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Articles |
1
Department of Biotechnology, University of Turku, Tykistökatu 6A 6th Floor, FIN-20520 Turku, Finland.
2
Department of Clinical Chemistry, Lund University,
University Hospital, S-20502 Malmö, Sweden.
a Author for correspondence. Fax 358-2-3338050; e-mail pauliina.nurmikko{at}utu.fi
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Hybridoma cell lines were screened with different methods that aimed at finding antibodies specific for the forms of free PSA produced by LNCaP cell line. Obtained antibodies were further studied for their characteristics related to previously characterized monoclonal antibodies.
Results: Numerous anti-PSA antibodies were obtained, of which four represented unique epitopes previously unrecognized by us. One free-PSA-specific antibody was bound to PSA on two distinct epitopes, and one antibody was bound to the carboxyl-terminal peptide of PSA. Two antibodies were found to bind to the peptide sequence adjacent to the internal cleavage site Lys145-Lys146. These antibodies failed to recognize internally cleaved PSA at Lys145-Lys146. We could not find anti-proPSA antibodies despite the fact that LNCaP PSA contained more than one-half of the zymogen form of PSA.
Conclusions: We report, for the first time, novel anti-PSA antibodies that do not recognize internally cleaved PSA at Lys145-Lys146 and thus are specific for intact, unclipped PSA.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antichymotrypsin (ACT), whereas a small
fraction remains free in the circulation (2)(3).
It has been shown that the proportions of free PSA and PSA-ACT complex
vary according to disease. The percentage of free PSA has been reported
to be significantly lower in PCa patients than in BPH patients
(3)(4). The gene that encodes for PSA is located on the long arm of chromosome 19 and has >84% nucleotide sequence homology with the gene that encodes for human glandular kallikrein (hK2). The amino acid sequence homology between these two proteins is 79% (5)(6). PSA is synthesized as a 261-amino acid preproform from which the 17-amino acid signal peptide is cleaved in the secretion process (5). The remaining zymogen form of PSA is activated to an active serine protease by cleavage of the 7-amino acid propeptide. hK2 is a likely physiological activator of proPSA because it has been shown in vitro that hK2 can cleave the prosequence of PSA (7)(8)(9).
LNCaP (lymph node cancer of the prostate) is a human metastatic prostate adenocarcinoma cell line that was isolated in 1977 from a needle aspiration biopsy of a patient with confirmed metastatic PCa (10). Various forms of free PSA have been found in the spent cell culture medium of LNCaP cells. Corey et al. (11) and Väisänen et al. (12) reported that LNCaP cells produce zymogen forms of PSA and a mature intact form of PSA. The zymogen form of PSA has also been found in the serum of patients with PCa (13). Because the zymogen form of PSA is enzymatically inactive (7)(8), it cannot form complexes with serpins and is likely to remain in a free form in the circulation. There are also other, controversial reports about the nature of serum free PSA, stating that it is an internally cleaved, inactive form produced by internal cleavage (14) or that it represents an unclipped mature but enzymatically inactive form of PSA (15).
Immunizations with purified PSA have produced monoclonal antibodies (MAbs) against PSA and hK2. Many MAbs cross-react with PSA and hK2 because of the high degree of homology between these two proteins (16)(17). However, specific immunoassays that measure free PSA, complexed PSA, and hK2 have been developed by us and others. At present, there are no immunoassays available that specifically recognize various candidate forms of free PSA. The aim of this study was to develop anti-PSA antibodies against the various forms of free PSA produced by the metastatic cancer cell line LNCaP.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Amino-terminal sequence analysis was performed with an Applied Biosystems model 477A pulsed-liquid sequencer connected to an online Applied Biosystems model 120A phenylthiohydantoin amino acid analyzer (Perkin-Elmer). MAbs 5A10, 2E9, 2H11, 3C1, 4H5, and 2C1 have previously been characterized (2)(18). MAbs 66 and 10 were a kind gift of from Dr. O. Nilsson (CanAg Diagnostics, Göteborg, Sweden). MAbs H117, H179, H164, and H50 were obtained from Abbott Laboratories. Antibody E73 was a kind gift from Dr. Elisabeth Paus (The Radium Hospital, Oslo, Norway).
