ADRENOMEDULLIN (ADM) FOR DIAGNOSIS AND/OR PREDICTION OF DEMENTIA AND ANTI-ANDRENOMEDULLIN BINDER FOR USE IN THERAPY OR PREVENTION OF DEMENTIA
20230221339 · 2023-07-13
Assignee
Inventors
Cpc classification
G01N33/74
PHYSICS
C07K16/22
CHEMISTRY; METALLURGY
C07K2317/34
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
G01N2800/52
PHYSICS
International classification
Abstract
Subject matter of the present invention is a method for diagnosing dementia, or determining the risk of getting dementia in a subject that does not have dementia, or monitoring therapy or monitoring or guiding intervention in a subject that has dementia, or monitoring therapy or monitoring or guiding preventive intervention in a subject that is at risk of getting dementia.
Claims
1-14. (canceled)
15. A method of treatment comprising: administering an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold to a subject, wherein said subject has a level of mature ADM-NH.sub.2 according to SEQ ID No.: 4 that is below a threshold level and/or has a marker ratio that is the ratio of the level of mature ADM-NH.sub.2 according to SEQ ID No.: 4 to the level of pro-Adrenomedullin or a fragment thereof wherein said marker level ratio is below a predetermined ratio threshold, wherein said anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal part (aa 1-21) of adrenomedullin: YRQSMNNFQGLRSFGCRFGTC (SEQ ID No. 21).
16. The method of claim 15 wherein, said fragment of pro-Adrenomedullin is selected from a group comprising PAMP (SEQ ID No. 2), MR-proADM (SEQ ID No. 3), ADM-Gly (SEQ ID No.: 5) and CT-proADM (SEQ ID No. 6).
17. The method of claim 15, wherein the threshold level of mature ADM-NH.sub.2 according to SEQ ID No.: 4 is below 15 pg/ml.
18. The method of claim 15, wherein the marker level ratio is in a range 0.2 to 0.75.
19. The method of claim 15, wherein the sample of bodily fluid is selected from the group of blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.
20. A method of treatment comprising: administering an Anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold to a subject, wherein said subject has a level of mature ADM-NH.sub.2 according to SEQ ID No.: 4 which is below a predetermined threshold level.
21. The method of treatment of claim 20 wherein the predetermined threshold level is 15 pg/ml.
22. The method of treatment of claim 20 wherein the subject also has a level of pro-Adrenomedullin or a fragment thereof which is not mature ADM-NH.sub.2 according to SEQ ID No. 4 which is is at or above a predetermined threshold level.
23. The method of claim 22 wherein the predetermined threshold for pro-Adrenomedullin or a fragment thereof which is not mature ADM-NH.sub.2 according to SEQ ID No.: 4 is normal levels.
Description
FIGURE DESCRIPTION
[0237]
[0238]
[0239]
[0240]
[0241]
[0242]
[0243]
[0244]
EXAMPLES
Example 1
[0245] Generation of Antibodies and Determination of their Affinity Constants
[0246] Several human and murine antibodies were produced and their affinity constants were determined (see Table 1).
[0247] Peptides/Conjugates for Immunization:
[0248] Peptides for immunization were synthesized, see Table 1, (JPT Technologies, Berlin, Germany) with an additional N-terminal Cystein (if no Cystein is present within the selected ADM-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. The murine antibodies were generated according to the following method:
[0249] A Balb/c mouse was immunized with 100 μg Peptide-BSA-Conjugate at day 0 and 14 (emulsified in 100 μl complete Freund's adjuvant) and 50 μg at day 21 and 28 (in 100 μl incomplete Freund's adjuvant). Three days before the fusion experiment was performed, the animal received 50 μg of the conjugate dissolved in 100 μl saline, given as one intraperitoneal and one intra-venous injection.
[0250] Splenocytes from the immunized mouse 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 two weeks the HAT medium is replaced with HT Medium for three passages followed by returning to the normal cell culture medium.
[0251] The cell culture supernatants were primary screened for antigen specific IgG antibodies three weeks after fusion. The positive tested microcultures were transferred into 24-well plates for propagation. After retesting, the selected cultures were cloned and recloned using the limiting-dilution technique and the isotypes were determined (see also Lane, R. D. 1985. J. Immunol. Meth. 81: 223-228; Ziegler et al. 1996. Horm. Metab. Res. 28: 11-15).
[0252] Mouse Monoclonal Antibody Production:
[0253] Antibodies were produced via standard antibody production methods (Marx et al, 1997. Monoclonal Antibody Production, ATLA 25, 121) and purified via Protein A. The antibody purities were >95% based on SDS gel electrophoresis analysis.
[0254] Human Antibodies:
[0255] Human Antibodies were produced by means of phage display according to the following procedure:
[0256] The human naive antibody gene libraries HALT/8 were used for the isolation of recombinant single chain F-Variable domains (scFv) against ADM peptide. The antibody gene libraries were screened with a panning strategy comprising the use of peptides containing a biotin tag linked via two different spacers to the ADM peptide sequence. A mix of panning rounds using non-specifically bound antigen and streptavidin bound antigen were used to minimize background of non-specific binders. The eluted phages from the third round of panning have been used for the generation of monoclonal scFv expressing E. coli strains. Supernatant from the cultivation of these clonal strains has been directly used for an antigen ELISA testing (see also Hust et al. 2011. Journal of Biotechnology 152, 159-170; Schütte et al. 2009. PLoS One 4, e6625).
[0257] Positive clones have been selected based on positive ELISA signal for antigen and negative for streptavidin coated micro titer plates. For further characterizations the scFv open reading frame has been cloned into the expression plasmid pOPE107 (Hust et al. 2011. Journal of Biotechnology 152, 159-170), captured from the culture supernatant via immobilised metal ion affinity chromatography and purified by a size exclusion chromatography.
[0258] Affinity Constants:
[0259] To determine the affinity of the antibodies to ADM, the kinetics of binding of ADM to immobilized antibody was determined by means of label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the antibodies was performed using an anti-mouse Fc antibody covalently coupled in high density to a CMS sensor surface according to the manufacturer's instructions (mouse antibody capture kit; GE Healthcare) (Lorenz et al. 2011. Antimicrob Agents Chemother. 55(1): 165-173).
[0260] The monoclonal antibodies were raised against the below depicted ADM regions of human and murine ADM, respectively. The following table represents a selection of obtained antibodies used in further experiments. Selection was based on target region:
TABLE-US-00019 TABLE 1 Affinity Sequence ADM constants Number Antigen/Immunogen Region Designation Kd(M) SEQ ID: 21 YRQSMNNFQGLRSFGCRFGTC 1-21 NT-H 5.9 × 10.sup.−9 SEQ ID: 22 CTVQKLAHQIYQ 21-32 MR-H 2 × 10.sup.−9 SEQ ID: 23 CAPRSKISPQGY-NH.sub.2 C-42-52 CT-H 1.1 × 10.sup.−9 SEQ ID: 24 YRQSMNQGSRSNGCRFGTC 1-19 NT-M 3.9 × 10.sup.−9 SEQ ID: 25 CTFQKLAHQIYQ 19-31 MR-M 4.5 × 10.sup.−10 SEQ ID: 26 CAPRNKISPQGY-NH.sub.2 C-40-50 CT-M 9 × 10.sup.−9
[0261] Generation of Antibody Fragments by Enzymatic Digestion:
[0262] The generation of Fab and F(ab)2 fragments was done by enzymatic digestion of the murine full-length antibody NT-M. Antibody NT-M was digested using a) the pepsin-based F(ab)2 Preparation Kit (Pierce 44988) and b) the papain-based Fab Preparation Kit (Pierce 44985). The fragmentation procedures were performed according to the instructions provided by the supplier. Digestion was carried out in case of F(ab)2-fragmentation for 8 h at 37° C. The Fab-fragmentation digestion was carried out for 16 h, respectively.
[0263] Procedure for Fab Generation and Purification:
[0264] The immobilized papain was equilibrated by washing the resin with 0.5 ml of Digestion Buffer and centrifuging the column at 5000×g for 1 minute. The buffer was discarded afterwards. The desalting column was prepared by removing the storage solution and washing it with digestion buffer, centrifuging it each time afterwards at 1000×g for 2 minutes. 0.5 ml of the prepared IgG sample where added to the spin column tube containing the equilibrated Immobilized Papain. Incubation time of the digestion reaction was done for 16 h on a tabletop rocker at 37° C. The column was centrifuged at 5000×g for 1 minute to separate digest from the Immobilized Papain. Afterwards the resin was washed with 0.5 ml PBS and centrifuged at 5000×g for 1 minute. The wash fraction was added to the digested antibody that the total sample volume was 1.0 ml. The NAb Protein A Column was equilibrated with PBS and IgG Elution Buffer at room temperature. The column was centrifuged for 1 minute to remove storage solution (contains 0.02% sodium azide) and equilibrated by adding 2 ml of PBS, centrifuge again for 1 minute and the flow-through discarded. The sample was applied to the column and resuspended by inversion. Incubation was done at room temperature with end-over-end mixing for 10 minutes. The column was centrifuged for 1 minute, saving the flow-through with the Fab fragments. (References: Coulter and Harris 1983. J. Immunol. Meth. 59, 199-203; Lindner et al. 2010. Cancer Res. 70, 277-87; Kaufmann et al. 2010. PNAS. 107, 18950-5; Chen et al. 2010. PNAS. 107, 14727-32; Uysal et al. 2009 J. Exp. Med. 206, 449-62; Thomas et al. 2009. J. Exp. Med. 206, 1913-27; Kong et al. 2009 J. Cell Biol. 185, 1275-840).
[0265] Procedure for Generation and Purification of F(Ab′)2 Fragments:
[0266] The immobilized Pepsin was equilibrated by washing the resin with 0.5 ml of Digestion Buffer and centrifuging the column at 5000×g for 1 minute. The buffer was discarded afterwards. The desalting column was prepared by removing the storage solution and washing it with digestion buffer, centrifuging it each time afterwards at 1000×g for 2 minutes. 0.5 ml of the prepared IgG sample where added to the spin column tube containing the equilibrated Immobilized Pepsin. Incubation time of the digestion reaction was done for 16 h on a tabletop rocker at 37° C. The column was centrifuged at 5000×g for 1 minute to separate digest from the Immobilized Papain. Afterwards the resin was washed with 0.5 mL PBS and centrifuged at 5000×g for 1 minute. The wash fraction was added to the digested antibody that the total sample volume was 1.0 ml. The NAb Protein A Column was equilibrated with PBS and IgG Elution Buffer at room temperature. The column was centrifuged for 1 minute to remove storage solution (contains 0.02% sodium azide) and equilibrated by adding 2 mL of PBS, centrifuge again for 1 minute and the flow-through discarded. The sample was applied to the column and resuspended by inversion. Incubation was done at room temperature with end-over-end mixing for 10 minutes. The column was centrifuged for 1 minute, saving the flow-through with the Fab fragments. (References: Mariani et al. 1991. Mol. Immunol. 28: 69-77; Beale 1987. Exp Comp Immunol 11:287-96; Ellerson et al. 1972. FEBS Letters 24(3):318-22; Kerbel and Elliot 1983. Meth Enzymol 93:113-147; Kulkarni et al. 1985. Cancer Immunol Immunotherapy 19:211-4; Lamoyi 1986. Meth Enzymol 121:652-663; Parham et al. 1982. J Immunol Meth 53:133-73; Raychaudhuri et al. 1985. Mol Immunol 22(9):1009-19; Rousseaux et al. 1980. Mol Immunol 17:469-82; Rousseaux et al. 1983. J Immunol Meth 64:141-6; Wilson et al. 1991. J Immunol Meth 138:111-9).
[0267] NT-H-Antibody Fragment Humanization:
[0268] The antibody fragment was humanized by the CDR-grafting method (Jones et al. 1986. Nature 321, 522-525).
[0269] The following steps where executed to achieve the humanized sequence: [0270] Total RNA extraction: Total RNA was extracted from NT-H hybridomas using the Qiagen kit. [0271] First-round RT-PCR: QIAGEN® OneStep RT-PCR Kit (Cat No. 210210) was used. RT-PCR was performed with primer sets specific for the heavy and light chains. For each RNA sample, 12 individual heavy chain and 11 light chain RT-PCR reactions were set up using degenerate forward primer mixtures covering the leader sequences of variable regions. Reverse primers are located in the constant regions of heavy and light chains. No restriction sites were engineered into the primers. [0272] Reaction Setup: 5×QIAGEN® OneStep RT-PCR Buffer 5.0 μl, dNTP Mix (containing 10 mM of each dNTP) 0.8 μl, Primer set 0.5 μl, QIAGEN® OneStep RT-PCR Enzyme Mix 0.8 μl, Template RNA 2.0 μl, RNase-free water to 20.0 μl, Total volume 20.0 μl PCR condition: Reverse transcription: 50° C., 30 min; Initial PCR activation: 95° C., 15 min Cycling: 20 cycles of 94° C., 25 sec; 54° C., 30 sec; 72° C., 30 sec; Final extension: 72° C., 10 min Second-round semi-nested PCR: The RT-PCR products from the first-round reactions were further amplified in the second-round PCR. 12 individual heavy chain and 11 light chain RT-PCR reactions were set up using semi-nested primer sets specific for antibody variable regions. [0273] Reaction Setup: 2×PCR mix 10 μl; Primer set 2 μl; First-round PCR product 8 μl; Total volume 20 μl; Hybridoma Antibody Cloning Report PCR condition: Initial denaturing of 5 min at 95° C.; 25 cycles of 95° C. for 25 sec, 57° C. for 30 sec, 68° C. for 30 sec; Final extension is 10 min 68° C. [0274] After PCR is finished, run PCR reaction samples onto agarose gel to visualize DNA fragments amplified. After sequencing more than 15 cloned DNA fragments amplified by nested RT-PCR, several mouse antibody heavy and light chains have been cloned and appear correct. Protein sequence alignment and CDR analysis identifies one heavy chain and one light chain. As the amino acids on positions 26, 40 and 55 in the variable heavy chain and amino acid on position 40 in the variable light are critical to the binding properties, they may be reverted to the murine original. The resulting candidates are depicted below. (Padlan 1991. Mol. Immunol. 28, 489-498; Harris and Bajorath. 1995. Protein Sci. 4, 306-310).
[0275] Annotation for the antibody fragment sequences (SEQ ID No.: 13-22): bold and underline are the CDR 1, 2, 3 in chronologically arranged; italic are constant regions; hinge regions are highlighted with bold letters and the histidine tag with bold and italic letters.
TABLE-US-00020 SEQ ID No.: 13 (AM-VH-C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSG STNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYC WGQGTTLT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK
SEQ ID No.: 14 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGS GSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC
WGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK
SEQ ID No.: 15 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGS GSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC
WGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK
SEQ ID No.: 16 (AM-VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGS GSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC
WGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK
SEQ ID No.: 17 (AM-VH4-T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGS GSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC
WGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK
SEQ ID No.: 18 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSN RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No.: 19 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSN RDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No.: 20 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSN RDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Example 2
[0276] Development of an Immunoassay
[0277] An immunoassay was developed using antibodies generated against human ADM peptides (NT-H, MR-H and CT-H; see table 1).
[0278] Labelling Procedure (Tracer):
[0279] 100 μg (100 μl) of antibody (1 mg/ml in PBS, pH 7.4) was mixed with 10 μl Akridinium NHS-ester (1 mg/ml in acetonitrile, InVent GmbH, Germany) (EP 0 353 971) and incubated for 20 min at room temperature. Labelled CT-H was purified by Gel-filtration HPLC on Bio-Sil® SEC 400-5 (Bio-Rad Laboratories, Inc., USA). The purified labeled antibody was diluted in (300 mmol/L potassiumphosphate, 100 mmol/L NaCl, 10 mmol/L Na-EDTA, 5 g/L Bovine Serum Albumin, pH 7.0). The final concentration was approx. 800.000 relative light units (RLU) of labelled compound (approx. 20 ng labeled antibody) per 200 μL. Akridiniumester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).
[0280] Solid Phase:
[0281] Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated (18h at room temperature) with antibody (1.5 μg antibody/0.3 mL 100 mmol/L NaCl, 50 mmol/L TRIS/HCl, pH 7.8). After blocking with 5% bovine serum albumine, the tubes were washed with PBS, pH 7.4 and vacuum dried.
[0282] Calibrators:
[0283] Synthetic human ADM (hADM) (Bachem, Switzerland) was linearily diluted using 50 mM Tris/HCl, 250 mM NaCl, 0.2% Triton X-100, 0.5% BSA, 20 tabs/L Protease Complete Protease Inhibitor Cocktail Tablets (Roche AG); pH 7.8. Calibrators were stored at −20° C. before use.
[0284] ADM Immunoassay:
[0285] 50 μl of sample (or calibrator) was pipetted into coated tubes, after adding labeled second antibody (200 μl), the tubes were incubated for 2 h at room temperature. Unbound tracer was removed by washing 5 times (each 1 ml) with washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Tube-bound chemiluminescence was measured by using the LB 953 (Berthold Technologies GmbH & Co. KG). Antibodies were used in a sandwich immunoassay as coated tube and labeled antibody and combined in the following variations (see Table 2). Incubation was performed as described under hADM-Immunoassay. Results are given in ratio of specific signal (at 10 ng/ml ADM)/background (sample without ADM) signal.
TABLE-US-00021 TABLE 2 Signal/noise ratio NT-ADM tracer MR-ADM tracer CT-ADM tracer NT-ADM / 195 241 MR-ADM 204 / 904 CT-ADM 260 871 /
[0286] Surprisingly, we found the combination of MR-ADM and CT-ADM as combination for highest signal/noise ratio. Subsequently, we used this antibody-combination for further investigations to measure bio-ADM. We used anti-MR-ADM as solid phase antibody and anti-CT-ADM as labeled antibody. A typical dose/signal curve is shown in
Example 3
[0287] Stability of Human Adrenomedullin
[0288] Human ADM was diluted in human Citrate plasma (n=5, final concentration 10 ng ADM/ml) and incubated at 24° C. At selected time points, aliquots were frozen at −20° C. Immediately after thawing the samples hADM was quantified by using the hADM immunoassay described above.
[0289] Table 3 shows the stability of hADM in human plasma at 24° C.
TABLE-US-00022 Average ADM Relative loss Loss of recovery of immune immune Time (h) (N = 5) reactivity reactivity %/h 0 100 / / 2 99.2 0.8 0.4 4 96.4 3.6 0.8 8 88.2 11.8 1.5 Average: 0.9%/h
[0290] Surprisingly, using the antibody-combinations MR-ADM and CT-ADM in a sandwich immune assay, the pre-analytical stability of the analyte is high (only 0.9%/h average loss of immune reactivity). In contrast, using other assay methods, a plasma half-life of only 22 min was reported (Hinson et al. 2000 Endocrine Reviews 21(2):138-167). Since the time from taking sample to analysis in hospital routine is less than 2h, the used ADM detection method is suitable for routine diagnosis. It is remarkable, that any non-routine additives to samples (like aprotinin, (Ohta et al. 1999. Clin Chem 45 (2): 244-251)) are not needed to reach acceptable ADM-immune reactivity stabilities.
Example 4
[0291] Reproducibility of Calibrator-Preparations and Interpreparation Variation of Calibrators
[0292] We found a high variation of results, preparing calibrators for ADM assays (average CV 8.5%, see Table 4). This may be due to high adsorption of hADM to plastic and glass surfaces (Lewis et al. 1998. Clinical Chemistry 44 (3): 571-577). This effect was only slightly reduced by adding detergents (up to 1% Triton×100 or 1% Tween 20), protein (up to 5% BSA) and high ionic strength (up to 1M NaCl) or combinations thereof. Surprisingly, if a surplus of anti-NT-ADM antibody (10 μg/ml) is added to the calibrator dilution buffer, the recovery and reproducibility of ADM assay calibrator-preparations was substantially improved to <1% of inter preparation CV (Table 4). Coefficients of variation are given from 5 independent preparation runs. The calibrators were measured using the ADM assay described above (s/n-r=signal to noise ratio). For all following studies, we used an ADM assay, based on calibrators, prepared in the presence of 10 μg/ml of NT-ADM antibody and 10 μg/ml of NT-ADM antibody as supplement in the tracer buffer.
[0293] Fortunately, the presence of N-terminal antibodies did not affect the bio-ADM-signal generated by the combination of MR- and C-terminal antibodies (
TABLE-US-00023 TABLE 4 In the presence Inter Inter of NT-ADM preparation preparation antibody CV Without CV calibrator (10 μg/ml) (%) antibody (%) 100 ng/ml 3453 s/n-r 0.9 2842 s/n-r 2.8 10 ng/ml 1946 s/n-r 0.8 1050 s/n-r 7.9 1 ng/ml 179 s/n-r 1.1 77 s/n-r 14.8 Average: 0.93 Average: 8.5
Example 5
[0294] Sensitivity
[0295] The goal of assay sensitivity was to completely cover the ADM concentration of healthy subjects and considerably lower concentrations.
[0296] Bio-ADM Concentration in Healthy Subjects
[0297] Healthy subjects (n=88, 57 females, 31 males, mean age: 42.2 years) were measured using the bio-ADM assay (Weber et al. 2017. JALM, 2(2): 222-233). The median interquartile range (IQR) was 13.7 (9.6-18.7) pg/mL and mean (SD) was 15.6 (9.2) pg/mL. Since the assay sensitivity (limit of detection) was 3 pg/ml, 100% of healthy subjects were detectable using the described bio-ADM assay.
Example 6
[0298] Study Design and Population MPP
[0299] 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. Bio-ADM was measured from plasma collected during baseline evaluation in 4,364 patients. 34 of the patients were already diagnosed as having Alzheimer's disease at the time of blood-sampling. A number of 187 patients developed AD within the following 7 years (incident AD). An informed consent was obtained from all participants and the Ethical Committee of Lund University, Lund, Sweden, approved the study protocol.
[0300] 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 F00, 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. We have chosen a data set for a case control study and MR-proADM was measured in a sub-cohort of MPP (n=250 controls and n=150 subjects with incident AD). Moreover, bio-ADM was measured in an independent cohort of patients that have already been diagnosed with Alzheimer's disease at the time of blood sampling (=prevalent AD; n=150).
[0301] A commercial fully automated homogeneous time-resolved fluoroimmunoassay 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).
[0302] Statistical Analysis:
[0303] 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 analyse 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 bio-ADM from clinical variables we used the likelihood ratio chi-square test for nested models.
[0304] All statistical tests were 2-tailed and a two-sided p-value of 0.05 was considered for significance. The statistical analyses were performed using R version 2.5.1 (http://www.r-project.org, library Design, Hmisc, ROCR) and Statistical Package for the Social Sciences (SPSS) version 22.0 (SPSS Inc., Chicago, Ill., USA).
[0305] Results:
[0306] Baseline characteristics of the cohort are shown in table 5.
TABLE-US-00024 Variable n = 4364 Age 69.4 (6.2) gender male 3008 (68.9%) Current Smoking 835 (19.1%) AHT 1476 (33.8%) HDL 1.4 (0.4) LDL 3.7 (1.0) BMI 27.1 (6.2) SBP 146.6 (20.3) prevalent Diabetes 466 (10.7%) Incident AD 187 (4.3%) Prevalent AD 34 (0.8%)
[0307] The bio-ADM concentrations in the MPP cohort and in an independent Alzheimer disease cohort are shown in
[0308] Low bio-ADM plasma concentration strongly predicts Alzheimer's disease with a Hazard Ratio (HR) of 0.73 (CI 0.6-0.87; p<0.001).
[0309] We created a data set for case-control choosing 400 patients from the MPP cohort (free from prior CVD and AD) with n=250 subjects who did not develop AD and n=150 subjects who did develop AD within the follow-up time of 7 years. Again, bio-ADM concentrations were significantly lower (p<0.0001 for all comparisons) in patients with incident AD and in patients with prevalent AD (independent cohort) compared to the non-AD group (
[0310] In a next step, we combined both biomarkers, bio-ADM and MR-proADM. For the calculation of the ratio, the concentration of the two markers has to be preferably expressed in the same unit (e.g. pmol/L). Therefore, in terms of calculating the ratio, concentrations for bio-ADM were calculated in pmol/L. The ratio of bio-ADM and MR-proADM is significantly decreased in subjects with incident AD when compared to non-AD subjects (p<0.0001;
Example 7—Administration of NT-H in Healthy Humans
[0311] The study was conducted in healthy male subjects as a randomized, double-blind, placebo-controlled, study with single escalating doses of NT-H antibody administered as intravenous (i.v.) infusion in 3 sequential groups of 8 healthy male subjects each (1st group 0.5 mg/kg, 2nd group 2 mg/kg, 3rd group 8 mg/kg) of healthy male subjects (n=6 active, n=2 placebo for each group).
[0312] The main inclusion criteria were written informed consent, age 18-35 years, agreement to use a reliable way of contraception and a BMI between 18 and 30 kg/m.sup.2.
[0313] Subjects received a single i.v. dose of NT-H antibody (0.5 mg/kg; 2 mg/kg; 8 mg/kg) or placebo by slow infusion over a 1-hour period in a research unit.
[0314] The baseline bio-ADM-values in the 4 groups did not differ. Median bio-ADM values were 7.1 pg/mL in the placebo group, 6.8 pg/mL in the first treatment group (0.5 mg/kg), 5.5 pg/mL in second treatment group (2 mg/kg) and 7.1 pg/mL in the third treatment group (8 mg/mL).
[0315] The results show, that bio-ADM-values rapidly increased within the first 1.5 hours after administration of NT-H antibody in healthy human individuals, then reached a plateau and slowly declined (