMC of patient GL. Full-length light chain and the Fd fragment of the heavy chain were amplified using degenerate oligonucleotide primers and ligated sequentially into the pCOMB3 vector (provided by Dr. Carlos Barbas, Scripps Research Institute, La Jolla, CA) as described (10). The presence of human Fab on the surface of the phage was determined by immunoblotting using mouse anti-human Fab-specific IgG and donkey anti-mouse IgG conjugated to HRP.
Phage biopanning
Pools of phage (108 PFU) were absorbed to confluent monolayers of adherent normal melanocytes in a T75 flask for 2.5 h at room temperature. Unbound phage in the supernatants were collected, titered in Escherichia coli XL-1 blue cells, and subjected to five rounds of selection for binding to HLA nonmatched (with respect to vaccine cell lines) melanoma lines A375 and DM196. Bound phage were eluted at pH 2.2 and selected for binding to the four vaccine lines Mel-2, Mel-3, Mel-4, and Mel-B. Finally, bound phage were eluted at pH 2.2 and selected for binding to tunicamycin-treated (50 µg/ml in DMSO, for 2 h at 37°C) melanoma cell lines DM196 and Mel-B. Phage were eluted and amplified by infection of E. coli XL-1 blue cells. Individual phage were isolated for further testing.
Phage cell binding screening
Cultured tumor or normal cells in suspension (5 x 105 cells/microtiter plate well) were incubated for 30 min at 4°C with 5% BSA or human AB serum (EBV-B cells only) in HBSS and with 108 PFU of specific phage or control VCS M13 phage. Cells were washed in HBSS supplemented with 0.1% BSA or 0.1% human AB serum (EBV-B cells only), and binding was determined by flow cytometry using biotinylated sheep anti-M13 Ab (1:100 dilution) and streptavidin-PE (1:75 dilution). Binding of specific phage to cells was considered significant at levels at least 4 times those of the control phage and with SD of the mean less than 10%.
DNA preparation and sequencing of clone A10-5
Phagemid DNA was prepared essentially as described (11). Briefly, E. coli XL-1 blue cells (Stratagene, La Jolla, CA), infected with A10-5 phagemid, were grown overnight at 37°C in super broth (SB3; 30 g tryptone, 20 g yeast extract, 10 g MOPS (Sigma, Denver, CO) per liter, pH 7.0), containing 1% glucose and 50 µg/ml carbenicillin. Phagemid DNA was purified using a Qiagen Maxi-prep kit (Qiagen, Valencia, CA) and both genes were sequenced using the following sequencing primers: sense, 5'-CCCAGGTGAAACTGCTCGAGTC-3'; antisense, 5'-TGGTCAGGGCACCTGAGTTCCACGACACCG-3' (VH); sense, 5'-CACGAATTCTAATCTAGTCGCC-3'; and antisense, 5'-GACCTTCTCCCCTGGAGA-3' (VL).
A10-5 Fab production and purification
Following phage screening, phage clone A10-5 was amplified by infection of E. coli XL-1 blue cells. Phage-infected cells were cultured overnight at 37°C in SB containing 50 µg/ml carbenicillin and 1% glucose. Phagemid DNA was purified using a Qiagen kit. To excise gene III and juxtapose the hexahistidine tag at the C terminus of the heavy chain, 10 µg phagemid DNA was digested with SpeI and NheI. After gel purification, fragments containing the heavy and light chains were self-ligated overnight at 16°C using a ligation kit (Stratagene). The ligated DNA was purified by phenol/chloroform extraction and used to transform E. coli JM109. Single colonies were selected, and the presence of heavy and light chains was confirmed by restriction enzyme digestion and sequencing.