immunogens
Two immunogen structures were used to develop antibodies against
free forms of PSA. LNCaP PSA has been purified previously with affinity
chromatography from spent cell culture medium (12). Briefly,
the supernatant was passed through a column containing immobilized MAb
5A10. The washing buffer was 50 mmol/L Tris, pH 7.5, containing 0.5
mol/L NaCl, and the elution buffer was 0.2 mol/L glycine, pH 3.0,
containing 0.5 mol/L NaCl. Eluted protein was immediately neutralized
by the addition of 2 mol/L Tris, pH 8 (1:10, by volume). After
purification, approximately one-half of the protein was in the mature
single-chain form of PSA, and one-half was in the -5 or -7
zymogen form. The second immunogen structure consisted of a 14-amino
acid synthetic peptide including the prosequence of PSA and the first
seven amino acids from the amino-terminal sequence (APLILSRIVGGWEC).
This peptide was coupled to keyhole limpet hemocyanin and bovine serum
albumin using the Imject Immunogen EDC Conjugation reagent set
(Pierce). The immunizations and fusions with the peptide were done
essentially as described below for LNCaP PSA.
immunizations
The immunizations that were made are summarized in Table 1
. Balb/c mice were immunized by intraperitoneal injection with various
amounts of the immunogen emulsified with Freund’s complete adjuvant
(Sigma). Booster doses were given at 3- to 4-week intervals. The total
immunization times varied from 2 to 10 months. A final booster was
given 3 days before the mice were killed. The splenic lymphoid cells
were fused with Sp2/0 myeloma cells at a 1:1 ratio as described
previously (2)(19). The fused cells were
harvested in 96-well cell culture plates in Optimem containing 200
mL/L fetal calf serum and HAT supplement. Approximately eight
plates were obtained from each fusion.
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screening methods
Several different screening methods were used. Common for all of
the methods was that they were designed to recognize antibodies that
detect PSA produced by LNCaP cells somewhat differently than PSA
purified from seminal plasma. Four of the screening methods are shown
in Fig. 1
. In all methods, hybridoma supernatants were incubated overnight at
4 °C either in microtitration wells coated with rabbit anti-mouse
antibody (methods 1, 2, and 4) or in microtitration wells coated with
streptavidin and biotinylated synthetic peptide (method 3). After
incubation, plates were washed four times. Bound antibody was detected
as described in the legend for Fig. 1
. For signal development, Delfia
enhancement solution was used at 200 µL/well. The signals were
measured with a 1234 Delfia fluorometer.
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antibody characteristics
Purified proteins.
LNCaP PSA was produced and purified as
described by Väisänen et al. (12). LNCaP proPSA
and mature PSA forms were separated by chromatofocusing, which
separates these PSA forms based on their different pI values. The pH
gradient was from 8.5 to 6, and the buffers used were 0.025 mol/L
ethanolamine-acetic acid, pH 8.5, and Polybuffer, pH 6 (diluted
1:10). Chromatofocusing was performed using a C 10/40 column packed
with 30 mL of Polybuffer exchanger gel, and the ÄKTAexplorer 100
system (Amersham Pharmacia Biotech). The flow rate was 0.3 mL/min, and
3.6-mL fractions were collected. Before fractions were collected, 0.4
mL of 2 mol/L Tris-HCl, pH 8, was added to each fraction tube.
The PSA concentration in each fraction was measured by the Prostatus
PSA free/total method (Perkin-Elmer Life Sciences). Fractions
containing PSA from one peak area were pooled, and the amino-terminal
regions were sequenced.
Separate pools of purified seminal plasma PSA were a generous gift from
Dr. U-H. Stenman (Helsinki University Central Hospital, Helsinki,
Finland). Pools A, B, C, D, and E contained different amounts
of internally cleaved PSA as described by Zhang et al. (20).
