ANTI-ADRENOMEDULLIN (ADM) ANTIBODY OR ANTI-ADM ANTIBODY FRAGMENT OR ANTI-ADM NON-IG SCAFFOLD FOR USE IN THERAPY OR PREVENTION OF SHOCK
20230104578 · 2023-04-06
Assignee
Inventors
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
C07K16/22
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K16/26
CHEMISTRY; METALLURGY
A61K2039/545
HUMAN NECESSITIES
C07K2317/24
CHEMISTRY; METALLURGY
International classification
Abstract
The present application is directed to an anti-Adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of shock in a patient, wherein said patient is characterized by having a level of dipeptidyl peptidase 3 (DPP3) in a sample of bodily fluid below a threshold and said anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal part (amino acid 1-21) of ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID No. 14).
Claims
1. A method for therapy or prevention of shock in a patient, comprising administering an effective amount of an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold to said patient in need of therapy or prevention of shock, wherein said patient is characterized by having a level of dipeptidyl peptidase 3 (DPP3) in a sample of bodily fluid below a threshold and said anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal part (amino acid 1-21) of ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID No. 14).
2. A method according to claim 1, wherein said shock is selected from the group comprising shock due to hypovolemia, cardiogenic shock, obstructive shock and distributive shock, in particular cardiogenic shock or septic shock.
3. A method according to claim 1, wherein in case of cardiogenic shock said patient may have suffered an acute coronary syndrome (e.g. acute myocardial infarction) or wherein said patient has heart failure (e.g. acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordal rupture or massive pulmonary embolism, or in case of hypovolemic shock said patient may have suffered a hemorrhagic disease including gastrointestinal bleed, trauma, vascular etiologies (e.g. ruptured abdominal aortic aneurysm, tumor eroding into a major blood vessel) and spontaneous bleeding in the setting of anticoagulant use or a non-hemorrhagic disease including vomiting, diarrhea, renal loss, skin losses/insensible losses (e.g. burns, heat stroke) or third-space loss in the setting of pancreatitis, cirrhosis, intestinal obstruction, trauma, or in case of obstructive shock said patient may have suffered a cardiac tamponade, tension pneumothorax, pulmonary embolism or aortic stenosis, or in case of distributive shock said patient may have septic shock, neurogenic shock, anaphylactic shock or shock due to adrenal crisis.
4. A method according to claim 1, wherein said threshold of DPP3 in a sample of bodily fluid of said patient is between 20 and 120 ng/mL, more preferred between 30 and 80 ng/mL, even more preferred between 40 and 60 ng/mL, most preferred said threshold is 50 ng/mL.
5. (canceled)
6. A method according to claim 1, wherein the bodily fluid is selected from whole blood, plasma and serum, preferably plasma.
7. (canceled)
8. A method according to claim 1, wherein the level of DPP3 is determined by contacting said sample of bodily fluid with a capture binder that binds specifically to DPP3, preferably wherein the capture binder is an antibody.
9. (canceled)
10. A method according to claim 1, wherein either the level of DPP3 protein and/or the level of active DPP3 is determined and compared to a predetermined threshold; or wherein said patient is additionally characterized by having a level of ADM-NH.sub.2 above a threshold; or wherein said threshold of ADM-NH.sub.2 in a sample of bodily fluid of said patient is between 40 and 100 pg/mL, more preferred between 50 and 90 pg/mL, even more preferred between 60 and 80 pg/mL, most preferred said threshold is 70 pg/mL; or wherein said threshold of ADM-NH2 in a sample of bodily fluid of said patient is 70 pg/mL.
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. A method according to claim 10, wherein the level of ADM-NH.sub.2 is determined by contacting said sample of bodily fluid with a capture binder that binds specifically to ADM-NH.sub.2, preferably wherein the capture binder is an antibody.
17. (canceled)
18. A method according to claim 1, wherein the sample of bodily fluid of said patient is selected from the group of blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.
19. A method according to claim 1, wherein said anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to the N-terminal end (amino acid 1) of ADM-Gly and/or ADM-NH.sub.2.
20. A method according to claim 1, wherein said antibody, antibody fragment or non-Ig scaffold does not bind to the C-terminal portion of ADM, having the sequence amino acid 43-52 of ADM: PRSKISPQGY-NH.sub.2 (SEQ ID NO: 24); or wherein said antibody or fragment or scaffold blocks the bioactivity of ADM not more than 80%, preferably not more than 50%.
21. (canceled)
22. A method according to claim 1, wherein said antibody or fragment is a monoclonal antibody or fragment that binds to ADM or an antibody fragment thereof, wherein the heavy chain comprises the sequences: TABLE-US-00045 CDR1: SEQ ID NO: 1 GYTFSRYW CDR2: SEQ ID NO: 2 ILPGSGST CDR3: SEQ ID NO: 3 TEGYEYDGFDY and wherein the light chain comprises the sequences: TABLE-US-00046 CDR1: SEQ ID NO: 4 QSIVYSNGNTY CDR2: RVS CDR3: SEQ ID NO: 5 FQGSHIPYT.
