Preclinical efficacy of hK2 targeted [177Lu]hu11B6 for prostate cancer theranostics.

Androgen ablating drugs increase life expectancy in men with metastatic prostate cancer, but resistance inevitably develops. In a majority of these recurrent tumors, the androgen axis is reactivated in the form of increased androgen receptor (AR) expression. Targeting proteins that are expressed as a down-stream effect of AR activity is a promising rationale for management of this disease. The humanized IgG1 antibody hu11B6 internalizes into prostate and prostate cancer (PCa) cells by binding to the catalytic cleft of human kallikrein 2 (hK2), a prostate specific enzyme governed by the AR-pathway. In a previous study, hu11B6 conjugated with Actinium-225 (225Ac), a high linear energy transfer (LET) radionuclide, was shown to generate an AR-upregulation driven feed-forward mechanism that is believed to enhance therapeutic efficacy. We assessed the efficacy of hu11B6 labeled with a low LET beta-emitter, Lutetium-177 (177Lu) and investigated whether similar tumor killing and AR-enhancement is produced. Moreover, single-photon emission computed tomography (SPECT) imaging of 177Lu is quantitatively accurate and can be used to perform treatment planning. [177Lu]hu11B6 therefore has significant potential as a theranostic agent. Materials and Methods: Subcutaneous PCa xenografts (LNCaP s.c.) were grown in male mice. Biokinetics at 4-336 h post injection and uptake as a function of the amount of hu11B6 injected at 72 h were studied. Over a 30 to 120-day treatment period the therapeutic efficacy of different activities of [177Lu]hu11B6 were assessed by volumetric tumor measurements, blood cell counts, molecular analysis of the tumor as well as SPECT/CT imaging. Organ specific mean absorbed doses were calculated, using a MIRD-scheme, based on biokinetic data and rodent specific S-factors from a modified MOBY phantom. Tumor tissues of treated xenografts were immunohistochemically (IHC) stained for Ki-67 (proliferation) and AR, SA-ß-gal activity (senescence) and analyzed by digital autoradiography (DAR). Results: Organ-to-blood and tumor-to-blood ratios were independent of hu11B6 specific activity except for the highest amount of antibody (150 µg). Tumor accumulation of [177Lu]hu11B6 peaked at 168 h with a specific uptake of 29 ± 9.1 percent injected activity per gram (%IA/g) and low accumulation in normal organs except in the submandibular gland (15 ± 4.5 %IA/g), attributed to a cross-reaction with mice kallikreins in this organ, was seen. However, SPECT imaging with therapeutic amounts of [177Lu]hu11B6 revealed no peak in tumor accumulation at 7 d, probably due to cellular retention of 177Lu and decreasing tumor volumes. For [177Lu]hu11B6 treated mice, tumor decrements of up to 4/5 of the initial tumor volume and reversible myelotoxicity with a nadir at 12 d were observed after a single injection. Tumor volume reduction correlated with injected activity and the absorbed dose. IHC revealed retained expression of AR throughout treatment and that Ki-67 staining reached a nadir at 9-14 d which coincided with high SA- ß-gal activity (14 d). Quantification of nuclei staining showed that Ki-67 expression correlated negatively with activity uptake. AR expression levels in cells surviving therapy compared to previous timepoints and to controls at 30 d were significantly increased (p = 0.017). Conclusions: This study shows that hu11B6 labeled with the low LET beta-emitting radionuclide 177Lu can deliver therapeutic absorbed doses to prostate cancer xenografts with transient hematological side-effects. The tumor response correlated with the absorbed dose both on a macro and a small scale dosimetric level. Analysis of AR staining showed that AR protein levels increased late in the study suggesting a therapeutic mechanism, a feed forward mechanism coupled to AR driven response to DNA damage or clonal lineage selection, similar to that reported in high LET alpha-particle therapy using 225Ac labeled hu11B6, however emerging at a later timepoint.

Comparing Pathways of Bradykinin Formation in Whole Blood From Healthy Volunteers and Patients With Hereditary Angioedema Due to C1 Inhibitor Deficiency.

