METHODS FOR DETERMINING PEPTIDYLGLYCINE ALPHA-AMIDATING MONOOXYGENASE (PAM) AND ITS USE FOR DIAGNOSTIC PURPOSE
20230097988 · 2023-03-30
Assignee
Inventors
Cpc classification
International classification
Abstract
The present invention is directed to a method for diagnosis or prognosis of a disease in a subject and/or predicting a risk of getting a disease or adverse event in a subject and/or monitoring a disease or adverse event in a subject by determining the level of peptidylglycine alpha-amidating monooxygenase (PAM) and/or its isoforms and/or fragments thereof in a sample of bodily fluid of said subject.
Claims
1. A method for diagnosis or prognosis of a disease in a patient and/or predicting a risk of getting a disease or an adverse event in a patient and/or monitoring a disease or an adverse event in a patient, comprising: determining the level of peptidylglycine alpha-amidating monooxygenase (PAM) and/or its isoforms and/or fragments thereof in a sample of bodily fluid of said patient, wherein the disease in said patient is selected from dementia, cardiovascular disorders, kidney diseases, cancer, inflammatory or infectious diseases and/or metabolic diseases, and wherein the adverse event is selected from a cardiac event, a cardiovascular event, a cerebrovascular event, a cancer, diabetes, infections, serious infections, sepsis-like systemic infections, sepsis and death due to all causes.
2. A method for diagnosis or prognosis of a disease in a patient and/or predicting a risk of getting a disease or an adverse event in a patient and/or monitoring a disease or adverse event in a patient by determining the level of peptidylglycine alpha-amidating monooxygenase (PAM) and/or its isoforms and/or fragments thereof in a sample of bodily fluid of said patient, the method comprising: determining the level of PAM and/or its isoforms and/or fragments thereof in a sample of bodily fluid of said patient, and comparing said determined amount to a predetermined threshold, wherein said patient is diagnosed as having a disease if said determined amount is below or above said predetermined threshold, or wherein an outcome of a disease is prognosticated if said determined amount is below or above said predetermined threshold, or wherein the risk of getting a disease or an adverse event is predicted in said patient if said determined amount is below or above said predetermined threshold, or wherein a disease or an adverse event of said patient is monitored.
3. A method according to claim 1, wherein the level of PAM and/or its isoforms and/or fragments thereof is the total concentration of PAM and/or its isoforms and/or fragments thereof having at least 12 amino acids or the activity of PAM and/or its isoforms and/or fragments thereof in said sample of bodily fluid of said patient.
4. A method according to claim 3, wherein the activity of PAM and/or its isoforms and/or fragments thereof is selected from the sequences SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 10.
5. A method according to claim 3, wherein the total concentration of PAM and/or its isoforms and/or fragments thereof having at least 12 amino acids is detected with an immunoassay.
6. A method according to claim 3, wherein the activity of PAM and/or its isoforms and/or fragments thereof is detected using a peptide-Gly as substrate.
7. A method according to claim 6, wherein the peptide-Gly substrate is selected from adrenomedullin (ADM), adrenomedullin-2, intermedin-short, pro-adrenomedullin N-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide gamma, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropin-releasing hormone (TRH), oxytocin and vasopressin.
8. A method for diagnosis or prognosis of a disease in a patient and/or predicting a risk of getting a disease or adverse event in a patient and/or monitoring a disease or adverse event in a patient by determining the level of PAM and/or its isoforms and/or fragments thereof in a sample of bodily fluid of said patient according to claim 1, wherein the PAM and/or its isoforms and/or fragments thereof is selected from the group comprising SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 10.
9. A method for diagnosis or prognosis of a disease in a patient and/or predicting a risk of getting a disease or adverse event in a patient and/or monitoring a disease or adverse event in a patient, comprising: determining the level of PAM and/or its isoforms and/or fragments thereof in a sample of bodily fluid of said patient according to claim 1, wherein the risk of getting a disease of a patient is determined, wherein said patient is a healthy patient.
10. A method according to claim 9, wherein said disease is selected from Alzheimer's disease, colorectal cancer and pancreatic cancer.
11. A method for determining the level of PAM and/or isoforms and/or fragments thereof in a bodily fluid sample using an assay, wherein said assay comprises two binders that bind to two different regions of PAM, wherein the two binders are directed to an epitope of at least 5 amino acids, preferably at least 4 amino acids in length, wherein said two binders are directed to an epitope comprised within the following sequences of PAM: peptide 1 (SEQ ID No. 11), peptide 2 (SEQ ID No. 12), peptide (SEQ ID No. 13), peptide 4 (SEQ ID No. 14), peptide 5 (SEQ ID No. 15), peptide 6 (SEQ ID No. 16), peptide 7 (SEQ ID No. 17), peptide 8 (SEQ ID No. 18), peptide 9 (SEQ ID No. 19), peptide 10 (SEQ ID No. 20), peptide 11 (SEQ ID No. 21), peptide 12 (SEQ ID No. 22), peptide 13 (SEQ ID No. 23) peptide 14 (SEQ ID No. 24) and recombinant PAM (SEQ ID No. 10).
12. A method for determining the activity of PAM and/or isoforms or fragments thereof in a bodily fluid sample of a patient comprising: contacting said sample with a capture-binder that binds specifically to active full-length PAM, its isoforms and/or active fragments thereof, separating PAM bound to said capture-binder, adding a substrate of PAM to said separated PAM, and quantifying PAM activity by measuring the conversion of the substrate of PAM.
13. A method for determining the activity of PAM and/or isoforms and/or fragments thereof in a bodily fluid sample of a patient, comprising: contacting said sample with a substrate (peptide-Gly) of PAM for an interval of time at t=0 min and t=n+1 min, detecting the reaction product (alpha-amidated peptide) of PAM in said sample at t=0 min and t=n+1 min, and quantifying the activity of PAM by calculating the difference of the reaction product between t=0 and t=n+1.
14. A method according to claim 13, wherein the peptide-Gly substrate is selected from adrenomedullin (ADM), adrenomedullin-2, intermedin-short, pro-adrenomedullin N-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide gamma, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropin-releasing hormone (TRH), oxytocin and vasopressin.
15. (canceled)
16. A kit for the determination of the level of PAM and/or its isoforms and/or fragments thereof, comprising one or more antibodies binding to PAM sequences selected from the group comprising recombinant PAM (SEQ ID No. 10), peptide 1 (SEQ ID No. 11), peptide 2 (SEQ ID No. 12), peptide (SEQ ID No. 13), peptide 4 (SEQ ID No. 14), peptide 5 (SEQ ID No. 15), peptide 6 (SEQ ID No. 16), peptide 7 (SEQ ID No. 17), peptide 8 (SEQ ID No. 18), peptide 9 (SEQ ID No. 19), peptide 10 (SEQ ID No. 20), peptide 11 (SEQ ID No. 21), peptide 12 (SEQ ID No. 22), peptide 13 (SEQ ID No. 23) and peptide 14 (SEQ ID No. 24.