For Fab purification, the clone containing heavy and light chains was incubated overnight at 37°C in 100 ml SB with 50 µg/ml carbenicillin and 1% glucose. The cell pellet was resuspended in 1 liter of SB containing 50 µg/ml carbenicillin and 10 mM isopropylthiogalactoside and cultured overnight at 37°C. The cell pellet was suspended in 100 ml of 100 mM NaCl and 20 mM Tris (pH 8.0), and lysed by sonication. Cell debris was removed by centrifugation at 10,000 x g for 5 min. NaCl was added to 0.8 M followed by 0.5 ml of 50% nitrilotriacetic acid slurry (Qiagen) and the sample was incubated on a rotator for 2 h at room temperature. The Fab-bound resin was pelleted by centrifugation at 10,000 x g for 15 min at 4°C. The pellet was washed once with 10 ml phosphate buffer (100 mM sodium phosphate, 30% glycerol (pH 8.0)), and Fab was eluted by incubation in 0.5 ml of 300 mM imidazole (Sigma) for 30 min at room temperature. After centrifugation, the supernatant containing Fab was dialyzed extensively against PBS overnight at 4°C. The Fab was analyzed by immunoblotting using HRP-labeled goat anti-human IgG Fab (Cappel, Chester, PA). Approximately 50% purity of the Fab was achieved by the one-step nitrilotriacetic acid purification procedure.
Biotinylation of phages and Fab
Phage (109 PFU/ml) or Fab (1 mg/ml) were suspended in biotinylation buffer (150 mM sodium borate, 1 M NaCl (pH 9.5)) containing 5 µl Biotin-7-normal human serum (20 mg/ml in DMSO; Boehringer Mannheim, Indianapolis, IN) and incubated for 15 min at room temperature. The reaction was stopped with 15 µl of stop solution (3 M NH4Cl; final concentration of 50 mM). Labeled phage or Fab were dialyzed against PBS overnight at 4°C.
Screening for Fab cell binding
For detection of Fab binding to the surfaces of cultured tumor and normal cells, 5 x 105 cells were incubated with specific A10-5 Fab or control 43 Fab (both at 2.5 µg/ml) in 1% BSA in PBS for 30 min at 4°C. Control 43 Fab binds to respiratory syncytial virus (12). Cells were washed in PBS/1% BSA and Fab binding to cells was determined by flow cytometry using PE-labeled streptavidin (1:75 dilution; ImmunoResearch, Grove, PA). To determine inhibition of binding of biotinylated A10-5 Fab to Mel-B melanoma cells, the cells (105/well) were incubated with serum (reciprocal serum dilutions 2, 6, 18, 54) derived from patient GL after vaccination, or with human AB serum pool derived from healthy donors for 2 h on ice. Biotinylated A10-5 Fab (40 µg/ml) was then added to the wells for 2 h on ice. To detect binding of A10-5 Fab to Mel-B cells, PE-labeled streptavidin (1:75 dilution) was added to the wells and inhibition of binding of biotinylated A10-5 Fab to the cells was determined relative to buffer controls. For detection of Fab binding to intracellular structures, cells were grown on 12-mm coverslips, washed with PBS, and fixed with 1% paraformaldehyde in PBS for 5 min on ice. Coverslips were washed in PBS and cells were permeabilized with 0.25% (v/v) Triton X-100 in PBS for 20 min on ice. Cells were incubated with biotinylated A10-5 Fab or control 43 Fab (both at 50 µg/ml) for 1 h at room temperature and rinsed with PBS. Cells were then incubated with streptavidin-fluoresceine isothiocyanate (diluted 1:100 in PBS; Lifetech) for 30 min at room temperature in the dark and rinsed with PBS. Coverslips were mounted onto glass slides with 5 µl Fluoromount G (Fisher Scientific, Pittsburgh, PA) and analyzed with a Leica (Deerfield, IL) TCS 4D confocal microscope.
To determine Fab binding to tissue, formalin-fixed, paraffin-embedded sections were cut (5-µm thickness), mounted on ProbeOn Plus slides (Fisher Scientific), and air-dried in a fume hood (7). The assay was performed by manual capillary action using the MicroProbe (13, 14). Briefly, tissue sections were rapidly dewaxed, cleared with absolute ethanol, and rehydrated with 1x Tris-based buffer, pH 7.4 (10x automation buffer; Biomeda, Foster, PA). Ag retrieval was conducted using an EDTA buffer heat-induced-epitope retrieval (HIER), 10 mM; NeoMarkers, Union City, CA) diluted to a final concentration of 1.0 mM. The manufacturer’s protocol was followed exactly.
