Immunoreactivity of Recombinant HumanGlandular Kallikrein Using Monoclonal

Antibodies Raised AgainstProstate-Specific Antigen

Riitta Eerola,1 Timo Piironen,1 Kim Pettersson,1 Janita Lovgren,2Markus Vehniainen,1 Hans Lilja,2 Barry Dowell,3 Timo Lovgren,1 and

Matti Karp1*1Department of Biotechnology, University of Turku, Turku, Finland

2Department of Clinical Chemistry, Lund University, University Hospital Malmo,Malmo, Sweden

3Abbott Laboratories, Abbott Park, Illinois

ABSTRACT: The gene encoding human glandular kallikrein (KLK2) was expressed inEscherichia coli, and the corresponding protein (hK2) was produced by fermentation. The hK2was characterized by Western blotting and epitope map using monoclonal antibodies (MAbs)specific for another protease, prostate-specific antigen (PSA) with high structural identity(80%). MAbs that recognized three different epitopes were bound to hK2, representing 7 outof 23 MAbs tested. One epitope was localized to the sequence region around amino acidposition 78, which is believed to be glycosylated in hK2. The affinities of MAbs recognizinghK2 were similar to those for PSA, suggesting that common epitopes seem to contain veryconserved structures. The results may help in designing specific diagnostic assays for theassessment of prostate cancer. Prostate 31:84–90, 1997. © 1997 Wiley-Liss, Inc.

KEY WORDS: cDNA; hK2; PSA; protease; Escherichia coli; diagnosis; cancer of theprostate

INTRODUCTION

Prostate-specific antigen (HK3, originally knownas PSA) is a 237-amino acid single-chain glycoproteinwith an apparent molecular mass of 33 kDa that be-longs to the family of serine proteases. It is structurallysimilar (80%) to human glandular kallikrein (hK2) [1].The expression of both proteins is androgen depen-dent and is restricted mainly to prostate tissue, al-though nonprostatic expression of both proteins hasalso recently been reported [2–4]. Circulating PSA inserum is the predominant tumor marker used inmonitoring patients diagnosed with cancer of theprostate (CAP). However, the value of PSA in detect-ing CAP is limited by the fact that serum PSA levelsmay also be elevated in subjects with benign prostatehyperplasia (BPH). In 1991 it was shown that PSAforms a complex with a1-antichymotrypsin (ACT) and

is the predominant form of PSA in serum describedindependently and simultaneously by Lilja et al. [5]and Stenman et al. [6]. Later it was demonstrated thatthe proportion of PSA-ACT to total PSA in serum wassignificantly higher in CAP than in BPH [6,7] and thatthe ratio of free to total PSA in serum was lower inCAP than in BPH and could provide a means to dis-tinguish CAP from BPH more reliably. Moreover, thepresent immunometric assays developed for PSAmeasurements may be cross-reacting with hK2. ThehK2 mRNA levels are estimated to be 10–50% of thatof PSA mRNA in prostatic tissue [8–10]; however, thepurification, characterization, and concentration of the

*Correspondence to: Dr. Matti Karp, Department of Biotechnology,University of Turku, FIN-20520 Turku, Finland.Received 13 February 1996; Accepted 7 March 1996

The Prostate 31:84–90 (1997)

© 1997 Wiley-Liss, Inc.

hK2 protein in body tissues and fluids have not beenreported.

In order to develop assays specific for PSA, we havepreviously reported the cloning of the cDNA codingfor pro-PSA and mature PSA by using a prokaryoticphagemid surface expression system [11]. We reportthe cloning of cDNA coding for hK2 in Escherichia coliand the results of extensive epitope mapping studiesusing the recombinant hK2 protein and several anti-PSA antibodies. The data based on phage surface ex-pression and on our previous results on epitope map-ping with PSA purified from human seminal fluid[12], in combination with the present results on hK2epitope mapping study, may permit the design ofmore specific diagnostic kits for both the specific de-tection and the monitoring of prostate cancer.