Pools A and B contained only intact PSA. Pools C and D contained the
intact form of PSA and PSA forms that had been internally cleaved at
Arg85-Phe86 and Lys145-Lys146. Pool D also contained a minor amount of
PSA cleaved at Lys182-Ser183. Pool E contained only the form internally
cleaved at Lys145-Lys146 and a minor amount of intact PSA. The staining
intensities in sodium dodecyl sulfate-polyacrylamide gel
electrophoresis suggested that the amounts of intact PSA in
pools C, D, and E were 
20%, 10% and <5%, respectively.
hK2 was produced with the baculovirus expression system and purified as described by Lövgren et al. (7). Preparation and purification of PSA-ACT in vitro has been described previously (18).
Epitope mapping.
Previously characterized MAbs were used in
sandwich assays in all possible combinations with the investigated MAbs
to determine the binding sites on the PSA molecule.
Peptide mapping.
Synthetic 15-mer peptides with 10-residue
overlaps covering the whole PSA sequence were used for the
determination of specific binding sites of antibodies that recognize
continuous epitopes. Europium-labeled MAbs were incubated with
biotinylated peptides attached to streptavidin plates as described by
Piironen et al. (21).
Specificity and binding to various PSA forms.
A suitable
partner antibody was selected to determine the specificities of the new
MAbs for PSA, hK2, and PSA-ACT complex. In addition, binding to various
PSA forms was determined using PSA pools A, B, C, D, and E, which were
purified from seminal plasma, and proPSA purified from LNCaP PSA. The
binding of labeled antibodies to various PSA forms was tested in a
three-step sandwich assay. Biotinylated suitable partner antibody (200
µL, containing 200 ng of antibody, per well) was incubated in
streptavidin-coated plates for 1 h, and plates were washed four
times. Pools A–E or proPSA (1 ng/well) were added, and plates were
incubated for 2 h and washed. Europium-labeled antibody (50
ng/well) was incubated for 2 h, and after a wash step, enhancement
solution was added and the signal was measured.
Affinity of MAbs.
The affinity constants of europium-labeled
MAbs were determined as described previously (18), using 2E9
or H117 as capture antibodies and PSA purified from seminal plasma, or
purified hK2. The affinities were calculated using the Scatchard method
(22).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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summarizes the immunizations that were made, the
number of PSA-positive cell lines, and the MAbs that were further
characterized from each fusion. All of the finally characterized MAbs
were from fusions where LNCaP PSA was used as an immunogen. As could be
expected, immunizations using a synthetic peptide gave fewer
PSA-positive cell lines. Some cell lines from peptide fusions were
positive for the peptide, but additional testing showed that antibodies
did not recognize the entire PSA molecule.
antibody characteristics
Epitope mapping.
MAbs were tested with various antibody
combinations to identify their binding sites on the PSA molecule. Based
on the results, a two-dimensional epitope map was constructed (Fig. 2
). The binding sites of anti-PSA MAbs are presented in relation to
previously characterized MAbs (21).
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The different binding regions of 83 anti-PSA MAbs have been described by Paus et al. (23) in the ISOBM study, where binding regions from 1 to 6 were mapped in two- and three-dimensional models (24). The binding regions of novel anti-PSA antibodies were compared to binding sites of previously characterized MAbs.
Antibody 5F12 mapped to group 1 free-PSA-specific antibodies, which bound to the same epitope as previously characterized MAb 5A10 [no. 25 in the ISOBM study (24)]. Interestingly, MAb 5F12 also blocked binding of MAb PSA10 (ISOBM no. 72), which belongs to antibody group 3a. Antibody 7G1 bound to an epitope close to H50 (ISOBM no. 57) and PSA10, which belong to antibody group 3a. Antibody 7G1 was also somewhat inhibited by antibody 2H11 (ISOBM no. 41), which belongs to antibody group 5b. Antibodies 5F7 and 5H6 bound to an area that overlapped the binding site for MAbs H50 and PSA10. Antibodies 7C4, 4D4, and 5C3 bound to an area close to the binding site for MAbs H164, H50, and 2H11, which is located near antibody group 5b.
Peptide mapping.
Biotinylated peptides 15-amino acid residues
in length (15-mers) and overlapping the entire PSA sequence were used
to study the binding of MAbs to linear PSA sequences (21).
Three of the seven MAbs tested bound to linear peptides (Table 1
).