The primary biotinylated A-10 Fab or control biotinylated 43 Fab (both at 10 µg/ml) were applied to the sections, and slides were incubated overnight at 4°C. The biotinylated complex was detected with an alkaline phosphatase-linked streptavidin complex (Boehringer Mannheim) at 1 U/ml in 10x automation buffer following a 10-min incubation at 45°C. The red signal was developed by incubating with an indol chromogen system, 5-bromo, 4-chloro, 3-indolylphosphate/iodoblue tetrazolium (BCIP/INT, Amresco, Solon, Ohio), for 10 min at 50°C. Sections were lightly counterstained with a 1:3 (v/v) solution of aqueous hematoxylin (Biomeda) in water for 10 s at room temperature, rinsed in water, mounted with Crystal Mount (Biomeda), and heated at 80°C to harden the medium.
Immunoaffinity purification of Ag
Protein extracts of Mel-B melanoma cells and FF2207 fibroblasts (106 cells each) were prepared in cold Nonidet P-40 lysis buffer (50 mM Tris-HCl (pH 7.5), containing 0.5% Nonidet P-40, 2 mM EGTA, 10 mM EDTA, 100 mM NaF, 1 mM PMSF, 25 mg/ml aprotinin, and 25 mg/ml leupeptin). Extracted proteins were absorbed to VCS M13 wild-type phage (107 PFU) and simultaneously incubated with biotinylated A10-5 phage (108 PFU) in 1% BSA/PBS on ice for 2 h. Streptavidin-agarose beads (50 µl; Sigma) were added to the absorbed phages and mixtures were incubated on a rotator at 30–50 rpm for 1 h at room temperature, followed by centrifugation at 10,000 rpm for 5 min at 4°C. Cell pellets were washed three times with PBS and protein was eluted with 50 µl of 0.1 N HCl. The eluate was separated from the beads by centrifugation (10,000 rpm) for 5 min at room temperature and 4 µl of 2 M Tris base (pH 10.5) was added. The purified protein was stored at -70°C.
Western blot analysis of purified protein
Purified protein samples (see above) were electrophoresed on a
10–16% SDS-PAGE (Bio-Rad, Richmond, CA) under nonreducing and
reducing (
-mercaptoethanol, Sigma; boiling for 5 min) conditions and
transferred to polyvinylidene difluoride membranes (Amersham,
Piscataway, NJ). After blocking with 5% BSA and 4% milk in PBS at
4°C overnight, membranes were washed in PT buffer (0.05% Tween
20/PBS), incubated with A10-5 Fab or control 43 Fab (both at 0.2
µg/ml) for 2 h at room temperature, washed again, and incubated
with HRP-labeled goat anti-human F(ab')2
(diluted 1:2000; Cappel) for 1 h at room temperature. Finally,
membranes were washed three times and developed using the ECL detection
kit (Amersham).
Expression cloning
For expression cloning of the Ag recognized by A10-5 Fab, a cDNA library was constructed from WM1158 melanoma cells in the CDM8 vector (15) and transfected into COS-7 monkey kidney cells using the Lipofectamine Plus kit (Lifetech). After 24 h, transfected COS-7 cells were detached with EDTA, washed, and immunoselected on microtiter plates coated sequentially with rabbit anti-human Fab and A10-5 Fab. Plasmid DNA was recovered from the bound COS-7 cells, amplified in E. coli MC1061/P3 cells (Lifetech), and reintroduced into COS-7 cells. Three rounds of enrichment were performed to isolate positive clones. The isolated cDNA was reintroduced into COS-7 cells and reactivity of the transfected cells with biotinylated A10-5 Fab was confirmed by flow cytometry using PE-labeled streptavidin. One cDNA clone (clone 2) was characterized in detail.
Nucleic acid sequencing and gene database analysis of the Ag
Nucleic acid sequencing was performed with a model 373A automated DNA sequencer (Applied Biosystems, Foster, CA) using the Applied Biosystems BioDye terminator cycle sequencing ready reaction kit. The sequencing primers for the Ag clones were 5'-ATGTCGTAACAACTCCGCCC-3' and 5'-AATCTCTGTAGGTAGTTTGT-3'. Nucleotide and amino acid sequences were analyzed using the DNASIS and BLAST software of GenBank and European Molecular Biology Laboratory databases.