MATERIALS AND METHODS

Plasmid Constructs and Fermentation

The gene encoding hK2 was initially cloned as apolymerase chain reaction (PCR) fragment into an E.coli vector pUC-19 [13]. The oligonucleotide primersfor PCR were designed to anneal in the initiation me-thionine region (58-end primer, at nt6–24 of EMBL Ac-cession no. S39329) of hK2 gene and in the terminationsignal region (38-end primer, nt848–863), and theirnucleotide composition was the following: 58-primer,58-AATTGGATCCTGTGTCAGCATGTGGGA-38,where ATG (in bold) denotes the initiation codon ofhK2 and the underlined sequence denotes the BamHI-recognition site; 3-primer, 58ATATAAGCTTGAGG-TAGGGGTGGGAC-38, where the underlined se-quence denotes the HindIII recognition sequence. PCRwas performed as described by Saiki et al. [14] using25 cycles and high-fidelity Vent™-polymerase (NewEngland Biolabs, Beverly, MA) and a human prostatecDNA [15] as a template. The PCR product was di-gested with restriction enzymes BamHI and HindIII(both from Promega, Madison, WI) and ligated usingT4-DNA-ligase (New England Biolabs) into a similarlycut vector, pUC-19. The resulting plasmid structure(pUC-19-hK2) obtained after transformation into E.coli XL-2 Blue (Stratagene, La Jolla, CA, USA) was con-firmed from plasmid minipreparations by restrictionenzyme digestions and thereafter sequencing the en-tire DNA fragment cloned from both strands. In orderto produce sufficient quantities of hK2, an expressionvector pKKtac-hK2 was created as follows: new PCRprimers carrying EcoRI and XbaI restriction sites weresynthesized: upstream oligonucleotide 58-ATATGAATTCATGTGGGACCTGGT-38 and downstreamoligonucleotide 58-ATATTCTAGAGAGGTAGGGG-TGGGAC-38, where underlined sequences denote therecognition sites for EcoRI and XbaI, respectively, and

bold text is the initiation codon of hK2. PCR was madeas described above, but now only 15 cycles and theamount of the template DNA pUC-19-hK2 was 20 ng.The product was digested with EcoRI and XbaI andligated into a similarly cut vector, pAFP-Fab designedfor secretion of recombinant antibodies, a derivative ofvector pKKtac [16]. The correct structure (pKKtac-hK2)was verified from plasmid minipreparations by re-striction enzyme digestions.

Plasmid pKKtac-hK2 was transformed into E. coliRV308 strain (su−, DlacX74, galISII::OP308, strA), andthe resulting strain was cultivated in a New Bruns-wick BioFlo 3000 (New Brunswick Scientific Com-pany, Edison, NJ) 5 l fermentor with a fed-batch pro-tocol developed by Vehniainen et al. (unpublisheddata). The fermentation was induced by 200 mM iso-propyl-b-D-thiogalactopyranoside at an optical den-sity (OD 600 nm) of 20 and at OD 600 of 74 the cellswere collected by Millipore Pellicon 10000 NMWL cut-off filtration/concentration (Millipore Corp., Bedford,MA). The different cell compartments (supernatant,periplasmic, cell membrane, and cytoplasmic frac-tions) were prepared as described [17] for checking thelocalization of hK2 produced.

Enzymatic Deglycosylation

Crude cell culture supernatant containing recombi-nant hK2 expressed in mammalian BHK-21 cells usingthe Semliki forest virus (SFV) expression system[18,19] was diluted in incubation buffer (20 mMNaHPO4, pH 7.5, 50 mM EDTA, 0.02% Na-azide). Thebacterially expressed (E. coli RV308/pKKtac-hK2)periplasmic fraction of nonglycosylated rec-hK2 wasdiluted in the same buffer; PSA purified from humanseminal plasma [20] was used as a control in the de-glycosylation reaction. Endo F1 PNGase (Oxford Gly-cosystems, Abington, UK) was added to a final con-centration of 27 U/ml. Samples were incubated at37°C for different times and tested for immunoreac-tivity with two different monoclonal antibody (MAb)combinations, H117/H50 and H117/H179, as de-scribed [12,19] and below.