23. A method according to claim 1, wherein said antibody or fragment comprises a sequence selected from the group comprising as a VH region: TABLE-US-00047 (AM-VH-C) SEQ ID NO: 6 QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGS GSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQ GTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK (AM-VH1) SEQ ID NO: 7 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPG SGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK (AM-VH2-E40) SEQ ID NO: 8 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPG SGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK (AM-VH3-T26-E55) SEQ ID NO: 9 QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPG SGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK (AM-VH4-T26-E40-E55) SEQ ID NO: 10 QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPG SGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK and comprises a sequence selected from the group comprising the following sequence as a VL region: TABLE-US-00048 (AM-VL-C) SEQ ID NO: 11 DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (AM-VL1) SEQ ID NO: 12 DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVS NRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (AM-VL2-E40) SEQ ID NO: 13 DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVS NRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
24. A method according to claim 1, wherein said antibody or fragment comprises the following sequence as a heavy chain: TABLE-US-00049 SEQ ID NO: 32 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGE ILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK or a sequence that is >95% identical to it, and comprises the following sequence as a light chain: TABLE-US-00050 SEQ ID NO: 33 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPR LLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC or a sequence that is >95% identical to it.
25. A method according to claim 1, wherein the anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold binds to the N-terminal part (amino acid 1-10) of ADM: YRQSMNNFQG (SEQ ID No. 26).
26. A method according to claim 1, wherein said antibody or fragment or scaffold exhibits a binding affinity to ADM of at least 10.sup.−7 M by label-free surface plasmon resonance using a Biacore 2000 system; or wherein the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold exhibits an affinity towards human ADM is between 1×10.sup.−9 to 3×10.sup.−9 by label-free surface plasmon resonance using a Biacore 2000 system.
27. (canceled)
28. A method according to claim 25, wherein the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is an IgG1 antibody.
29. A method according to claim 1, wherein the anti-adrenomedullin (ADM) antibody or an anti-ADM antibody fragment or anti-ADM non-Ig scaffold is in a pharmaceutical formulation with a pharmaceutically acceptable excipient.
30. A method according to claim 29, wherein said pharmaceutical formulation is a solution, preferably a ready-to-use solution; or wherein said pharmaceutical formulation is in a freeze-dried state.
31. (canceled)
32. A method according to claim 29, wherein said pharmaceutical formulation is administered intra-muscular; or wherein said pharmaceutical formulation is administered intra-vascular; or wherein said pharmaceutical formulation is administered via infusion; wherein said pharmaceutical formulation is to be administered systemically.
33. (canceled)
34. (canceled)
35. (canceled)
Description
FIGURE DESCRIPTION
[0297]
[0298] Illustration of antibody formats—Fv and scFv-Variants.
[0299]
[0300] Illustration of antibody formats—heterologous fusions and bifunctional antibodies.
[0301]
[0302] Illustration of antibody formats—bivalental antibodies and bispecific antibodies.
[0303]
[0316]
[0317] This figure shows a typical hADM dose/signal curve. And an hADM dose signal curve in the presence of 100 μg/mL antibody NT-H.
[0318]
[0319] This figure shows the stability of hADM in human plasma (citrate) in absence and in the presence of NT-H antibody.
[0320]
[0321] Alignment of the Fab with homologous human framework sequences.
[0322]
[0323]
[0324]
[0325]
[0326]
[0327]
EXAMPLES
[0328] Example 1—Generation of Antibodies and determination of their affinity constants Several human and murine antibodies were produced and their affinity constants were determined (see tables 1 and 2). It should be emphasized that the antibodies, antibody fragments and non-Ig scaffolds of the example portion in accordance with the invention are binding to ADM, and thus should be considered as anti-ADM antibodies/antibody fragments/non-Ig scaffolds.
[0329] Peptides/Conjugates for Immunization:
[0330] 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.
[0331] Mouse Monoclonal Antibody Production:
[0332] 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. 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. 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 re-cloned 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. Horn. Metab. Res. 28: 11-15).
[0333] 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.
[0334] Human Antibodies:
[0335] Human Antibodies were produced by means of phage display according to the following procedure: The human naive antibody gene libraries HAL7/8 were used for the isolation of recombinant single chain F-Variable domains (scFv) against adrenomedullin 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 adrenomedullin 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; Schiitte et al. 2009. PLoS One 4, e6625). 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., J. Biotechn. 2011), captured from the culture supernatant via immobilized metal ion affinity chromatography and purified by a size exclusion chromatography.
[0336] Affinity Constants: 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 CM5 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).