Antibodies 4D4 and 5C3 bound to the 15-mers 130ASGWGSIEPEEFLTP144 and 135SIEPEEFTLTPKKLQC149, respectively. The most common internal PSA cleavage site, at amino acids Lys145-Lys146, renders PSA inactive (25).
Antibody 5H6 bound to the carboxyl-terminal peptide of PSA (225YRKWIKDTIVANP237). Another antibody (E73) has been characterized as binding close to the carboxyl-terminal region of the PSA molecule (data not shown). MAb E73 bound to the 15-mer peptide (215RPSLYTKVVHYRKWI229) adjacent to the site recognized by 5H6.
Results from the peptide-binding studies were combined with the data
presented by Piironen et al. (21) to create a
three-dimensional epitope map showing seven independent antigenic
domains on the PSA moiety (Fig. 3
).
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Specificity of the MAbs.
MAb 5F12 was a free-PSA-specific
antibody. MAbs 7C4, 4D4, 5C3, 5F7, and 5H6 recognized free PSA and the
PSA-ACT complex with similar affinity, but not hK2, and were thus
designed as total-PSA-specific antibodies. MAb 7G1 recognized hK2 and
PSA complexed with ACT with similar affinity.
Binding to various PSA forms.
Binding of the new MAbs to
different PSA forms was tested using sandwich assay formats with
different capture antibodies and the new MAbs as the detection
antibodies. Binding to mature intact PSA, mature internally
cleaved PSA forms, and proPSA was studied.
MAbs 4D4 and 5C3 recognized seminal plasma PSA less than proPSA when
screening method 4 was used. These antibodies were further tested with
pools of seminal plasma PSA that contained various amounts of
differently cleaved PSA forms (20). The amount of antibody
(4D4 or 5C3) that bound to pool E, which contains mostly Lys145-Lys146
internally cleaved PSA, was <5% compared with the amount of antibody
that bound to pools A and B, which contained only intact PSA. It seems
that these antibodies recognized only PSA forms where the Lys145-Lys146
cleavage site is intact, such as in LNCaP PSA, which was used as an
immunogen. Fig. 4
illustrates the reactivity of different antibodies with pools A–E of
seminal plasma PSA. Antibodies were designated according to different
binding regions illustrated in the ISOBM study (24).
Significant differences in the binding to the various PSA forms were
seen only for MAbs 4D4 and 5C3.
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Affinity of MAbs.
The affinity constants of the MAbs are
listed in Table 2
. For MAbs 5F7, 7C4, 5H6, and 7G1, seminal plasma PSA was used in the
affinity determination. In addition, the affinity of MAb 7G1 for hK2
was also determined. MAb 7G1 had very high affinity for both seminal
plasma PSA and hK2 (Ka = 2 x
1010 L/mol). For MAbs 4D4 and 5C3, the
affinity constants were determined using intact forms of seminal plasma
PSA (pools A and B). The Kas were
2.5 x 109 and 2.7 x
109 L/mol for 4D4 and 5C3, respectively. Similar
affinity constants were obtained for proPSA with these antibodies. In
addition, MAbs 4D4 and 5C3 were tested for their affinity for pools of
seminal plasma PSA that contained internally cleaved forms (pools C, D,
and E). The affinity constants of these two MAbs decreased with
increasing amounts of internally cleaved PSA forms. The affinities of
MAbs 4D4 and 5C3 for pool E PSA could not be determined by the
Scatchard method because the affinities were very low (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The purified LNCaP PSA consisted of approximately one-half single-chain mature and one-half zymogen forms of PSA. From eight LNCaP PSA fusions, 125 positive cell lines were grown and tested with several different methods. Most of the antibodies were highly similar to the previously characterized anti-PSA antibodies (21)(24). Seven antibodies were characterized further, and four of these were shown to have epitopes previously unknown. Two novel antibodies (4D4 and 5C3) bound epitopes that overlapped the most common internal cleavage site. One antibody (5H6) bound to the carboxyl-terminal peptide of the protein. MAb 5F12 was specific for free PSA and recognized two separate epitopes on PSA. Synthetic peptide fusions did not produce anti-PSA antibodies.