Immunoreactivity, Determination of AffinityConstants, Epitope Mapping, and

Western Blotting

The production and characterization of MAbs5A10, 9B10, 2C1, 3C1, 10D7, 9E8, 2E9, 5F11, 7H2, 4H5,7H10, 2H11, and 2H12 used in this study have beendescribed earlier [5,12,19]. MAbs H50, H68, H117,H164, and H179 were obtained from Abbott Labora-tories (Abbott Park, IL), antibody F5 was from Dr. Chu[21] and antibodies 6, 10, 30, and 36 were from CanAg(CanAg Diagnostics, Goteborg, Sweden). Immunore-activity and affinity constants were determined using

Immunoreactivity of Recombinant hK2 85

different two-site combinations of capture antibodyand Europium chelate-labeled antibody with time-resolved fluorometry as described by Pettersson et al.[12]. Briefly, to measure the affinity of a MAb, it islabeled with a Europium chelate and combined withcapture antibody that recognizes an epitope indepen-dent of that of the tracer antibody. High-affinity cap-ture MAbs are selected and used in high excess overthe amount of antigen used to ensure virtually com-plete binding. Increasing amounts of the labeled anti-body are used to titrate the immobilized antigen. Fromthe saturation curve thus obtained, the affinity con-stant is measured according to the method of Scatchard[22]. The labeling degree of the tracer antibody is usedto convert the measured signal into molar units. Theaffinity constants thus measured are highly reproduc-ible—generally more than two- to threefold differ-ences in Ka values in (M−1) obtained with the samelabeled preparation can be regarded as statisticallysignificant. The PSA purified from seminal fluid wasobtained as described earlier [20]. Western blottingwas conducted as described earlier for phage-ex-pressed PSA [11].

RESULTS AND DISCUSSION

We have expressed the hK2 gene, a PSA homo-logue, in E. coli in order to produce the hK2 protein insufficient quantities to determine in detail the bindingspecificities of anti-PSA antibodies to hK2. The pro-duction system was based on a vector, pKKtac, whichcontains a strong tac-promoter. The hK2 gene contain-ing the initiation codon followed by prepro-sequenceof its own was cloned as a PCR fragment; the entirefragment was sequenced on both strands. The nucleo-tide sequence was found to match with the previouslypublished sequence [23], except for two positions at

nucleotides 600 (a G-to-A change) and 663 (a G-to-Tchange), which did not change the structure of thecorresponding translation product (gly-residue atboth positions). The hK2 protein was produced by fer-mentation of E. coli RV308/pKKtac-hK2 at the level of110 mg/L based on the immunoreactivity with thecombination of the anti-PSA MAbs H117/H50 usingpurified PSA as a control. The hK2 protein was mainlylocated in the periplasmic space, as judged by the im-munoreactivity found after specific fractionation ofcell compartments (data not shown). The low level ofproduction might be due to the short eukaryotic signalpeptide sequence (17 amino acids) and the propeptide(7 amino acids), causing excessive jamming of secre-tion in the cytoplasmic membrane of E. coli. Shakeflask cultures yielded hK2 at the level of 6.0 mg/L. Thecrude fermentation preparation was used for epitopemapping studies and for the Western blotting experi-ments.

The accessible epitopes on the E. coli produced hK2were mapped by using all possible two-site combina-tions of PSA-specific MAbs in a time-resolved immu-nofluorometric assay format. We have previously

Fig. 1. Western blot of recombinant Escherichia coli-expressedproteins. E. coli RV308 carrying the plasmid pKKtac-hK2 wasgrown, induced and prepared as described in the Methods section.The SDS-PAGE gel was loaded with proteins of the periplasmicpreparate as well as with a PSA control, electrotransferred, andimmunodetected as described in the text. Combination of twoimmunodetections either with anti-PSA antibody H117 (left) orwith the F5 antibody (right).