[0337] 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-00036 TABLE 1 immunization peptides Affinity Sequence ADM constants Number Antigen/Immunogen Region Designation Kd (M) SEQ ID: 14 YRQSMNNFQGLRSFGCRFGTC 1-21 NT-H 5.9 × 10.sup.−9 SEQ ID: 15 CTVQKLAHQIYQ 21-32 MR-H 2 × 10.sup.−9 SEQ ID: 16 CAPRSKISPQGY-NH.sub.2 C-42-52 CT-H 1.1 × 10.sup.−9 SEQ ID: 17 YRQSMNQGSRSNGCRFGTC 1-19 NT-M 3.9 × 10.sup.−9 SEQ ID: 18 CTFQKLAHQIYQ 19-31 MR-M 4.5 × 10.sup.−10 SEQ ID: 19 CAPRNKISPQGY-NH.sub.2 C-40-50 CT-M 9 × 10.sup.−9
[0338] The following is a list of further obtained monoclonal antibodies:
TABLE-US-00037 TABLE 2 max inhibition Clone Affinity bioassay (%) Target Source number (M) (see example 2) NT-M Mouse ADM/63 5.8 × 10.sup.−9 45 Mouse ADM/364 2.2 × 10.sup.−8 48 Mouse ADM/365 3.0 × 10.sup.−8 Mouse ADM/366 1.7 × 10.sup.−8 Mouse ADM/367 1.3 × 10.sup.−8 Mouse ADM/368 1.9 × 10.sup.−8 Mouse ADM/369 2.0 × 10.sup.−8 Mouse ADM/370 1.6 × 10.sup.−8 Mouse ADM/371 2.0 × 10.sup.−8 Mouse ADM/372 2.5 × 10.sup.−8 Mouse ADM/373 1.8 × 10.sup.−8 Mouse ADM/377 1.5 × 10.sup.−8 Mouse ADM/378 2.2 × 10.sup.−8 Mouse ADM/379 1.6 × 10.sup.−8 Mouse ADM/380 1.8 × 10.sup.−8 Mouse ADM/381 2.4 × 10.sup.−8 Mouse ADM/382 1.6 × 10.sup.−8 Mouse ADM/383 1.8 × 10.sup.−8 Mouse ADM/384 1.7 × 10.sup.−8 Mouse ADM/385 1.7 × 10.sup.−8 Mouse ADM/403 1.2 × 10.sup.−8 Mouse ADM/395 1.2 × 10.sup.−8 Mouse ADM/396 3.0 × 10.sup.−8 Mouse ADM/397 1.5 × 10.sup.−8 MR-M Mouse ADM/38 .sup. 4.5 × 10.sup.−10 68 MR-M Mouse ADM/39 5.9 × 10.sup.−9 72 CT-M Mouse ADM/65 9.0 × 10.sup.−9 100 CT-M Mouse ADM/66 1.6 × 10.sup.−8 100 NT-H Mouse ADM/33 5.9 × 10.sup.−8 38 NT-H Mouse ADM/34 1.6 × 10.sup.−8 22 MR-H Mouse ADM/41 1.2 × 10.sup.−8 67 MR-H Mouse ADM/42 <1 × 10.sup.−8 MR-H Mouse ADM/43 2.0 × 10.sup.−9 73 MR-H Mouse ADM/44 <1 × 10.sup.−8 CT-H Mouse ADM/15 <1 × 10.sup.−8 CT-H Mouse ADM/16 1.1 × 10.sup.−9 100 CT-H Mouse ADM/17 3.7 × 10.sup.−9 100 CT-H Mouse ADM/18 <1 × 10.sup.−8 hADM Phage display ADM/A7 <1 × 10.sup.−8 Phage display ADM/B7 <1 × 10.sup.−8 Phage display ADM/C7 <1 × 10.sup.−8 Phage display ADM/G3 <1 × 10.sup.−8 Phage display ADM/B6 <1 × 10.sup.−8 Phage display ADM/B11 <1 × 10.sup.−8 Phage display ADM/D8 <1 × 10.sup.−8 Phage display ADM/D11 <1 × 10.sup.−8 Phage display ADM/G12 <1 × 10.sup.−8
[0339] Generation of antibody fragments by enzymatic digestion: The generation of Fab and F(ab).sub.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).sub.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).sub.2-fragmentation for 8 h at 37° C. The Fab-fragmentation digestion was carried out for 16 h, respectively.
[0340] Procedure for Fab Generation and Purification: 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).
[0341] Procedure for generation and purification of F(ab′12 Fragments: 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).
[0342] NT-H-Antibody Fragment Humanization: The antibody fragment was humanized by the CDR-grafting method (Jones et al. 1986 Nature 321, 522-525).
[0343] The following steps where done to achieve the humanized sequence:
[0344] Total RNA extraction: Total RNA was extracted from NT-H hybridomas using the Qiagen kit. 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.
[0345] 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.
[0346] 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.
[0347] After PCR was finished, PCR reaction samples were run 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. After alignment with homologous human framework sequences the resulting humanized sequence for the variable heavy chain is the following: see
[0348] Annotation for the antibody fragment sequences (SEQ ID No.: 6-13; 32 and 33): bold and underlined are the CDR 1, 2, 3 in italic are constant regions; hinge regions are highlighted with bold letters; framework point mutation have a grey letter-background.
TABLE-US-00038 SEQ ID No.: 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYN EKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYC WGQGTTLTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPK SEQ ID No.: 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNY AQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC
WGQGTTVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPK SEQ ID No.: 8 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNY AQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC
WGQGTTVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPK SEQ ID No.: 9 (AM-VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNY AQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC
WGQGTTVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPK SEQ ID No.: 10 (AM-VH4-T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNY AQKFQGRVTITDESTSTAYMELSSLRSEDTAVYYC
WGQGTTVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPK SEQ ID No.: 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVP DRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No.: 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGV PDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No.: 13 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGV PDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 32 (Adrecizumab heavy chain) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYN QKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 33 (Adrecizumab light chain) DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC
Example 2—Effect of Selected Anti-ADM-Antibodies on Anti-ADM-Bioactivity
[0349] The effect of selected ADM-antibodies on ADM-bioactivity was tested in a human recombinant Adrenomedullin receptor cAMP functional assay (Adrenomedullin Bioassay).