MAbs 4D4 and 5C3 bound to two adjacent 15-mer linear peptide sequences,
which covered amino acids 130–144 and 135–149, respectively. These
antibodies were very similar in their specificities and affinities,
although they were produced by different clones. They did not recognize
hK2. MAbs 4D4 and 5C3 inhibited the activity of PSA toward chromogenic
peptide substrate (data not shown), which was expected because the
catalytically active site of PSA was mapped next to the binding sites
of MAbs 4D4 and 5C3 (Fig. 3
). When these antibodies were tested with
seminal plasma PSA pools that contained different amounts of internally
cleaved PSA forms (20), it could be seen that these
antibodies did not recognize PSA that was cleaved internally between
Lys145 and Lys146. The exact peptides involved in the 4D4 and 5C3
binding site are not known, and thus it is not known whether these
antibodies bind directly at the cleavage site. However, cleavage of the
peptide bond at Lys145-Lys146 produces two loose ends that most likely
affect protein conformation. This leads to loss of epitopes over a
larger area, so that 4D4 and 5C3 are no longer capable of binding to
internally cleaved PSA at Lys145-Lys146.
MAb 5H6, another novel antibody, bound to the carboxyl-terminal peptide containing the last 15 amino acids on the PSA molecule. This peptide is helical in its native form and is located on the surface of the molecule (21). Amino acid 234 in the PSA molecule is valine, but in hK2 it is alanine. Because of this difference in one amino acid, this antibody does not recognize hK2. Another antibody, E73 from Dr. E. Paus, was also mapped to the carboxyl-terminal part of PSA, but the peptide sequence only partially overlaps the 5H6 binding site.
An immunoassay was constructed that used 5H6 as the detection antibody. The idea of this assay was to study changes in the carboxyl-terminal part of PSA. Because 5H6 binds to the carboxyl-terminal peptide of PSA, it was thought that cleavage of amino acids at the carboxy terminus might decrease the binding of 5H6 to PSA. Väisänen et al. (12) reported that mature LNCaP PSA grown with serum is inactive for unknown reasons. Corey et al. (11) reported similar results, showing that part of the inactive fraction of PSA could be activated with trypsin, but part remained in the inactive form. Cleavage of the amino acids at the carboxy terminus of PSA could change the conformation of the protein and possibly render PSA inactive. LNCaP PSA forms from the spent culture medium of LNCaP cells grown with or without serum (12) were separated after affinity purification by chromatofocusing into the proform and mature form of the protein. Different LNCaP PSA forms were tested with an immunoassay that used H117 as the capture antibody and 5H6 as the detection antibody. We wanted to see whether these different LNCaP PSA forms differ in their carboxyl-terminal amino acid sequence. Immunoassay with 5H6 did not show differences among these different PSA forms (data not shown).
Increased serum PSA may result from various urological problems other than PCa, and thus PSA is not cancer specific. However, the proportion of free PSA to PSA-ACT complex in serum has been shown to be significantly higher in BPH than in PCa (3)(4). The mechanisms that produce the increased fraction of serum free PSA in BPH are not known. Björk et al. (26) reported a lack of ACT production in PSA-containing BPH nodules in contrast to cancerous tissues, where production of both PSA and ACT could be detected. This could lead to increased formation of PSA-ACT complex in cancer and thus explain the differences in the amounts of free PSA in BPH and PCa. However, Jung et al. (27) demonstrated that the amounts of different forms of PSA in prostatic tissue do not correlate with amounts or ratios of different PSA forms in serum. Thus, the patterns of PSA forms seen in serum might not be a simple reflection of the patterns in tissue. Instead, release of different proportions of enzymatically active or inactive forms of free PSA from neoplastic and benign cells might produce the differences in the free-to-total PSA ratio in PCa and BPH.