TABLE I. Affinity Constants (M−1) of VariousMonoclonal Antibodies for Seminal Fluid PSA andRecombinant Human Glandular Kallikrein (hK2)

Produced by Escherichia coli and BHK21 Cell Line

MAbPSAM−1

rec-hK2

E. coliM−1

BHK21M−1

4H5 2.0 × 109 1.0 × 109 1.8 × 109

3C1 5.0 × 108 2.0 × 109 1.9 × 109

F5 8.0 × 109 2.0 × 108 1.7 × 108

H50 6.0 × 108 5.0 × 108 2.6 × 109

H117 1.0 × 1010 5.0 × 109 1.4 × 1010

H179 4.0 × 109 2.0 × 109 a

5F11 3.0 × 108 4.0 × 108 7.4 × 108

aBelow detection limit (<1.0 × 106 M−1).

86 Eerola et al.

made similar epitope mapping studies for PSA puri-fied from seminal fluid [12]. Only seven MAbs out of23 tested (all reacting with PSA purified from seminalplasma) cross-reacted with hK2. All seven MAbs alsorecognized total PSA. Nine MAbs specific for free PSA(7H10, 9B10, 10D7, 6, 2H12, 30, 5A10, 2E9, H68), aswell as seven MAbs that also recognize PSA com-plexed to ACT (2C1, 7H2, 2H11, 9E8, H164, 36, 10),failed to cross-react with hK2 (not shown). The num-ber of hK2-cross-reacting MAbs is unexpectedly lowin view of the close structural homology between thetwo proteins as we assumed that most of the anti-PSAantibodies would also react with hK2. The inhibitionof formation of antibody–protein sandwiches by com-

peting antibodies was also studied to locate the bind-ing of different antibodies relative to each other. It wasfound that antibodies H117, 3C1, 5F11, and 4H5 rec-ognize same epitope area; i.e., interfere with eachother in the binding to hK2. Furthermore, the locationof the epitope defined by the F5 antibody completelyoverlapped this epitope. MAb H50 recognized a com-pletely independent epitope, which was partiallyoverlapped by MAb H179.

Table I shows the affinity constants of the sevencross-reacting monoclonal anti-PSA antibodies. Theaffinity constants are approximately the same for thetwo proteins, except in the case of MAb F5, which hasa hundredfold higher affinity for PSA compared to

Fig. 2. Role of glycosylation for immunoreactivity. Different supernatant preparations were treated with endo F1 PNGase for 0–20 hrand tested for immunoreactivity using two-site immunofluorometric assays as described under Methods. At the top of each figure themethod of expression (host/method) is indicated as well as the Mab sandwich used for the detection. The results are expressed in termsof immunoreactivity found (in µg/L) (using purified PSA as standard) as a function of deglycosylation time. A: Note the different scale. Blackbars represent the untreated controls; gray bars denote the deglycosylation samples.

Immunoreactivity of Recombinant hK2 87

recombinant hK2. The structural basis of the reducedaffinity of F5 in recognition of hK2 remains to be elu-cidated in more detailed studies. There was no detect-able affinity when hK2 produced by the mammaliancell line BHK21 was reacted with MAb H179, whichfact is presumably due to differences in glycosylation.