[0350] Testing of Antibodies Targeting Human or Mouse Adrenomedullin in Human Recombinant Adrenomedullin Receptor cAMP Functional Assay (Adrenomedullin Bioassay)
[0351] Materials: Cell line CHO-K1, Receptor Adrenomedullin (CRLR+RAMP3), Receptor Accession Number Cell line: CRLR: U17473; RAMP3: AJ001016
[0352] CHO-K1 cells expressing human recombinant adrenomedullin receptor (FAST-027C) grown prior to the test in media without antibiotic were detached by gentle flushing with PBS-EDTA (5 mM EDTA), recovered by centrifugation and resuspended in assay buffer (KRH: 5 mM KCl, 1.25 mM MgSO.sub.4, 124 mM NaCl, 25 mM HEPES, 13.3 mM Glucose, 1.25 mM KH.sub.2PO.sub.4, 1.45 mM CaCl.sub.2, 0.5 g/l BSA).
[0353] Dose response curves were performed in parallel with the reference agonists (hADM or mADM).
[0354] Antagonist test (96well): For antagonist testing, 6 μl of the reference agonist (human (5.63 nM) or mouse (0.67 nM) adrenomedullin) was mixed with 6 μl of the test samples at different antagonist dilutions; or with 6 μl buffer. After incubation for 60 min at room temperature, 12 μl of cells (2,500 cells/well) were added. The plates were incubated for 30 min at room temperature. After addition of the lysis buffer, percentage of DeltaF will be estimated, according to the manufacturer specification, with the HTRF kit from Cis-Bio International (cat n° 62AM2 PEB) hADM 22-52 was used as reference antagonist.
[0355] Antibodies Testing cAMP-HTRF Assay
[0356] The anti-h-ADM antibodies (NT-H, MR-H, CT-H) were tested for antagonist activity in human recombinant adrenomedullin receptor (FAST-027C) cAMP functional assay in the presence of 5.63 nM Human ADM 1-52, at the following final antibody concentrations: 100 μg/ml, 20 μg/ml, 4 μg/ml, 0.8 μg/ml, 0.16 μg/ml.
[0357] The anti-m-ADM antibodies (NT-M, MR-M, CT-M) were tested for antagonist activity in human recombinant ADM receptor (FAST-027C) cAMP functional assay in the presence of 0.67 nM Mouse ADM 1-50, at the following final antibody concentrations: 100 μg/ml, 20 μg/ml, 4 μg/ml, 0.8 μg/ml, 0.16 μg/ml. Data were plotted relative inhibition vs. antagonist concentration (see
TABLE-US-00039 TABLE 3 maximal inhibition of the bio-ADM activity Maximal inhibition of ADM bioactivity Antibody (ADM-Bioassay) (%) NT-H 38 MR-H 73 CT-H 100 NT-M FAB 26 NT-M FAB2 28 NT-M 45 MR-M 66 CT-M 100 Non specific 0 mouse IgG
Example 3—Stabilization of hADM by the Anti-ADM Antibody
[0358] The stabilizing effect of human ADM by human ADM antibodies was tested using a hADM immunoassay.
[0359] Immunoassay for the Quantification of Human Adrenomedullin
[0360] The technology used was a sandwich coated tube luminescence immunoassay, based on Acridinium ester labelling.
[0361] Labelled compound (tracer): 100 μg (100 μl) CT-H (1 mg/ml in PBS, pH 7.4, AdrenoMed AG Germany) was mixed with 10 μl Acridinium NHS-ester (1 mg/ml in acetonitrile, InVent GmbH, Germany) (EP 0353971) 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 CT-H was diluted in (300 mmol/L potassium phosphate, 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. Acridiniumester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).
[0362] Solid phase: Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated (18 h at room temperature) with MR-H (AdrenoMed AG, Germany) (1.5 μg MR-H/0.3 mL 100 mmol/L NaCl, 50 mmol/L TRIS/HCl, pH 7.8). After blocking with 5% bovine serum albumin, the tubes were washed with PBS, pH 7.4 and vacuum dried.
[0363] Calibration: The assay was calibrated, using dilutions of hADM (BACHEM AG, Switzerland) in 250 mmol/L NaCl, 2 g/L Triton X-100, 50 g/L Bovine Serum Albumin, 20 tabs/L Protease Inhibitor Cocktail (Roche Diagnostics AG, Switzerland).
[0364] hADM Immunoassay: 50 μl of sample (or calibrator) was pipetted into coated tubes, after adding labeled CT-H (200 μl), the tubes were incubated for 4 h at 4° C. 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).
[0365] Tube-bound chemiluminescence was measured by using the LB 953:
[0366] Stability of human Adrenomedullin: Human ADM was diluted in human Citrate plasma (final concentration 10 nM) and incubated at 24° C. At selected time points, the degradation of hADM was stopped by freezing at −20° C. The incubation was performed in absence and presence of NT-H (100 μg/ml). The remaining hADM was quantified by using the hADM immunoassay described above.