RT-PCR
To confirm the presence of sequences of the cloned cDNA (clone 2) in WM1158 melanoma cells, RT-PCR was performed using primers (sense, 5'-AGTCACTGCCTCCCGAAGTTGC-3' and antisense, 5'-AGCCAGCACAATGGATCTCGAGGGA-3') derived from the nucleotide sequence of clone 2. The RT-PCR product was sequenced.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pools of phage from the combinatorial Fab library were absorbed to normal melanocytes followed by panning against and elution from melanoma cells. To eliminate phage binding to N-linked carbohydrate Ags, the phage were bound to and eluted from tunicamycin-treated melanoma cells (7). Greater than 106-fold enrichment for melanoma-specific phage was achieved. Four phage were selected for detailed binding specificity studies because they showed high-level binding to cultured melanoma cells (MEL-2, -3, -4, and -B) used to vaccinate the patient, and to HLA nonmatched (with respect to the vaccine cell lines) melanoma cells A375, DM196, WM793, WM35, and WM1552C (results not shown). Phage pool A10 did not bind to fibroblasts and melanocytes and, therefore, this phage population was cloned. Clone A10-5 was selected for detailed study because it showed high-level binding to melanoma cells. The deduced amino acid sequence of the heavy chain of the Fab is 94.2% identical with a published human anti-HSV heavy chain (16), and the light chain is 100% homologous with a human anti-staphylococcus protein A light chain (17).
Binding reactivity of A10-5 phage and Fab to cultured cell lines
Flow cytometry indicated significant binding of phage A10-5
to the cell surface of all nine melanoma cell lines tested, but not to
two breast cancer lines. The phage showed low-level binding to
colorectal cancer cell line SW1116 and glioma cell line U87 MG, and no
significant binding to one EBV-B cell line, to three melanocyte and two
fibroblast cell lines (Table I
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Expressed A10-5 Fab, but not control 43 Fab, bound to the surface of
WM1158 melanoma cells; neither Fab bound to FF2207 fibroblasts (Fig. 1). A10-5 Fab bound to intracytoplasmic
structures and nuclei of Mel-B melanoma cells as well as FF2207
fibroblasts (Fig. 2). Control 43 Fab did
not bind to these cells (results not shown). The binding of A10-5 Fab
to Mel-B melanoma cells was inhibited by GL patient’s serum (diluted
1:3) by 82%, whereas the AB serum pool inhibited this reaction by
11%. In this assay, circulating Ag most likely does not interfere
because serum-free supernatant derived from Mel-B cells did not
significantly inhibit binding of A10-5 Fab to the cells (not shown).
Thus, the Ag recognized by A10-5 Fab is not shed by melanoma cells in
vitro and therefore, it most likely is also not shed into the
patient’s circulation in vivo.
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Expressed A10-5 Fab, but not 43 Fab, bound to all of 10 metastatic
melanoma lesions (Fig. 3A) and
to three of five vertical growth phase (VGP) primary melanoma lesions
(Fig. 3A). There was strong cytoplasmic staining of the
cells. However, A10-5 Fab did not bind to any of five radial growth
phase (RGP) primary melanoma or five compound nevic tissues tested
(Fig. 3A). Up to 60% of the cells within metastatic lesions
specifically bound A10-5 Fab, whereas only up to 20% of the cells
within VGP lesions specifically bound the Fab (Fig. 3B).
Fig. 4 shows the staining of metastatic
melanoma (A) and VGP primary melanoma (C)
lesions, and the absence of staining of RGP primary melanoma lesion
(D) with A10-5 Fab. None of the lesions demonstrated
staining with control 43 Fab, as shown for metastatic melanoma in Fig. 4B. In some tissues, fibroblasts and infiltrating
macrophages were stained, whereas endothelial cells, melanocytes, and
keratinocytes were not stained.