17. A method according to claim 2, wherein the level of PAM and/or its isoforms and/or fragments thereof is the total concentration of PAM and/or its isoforms and/or fragments thereof having at least 12 amino acids or the activity of PAM and/or its isoforms and/or fragments thereof in said sample of bodily fluid of said patient.
18. A method according to claim 17, wherein the activity of PAM and/or its isoforms and/or fragments thereof is selected from the sequences SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 10.
19. A method according to claim 17, wherein the total concentration of PAM and/or its isoforms and/or fragments thereof having at least 12 amino acids is detected with an immunoassay.
20. A method according to claim 17, wherein the activity of PAM and/or its isoforms and/or fragments thereof is detected using a peptide-Gly as substrate.
21. A method according to claim 20, wherein the peptide-Gly substrate is selected from adrenomedullin (ADM), adrenomedullin-2, intermedin-short, pro-adrenomedullin N-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide gamma, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropin-releasing hormone (TRH), oxytocin and vasopressin.
Description
FIGURE DESCRIPTION
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EXAMPLES
Example 1— Production of Recombinant PAM
[0211] PAM cDNA was synthesized according to Uniprot Accession No. P19021 encoding amino acids 21-834 of the PAM protein involving codon optimization for expression in mammalian cells. The signal sequence of PAM was replaced with human serum albumin signal sequence (MKWVTFISLLFLFSSAYSFR [SEQ ID No. 9]). At the C-terminus of PAM a hexa-histidine tag was added linked via a GS linker to PAM. The sequence of recombinant PAM (amino acids 21-834 of PAM without signal sequence and hexa-histidine tag) is shown in SEQ ID No. 10. The cDNA was cloned into an expression vector (plasmid DNA) using a 5′-NotI and a 3′ HindIII restriction site. The expression vector harboring the cDNA for PAM expression was replicated in- and prepared from E. coli. as a low-endotoxin preparation.
[0212] HEK-INV cells were transfected with the expression vector using INVect transfection reagents in serum free suspension culture. The transfection rate was controlled via co-transfection with a GFP-(green fluorescent protein) containing expression vector. Cultivation of cells was carried out in presence of valproic acid and Penicillin-Streptomycin at 37° C. and 5% CO.sub.2. Cells were harvested via centrifugation when viability reached <60% (>2000 g, 30-45 min, 2-8° C.). Cell culture supernatant (CCS) was washed 5 times with 100 mM Tris/HCL pH 8.0 via tangential flow filtration (TFF, 30 kDa cut-off).
[0213] Purification of recombinant PAM included application of buffer exchanged CCS on a Q-sepharose fast flow resin (GE Healthcare) with a NaCl gradient (up to 2 M) elution. Amidating activity containing fractions were pooled and applied onto a Superdex 200 pg (GE Healthcare) size exclusion chromatography column with a 100 mM Tris/HCl, 200 mM NaCl, pH8.0 elution buffer. Amidating activity containing fractions were pooled, dialyzed against 100 mM Tris HCl, 200 mM NaCl, pH 8.0, sterile filtered (0.2 μm). Endotoxin load was determined by Charles River PTS Endosafe system and was below 5 EU/mL.
Example 2—Production of Antibodies
[0214] Anti-PAM antibodies according to the present invention may be synthesised as follows:
[0215] PAM peptides for immunization were synthesized, see Table 1, (Peptides & Elephants, Hennigsdorf, Germany) with an additional C-terminal cysteine (if no cysteine is present within the selected PAM-sequence) residue for conjugation of the peptides to Bovine Serum Albumin (BSA). The peptides were covalently linked to BSA by using Sulfolink-coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the manual of Perbio. Recombinant PAM was produced by InVivo Biotech Services, Hennigsdorf, as described in example 1.
TABLE-US-00001 TABLE 1 PAM immunization peptides Name (amino acid position*) Sequence Peptide 1 (aa 42-56) (SEQ ID No. 11) CLGTTRPVVPIDSSD Peptide 2 (aa 109-128) (SEQ ID No. 12) CNMPSSTGSYWFCDEGTCTD Peptide 3 (aa 168-180) (SEQ ID No. 13) YGDISAFRDNNKD Peptide 4 (aa 204-216) (SEQ ID No. 14) SVDTVIPAGEKVV Peptide 5 (aa 329-342) (SEQ ID No. 15) CTQNVAPDMFRTIP Peptide 6 (aa 291-310) (SEQ ID No. 16) TGEGRTEATHIGGTSSDEMC Peptide 7 (aa 234-244) (SEQ ID No. 17) YRVHTHHLGKV Peptide 8 (aa 261-276) (SEQ ID No. 18) QSPQLPQAFYPVGHPV Peptide 9 (aa 530-557) (SEQ ID No. 19) RGDHVWDGNSFDSKFVYQQIGLGPIEED Peptide 10 (aa 611-631) (SEQ ID No. 20) EGPVLILGRSMQPGSDQNHFC Peptide 11 (aa 562-579 (SEQ ID No. 21) IDPNNAAVLQSSGKNLFY Peptide 12 (aa 745-758) (SEQ ID No. 22) NGKPHFGDQEPVQG Peptide 13 (aa 669-687) (SEQ ID No. 23) WGEESSGSSPLPGQFTVPH Peptide 14 (aa 710-725) (SEQ ID No. 24) CFKTDTKEFVREIKHS Recombinant PAM SEQ ID No. 10 *according to SEQ ID No. 1; amino acid (aa)
[0216] Balb/c mice were intraperitoneally (i.p.) injected with 100 μg recombinant PAM or 100 μg PAM-peptide-BSA-conjugates at day 0 (emulsified in TiterMax Gold Adjuvant), 100 μg and 100 μg at day 14 (emulsified in complete Freund's adjuvant) and 50 μg and 50 μg at day 21 and 28 (in incomplete Freund's adjuvant). The animal received an intravenous (i.v.) injection of 50 μg recombinant PAM at day 40 or 50 μg PAM-peptide-BSA-conjugates dissolved in saline at day 45. Three days later the mice were sacrificed and the immune cell fusion was performed.
[0217] Splenocytes from the immunized mice and cells of the myeloma cell line SP2/0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37° C. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium (RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT-Supplement). After one week, the HAT medium was replaced with HT Medium for three passages followed by returning to the normal cell culture medium.