There have been controversial reports about the nature of free PSA in serum. A zymogen form of PSA starting at amino acid -4 in the serum of PCa patients was reported by Mikolajczyk et al. (13). Recently, -2 and -4 proforms of PSA were also shown to be more highly correlated with PCa tissue than with benign prostate tissue (28). LNCaP cells have been shown to produce proforms of PSA starting at amino acid -7 or -5 (12). These proforms have high pI values that, according to Väisänen et al. (12), disappeared after incubation with hK2. These high pI values have also been found in the serum of patients with advanced PCa, but not patients with BPH (29). Noldus et al. (14), however, did not detect any zymogen forms in the sera of patients with high-grade PCa. Their purification methods did not exclude hK2, which could possibly cleave proPSA into the mature form during purification.
Characterization of the different forms of free PSA could add new discriminatory information to the diagnosis of PCa. An anti-proPSA antibody would enable specific and sensitive measurement of the zymogen forms of the protein. Despite the highly immunogenic nature of LNCaP PSA, we could not find antibodies specific for or with a stronger preference for the zymogen form of PSA.
There could be many reasons for not obtaining anti-proPSA antibodies. It has been shown that the PSA prosequence of mouse kallikreins is similar to kallikrein prosequences in human (30). This could mean that the propeptide is not immunogenic in mice. In addition, because of the homology, mouse kallikreins conceivably might be able to cleave the human proPSA to mature PSA, leading to the loss of the prosequence. Additionally, the orientation of the prosequence in the PSA molecule is not known, and it could be partially buried.
The characterization of various forms of free PSA from seminal plasma
and prostate tissue has been one approach in understanding the
different molecular forms of free PSA and their relevance in different
prostatic diseases. One explanation for the inactive free-PSA forms are
internally cleaved forms of PSA. Seminal plasma PSA has been shown to
contain 30% internally cleaved PSA, in which the most common
internal cleavage site is at Lys145-Lys146 (25). Noldus et
al. (14) detected this internally cleaved PSA form in the
sera of patients with high-grade PCa. Charrier et al. (31)
used two-dimensional electrophoresis to compare the patterns of PSA
forms sera from patients with BPH and PCa, and demonstrated that BPH
sera contain more cleaved forms of free PSA than PCa sera. Internal
cleavage sites have also been identified between Arg85-Phe86 and
Lys182-Ser183 (20)(32). A recently characterized
novel form of PSA, "B-PSA", which was isolated from benign
transition zone tissue of BPH patients, contains the internal cleavage
site at Lys182-Ser183 (33). Chen et al. (34)
reported PSA forms with internal cleavage sites at His54-Ser55,
Phe57-His58, Lys145-Lys146, and Lys146-Leu147 in BPH nodule fluids. It
is not known whether cleavage at sites other than Lys145-Lys146
inactivates PSA.
Zhang et al. (20) reported an inactive mature, unclipped form of PSA in seminal fluid that could not complex with ACT. This intact, inactive PSA has also been found in serum (13)(14)(15) and in the spent medium of LNCaP cells (11)(12). At present, there is no explanation for this inactive, unclipped form of PSA.
There are separate antigenic areas on the PSA molecule (Fig. 3
). The
presence of these areas might lower the possibility of obtaining
antibodies against less immunogenic areas. In this study, however,
antibodies against new, previously unrecognized epitopes were found. An
antibody that does not recognize internally cleaved PSA at
Lys145-Lys146 has been used in the development of an immunoassay for
intact PSA. Optimization of the assay and testing of a large patient
sample panel are ongoing, and results from that study could give some
answers to the question of the nature of free PSA in the
circulation.
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Acknowledgments |
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Footnotes |
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1-antichymotrypsin; hK2, human glandular kallikrein; and MAb, monoclonal antibody. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antichymotrypsin is the major form of prostate-specific antigen in serum of patients with prostatic cancer: assay of the complex improves clinical sensitivity for cancer. Cancer Res 1991;51:222-226.1-antichymotrypsin as an indicator of prostate cancer. J Urol 1993;150:100-105.[ISI][Medline]
[Order article via Infotrieve]
(1)-antichymotrypsin complex in serum but not in prostatic fluid. Clin Chem 1997;43:352-359.1-antichymotrypsin complex. Clin Chem 1995;41:1480-1488.1-Antichymotrypsin production in PSA-producing cells is common in prostate cancer but rare in benign prostatic hyperplasia. Urology 1994;43:427-434.[ISI][Medline]
[Order article via Infotrieve]
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