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Western blot analysis under nonreducing conditions using A10-5 Fab
and immunoaffinity-purified Mel-B melanoma cell extract revealed an
intense 23-kDa band and a weaker 33-kDa band (Fig. 5). However, under reducing conditions,
only the 23-kDa band was visualized. In extracts of FF2207 fibroblasts,
A10-5 Fab reacted only with the 23-kDa protein under nonreducing
conditions. The control 43 Fab did not react with any of those
proteins. A10-5 Fab did not react with extracts of human melanocytes
(results not shown).
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Eight candidate cDNA clones were investigated after three rounds
of enrichment by panning for A10-5 Fab reactivity. One of these, clone
2, reacted specifically with A10-5 Fab after reintroduction into
transfected COS-7 cells as determined by flow cytometry (Table II). Thus, >88% of the clone
2-transfected COS-7 cells specifically reacted with A10-5 Fab, whereas
<5% of the cells reacted with 43 Fab. Furthermore, <5% of control
vector-transfected COS-7 cells reacted with A10-5 Fab. The nucleotide
sequence of clone 2 is 100% identical with the sequence of the human
ribosomal L13a (18) protein, previously termed p23
(19). p23 interacts with ErbB-3 (20) and is
the human homologue of the murine p198 transplantation protein
(21). Both proteins show 88% identity at the nucleotide
level and 94% identity at the amino acid level.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The deduced amino acid sequence of the heavy chain of the Fab is 94.2% identical with a published human anti-HSV heavy chain (16), and the light chain is 100% identical with a human anti-staphylococcus protein A light chain (17). Thus, the combination of a known heavy and light chain sequence results in an Ab with a new Ag specificity. Binding of the Fab to tumor cells was significantly inhibited by the patient’s serum, but not by control serum, suggesting that original light and heavy chain pairing occurred during the assembly of the combinatorial Fab, although we cannot exclude the possibility that the Fab and the serum Abs bind to spatially close, but distinct determinants.
The Ag recognized by A10-5 Fab is expressed on the surface of cultured melanoma cells and, to a lesser extent, on colorectal cancer and glioma cells, but is not surface-expressed by normal cultured melanocytes, fibroblasts, or EBV-transformed B cells. However, permeabilized cultured fibroblasts did reveal the Ag in the cytoplasm and nucleus. The Ag is also expressed in the cytoplasm and nucleus of cultured melanoma cells. In tissue sections, the Ag is expressed in metastatic and VGP primary melanomas, but not in RGP primary melanoma, nevi, or normal skin.
The Ag appeared as 33- and 23-kDa protein bands in Western blots of nonreduced melanoma cell extracts, but as a single 23-kDa protein in reduced extracts. In fibroblast extracts, only the 23-kDa protein was detected under nonreducing conditions. Thus, in melanoma cells, the Ag consists of a 23-kDa protein that might be disulfide-linked to a 10-kDa protein or might be presented to the cell surface by electrostatic interaction with acidic proteins, because the Ag is a highly basic protein. Alternatively, it is possible that the 33- and 23-kDa moieties are not related, but both express the determinant recognized by A10-5 Fab. In contrast, the Ag in fibroblasts is expressed only in the cytoplasm and only the 23-kDa protein is found. Thus, processing and/or association of the protein with other proteins in melanoma cells differs from that in fibroblasts. Differences in protein processing in tumor cells vs normal cells also have been described in the cathepsin system (22, 23). Ovarian carcinoma patients raised Abs to both the immature (procathepsin D) and the mature (cathepsin) form of the enzyme (24, 25).
The detection of p23 expression on the tumor cell surface was unexpected because p23 lacks a transmembrane domain. However, it is possible that association of p23 with a 10-kDa protein targets p23 to the membrane, although this has not been directly investigated. Moreover, transfection of COS cells with p23 cDNA resulted in membrane expression of the protein, as evidenced by A10-5 Fab binding to the surface of transfected COS cells. Again, p23 may be associated with another anchoring-facilitating protein in COS cells.