[0218] The cell culture supernatants were primarily screened for recombinant PAM binding IgG antibodies two weeks after fusion. Therefore, recombinant PAM (SEQ ID No. 10) was immobilized in 96-well plates (100 μg/well) and incubated with 50 μl cell culture supernatant per well for 2 hours at room temperature. After washing of the plate, 50 μl/well POD-rabbit anti mouse IgG was added and incubated for 1 h at RT.
[0219] After a next washing step, 50 μl of a chromogen solution (3.7 mM o-phenylene-diamine in citrate/hydrogen phosphate buffer, 0.012% H.sub.2O.sub.2) were added to each well, incubated for 15 minutes at RT and the chromogenic reaction stopped by the addition of 50 μl 4N sulfuric acid. Absorption was detected at 490 mm.
[0220] The positive tested microcultures were transferred into 24-well plates for propagation. After retesting the selected cultures were cloned and re-cloned using the limiting-dilution technique and the isotypes were determined.
[0221] Antibodies raised against recombinant human PAM or PAM-peptides were produced via standard antibody production methods (Marx et al. 1997) and purified via Protein A. The antibody purities were >90% based on SDS gel electrophoresis analysis.
Example 3—PAM Activity Assay
[0222] Human serum or Li-Heparin plasma from self-reported healthy volunteers was used as source of human native PAM. Each sample (200) was diluted two-fold in 100 mM Tris-HCl in duplicate. The amidation reaction was initiated by addition of 160 μl of PAM-reaction buffer (100 mM Tris-HCl, pH 7.5, 6.25 μM CuSO.sub.4, 2.5 mM L-ascorbate, 125 μg/mL catalase, 62.5 μM amastatin, 250 μM leupeptin, 36 μg/mL synthetic ADM-Gly and 375 μg/mL NT-ADM antibody). Afterwards, 100 μl of each individual reaction of duplicated samples were combined and transferred into 20 μl of 200 mM EDTA to terminate the amidation reaction and to generate t=0 minutes reaction time-point followed by incubation at 37° C. for 40 minutes. Afterwards the non-terminated reactions were stopped with 10 μl of 200 mM EDTA. To determine the PAM activity, bio-ADM as reaction product was quantified in each sample using the Sphingotest® bio-ADM immunoassay (Weber et al. 2017). The amidation assay was calibrated using a 6-point calibration curve generated with human recombinant PAM of known activity. Samples and calibrators were treated in the same manner. Relative light units (RLU t40 min-t0 min) determined via Sphingotest® bio-ADM immunoassay for each sample were fitted against the RLU (t40 min-t0 min) of the calibrator to determine the PAM activity in the samples. PAM activity is described as “adrenomedullin maturation activity” (AMA) in μg bio-ADM formed per hour and L of sample.
[0223] A typical PAM calibration curve is shown in
Example 4 PAM Immunoassays
[0224] Antibodies against recombinant PAM (SEQ ID No. 10) and PAM peptides (SEQ ID No. 11 to 24) were raised as described in example 1.
[0225] The technology used was a sandwich luminescence immunoassay, based on Akridinium ester labelling.
[0226] 4.1. Labelled Compound (Tracer)
[0227] Purified antibodies (0.2 g/L) were labelled by incubation in 10% labelling buffer (500 mmol/L sodium phosphate, pH 8.0) with 1:5 mol/L ratio of MACN-acridinium-NHS-ester (1 g/L, InVent GmbH) for 20 min at 22° C. After adding 5% 1 mol/L Tris-HCl, pH 8.0, for 10 min, the respective antibody was separated from free label via CentriPure P10 columns (emp Biotech GmbH). The purified labelled antibody was diluted in 300 mmol/1 potassium phosphate, 100 mmol/1 NaCl, 10 mmol/1 Na-EDTA, 5 g/l Bovine Serum Albumin (pH 7.0). The final concentration was approximately 20 μg of labelled antibody per 150 μL.
[0228] 4.2. Solid Phase
[0229] White polystyrene microtiter plates (Greiner Bio-One International AG) were coated (18 h at 20° C.) with the respective antibody (2 μg/0.2 mL per well 50 mmol/L Tris-HCl, 100 mmol/L NaCl, pH 7.8). After blocking with 30 g/L Karion, 5 g/L BSA (protease free), 6.5 mmol/L monopotassium phosphate, 3.5 mmol/L sodium dihydrogen phosphate (pH 6.5), the plates were vacuum-dried.
[0230] 4.3 Calibration
[0231] The assay was calibrated, using dilutions of recombinant PAM as described in Example 1. The typical concentration range was within of 5-5,000 μg/mL.
[0232] 4.4. Pam Immunoassays:
[0233] 4.4.1. PAM-LIA
[0234] One-Step version: 50 μL, of samples/calibrators were pipetted into pre-coated microtiter plates. After adding 200 μL, of labelled antibody in buffer (300 mmol/L potassium phosphate, 100 mmol/L NaCl, 10 mmol/L Na-EDTA, 50 μmol/L amastatin, 100 μmol/L leupeptin, 0.1% bovine IgG, 0.02% mouse IgG, 0.5% BSA, pH 7.0), the microtiter plates were incubated for 20 h at 2-8° C. under agitation at 600 rpm. Unbound tracer was removed by washing 5 times (each 350 μL, per well) with washing solution (20 mmol/L PBS, 1 g/L Triton X-100, pH 7.4). Wellbound chemiluminescence was measured for 1 s per well by using the Centro LB 960 microtiter plate luminescence reader (Berthold Technologies).
[0235] Two-Step version: 50 μL of samples/calibrators were pipetted into pre-coated microtiter plates. After adding 200 μL of buffer (as described in one-step version), the microtiter plates were incubated for 15-20 h at 2-8° C. under agitation at 600 rpm. Unbound sample was removed by washing 4 times (each 350 μL per well) with washing solution with subsequent addition of 200 μl of tracer material and incubation of microtiter plates at room temperature for 2 h. Unbound tracer was removed by washing 4 times (each 350 μL per well) with washing solution. Well-bound chemiluminescence was measured for 1 s per well by using the Centro LB 960 microtiter plate luminescence reader (Berthold Technologies).