[0367]
Example 4—Sepsis Mortality
[0368] a) Early Treatment of Sepsis
[0369] Animal model: 12-15 week-old male C57Bl/6 mice (Charles River Laboratories, Germany) were used for the study. Peritonitis had been surgically induced under light isofluran anesthesia. Incisions were made into the left upper quadrant of the peritoneal cavity (normal location of the cecum). The cecum was exposed and a tight ligature was placed around the cecum with sutures distal to the insertion of the small bowel. One puncture wound was made with a 24-gauge needle into the cecum and small amounts of cecal contents were expressed through the wound. The cecum was replaced into the peritoneal cavity and the laparotomy site was closed. Finally, animals were returned to their cages with free access to food and water. 500 μl saline were given s.c. as fluid replacement.
[0370] Application and dosage of the compound (NT-M, MR-M, CT-M): Mice were treated immediately after CLP (early treatment). CLP is the abbreviation for cecal ligation and puncture (CLP).
[0371] Study groups: Three compounds were tested versus: vehicle and versus control compound treatment. Each group contained 5 mice for blood drawing after 1 day for BUN (serum blood urea nitrogen test) determination. Ten further mice per each group were followed over a period of 4 days.
[0372] Group Treatment (10 μl/g bodyweight) dose/Follow-Up: [0373] 1 NT-M, 0.2 mg/ml survival over 4 days [0374] 2 MR-M, 0.2 mg/ml survival over 4 days [0375] 3 CT-M, 0.2 mg/ml survival over 4 days [0376] 4 non-specific mouse IgG, 0.2 mg/ml survival over 4 days [0377] 5 control—PBS 10 μl/g bodyweight survival over 4 days
[0378] Clinical chemistry: Blood urea nitrogen (BUN) concentrations for renal function were measured baseline and day 1 after CLP. Blood samples were obtained from the cavernous sinus with a capillary under light ether anaesthesia. Measurements were performed by using an AU 400 Olympus Multianalyser. The 4-day mortality and the average BUN concentrations are given in table 4.
TABLE-US-00040 TABLE 4 4-day mortality and BUN concentrations survival BUN pre CLP BUN day 1 4-day mortality (%) (mM) (mM) PBS 0 8.0 23.2 non-specific mouse IgG 0 7.9 15.5 CT-M 10 7.8 13.5 MR-M 30 8.1 24.9 NT-M 70 8.8 8.2
[0379] It can be seen from Table 4 that the NT-M antibody reduced mortality considerably. After 4 days 70% of the mice survived when treated with NT-M antibody. When treated with MR-M antibody 30% of the animals survived and when treated with CT-M antibody 10% of the animals survived after 4 days. In contrast thereto all mice were dead after 4 days when treated with unspecific mouse IgG. The same result was obtained in the control group where PBS (phosphate buffered saline) was administered to mice. The blood urea nitrogen or BUN test is used to evaluate kidney function, to help diagnose kidney disease, and to monitor patients with acute or chronic kidney dysfunction or failure. The results of the S-BUN Test revealed that the NT-M antibody was the most effective to protect the kidney.
[0380] b) Late Treatment of Sepsis
[0381] Animal model: 12-15 week-old male C57Bl/6 mice (Charles River Laboratories, Germany) were used for the study. Peritonitis had been surgically induced under light isofluran anesthesia. Incisions were made into the left upper quadrant of the peritoneal cavity (normal location of the cecum). The cecum was exposed and a tight ligature was placed around the cecum with sutures distal to the insertion of the small bowel. One puncture wound was made with a 24-gauge needle into the cecum and small amounts of cecal contents were expressed through the wound. The cecum was replaced into the peritoneal cavity and the laparotomy site was closed. Finally, animals were returned to their cages with free access to food and water. 500 μl saline were given s.c. as fluid replacement.
[0382] Application and dosage of the compound (NT-M FAB2): NT-M FAB2 was tested versus: vehicle and versus control compound treatment. Treatment was performed after full development of sepsis, 6 hours after CLP (late treatment). Each group contained 4 mice and were followed over a period of 4 days.
[0383] Group Treatment (10 μl/g bodyweight) dose/Follow-Up: [0384] 1 NT-M, FAB2 0.2 mg/ml survival over 4 days [0385] 2 control non-specific mouse IgG, 0.2 mg/ml survival over 4 days [0386] 3 vehicle:—PBS 10 μl/g bodyweight survival over 4 days
TABLE-US-00041 TABLE 5 4-day mortality 4 day mortality survival (%) PBS 0 Non-specific mouse IgG 0 NT-M FAB2 75
[0387] It can be seen from Table 5 that the NT-M FAB 2 antibody reduced mortality considerably. After 4 days 75% of the mice survived when treated with NT-M FAB 2 antibody. In contrast thereto all mice were dead after 4 days when treated with non-specific mouse IgG. The same result was obtained in the control group where PBS (phosphate buffered saline) was administered to mice.