The periplasmic fractions were probed with twodifferent anti-PSA antibodies, F5 and H117, and theWestern blot of this experiment is shown in Figure 1.The control, PSA purified from human seminal fluid,has an electrophoretic mobility corresponding to ap-proximately 32 kDa that was roughly the same as thatof hK2. PSA and hK2 are known to be highly homolo-gous glycoproteins with one asparagine-linked carbo-hydrate side chain [24]. The location of these sidechains is different and is positioned at Asn-45 in thecase of PSA and Asn-78 in hK2 protein [25]. However,recombinant hK2 produced by E. coli cannot be glyco-sylated due to inability of the prokaryotic protein syn-thesis machinery. As can be seen from the Westernblot shown in Figure 1 there is a faint band at around29 kDa detected by the H117 antibody, which is thesize of deglycosylated native HK2 protein [26]. Thisband is not seen when probed with anti-PSA antibodyF5. A presumable degradation product of about 14kDa indicates excessive autocatalysis or E. coli nonspe-

cific protease action. We suggest that the hK2 bandseen at around 32 kDa is the full-length prepro en-zyme due to the reduced capability of E. coli to splicethe short eukaryotic signal peptide sequence and thepropeptide. A faint band at 67 kDa appears to exist aswell, which might be an indication of a dimeric struc-ture of hK2. This finding is consistent with a recentreport on recombinant insect cell-producing systemdescribing polymorphic forms for hK2 [27].

The role of glycosylation of hK2 on the recognitionby different anti-PSA antibodies was evaluated bycomparing the immunoreactivities of E. coli and mam-malian cell line (BHK21 using the SFV expression sys-tem with full-length prepro-hK2 coding sequence)[18,19] expressed proteins. These proteins togetherwith a PSA control (purified from seminal fluid) wereincubated in the presence of Endo F1 glycosidase forvarying times. After the reactions were stopped, theremaining immunoreactivity was assayed using time-resolved fluorescence with two anti-PSA-specific MAbcombinations recognizing hK2. As shown in Figure 2,the E. coli-expressed hK2 reacts readily (to the sameextent as the PSA standard) with two-site combina-tions of MAbs H117/H50 and H117/H179, whereasSFV-expressed hK2 is not effectively recognized bythe latter combination. Endo F treatment of the hK2

Fig. 3. Epitope map of PSA and hK2, showing the relationship of 23 anti-PSA MAbs. Overlapping circles indicate no sandwich formationin the immunoreactions (see Methods, for details). Touching circles indicate interfering sandwich formation. Separate circles indicateindependent epitopes. Left, schematic representation of PSA molecule; right, hK2 molecule.

88 Eerola et al.

protein did not change the immunoreactivity in anyother cases than for the SFV-expressed hK2. In thiscase, roughly 20% of immunoreactivity (compared tothe assay, in which detection was with MAb pairH117/H50) was recovered subsequent to 20 hr of in-cubation with Endo F. Thus, it can be concluded thatthe anti-PSA specific antibody H179 reacts with anepitope in the PSA protein at amino acid position 78 ornearby in the tertiary structure, the site of which isblocked in hK2 by glycosylation and which is open inE. coli-expressed HK2 and partially revealed by enzy-matic deglycosylation of hK2 expressed in eukaryoticcells.

Based on the results discussed above, we were ableto draw an epitope map of PSA and hK2 showing therelationship of 23 different anti-PSA MAbs summa-rized schematically in Figure 3. Based on the epitopemap one could easily design an hK2-specific immu-noassay by first blocking cross-reacting PSA with anexcess of PSA-specific MAb, such as 2H11, and there-after measure the hK2 with a pair of MAbs recogniz-ing different epitopes (i.e., H50 and H117).

We conclude that the information presented here,regarding the hK2 cross-reactivity of antibodies raisedagainst PSA, will be helpful in the development ofassays of a more precisely defined specificity. It alsoopens up the perspective for assessing the value ofhK2 as an independent tumor marker. Preliminary re-sults (unpublished data) in clinically nondefined se-rum samples indicate that the concentration of hK2relative to PSA is generally low but significantly in-creased in a small number of serum samples.

ACKNOWLEDGMENTS

Financial support from the University Foundationof Turku is gratefully acknowledged. We thank Dr.David Robertson for helpful discussions and for revis-ing the text.

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