[0236] Results: Antibodies bound to the solid phase and labelled antibodies directed to the different PAM immunization peptides as well as full-length (recombinant) PAM (see example 2) were tested with recombinant PAM as well as blood samples. Exemplary standard curves for different antibody combinations are shown in
[0237] 4.4.2. Enzyme Capture Assay (ECA) for the Detection of PAM Activity
[0238] Enzyme capture assays were established to detect the activity of PAM. 50 μL of samples/calibrators were pipetted into pre-coated microtiter plates (as described in 4.2.). After adding 200 μL of buffer (300 mmol/L potassium phosphate, 100 mmol/L NaCl, 50 μmol/L amastatin, 100 μmol/L leupeptin, 0.1% bovine IgG, 0.02% mouse IgG, 0.5% BSA, pH 7.0) the microtiter plates were incubated for 1 h at room temperature under agitation at 600 rpm. Unbound sample was removed by washing 4 times (each 350 μL per well) with washing solution with subsequent addition of 200 μl reaction buffer per well and incubation at 37° C. Reaction buffer including all components and final concentrations were as described in Example 3, with the exceptions that 100 μg/mL NT-ADM-antibody and 288 μg/mL ADM-Gly were used. Reaction was terminated at several time-points by transferring 10 μl of each individual reaction into 190 μl of EDTA containing buffer (300 mmol/L potassium phosphate, 100 mmol/L NaCl, 10 mmol/L Na-EDTA, 50 μmol/L amastatin, 100 μmol/L leupeptin, 0.1% bovine IgG, 0.02% mouse IgG, 0.5% BSA, pH 7.0). Terminated reactions were applied onto the Sphingotest® bio-ADM immunoassay for quantification of produced bio-ADM. A typical standard curve using an antibody directed to PAM immunization peptide 10 (SEQ ID No. 20) as solid phase is shown in
[0239] In a further step, PAM activity (as described in example 3) and PAM concentration using a PAM-LIA (solid phase antibody directed against full-length PAM, tracer antibody directed against peptide 13 [SEQ ID No. 23]) were determined in heparin samples from healthy volunteers (n=26). PAM activity and PAM concentration correlated significantly as shown in
Example 5— ADM-Gly Immunoassay
[0240] ADM-Gly was quantified as based on Weber et al. (Weber et al. 2017. JALM 2(2): 222-233) for bioactive ADM with the following modifications: the tracer-antibody used for ADM-Gly detection, labelled with MACN-acridinium-NHS, was directed to the C-terminal glycine of ADM-Gly. The assay was calibrated with synthetic ADM-Gly. The limit of detection (LOD) was 10 pg/mL of ADM-Gly. Cross-reactivity of antibody directed to the C-terminal glycine of ADM with bio-ADM was in the range between 6 and 50% in a concentration dependent manner. All determined ADM-Gly concentrations were corrected for cross-reactivity as follows: For each ADM-Gly quantification additional quantification of bio-ADM in corresponding samples was performed using the Sphingotest® bio-ADM immunoassay. The corresponding bio-ADM values were used to determine the signal (RLU) generated with the antibody directed to C-terminal glycine of ADM on a bio-ADM calibration curve. The determined signal (RLU) was used to calculate the false-positive ADM-Gly concentration (pg/mL) using the ADM-Gly calibration curve. This concentration was subtracted from the initially determined ADM-Gly concentration. A typical standard curve is shown in
Example 6— Prediction of Diseases in Healthy Subjects
[0241] 6.1. Study Cohort
[0242] The Malmo Preventive Project (MPP) was funded in the mid-1970s to explore CV risk factors in general population, and enrolled 33,346 individuals living in Malmo (Fedorowski et al. 2010. Eur Heart J 31: 85-91). Between 2002 and 2006, a total of 18,240 original participants responded to the invitation (participation rate, 70.5%) and were screened including a comprehensive physical examination and collection of blood samples (Fava et al. 2013. Hypertension 2013; 61: 319-26). The re-examination in MPP is in the present study regarded as the baseline. Subjects with prior CVD at baseline were excluded.
[0243] An informed consent was obtained from all participants and the Ethical Committee of Lund University, Lund, Sweden, approved the study protocol.
[0244] A commercial fully automated homogeneous time-resolved fluoro-immunoassay was used to measure MR-proADM in plasma (BRAHMS MR-proADM KRYPTOR; BRAHMS GmbH, Hennigsdorf, Germany) (Caruhel et al. 2009. Clin Biochem. 42 (7-8): 725-8).
[0245] Bio-ADM was measured as described by Weber et al. 2017 (Weber et al. 2017. JAMA 2(2): 222-233). AMA was determined in 4942 serum samples from MPP as described in example 3. Each sample was measured in duplicate. Samples, controls and calibrators were treated in the same manner. Baseline clinical characteristics of AMA after stratification to Quartiles is shown in table 2.
TABLE-US-00002 TABLE 2 Baseline clinical characteristics according to quartile (Q) of AMA at baseline of subjects analysed Q1 Q2 Q3 Q4 (n = 1235) (n = 1236) (n = 1236) (n = 1235) P AMA in μg/(L*h) (SD) 9.416 (1.21) 11.66 (0.46) 13.39 (0.57) 17 (3) N/A AMA range 3.8-10.86 10.86-12.47 12.47-14.47 14.47-72.15 N/A Age in years (SD) 68.97 (6.18) 69.16 (6.28) 69.34 (6.38) 70.45 (6.07) <0.0001 Current smoking, n (%) 188 (15.2) 217 (17.6) 255 (20.6) 287 (23.2) <0.0001 Systolic blood pressure 144 (19.77) 145.1 (19.83) 144.8 (20.33) 147.6 (21.34) <0.0002 in mmHg (SD) Diastolic blood pressure 82.83 (10.12) 84.04 (10.83) 83.12 (10.61) 84.45 (11.51) 0.0041 mmHg (SD) Diabetes Mellitus, n (%) 166 (13.4) 127 (10.3) 113 (9.1) 127 (10.3) 0.0043 Glucose in mmol/L (SD) 6.024 (1.95) 5.78 (1.21) 5.794 (1.37) 5.753 (1.28) 0.0299 N/A: not applicable
[0246] Statistical analysis: Values are expressed as means and standard deviations, medians and interquartile ranges (IQR), or counts and percentages as appropriate. Group comparisons of continuous variables were performed using the Kruskal-Wallis test. Biomarker data were log-transformed. Cox proportional-hazards regression was used to analyze the effect of risk factors on survival in uni- and multivariable analyses. The assumptions of proportional hazard were tested for all variables. For continuous variables, hazard ratios (HR) were standardized to describe the HR for a biomarker change of one IQR. 95% confidence intervals (CI) for risk factors and significance levels for chi-square (Wald test) are given. The predictive value of each model was assessed by the model likelihood ratio chi-square statistic. The concordance index (C index) is given as an effect measure. It is equivalent to the concept of AUC adopted for binary outcome. For multivariable models, a bootstrap corrected version of the C index is given. Survival curves plotted by the Kaplan-Meier method were used for illustrative purposes. To test for independence of PAM from clinical variables we used the likelihood ratio chi-square test for nested models. All statistical tests were 2-tailed and a two-sided p-value of 0.05 was considered for significance.