Example 5—Administration of NT-H in Healthy Humans
[0388] 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). 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. 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. The baseline ADM-values in the 4 groups did not differ. Median 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). The results show, that 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 (
Example 6—Methods for the Measurement of DPP3 Protein and DPP3 Activity
[0389] Generation of antibodies and determination DPP3 binding ability: Several murine antibodies were produced and screened by their ability of binding human DPP3 in a specific binding assay (see Table 6).
[0390] Peptides/conjugates for immunization: DPP3 peptides for immunization were synthesized, see Table 6, (JPT Technologies, Berlin, Germany) with an additional N-terminal cystein (if no cystein is present within the selected DPP3-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 GST-hDPP3 was produced by USBio (United States Biological, Salem, Mass., USA).
[0391] Immunization of mice, immune cell fusion and screening: Balb/c mice were intraperitoneally (i.p.) injected with 84 μg GST-hDPP3 or 100 μg DPP3-peptide-BSA-conjugates at day 0 (emulsified in TiterMax Gold Adjuvant), 84 μg or 100 μg at day 14 (emulsified in complete Freund's adjuvant) and 42 μg or 50 μg at day 21 and 28 (in incomplete Freund's adjuvant). At day 49 the animal received an intravenous (i.v.) injection of 42 μg GST-hDPP3 or 50 μg DPP3-peptide-BSA-conjugates dissolved in saline. Three days later the mice were sacrificed and the immune cell fusion was performed.
[0392] 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. The cell culture supernatants were primarily screened for recombinant DPP3 binding IgG antibodies two weeks after fusion. Therefore, recombinant GST-tagged hDPP3 (USBiologicals, Salem, USA) was immobilized in 96-well plates (100 ng/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. After a next washing step, 50 μl of a chromogen solution (3.7 mM o-phenylen-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.
[0393] 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.
[0394] Mouse Monoclonal Antibody Production
[0395] Antibodies raised against GST-tagged human DPP3 or DPP3-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.
[0396] Characterization of Antibodies—Binding to hDPP3 and/or Immunization Peptide
[0397] To analyze the capability of DPP3/immunization peptide binding by the different antibodies and antibody clones a binding assay was performed:
[0398] Solid phase: Recombinant GST-tagged hDPP3 (SEQ ID NO. 34) or a DPP3 peptide (immunization peptide, SEQ ID NO. 35) was immobilized onto a high binding microtiter plate surface (96-Well polystyrene microplates, Greiner Bio-One international AG, Austria, 1 μg/well in coupling buffer [50 mM Tris, 100 mM NaCl, pH7.8], 1 h at RT). After blocking with 5% bovine serum albumin, the microplates were vacuum dried.
[0399] Labelling procedure (tracer): 100 μg (100 μl) of the different antiDPP3 antibodies (detection antibody, 1 mg/ml in PBS, pH 7.4) were mixed with 10 μl acridinium NHS-ester (1 mg/ml in acetonitrile, InVent GmbH, Germany; EP 0 353 971) and incubated for 30 min at room temperature. Labelled antiDPP3 antibody was purified by gel-filtration HPLC on Shodex Protein 5 μm KW-803 (Showa Denko, Japan). The purified labelled antibody was diluted in assay buffer (50 mmol/l potassium phosphate, 100 mmol/l NaCl, 10 mmol/l Na.sub.2-EDTA, 5 g/l bovine serum albumin, 1 g/l murine IgG, 1 g/l bovine IgG, 50 μmol/l amastatin, 100 μmol/l leupeptin, pH 7.4). The final concentration was approx. 5-7*10.sup.6 relative light units (RLU) of labelled compound (approx. 20 ng labelled antibody) per 200 μl. acridinium ester chemiluminescence was measured by using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).
[0400] hDPP3 binding assay: the plates were filled with 200 μl of labelled and diluted detection antibody (tracer) and incubated for 2-4 h at 2-8° C. Unbound tracer was removed by washing 4 times with 350 μl washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Well-bound chemiluminescence was measured by using the Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).
[0401] Characterization of Antibodies—hDPP3-Inhibition Analysis
[0402] To analyze the capability of DPP3 inhibition by the different antibodies and antibody clones a DPP3 activity assay with known procedure (Jones et al., 1982) was performed. Recombinant GST-tagged hDPP3 was diluted in assay buffer (25 ng/ml GST-DPP3 in 50 mM Tris-HCl, pH7.5 and 100 μM ZnCl.sub.2) and 200 μl of this solution incubated with 10 μg of the respective antibody at room temperature. After 1 hour of pre-incubation, fluorogenic substrate Arg-Arg-βNA (20 μl, 2 mM) was added to the solution and the generation of free βNA over time was monitored using the Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG) at 37° C. Fluorescence of βNA is detected by exciting at 340 nm and measuring emission at 410 nm. Slopes (in RFU/min) of increasing fluorescence of the different samples are calculated. The slope of GST-hDPP3 with buffer control is appointed as 100% activity. The inhibitory ability of a possible capture-binder is defined as the decrease of GST-hDPP3 activity by incubation with said capture-binder in percent.
[0403] The following table represents a selection of obtained antibodies and their binding rate in Relative Light Units (RLU) as well as their relative inhibitory ability (%; table 6). The monoclonal antibodies raised against the below depicted DPP3 regions, were selected by their ability to bind recombinant DPP3 and/or immunization peptide, as well as by their inhibitory potential.