In light of the preferential expression of the combinatorial Fab-defined Ag by metastatic melanoma, this Ag may be of prognostic and/or therapeutic value. Expression of the Ag by cultured fibroblasts and fibroblasts in tissue sections does not preclude its use as a therapeutic target because the Ag was not expressed on the surface of these cells. Molecular cloning of the Ag by screening a melanoma cDNA library with A10-5 Fab showed that the cloned Ag is identical with the human ribosomal protein L13a, located on chromosome 1a (18) and previously termed p23 (Ref. 19 ; GenBank no. X56932). p23 is expressed by breast and lung carcinomas and sarcomas (20). Here, we demonstrate that p23 is expressed in human melanoma cells, both in isolated form and in association with a 10-kDa protein. p23 binds with its N-terminal end to the 26-aa juxtamembrane cytoplasmic domain of ErbB-3 (20). Melanoma cells express ErbB-3 (26), and p23 may bind to ErbB-3 in these cells. The interaction of p23 with ErbB-3 has been proposed to play a role in negative growth regulation of ErbB-3-expressing cells, based on observations that human breast cancer cells transfected with p23 show suppressed colony formation of ErbB-3-overexpressing cells, decreased growth rate, and induction of differentiation and apoptosis (20). Our findings implicating preferential expression of p23 in metastatic vs primary melanomas contrast with those findings; however, it is possible that the association of p23 with the 10-kDa protein in melanoma cells inhibits its growth-suppressive function. The identity of the 10-kDa species is presently unknown.
Human p23 demonstrates >88% amino acid sequence identity with the murine p198 transplantation Ag (21), and the bovine (19) and rat (27) p23 L13a. A mutated epitope of murine p198 is the target of cytolytic T cells in the rejection of murine P815 mastocytoma in vivo (21). Here, we demonstrate the immunogenicity of the human p23 homologue in a vaccinated melanoma patient, as we have successfully isolated a Fab from the B cell library of this patient. Both the human p23 of melanoma cells described here and the murine p198 homologue are expressed on the human and murine tumor cell surface, respectively. Although p23 mRNA has been detected in human breast and lung carcinomas and in sarcomas (20), that study did not investigate cell surface expression of p23.
In addition to the ribosomal protein p23, other tumor-associated ribosomal proteins (e.g., the cell surface-expressed 67-kDa protein with a 37-kDa core protein (28, 29) and the ribosomal protein L7 (30)) are associated with tumor cells. Thus, ribosomal proteins can be expressed on the tumor cell surface. In addition, p23 is also found in the nucleus of permeabilized melanoma cells and fibroblasts, consistent with the previously demonstrated nuclear location of ribosomal proteins (31).
p23 mRNA was detected in normal human tissues, including heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas (20). However, we did not find p23 protein expression in normal tissues of the skin, colon, stomach, and endometrium using the A10-5 phage in immunohistochemical analysis of these tissues (not shown), although we did not determine RNA levels in those tissues. This finding is surprising because ribosomal proteins are expected to be expressed in most, if not all, normal tissues. It is possible that protein expression in normal tissues is below the level of detection by A10-5 Fab/phage binding in immunohistochemistry. The one-step immunohistochemical technique used may have been insufficiently sensitive to detect low protein levels in situ. In contrast, the protein is expressed at relatively high levels in tumor cells, allowing its detection by A10-5 Fab.
Other investigators (32, 33) have isolated Ab fragments
with melanoma cell reactivity from the B cell libraries of melanoma
patients using the Ab phage display approach. However, the tissue
specificity of the Fab was not determined and the Ags recognized by the
Fab were not identified in those studies. To our knowledge, our study
presents the first successful cloning of an Ag using phage-displayed Ab
fragments. The p23 Ag is an immunotherapeutic target on melanoma cells
for B cells (because it is recognized by A10-5 Fab) and, possibly, for
T cells (analogous to the murine p198 homologue, a CTL target Ag; Ref.
21), making p23 a vaccine candidate Ag for melanoma
patients. Furthermore, p23, like integrin 3
(34), is a marker that may allow the distinction between
RGP and VGP primary melanoma. Additional studies with higher numbers of
tissues using sensitive Fab staining techniques are required to
critically evaluate the potential of p23 as a diagnostic marker and
therapeutic target.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dorothee Herlyn, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104.
3 Abbreviations used in this paper: SB, super broth; VGP, vertical growth phase; RGP, radial growth phase.
Received for publication June 21, 2000. Accepted for publication October 4, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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