[0247] 6.2. Prediction of Alzheimer's Disease
[0248] 3954 samples with information about dementia diagnosis refilling efficiency in hemodialysed CKD patients were selected (n=174 with incident AD). Information about dementia diagnoses was requested from the Swedish National Patient Register (SNPR). The diagnoses in the register were collected according to different revisions of the International Classification of Diseases (ICD) codes 290, 293 (ICD-8), 290, 331 (ICD-9) or FOO, F01, F03, G30 (ICD-10). Since 1987, SNPR includes all in-patient care in Sweden and, in addition, contains data on outpatient visits including day surgery and psychiatric care from both private and public caregivers recorded after 2000. All-cause dementia was diagnosed according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders (DSM)-III revised edition, whilst the DSM-IV criteria were applied for the Alzheimer's disease and vascular dementia diagnoses. Diagnoses were validated by a thorough review of medical records as well as neuroimaging data when available. A research physician assigned the final diagnosis for each patient and a geriatrician specialized in cognitive disorders was consulted in unresolved cases. The PAM activity (AMA) was determined as described in example 3. AMA in the MPP cohort is shown in
[0249] Reduced serum AMA strongly predicts Alzheimer's disease with a Hazard Ratio (HR) of 0.74 (CI 0.6-0.88; p<0.001) and a HR of 0.72 (CI 0.6-0.85) when adjusted for age (table 3).
[0250] Furthermore, AMA as a predictor of AD was independent from bio-ADM concentrations. Both markers contribute to AD prediction. While the C-Index for AMA alone is 0.571 (CI 0.525-0.616; Chi.sup.2 10.97) the C-index for both combined markers, i.e. AMA and bio-ADM is 0.595 (Chi.sup.2 18.96; p<0.0001).
[0251] Moreover, AMA in combination with bio-ADM and MR-proADM concentrations further improve the prediction of incident Alzheimer. While MR-proADM alone had no predictive value for AD, the combination of AMA, bio-ADM and MR-proADM showed a C-index of 0.622 (Chi.sup.2 26.73; p=0.00001).
TABLE-US-00003 TABLE 3 Prediction of Alzheimer's disease Hazard Ratio Biomarker (HR) (CI) p-Value C-Index (CI) Chi.sup.2 AMA 0.72 (0.6-0.85) p < 0.001 0.571 (0.525-0.616) 10.97 AMA, bio-ADM p < 0.0001 0.595 18.96
[0252] 6.3. Prediction of Colorectal Cancer (CRC)
[0253] AMA of subjects with and without incident CRC is shown in
[0254] Results for the single markers and marker combinations are shown in Table 3. Reduced serum AMA (age-adjusted) strongly predicts development of CRC with a Hazard Ratio (HR) of 0.68 (p<0.0001).
[0255] Increased MR-proADM concentrations predict development of CRC with a HR of 1.36 p<0.05). The highest quartile is associated with the highest risk of CRC development (p=0.051).
[0256] While bio-ADM concentrations were not predictive for development of CRC, a combination of bio-ADM and AMA showed an improved CRC prediction (see table 4). In addition, a combination of AMA and MR-proADM further improved the prediction of CRC development.
[0257] In summary, reduced AMA values predict development of CRC. Increased MR-proADM concentrations also predict development of CRC. A combination of AMA with bio-ADM or MR-proADM enhances the predictive value for CRC.
TABLE-US-00004 TABLE 4 Prediction of colorectal cancer Hazard Ratio Biomarker (HR) (CI) p-Value C-Index (CI) Chi.sup.2 AMA 0.68 (0.6-0.85) p < 0.00001 0.588 (0.535-0.641) 8.51 AMA, bioADM p < 0.002 0.598 12.48 MR-proADM 1.36 (1.08-1.72) p < 0.05 0.587 (0.532-0.642) 6.27 AMA, MR-proADM p < 0.0005 0.612 16.51
[0258] 6.4. Prediction of Pancreatic Cancer
[0259] Moreover, AMA is increased in incident pancreatic cancer compared to subjects without pancreatic cancer (p<0.005) (
[0260] 6.5. Prediction of all-Cause and Cardiovascular Mortality
[0261] Mortality analyses were performed in 4942 samples with information about death and cardiovascular events from the MPP cohort. Information about cardiovascular events and diagnoses was requested from the Swedish National Patient Register (SNPR). The diagnoses in the register were collected according to different revisions of the International Classification of Diseases (ICD) codes. Since 1987, SNPR includes all in-patient care in Sweden and, in addition, contains data on outpatient visits including day surgery and psychiatric care from both private and public caregivers recorded after 2000. The PAM activity (AMA) was determined as described in example 3. Within of the total set of 4942 serum samples from the MPP study cohort 1361 subjects died (all-cause mortality) during follow-up period of 12.8 years. From the total number of 1361 death-events 480 events where accounted to as cardiovascular mortality.
[0262] Elevated serum AMA strongly predicts all-cause mortality with a Hazard Ratio (HR) of 1.354 (CI 1.197-1.531; p<0.0001) (Table 5). The predictive value of AMA was independent of the common cardiovascular risk factors (age, gender, blood-pressure, body-mass index, antihypertensive medication, low- and high-density lipoproteins and history of diabetes).
[0263] Elevated serum AMA strongly predicts cardiovascular mortality with a Hazard Ratio (HR) of 1.6 (CI 1.3-1.969; p<0.0001) (Table 5). The predictive value of AMA was independent of the common cardiovascular risk factors (age, gender, blood-pressure, body-mass index, antihypertensive medication, low- and high-density lipoproteins and history of diabetes).
TABLE-US-00005 TABLE 5 Prediction of all-cause and cardiovascular mortality by PAM activity Q1 Q2 (n = 3707) (n = 1235) AMA in μg/(L*h) (SD) 11.49 (1.82) 17 (3) AMA range 3.8-14.47 14.47-72.15 All-Cause Mortality Number of Events 943 418 Logrank Hazard Ratio (ref) 1.354 (1.197-1.531) (95% CI) Chi.sup.2 26.8 p-value <0.0001 Cardiovascular mortality Number of Events 314 166 Logrank Hazard Ratio (ref) 1.6 (1.3-1.969) (95% CI) Chi.sup.2 24.38 p-value <0.0001
[0264] 6.6. Prediction of Cardiovascular Disorders
[0265] Cardiovascular disorder analyses were performed in 4942 samples with information about death- and cardiovascular events from the MPP cohort. Information about cardiovascular events and diagnoses was requested from the Swedish National Patient Register (SNPR). The diagnoses in the register were collected according to different revisions of the International Classification of Diseases (ICD) codes. Since 1987, SNPR includes all in-patient care in Sweden and, in addition, contains data on outpatient visits including day surgery and psychiatric care from both private and public caregivers recorded after 2000. The PAM activity (AMA) was determined as described in example 3. Within of the total set of 4942 serum samples from the MPP study cohort 278 subjects developed heart failure (incident heart failure) and 633 subjects developed atrial fibrillation (incident atrial fibrillation) during follow-up period of 12.8 years.