[0404] All antibodies raised against the GST-tagged, full length form of recombinant hDPP3 show a strong binding to immobilized GST-tagged hDPP3. Antibodies raised against the SEQ ID NO.: 35 peptide bind as well to GST-hDPP3. The SEQ ID NO.: 35 antibodies also strongly bind to the immunization peptide.
TABLE-US-00042 TABLE 6 list of antibodies raised against full-length or sequences of hDPP3 and their ability to bind hDPP3 (SEQ ID NO.: 34) or immunization peptide (SEQ ID NO.: 35) in RLU, as well as the maximum inhibition of recombinant GST-hDPP3. hDPP3 immunization Max. Sequence hDPP3 binding peptide inhibition number Antigen/ Immunogen region Clone [RLU] binding [RLU] of hDPP3 SEQ ID GST tagged recombinant FL-hDPP3 1-737 2552 3.053.621 0 65% NO.: 34 2553 3.777.985 0 35% 2554 1.733.815 0 30% 2555 3.805.363 0 25% SEQ ID CETVINPETGEQIQSWYRSGE 474-493 1963 141.822 2.163.038 60% NO.: 35 1964 100.802 2.041.928 60% 1965 99.493 1.986.794 70% 1966 118.097 1.990.702 65% 1967 113.736 1.909.954 70% 1968 105.696 2.017.731 65% 1969 82.558 2.224.025 70%
[0405] The development of a luminescence immunoassay for the quantification of DPP3 protein concentrations (DPP3-LIA) as well as an enzyme capture activity assay for the quantification of DPP3 activity (DPP3-ECA) have been described recently (Rehfeld et al. 2019. JALM 3(6). 943-953), which is incorporated here in its entirety by reference.
Example 7—DPP3 in Shock
[0406] DPP3 concentration in plasma of patients with sepsis/septic shock and cardiogenic shock was determined and related to the short term-mortality of the patients.
[0407] a) Study Cohort—Sepsis/Septic Shock
[0408] In 574 plasma samples from patients of the Adrenomedullin and Outcome in Severe Sepsis and Septic Shock (AdrenOSS-1) study DPP3 was measured. AdrenOSS-1 is a prospective, observational, multinational study including 583 patients admitted to the intensive care unit with sepsis or septic shock (Hollinger et al., 2018 Aug. 22;3(6):1424-1433). 292 patients were diagnosed with septic shock.
[0409] b) Study Cohort—Cardiogenic Shock
[0410] Plasma samples from 108 patients that were diagnosed with cardiogenic shock were screened for DPP3. Blood was drawn within 6 h from detection of cardiogenic shock. Mortality was followed for 7 days.
[0411] hDPP3 immunoassay: An immunoassay (LIA) or an activity assays (ECA) detecting the amount of human DPP3 (LIA) or the activity of human DPP3 (ECA), respectively, was used for determining the DPP3 level in patient plasma. Antibody immobilization, labelling and incubation were performed as described in Rehfeld et al. (Rehfeld et al. 2019. JALM 3(6): 943-953).
[0412] Results: Short-term patients' survival in sepsis patients was related to the DPP3 plasma concentration at admission. Patients with DPP3 plasma concentration above 40.5 ng/mL (3rd Quartile) had an increased mortality risk compared to patients with DPP3 plasma concentrations below this threshold (
Example 8—NT-ADM Antibodies in Patients with Septic Shock (AdrenOSS-2
[0413] AdrenOSS-2 is a double-blind, placebo-controlled, randomized, multicenter, proof of concept and dose-finding phase II clinical trial to investigate the safety, tolerability and efficacy of the N-terminal ADM antibody named Adrecizumab in patients with septic shock and elevated adrenomedullin (Geven et al. BMJ Open 2019;9:e024475). In total, 301 patients with septic shock and bio-ADM concentration >70 pg/mL were randomized (2:1:1) to treatment with a single intravenous infusion over approximately 1 hour with either placebo (n=152), adrecizumab 2 ng/kg (n=72) or adrecizumab 4 ng/kg (n=77). All-cause mortality within 28 (90) days after inclusion was 25.8% (34.8%). Mean age was 68.4 years and 61% were male. For the per protocol analysis, n=294 patients remained eligible, and 14-day all-cause mortality rate was 18.5%.
[0414] In patients treated with Adrecizumab (both doses combined, per protocol population), a trend to lower short term mortality (14 days post admission) was observed compared to placebo (Hazard ratio (HR) 0.701 [0.408-1.21], p=0.100) (
[0415] Treatment effects for different DPP3 thresholds (14-day mortality) are summarized in table 7.
TABLE-US-00043 TABLE 7 Hazard risks (HR) for 14-day mortality with different pre-dose DPP3 concentrations p-value n HR (1-sided, log rank) all 294 0.701 0.100 DPP3 < 90 273 0.606 0.049 DPP3 < 70 261 0.546 0.027 DPP3 < 60 254 0.449 0.008 DPP3 < 50 244 0.426 0.007 DPP3 < 40 227 0.385 0.005
Example 9
[0416] To show that the anti-ADM treatment works also in patients with bio-ADM levels lower than 70 pg/mL, it was investigated whether in the included patient population (all with bio-ADM >70 pg/mL) patients would exhibit a different treatment effect depending on their pre-dose bio-ADM concentration. No such interaction was detected which in other words means, that the beneficial treatment effect was detectable for all patients independent of their pre-dose bio-ADM concentration. This finding strongly suggests, that treatment works also in patients with bio-ADM levels lower than 70 pg/mL, with the likely (and acceptable) restriction that it such effect might not be expected in patients with bio-ADM concentrations in the healthy normal range.