Multiple pathways have been proposed to generate bradykinin (BK)-related peptides from blood. We applied various forms of activation to fresh blood obtained from 10 healthy subjects or 10 patients with hereditary angioedema (HAE-1 or -2 only) to investigate kinin formation. An enzyme immunoassay for BK was applied to extracts of citrated blood incubated at 37°C under gentle agitation for 0-2 h in the presence of activators and/or inhibitory agents. Biologically active kinins in extracts were corroborated by c-Fos accumulation in HEK 293a cells that express either recombinant human B2 or B1 receptors (B2R, B1R). Biological evidence of HAE diagnostic and blood cell activation was also obtained. The angiotensin converting enzyme inhibitor enalaprilat, without any effect per se, increased immunoreactive BK (iBK) concentration under active stimulation of blood. Tissue kallikrein (KLK-1) and Kontact-APTT, a particulate material that activates the contact system, rapidly (5 min) and intensely (>100 ng/mL) induced similar iBK generation in the blood of control or HAE subjects. Tissue plasminogen activator (tPA) slowly (≥1 h) induced iBK generation in control blood, but more rapidly and intensely so in that of HAE patients. Effects of biotechnological inhibitors indicate that tPA recruits factor XIIa (FXIIa) and plasma kallikrein to generate iBK. KLK-1, independent of the contact system, is the only stimulus leading to an inconsistent B1R stimulation. Stimulating neutrophils or platelets did not generate iBK. In the HAE patients observed during remission, iBK formation capability coupled to B2R stimulation appears largely intact. However, a selective hypersensitivity to tPA in the blood of HAE patients suggests a role of plasmin-activated FXIIa in the development of attacks. Proposed pathways of kinin formation dependent on blood cell activation were not corroborated.

Classification analyses for prostate cancer, benign prostate hyperplasia and healthy subjects by SERS-based immunoassay of multiple tumour markers.

Prostate cancer (PCa) is a leading cause of cancer-related death among males globally. To date, prostate-specific antigen (PSA), as a typical tumour marker, has been widely used in the early diagnosis of PCa. However, in practical clinical tests, high serum levels of PSA show a high probability for false-positive results, leading to misdiagnoses. In this study, we developed a new classification system for PCa, benign prostate hyperplasia (BPH) and healthy subjects by using a surface-enhanced Raman scattering (SERS)-based immunoassay of multiple tumour markers along with a support vector machine (SVM) algorithm. Silver nanoparticles (AgNPs) as immune probes and SiC@Ag@Ag-NPs SERS as immune substrates were constructed into a sandwich structure to serve as an ultrasensitive SERS-based immunoassay platform of tumour markers. With this assay, the limits of detection for PSA, prostate-specific membrane antigen (PSMA) and human kallikrein 2 (hK2) were as low as 0.46 fg mL-1, 1.05 fg mL-1 and 0.67 fg mL-1, respectively. Furthermore, the serum levels of PSA, PSMA and hK2 in clinical samples were successfully detected using the SERS-based immunoassay platform, and correct classifications of PCa, BPH and healthy subjects were feasible with help of the linear SVM algorithm. These results demonstrate the potential for improving the diagnostic accuracy of PCa. Overall, the linear SVM classification model with multiple tumour markers exhibited good classifications of PCa, BPH and healthy subjects, with a PCa diagnostic accuracy of 70% that was significantly superior to that of the linear SVM classification model based only on the serum level of PSA (50%). Therefore, combining the SERS-based immunoassay with pattern recognition technology can allow for comprehensive analyses of the serum levels of multiple tumour markers to effectively improve the diagnostic accuracy of cancer with potential applications in point-of-care testing.

Kallikrein-Kinin System Suppresses Type I Interferon Responses: A Novel Pathway of Interferon Regulation.

The Kallikrein-Kinin System (KKS), comprised of kallikreins (klks), bradykinins (BKs) angiotensin-converting enzyme (ACE), and many other molecules, regulates a number of physiological processes, including inflammation, coagulation, angiogenesis, and control of blood pressure. In this report, we show that KKS regulates Type I IFN responses, thought to be important in lupus pathogenesis. We used CpG (TLR9 ligand), R848 (TLR7 ligand), or recombinant IFN-α to induce interferon-stimulated genes (ISGs) and proteins, and observed that this response was markedly diminished by BKs, klk1 (tissue kallikrein), or captopril (an ACE inhibitor). BKs significantly decreased the ISGs induced by TLRs in vitro and in vivo (in normal and lupus-prone mice), and in human PBMCs, especially the induction of Irf7 gene (p < 0.05), the master regulator of Type I IFNs. ISGs induced by IFN-α were also suppressed by the KKS. MHC Class I upregulation, a classic response to Type I IFNs, was reduced by BKs in murine dendritic cells (DCs). BKs decreased phosphorylation of STAT2 molecules that mediate IFN signaling. Among the secreted pro-inflammatory cytokines/chemokines analyzed (IL-6, IL12p70, and CXCL10), the strongest suppressive effect was on CXCL10, a highly Type I IFN-dependent cytokine, upon CpG stimulation, both in normal and lupus-prone DCs. klks that break down into BKs, also suppressed CpG-induced ISGs in murine DCs. Captopril, a drug that inhibits ACE and increases BK, suppressed ISGs, both in mouse DCs and human PBMCs. The effects of BK were reversed with indomethacin (compound that inhibits production of PGE2), suggesting that BK suppression of IFN responses may be mediated via prostaglandins. These results highlight a novel regulatory mechanism in which members of the KKS control the Type I IFN response and suggest a role for modulators of IFNs in the pathogenesis of lupus and interferonopathies.

Indirect Back-Titration ELISA: A New Format for Estimation of Human Tissue Kallikreins.

Enzyme-linked immunosorbent assay (ELISA) is either based on sandwich, competitive, or inhibition type of format. However, these formats need 2 or 3 monoclonal antibodies (moAB) to estimate 1 antigen. To get a cost-effective, high throughput, ELISA for estimation of human tissue kallikreins we have now developed an indirect, back-titration style, Time Resolved ImmunoFluorometric (TRIF) ELISA that uses only 1 antigen-specific moAB and a general polyclonal antibody. Polystyrene microtiter plate wells coated with a capture antibody, a mouse moAB prepared against a specific human tissue kallikrein are allowed to interact either with the corresponding pure antigen, as the calibrator, or with the corresponding antigen present in a biological fluid or tissue extract. The detection antibody, anti-mouse IgG conjugated with alkaline phosphatase, is added to find the antigen-free immobilized capture moAB. Conjugated enzyme is allowed to hydrolyze diflunisal phosphate to produce a highly fluorescent complex. The fluorescence measured in TRIF mode corresponds to the antigen-free immobilized capture moAB and is used to quantify antigen-bound capture moAB. The detection antibody binds with the antigen-free capture moAB and strength of the signal correlates inversely with the amount of antigen bound to the capture moAB. With a minimum detection level of 20 ng/L the assay has no cross-reactivity with several test molecules. The method is sensitive, specific, applicable to a variety of biological samples, and cost-effective as it uses only 1 moAB and a polyclonal antibody. Using this assay, a single epitope can be estimated without purification.

Autoantibodies contribute to the immunopathogenesis of experimental dry eye disease.

PURPOSE: The purpose of this study was to determine if autoantibodies play a role in the immunopathogenesis of experimental dry eye disease. METHODS: Dry eye was induced by exposing female C57BL/6 wild-type mice or hen egg lysozyme B-cell receptor transgenic mice to desiccating stress (subcutaneous scopolamine [0.5 mg/0.2 mL] 3 times a day, humidity < 40%, and sustained airflow) for 3 weeks, allowing sufficient time for a humoral immune response. Serum or purified IgG isolated from dry-eye mice or untreated controls was passively transferred to nude recipient mice, which were evaluated for ocular surface inflammation 3 days after transfer. To determine if complement activation contributed to serum-induced dry eye disease, cobra venom factor was used to deplete complement activity. RESULTS: Autoantibodies against kallikrein 13 were identified in serum from dry-eye mice, but were undetectable in untreated controls. Autoantibody-containing serum or purified IgG from dry-eye mice was sufficient to mediate complement-dependent ocular surface inflammation. Serum or purified IgG caused marked inflammatory burden and tissue damage within the ocular surface tissues, including elevated Gr1+ neutrophil infiltration and proinflammatory cytokines/chemokines associated with goblet cell loss. Moreover, complement C3b deposition was found within the ocular surface tissues of mice receiving dry-eye serum, but not in recipients of control serum. Functionally, complement depletion attenuated the ability to transfer dry-eye-specific serum or IgG-mediated disease. CONCLUSIONS: These data demonstrate for the first time a complement-dependent pathogenic role of dry-eye-specific autoantibodies, and suggest autoantibody deposition within the ocular surface tissues contributes to the predominantly T-cell-mediated immunopathogenesis of dry eye disease.

Determination of kininogens levels and kallikrein/kininase II activities in patients with thromboangiitis obliterans.

Some components of the kinin system such as plasma kallikrein levels, the activities of tissue kallikrein (including saliva) and kininase II and the concentrations of kininogen fractions (low-molecular weight/LKg and high-molecular weight/HKg) were evaluated in the plasma of patients with thromboangiitis obliterans (TAO) presenting clinical symptoms of the condition. Twenty TAO were diagnosed by means of the traditional Shionoya and Olin criteria and later classified into non-smokers (n = 11) and active smokers (n = 9). Fifty-three normal, non-smoking/smoking individuals (control) were also studied. Kininogen levels were determined by ELISA; the activities of kallikreins and kininase II were determined using selective substrates. The levels of enzymes (kallikreins and kininase II) and protein (kininogens) were significantly higher in patients with TAO who were active smokers compared to the control groups (no matter whether control individuals were active smokers or non-smokers, P < 0.001 for all comparisons). Interestingly, regardless of the time of disease onset, a significant increase in the levels of these components of the kinin system was also observed in patients when TAO active smokers were compared with TAO ex-smokers (P < 0.01 for all analysed parameters). Activation of the kinin system in patients with TAO may indicate the involvement of vasodilatation in an attempt to control vascular changes, thereby favouring the deposition of immune complexes at the vascular level because of nicotine stimulation. Moreover, our results corroborate the idea that TAO can be an autoimmune disorder with specific mechanisms.

Specific inhibition of tissue kallikrein 1 with a human monoclonal antibody reveals a potential role in airway diseases.

KLK1 (tissue kallikrein 1) is a member of the tissue kallikrein family of serine proteases and is the primary kinin-generating enzyme in human airways. DX-2300 is a fully human antibody that inhibits KLK1 via a competitive inhibition mechanism (Ki=0.13 nM). No binding of DX-2300 to KLK1 was observed in a surface-plasmon-resonance biosensor assay when KLK1 was complexed to known active-site inhibitors, suggesting that DX-2300 recognizes the KLK1 active site. DX-2300 did not inhibit any of the 21 serine proteases that were each tested at a concentration of 1 microM. We validated the use of DX-2300 for specific KLK1 inhibition by measuring the inhibition of KLK1-like activity in human urine, saliva and bronchoalveolar lavage fluid, which are known to contain active KLK1. In human tracheobronchial epithelial cells grown at the air/liquid interface, DX-2300 blocked oxidative-stress-induced epidermal-growth-factor receptor activation and downstream mucus cell proliferation and hypersecretion, which have been previously shown to be mediated by KLK1. In an allergic sheep model of asthma, DX-2300 inhibited both allergen-induced late-phase bronchoconstriction and airway hyper-responsiveness to carbachol. These studies demonstrate that DX-2300 is a potent and specific inhibitor of KLK1 that is efficacious in in vitro and in vivo models of airway disease.

The lupus-susceptibility gene kallikrein downmodulates antibody-mediated glomerulonephritis.

Sle3 is a NZM2410/NZW-derived lupus-susceptibility interval on murine chromosome 7, which is associated with spontaneous lupus nephritis (SLN), and also anti-GBM-induced glomerulonephritis (GN). The tissue kallikrein gene cluster is located within the Sle3 interval and constitutes potential candidate genes for this locus. We have recently reported that renal kallikrein expression was upregulated by anti-GBM antibody challenge in a strain-specific manner and that it was significantly underexpressed in the anti-GBM-sensitive strains, including B6.Sle3. Further sequencing and functional studies reported earlier provided evidence that kallikreins could constitute disease genes in lupus. In this report, we have used an adenoviral vector to deliver the klk1 gene to B6.Sle3 congenics to directly test if kallikreins might have a protective effect against anti-GBM-induced nephritis. Our data show that klk1 gene delivery ameliorated anti-GBM-induced nephritis in B6.Sle3 congenics. Taken together with earlier studies, these findings indicate that kallikreins play an important protective role in autoantibody-initiated GN and could constitute potential candidate genes for anti-GBM-induced GN and SLN.

Prostatic kallikrein: a new major dog allergen.

BACKGROUND: Dog dander is an important cause of respiratory allergy, but the spectrum of known dog allergens appears incomplete. Two lipocalins, Can f 1 and Can f 2, and serum albumin, Can f 3, have been characterized in detail but do not fully account for the IgE antibody-binding activity of dog dander extract. Allergen activity has previously been detected in dog urine but not further characterized. OBJECTIVE: We sought to identify, characterize, and assess the importance of allergen components in dog urine. METHODS: Dog urine was fractionated by means of size exclusion chromatography and examined for IgE antibody binding. A protein present in one fraction displaying IgE antibody-binding activity was identified by means of N-terminal sequencing and mass spectrometry. A recombinant form of the protein was produced in Pichia pastoris. IgE antibody binding to dog allergen components among sera of 37 subjects with dog allergy was determined by means of ImmunoCAP analysis. RESULTS: An IgE antibody-binding protein was isolated from dog urine and identified as prostatic kallikrein. A closely related or identical protein was detected in dog dander. The recombinant prostatic kallikrein displayed immunologic and biochemical properties similar to those of the natural protein and bound IgE antibodies from 26 (70%) of 37 sera of subjects with dog allergy, 14 of which reacted to none of Can f 1, Can f 2, or Can f 3. The dog allergen identified here was found to cross-react with human prostate-specific antigen, a key culprit in IgE-mediated vaginal reactions to semen. CONCLUSION: Prostatic kallikrein is a new major dog allergen.

Intact free prostate-specific antigen and free and total human glandular kallikrein 2. Elimination of assay interference by enzymatic digestion of antibodies to F(ab')2 fragments.

Various blood constituents can interfere with immunoassays, usually by binding the Fc portion of antibodies. Our previously developed assays for intact free prostate-specific antigen (PSA), free human kallikrein 2 (hK2), and total hK2 frequently yielded falsely high results despite including an excess of scavenger antibodies. We investigated whether this interference could be eliminated by replacing monoclonal capture or tracer antibodies with F(ab')2 or recombinant Fab fragments. Female heparin plasma samples (n = 1092), which should have negligible PSA and hK2, and male samples (n = 957) were analyzed to identify samples manifesting interference, which then were used to optimize protocols for the immunoassays. We compared original assays (monoclonal antibodies) versus optimized assays (F(ab')2 fragments: denatured mouse IgG added as scavenger) using another set of EDTA plasma (n = 113), heparin plasma (n = 160), and serum samples (n = 171). With the original assays, the frequency of falsely elevated hK2 and intact free PSA was 15 and 13%, respectively. The optimized assays eliminated 70-85% of these falsely elevated results and importantly reduced the magnitude in the remainder. F(ab')2 fragmentation was the most important factor in reducing interference. The optimized intact free PSA, free hK2, and total hK2 assays manifested high accuracy close to the lower limit of detection.

Potential scope of action of tissue kallikreins in CNS immune-mediated disease.

The objective of this study was to define the potential scope of action of tissue kallikreins in T cell-mediated disease of the CNS. We demonstrate quantitatively the differential expression of all 15 human tissue kallikreins within brain, spinal cord and immune compartments. In human Jurkat T cells we demonstrate differential regulation of select kallikreins by CD3 receptor, Concanavilin A (Con A), interleukin 2 (IL2), and lipopolysaccharide (LPS)-mediated activation and by exposure to steroid hormones, dexamethasone, norgestrel, androstan and estradiol. The patterns of co-expression and co-regulation described point to novel effector roles for select tissue kallikreins in neurological disorders involving T cells, such as multiple sclerosis.

High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays.

A method was developed to rapidly identify high-affinity human antibodies from phage display library selection outputs. It combines high-throughput Fab fragment expression and purification with surface plasmon resonance (SPR) microarrays to determine kinetic constants (kon and koff) for 96 different Fab fragments in a single experiment. Fabs against human tissue kallikrein 1 (hK1, KLK1 gene product) were discovered by phage display, expressed in Escherichia coli in batches of 96, and purified using protein A PhyTip columns. Kinetic constants were obtained for 191 unique anti-hK1 Fabs using the Flexchip SPR microarray device. The highest affinity Fabs discovered had dissociation constants of less than 1 nM. The described SPR method was also used to categorize Fabs according to their ability to recognize an apparent active site epitope. The ability to rapidly determine the affinities of hundreds of antibodies significantly accelerates the discovery of high-affinity antibody leads.

The antiangiogenic tissue kallikrein pattern of endothelial cells in systemic sclerosis.

OBJECTIVE: Postnatal angiogenesis relies on a proper response of endothelial cells to angiogenic stimuli. In systemic sclerosis (SSc), endothelial cells are unresponsive to angiogenic factors. Since circumstantial and experimental evidence points to tissue kallikreins as powerful effectors of the angiogenic response, we undertook this study to investigate the kallikrein pattern of normal and SSc endothelial cells in order to identify differences that can account for defective angiogenesis. METHODS: Expression of 14 tissue kallikreins was studied by a microarray approach, by reverse transcription-polymerase chain reaction, and by Western blotting in endothelial cells isolated from the skin of clinically healthy subjects and SSc patients. Cell proliferation was quantified by direct cell counting. Invasion and capillary morphogenesis were evaluated in a Boyden chamber and in culture flasks layered with Matrigel. Cyclic nucleotide production was measured by enzyme immunoassay. MAP kinase and ERK activation were measured by Western blotting. RESULTS: Endothelial cells from SSc patients showed poor expression of kallikreins 9, 11, and 12 compared with endothelial cells from normal subjects. Antibodies against the relevant kallikreins on normal endothelial cells revealed that while kallikreins 9, 11, and 12 induced cell growth, only kallikrein 12 regulated invasion and capillary morphogenesis. Buffering of kallikrein 12 with antibodies resulted in the acquisition of an SSc-like pattern by normal cells in in vitro angiogenesis. Reduction of cAMP and cGMP production and of ERK phosphorylation upon administration of antikallikrein antibodies revealed that the activity of kallikreins 9, 11, and 12 was mediated by kinins. CONCLUSION: Reduction of tissue kallikreins 9, 11, and 12 may be relevant to reduced angiogenesis in SSc patients.

Autoimmunity against a tissue kallikrein in IQI/Jic Mice: a model for Sjogren's syndrome.

We have recently characterized IQI/Jic mice as a model for Sjogren's syndrome (SS), a chronic autoimmune disease in humans. In SS, local lymphocytic infiltrations into salivary and lacrimal glands frequently develop to the involvement of systemic exocrine and nonexocrine organs, and the mechanism for progression of this disease remains obscure. Herein, we report identification of an autoantigen shared by various target organs in IQI/Jic mice. Polypeptides identified based on immunorecognition by autoantibodies in sera from IQI/Jic mice affected with autoimmune disease (>12 weeks of age) were tissue kallikrein (Klk)-1 and -13 and were cross-reactive to the autoantibodies. Interestingly, Klk-13, but not Klk-1, caused a proliferative response of splenic T cells from IQI/Jic mice from the age of 4 weeks onward. In addition, remarkably enhanced expression of Klk-13 was observed in the salivary glands of the mice in accordance with the development of inflammatory lesions. These results indicate that Klk-13 acts as an autoantigen and may increase T cells responsive to organs commonly expressing Klk-13, playing a pivotal role in the etiology of progression of disease in IQI/Jic mice. Our findings provide insights into the contributions of autoantigens shared by multiple organs in the progress of SS from an organ-specific to a systemic disorder.

Development of sensitive immunoassays for free and total human glandular kallikrein 2.

BACKGROUND: Free and total human kallikrein 2 (hK2) might improve the discrimination between prostate cancer and benign prostatic hyperplasia. Concentrations of hK2 are 100-fold lower than concentrations of prostate-specific antigen (PSA); therefore, an hK2 assay must have a low detection limit and good specificity. METHODS: PSA- and hK2-specific monoclonal antibodies were used in solid-phase, two-site immunofluorometric assays to detect free and total hK2. The total hK2 assay used PSA-specific antibodies to block nonspecific signal. The capture antibody of the free hK2 assay did not cross-react with PSA. To determine the hK2 concentrations in the male bloodstream, total hK2 was measured in a control group consisting of 426 noncharacterized serum samples. Free and total hK2 were measured in plasma from 103 patients with confirmed prostate cancer. RESULTS: All 426 males in the control group had a total hK2 concentration above the detection limit of 0.0008 microg/L. The median total hK2 concentration was 0.022 microg/L (range, 0.0015-0.37 microg/L). hK2 concentrations were 0.1-58% of total PSA (median, 3.6%). hK2 concentrations were similar in men 41-50 and 51-60 years of age. The ratio of hK2 to PSA steadily decreased from 5-30% at PSA <1 microg/L to 1-2% at higher PSA concentrations. In 103 patients with prostate cancer, the median hK2 concentration in plasma was 0.079 microg/L (range, 0.0015-16.2 microg/L). The median free hK2 concentration was 0.070 (range, 0.005-12.2) microg/L. The proportion of free to total hK2 varied from 17% to 131% (mean, 85%). CONCLUSIONS: The wide variation in the free-to-total hK2 ratio suggests that hK2 in blood plasma is not consistently in the free, noncomplexed form in patients with prostate cancer. The new assay is sufficiently sensitive to be used to study the diagnostic accuracies of free and total hK2 for prostate cancer.

Generation of monoclonal antibodies specific for human kallikrein 2 (hK2) using hK2-expressing tumors.

BACKGROUND: Human kallikrein 2 (hK2) and prostate-specific antigen (PSA) are serine proteases in the human kallikrein gene family that are 80% identical at the protein level. Like PSA, hK2 is expressed primarily in the prostate, making it an attractive bio-marker for prostate cancer development. In addition, its potent enzymatic activity may functionally affect the biology of prostate cancer. In order to further elucidate the possible roles of hK2 in prostate cancer, we have generated a panel of hK2-specific, non-PSA cross-reactive monoclonal antibodies. METHODS: A novel tumor-immunization strategy was used to produce monoclonal antibodies. Human hK2 cDNA was transfected into a BALB/c tumor cell line and used to immunize both BALB/c and PSA-expressing BALB/c.PSA transgenic mice. Because the BALB/c.PSA transgenic mouse showed a biased response towards hK2, a B cell fusion was performed using spleen cells from a transgenic mouse immunized in this fashion. RESULTS: A panel of monoclonal antibodies was produced and shown to be hK2-specific using newly developed hK2-specific sandwich ELISA and ELIspot assays. One of the monoclonal antibodies (6B7) was used to detect hK2 in human prostate by immunohistochemistry. Interestingly, two of the antibodies affected the function of hK2. The 1F8 antibody enhanced the enzymatic activity of hK2 whereas the 3C7 antibody inhibited its function. CONCLUSIONS: These hK2-specific antibodies illustrate a novel approach for constructing B-cell hybridomas and provide useful reagents to examine the role of hK2 in the biology and detection of prostate cancer.

Development of a dual monoclonal antibody immunoassay for total human kallikrein 2.

BACKGROUND: Human kallikrein 2 (hK2) shares 80% sequence identity with prostate-specific antigen (PSA). Because both hK2 and hK2-alpha(1)-antichymotrypsin (hK2-ACT) complexes have been identified in patient sera, we devised an immunoassay for total hK2 [(thK2); hK2 and hK2-ACT] and evaluated it in healthy subjects and patients with prostate disease. METHODS: We developed monoclonal antibodies (mAbs) with high specificity for hK2 and hK2-ACT and minimal cross-reactivity to PSA. Using these mAbs, a sandwich assay was developed and its specificity for forms of hK2 was assessed. Serum samples (n = 1035) from healthy volunteers, patients with increased PSA, and men who had undergone radical prostatectomy were assayed for thK2. We also measured thK2 in samples before and after storage under common laboratory conditions. RESULTS: The minimum detectable concentration in the thK2 assay was 0.008 microg/L, and PSA cross-reactivity was <0.001%. The assay detected prohK2 and three different hK2-serum protease complexes. The median serum concentration of thK2 in control samples (0.013 microg/L) was significantly lower than the median in samples from patients with increased PSA concentrations (0.085 microg/L). Immunoreactive hK2 changed little in samples stored for up to 1 month at -70 degrees C. CONCLUSIONS: The thK2 assay recognizes all forms of hK2 that have been found in bodily fluids to date.

Expression of tissue kallikrein and kinin receptors in angiogenic microvascular endothelial cells.

Angiogenesis is the sprouting of new capillary blood vessels from pre-existing ones. The kinin family of vasoactive peptides, formed by the serine protease tissue kallikrein from its endogenous multifunctional protein substrate kininogen, is believed to regulate the angiogenic process. The aim of this study was to determine the expression of tissue kallikrein and kinin receptors in an in vitro model of angiogenesis. Microvascular endothelial cells from the bovine mature and regressing corpus luteum were used only if they reacted with known endothelial cell markers. At first the cultured endothelial cells began sprouting, and within four weeks formed three-dimensional, capillary-like structures. Immunolabelling for tissue prokallikrein and the mature enzyme was intense in the angiogenic endothelial cells derived from mature corpora lutea. Immunoreactivity was lower in non-angiogenic endothelial cells and least in angiogenic endothelial cultures of the regressing corpus luteum. Additionally, using specific antisense DIG-labelled probes, tissue kallikrein mRNA was demonstrated in cells of the angiogenic phenotype. Immunolabelled kinin B2 receptors, but not kinin B1 receptors, were visualised on angiogenic endothelial cells. Our results suggest an important regulatory role for kinins in the multiple steps of the angiogenic cascade that may occur in wound healing and cancer cell growth.

Reactivity of anti-PSA monoclonal antibodies with recombinant human kallikrein-2.

Seventy-nine monoclonal antibodies submitted to the ISOBM TD-3 PSA Workshop were tested for their reactivity with recombinant human kallikrein-2 (rhK2). A sandwich immunofluorometric assay using polyclonal anti-prostate-specific antigen (PSA) antiserum-coated plates was used to capture rhK2 and subsequently the test antibody. The response of each test antibody was compared with 3 reference antibodies (H50, H117 and 5E4) known to react with hK2. Nine antibodies from the workshop panel failed to react with purified PSA and rhK2 in this assay and were subsequently excluded. From the remaining panel of antibodies, 11/70 showed strong reactivity with rhK2, 9/70 showed weak reactivity with rhK2, while 50/70 antibodies did not react with rhK2 in this assay format. All antibodies binding to rhK2 recognized both free and complexed PSA.