[0266] Elevated serum AMA strongly predicts incident heart failure (83 prevalent HF cases were excluded from the analyses) with a Hazard Ratio (HR) of 1.537 (CI 1.169-2.021; p<0.0007) (Table 6).
[0267] Elevated serum AMA strongly predicts incident atrial fibrillation (267 prevalent AF cases were excluded from the analyses) with a Hazard Ratio (HR) of 1.459 (CI 1.214-1.752; p<0.0001) (Table 6).
TABLE-US-00006 TABLE 6 Prediction of cardiovascular disorders Q1 Q2 (n = 3707) (n = 1119) Heart failure Number of Events 186 92 Logrank Hazard Ratio (ref) 1.537 (1.169-2.021) (95% CI) Chi.sup.2 11.56 p-value =0.0007 Atrial fibrillation Q1 Q2 (n = 3534) (n = 1141) Number of Events 436 197 Logrank Hazard Ratio (ref) 1.459 (1.214-1.752) (95% CI) Chi.sup.2 19.59 p-value <0.0001
Example 7— Diagnosis of Diseases
[0268] 7.1. Diagnosis of Alzheimer's Disease
[0269] Serum samples from 27 individuals with diagnosed Alzheimer's disease were obtained from InVent Diagnostica GmbH. The AD diagnosis is based on cognitive tests (CERAD, DemTec, MMST and Clock-Drawing test) as well as on MRI (Magnetic resonance imaging) and CT-scans. As controls, 67 serum samples from self-reported healthy volunteers were used. AMA was detected as described in example 3.
[0270] As shown in
[0271] 7.2. Diagnosis of Cardiovascular and Metabolic Disorders
[0272] In the total set of 4942 serum samples from the MPP study cohort, 267 cases of prevalent atrial fibrillation, 83 cases of prevalent chronic heart failure and 533 cases of prevalent diabetes were present.
[0273] Significant elevation of serum AMA (p<0.0001) was observed in prevalent atrial fibrillation (mean AMA: 13.92 AMA-Units, n=267) when compared to individuals free of prevalent atrial fibrillation (mean AMA: 12.8 AMA-Units, n=4675).
[0274] Significant elevation of serum AMA (p=0.0019) was observed in prevalent chronic heart failure (mean AMA: 14.31 AMA-Units, n=83) when compared to individuals free of prevalent heart failure (mean AMA: 12.84 AMA-Units, n=4859).
[0275] Significant reduction of serum AMA (p=0.0035) was observed in prevalent diabetes (mean AMA: 12.69 AMA-Units, n=533) when compared to individuals free of prevalent diabetes (mean AMA: 12.89 AMA-Units, n=4409).
Example 8— Prognosis and Monitoring
[0276] 8.1. Study Cohort AdrenOSS-1
[0277] AdrenOSS-1 was a European prospective observational study. Twenty-four centers in five countries (France, Belgium, The Netherlands, Italy, and Germany) contributed to the trial achievement of 583 enrolled patients (recruited from June 2015 to May 2016). The study protocol was approved by the local ethics committees and was conducted in accordance with the Declaration of Helsinki. The study enrolled patients aged 18 years and older who were (1) admitted to the ICU for sepsis or septic shock or (2) transferred from another ICU in the state of sepsis and septic shock within less than 24 h after admission. Included patients were stratified by severe sepsis and septic shock based on definitions for sepsis and organ failure from 2001 (Levy et al. 2003. 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Crit Care Med. 31(4):1250-6). The term “sepsis” refers to the updated definition of Sepsis-3 (Singer et al. 2016 The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 315(8):801-10). Patients were treated according to local practice, and treatments as well as procedures were registered. The primary endpoint was 28-day mortality. Secondary endpoints concerned organ failure (as defined by the Sequential Organ Failure Assessment [SOFA] score) and organ support, vasopressor/inotrope use, fluid balance, and use of renal replacement therapy (RRT).
[0278] Upon admission, demographics (age, sex), body mass index, presence of septic shock, type of ICU admission, organ dysfunction scores (SOFA, Acute Physiologic Assessment and Chronic Health Evaluation II [APACHE II]), origin of sepsis, pre-existing comorbidities (i.e., treated within the last year), past medical history, laboratory values, and organ support were recorded, and blood was drawn for measurement of bio-ADM and other markers. After patient enrolment, the following data were collected daily during the first week: SOFA score, antimicrobial therapies, fluid balance, ventilation status, Glasgow Coma Scale score, central venous pressure, need for RRT, invasive procedures for sepsis control, and vasopressor/inotrope treatment. Moreover, discharge status and mortality were recorded on day 28 after ICU admission.
[0279] Blood for the central laboratory was sampled within 24 h after ICU admission and on day 2 (mean 47 h, SD 9 h) after the first sample. Samples were subsequently processed and stored at −80° C. The PAM activity (AMA) was measured in n=197 plasma samples, randomly selected from AdrenOSS-I cohort as described in example 3.
[0280] 8.2. Outcome Prognosis in Sepsis
[0281] The AMA in the AdrenOSS-I cohort as shown in
[0282] In addition, the ADM-Gly concentrations in the AdrenOSS-I cohort, also revealed a significantly higher concentration in non-survivors compared to survivors (p<0.0001). High ADM-Gly concentrations strongly predict 28-day mortality with a HR of 2.29 (p<0.005).
[0283] The outcome for the 28-day mortality for the single biomarkers AMA and ADM-Gly is shown in table 7. A cut-off for AMA and ADM-Gly concentration, respectively, was chosen to result in an equal sensitivity of 80.4%, while the specificity was 21.7% for AMA and 38.5% for ADM-Gly, respectively. When both markers were combined by, i.e. a ratio, the specificity for 28-day survival was increased to 43.4%. Furthermore, the Odds ratio (OR) was 1.13 and 2.56 for PAM and ADM Gly, respectively, while the combination of both markers resulted in an increased OR of 3.14.
TABLE-US-00007 TABLE 7 Cross-tables for the evaluation of 28 day mortality outcome. [μg/(L*h)] 28 Day Mortality 28 Day Survival AMA > 17.1 41 112 PPV: 26.8% AMA < 17.1 10 31 NPV: 75.6% Prevalence: 26.3% Sensitivity: 80.4% Specificity: 21.7% OR: 1.13 [pg/mL] 28 Day Mortality 28 Day Survival 15 ADM-Gly > 140 41 88 PPV: 31.8% ADM-Gly < 140 10 55 NPV: 84.6% Prevalence: 26.3% Sensitivity: 80.4% Specificity: 38.5% OR: 2.56 AMA/ADM-Gly ratio 28 D Mortality 28 D Survival AMA/ADM- 41 81 PPV: 33.6% Gly < 132 AMA/ADM- 10 62 NPV: 86.1% Gly > 132 Prevalence: 26.3% Sensitivity: 80.4% Specificity: 43.4% OR: 3.14
[0284] 9. Determination of PAM Activity in Human Saliva
[0285] Saliva was collected from 5 self-reported healthy subjects in separate sterile tubes. PAM activity in human saliva samples was tested as described in example 3. PAM activity could be measured in saliva samples (range from around 700 to 2000 μg/(L*h) (
TABLE-US-00008 SEQUENCES SEQ ID NO: 1-Prepro-PAM isoform 1 AS 1-973 10 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE 410 420 430 440 450 REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN 460 470 480 490 500 AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE 510 520 530 540 550 ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG 560 570 580 590 600 LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH 610 620 630 640 650 QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG 660 670 680 690 700 YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV 710 720 730 740 750 ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF 760 770 780 790 800 GDQEPVQGFV MNFSNGEIID IFKPVRKHFD MPHDIVASED GTVYIGDAHT 810 820 830 840 850 NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA EAVVETKMEN KPTSSELQKM 860 870 880 890 900 QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL LAIAIFIRWK KSRAFGDSEH 910 920 930 940 950 KLETSSGRVL GRFRGKGSGG LNLGNFFASR KGYSRKGFDR LSTEGSDQEK 960 970 EDDGSESEEE YSAPLPALAP SSS SEQ ID NO: 2-Prepro-PAM isoform 2 AS 1-868 10 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQDFH MEEALDWPGV 410 420 430 440 450 YLLPGQVSGV ALDPKNNLVI FHRGDHVWDG NSFDSKFVYQ QIGLGPIEED 460 470 480 490 500 TILVIDPNNA AVLQSSGKNL FYLPHGLSID KDGNYWVTDV ALHQVFKLDP 510 520 530 540 550 NNKEGPVLIL GRSMQPGSDQ NHFCQPTDVA VDPGTGAIYV SDGYCNSRIV 560 570 580 590 600 QFSPSGKFIT QWGEESSGSS PLPGQFTVPH SLALVPLLGQ LCVADRENGR 610 620 630 640 650 IQCFKTDTKE FVREIKHSSF GRNVFAISYI PGLLFAVNGK PHFGDQEPVQ 660 670 680 690 700 GFVMNFSNGE IIDIFKPVRK HFDMPHDIVA SEDGTVYIGD AHTNTVWKFT 710 720 730 740 750 LTEKLEHRSV KKAGIEVQEI KEAEAVVETK MENKPTSSEL QKMQEKQKLI 760 770 780 790 800 KEPGSGVPVV LITTLLVIPV VVLLAIAIFI RWKKSRAFGD SEHKLETSSG 810 820 830 840 850 RVLGRFRGKG SGGLNLGNFF ASRKGYSRKG FDRLSTEGSD QEKEDDGSES 860 EEEYSAPLPA LAPSSS SEQ ID No.: 3-Prepro-PAM isoform 3 AS (amino acids 829-896 of SEQ ID No. 1 missing) 10 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE 410 420 430 440 450 REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN 460 470 480 490 500 AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE 510 520 530 540 550 ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG 560 570 580 590 600 LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH 610 620 630 640 650 QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG 660 670 680 690 700 YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV 710 720 730 740 750 ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF 760 770 780 790 800 GDQEPVQGFV MNFSNGEIID IFKPVRKHFD MPHDIVASED GTVYIGDAHT 810 820 830 840 850 NTVWKFTLTE KLEHRSVKKA GIEVQEIKDS EHKLETSSGR VLGRFRGKGS 860 870 880 890 900 GGLNLGNFFA SRKGYSRKGF DRLSTEGSDQ EKEDDGSESE EEYSAPLPAL 905 APSSS SEQ ID No. 4-Prepro-PAM isoform 4 (amino acids 829-914 of SEQ ID No. 1 missing) 10 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE 410 420 430 440 450 REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN 460 470 480 490 500 AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE 510 520 530 540 550 ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG 560 570 580 590 600 LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH 610 620 630 640 650 QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG 660 670 680 690 700 YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV 710 720 730 740 750 ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF 760 770 780 790 800 GDQEPVQGFV MNFSNGEIID IFKPVRKHFD MPHDIVASED GTVYIGDAHT 810 820 830 840 850 NTVWKFTLTE KLEHRSVKKA GIEVQEIKGK GSGGLNLGNF FASRKGYSRK 860 870 880 890 GFDRLSTEGS DQEKEDDGSE SEEEYSAPLP ALAPSSS SEQ ID No. 5-Prepro-PAM Isoform 5 (Isoform 1 with an additional aa in position 896) 10 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE 410 420 430 440 450 REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN 460 470 480 490 500 AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE 510 520 530 540 550 ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG 560 570 580 590 600 LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH 610 620 630 640 650 QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG 660 670 680 690 700 YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV 710 720 730 740 750 ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF 760 770 780 790 800 GDQEPVQGFV MNFSNCEIID IFKPVRKHFD MPHDIVASED GTVYIGDAHT 810 820 830 840 850 NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA EAVVETKMEN KPTSSELQKM 860 870 880 890 900 QIKQKIIKIP GSGVPVVLIT TLLVIPVVVL LAIAIFIRWK KSRAFGADSE 910 920 930 940 950 HKLETSSGRV LGRFRGKGSG GLNLGNFFAS RKGYSRKGFD RLSTEGSDQE 960 970 KEDDGSESEE EYSAPLPALA PSSS SEQ ID No. 6-Prepro-PAM Isoform 6 (amino acids 897-914 of SEQ ID No. 1 missing) 10 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE 410 420 430 440 450 REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN 460 470 480 490 500 AILVRDRIHK FHRLVSTLRP PESRVISIQQ PPPGEGTWEP EHTGDFHMEE 510 520 530 540 550 ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG 560 570 580 590 600 LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH 610 620 630 640 650 QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG 660 670 680 690 700 YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV 710 720 730 740 750 ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF 760 770 780 790 800 GDQEPVQGFV MNFSNGEIID IFKPVRKHID MPHDIVASED GTVYIGDAHT 810 820 830 840 850 NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA EAVVETKMEN KPTSSELQKM 860 870 880 890 900 QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL LAIAIFIRWK KSRAFGGKGS 910 920 930 940 950 GGLNLGNFFA SRKGYSRKGF DRLSTEGSDQ EKEDDGSESE EEYSAPLPAL APSSS SEQ ID No. 7-PHM subunit of PAM 10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTKDA 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT 310 320 330 340 350 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE 360 370 380 390 400 EEEVLDQGDF YSLLSKLLGE REDVVHVHKY NPTEKAESES DLVAEIANVV 410 420 430 440 450 QKKDLGRSDA REGAEHERGN 460 PPPGEGTWEP EHTG SEQ ID No. 8-PAL subunit of PAM 10 20 30 40 50 DFHMEEALDW PGVYLLPGQV SGVALDPKNN LVIFHRGDHV WDGNSFDSKF 60 70 80 90 100 VYQQIGLGPI EEDTILVIDP NNAAVLQSSG KNLFYLPHGL SIDKDGNYWV 110 120 130 140 150 TDVALHQVFK LDPNNKEGPV LILGRSMQPG SDQNHFCQPT DVAVDPGTGA 160 170 180 190 200 IYVSDGYCNS RIVQFSPSGK FITQWGEESS GSSPLPGQFT VPHSLALVPL 210 220 230 240 250 LGQLCVADRE NGRIQCFKTD TKEFVREIKH SSFGRNVFAI SYIPGLLFAV 260 270 280 290 300 NGKPHFGDQE PVQGFVMNFS NGEIIDIFKP VRKHFDMPHD IVASEDGTVY 310 320 IGDAHTNTVW KFTLTEKLEH RSV SEQ ID No. 9-signal sequence human serum albumin 10 20 MKWVTFISLL FLFSSAYSFR SEQ ID No. 10-Sequence of recombinant human PAM 10 20 30 40 50 SPLSVFKRFK ETTRPFSNEC LGTTRPVVPI DSSDFALDIR MPGVTPKQSD 60 70 80 90 100 TYFCMSMRIP VDEEAFVIDF KPRASMDTVH HMLLFGCNMP SSTGSYWFCD 110 120 130 140 150 EGTCTDKANI LYAWARNAPP TRLPKGVGFR VGGETGSKYF VLQVHYGDIS 160 170 180 190 200 AFRDNNKDCS GVSLHLTRLP QPLIAGMYLM MSVDTVIPAG EKVVNSDISC 210 220 230 240 250 HYKNYPMHVF AYRVHTHHLG KVVSGYRVRN GQWTLIGRQS PQLPQAFYPV 260 270 280 290 300 GHPVDVSFGD LLAARCVFTG EGRTEATHIG GTSSDEMCNL YIMYYMEAKH 310 320 330 340 350 AVSFMTCTQN VAPDMFRTIP PEANIPIPVK SDMVMMHEHH KETEYKDKIP 360 370 380 390 400 LLQQPKREEE EVLDQGDFYS LLSKLLGERE DVVHVHKYNP TEKAESESDL 410 420 430 440 450 VAEIANVVQK KDLGRSDARE GAEHERGNAI LVRDRIHKFH RLVSTLRPPE 460 470 480 490 500 SRVFSLQQPP PGEGTWEPEH TGDFHMEEAL DWPGVYLLPG QVSGVALDPK 510 520 530 540 550 NNLVIFHRGD HVWDGNSFDS KFVYQQIGLG PIEEDTILVI DPNNAAVLQS 560 570 580 590 600 SGKNLFYLPH GLSIDKDGNY WVTDVALHQV FKLDPNNKEG PVLILGRSMQ 610 620 630 640 650 PGSDQNHFCQ PTDVAVDPGT GAIYVSDGYC NSRIVQFSPS GKFITQWGEE 660 670 680 690 700 SSGSSPLPGQ FTVPHSLALV PLLGQLCVAD RENGRIQCFK TDTKEFVREI 710 720 730 740 750 KHSSFGRNVF AISYIPGLLF AVNGKPHFGD QEPVQGFVMN FSNGEIIDIF 760 770 780 790 800 KPVRKHFDMP HDIVASEDGT VYIGDAHTNT VWKFTLTEKL EHRSVKKAGI 810 EVQEIKEAEA VVGS SEQ ID No. 11-Peptide 1 (aa 42-56 of PAM SEQ ID No. 1) 10 CLGTTRPVVP IDSSD SEQ ID No. 12-Peptide 2 (aa 109-128 of PAM SEQ ID No. 1) 10 CNMPSSTGSY WFCDEGTCTD SEQ ID No. 13-Peptide 3 (aa 168-180 of PAM SEQ ID No. 1) 10 YGDISAFRDN NKD SEQ ID No. 14-Peptide 4 (aa 204-216 of PAM SEQ ID No. 1) 10 SVDTVIPAGE KVV SEQ ID No. 15-Peptide 5 (aa 329-342 of PAM SEQ ID No. 1) 10 CTQNVAPDMF RTIP SEQ ID No. 16-Peptide 6 (aa 291-310 of PAM SEQ ID No. 1) 10 20 TGEGRTEATH IGGTSSDEMC SEQ ID No. 17-Peptide 7 (aa 234-244 of PAM SEQ ID No. 1) 10 YRVHTHHLGK V SEQ ID No. 18-Peptide 8 (aa 261-276 of PAM SEQ ID No. 1) 10 QSPQLPQAFY PVGHPV SEQ ID No. 19-Peptide 9 (aa 530-557 of PAM SEQ ID No. 1) 10 20 RGDHVWDGNS FDSKFVYQQI GLGPIEED SEQ ID No. 20-Peptide 10 (aa 611-631 of PAM SEQ ID No. 1) 10 20 EGPVLILGRS MQPGSDQNHF C SEQ ID No. 21-Peptide 11 (aa 562-579 of PAM SEQ ID No. 1) 10 IDPNNAAVLQ SSGKNLFY SEQ ID No. 22-Peptide 12 (aa 745-758 of PAM SEQ ID No. 1) 10 NGKPHFGDQE PVQG SEQ ID No. 23-Peptide 13 (aa 669-687 of PAM SEQ ID No. 1) 10 WGEESSGSSP LPGQFTVPH SEQ ID No. 24-Peptide 14 (aa 710-725 of PAM SEQ ID No. 1) 10 CFKTDTKEFV REIKHS