[0417] P-value for interaction term between treatment and pre-dose bio-ADM: [0418] Per Protocol population: p=0.279 [0419] Per Protocol population/with pre-dose DPP3<70 ng/mL: p=0.150
[0420] This is further illustrated in Kaplan Meier Plots, where the patient populations were separated by their pre-dose bio-ADM concentration (below/above bio-ADM median) (see
TABLE-US-00044 SEQUENCES SEQ ID No.: 1 GYTFSRYW SEQ ID No.: 2 ILPGSGST SEQ ID No.: 3 TEGYEYDGFDY SEQ ID No.: 4 QSIVYSNGNTY SEQUENCE “RVS” (not part of the Sequencing Listing): RVS SEQ ID No.: 5 FQGSHIPYT SEQ ID No.: 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNE KFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID No.: 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYA QKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID No.: 8 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYA QKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID No.: 9 (AM-VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYA QKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID No.: 10 (AM-VH4-T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYA QKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID No.: 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVP DRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No.: 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No.: 13 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No.: 14 (human ADM 1-21) YRQSMNNFQGLRSFGCRFGTC SEQ ID No.: 15 (human ADM 21-32) CTVQKLAHQIYQ SEQ ID No.: 16 (human ADM C-42-52) CAPRSKISPQGY-CONH.sub.2 SEQ ID No.: 17 (murine ADM 1-19) YRQSMNQGSRSNGCRFGTC SEQ ID No.: 18 (murine ADM 19-31) CTFQKLAHQIYQ SEQ ID No.: 19 (murine ADM C-40-50) CAPRNKISPQGY-CONH.sub.2 SEQ ID No.: 20 (mature human Adrenomedullin (mature ADM); amidated ADM; bio-ADM): amino acids 1-52 or amino acids 95 - 146 of pro-ADM YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY-CONH.sub.2 SEQ ID No.: 21 (Murine ADM 1-50) YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQLTDKDKDGMAPRNKISPQGY-CONH.sub.2 SEQ ID No.: 22 (1-21 of human ADM): YRQSMNNFQGLRSFGCRFGTC SEQ ID No.: 23 (1-42 of human ADM): YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA SEQ ID No.: 24 (aa 43 - 52 of human ADM) PRSKISPQGY-NH2 SEQ ID No.: 25 (aa 1-14 of human ADM) YRQSMNNFQGLRSF SEQ ID No.: 26 (aa 1-10 of human ADM) YRQSMNNFQG SEQ ID No.: 27 (aa 1-6 of human ADM) YRQSMN SEQ ID No.: 28 (aa 1-32 of human ADM) YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQ SEQ ID No.: 29 (aa 1-40 murine ADM) YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQLTDKDKDGMA SEQ ID No.: 30 (aa 1-31 murine ADM) YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQL SEQ ID No.: 31 (proADM: 164 amino acids (22 - 185 of preproADM)) ARLDVASEF RKKWNKWALS RGKRELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL SEQ ID NO: 32 (Adrecizumab heavy chain) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQ KFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 33 (Adrecizumab light chain) DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 34 - human DPP3 (amino acid 1-737) MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAAWYGGLAVLLQTSPEAPYIYALL SRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNYKSFGDTKFVPNLPKEKLE RVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLRHLGLGKEGITTYFSGNCTMEDAKLAQD FLDSQNLSAYNTRLFKEVDGEGKPYYEVRLASVLGSEPSLDSEVTSKLKSYEFRGSPFQVTRG DYAPILQKVVEQLEKAKAYAANSHQGQMLAQYIESFTQGSIEAHKRGSRFWIQDKGPIVESYI GFIESYRDPFGSRGEFEGFVAVVNKAMSAKFERLVASAEQLLKELPWPPTFEKDKFLTPDFTS LDVLTFAGSGIPAGINIPNYDDLRQTEGFKNVSLGNVLAVAYATQREKLTFLEEDDKDLYILW KGPSFDVQVGLHELLGHGSGKLFVQDEKGAFNFDQETVINPETGEQIQSWYRSGETWDKFS TIASSYEECRAESVGLYLCLHPQVLEIFGFEGADAEDVIYVNWLNMVRAGLLALEFYTPEAFN WRQAHMQARFVILRVLLEAGEGLVTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQ VLKSTGDVAGGRALYEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLE YEASAAGLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA SEQ ID No. 35 - human DPP3 (amino acid 474-493 (N-Cys)) - immunization peptide with additional N-terminal Cystein CETVINPETGEQIQSWYRSGE SEQ ID No. 36 - IGHV1-69*11 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGTANYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYYYYYGMDVWGQGTTVTVSS SEQ ID No. 37 - HB3 QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNE KFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSS