PRO-ADRENOMEDULLIN OR FRAGMENT THEREOF IN PATIENTS INFECTED WITH CORONA VIRUS AND TREATMENTS WITH BINDER AGAINST ADRENOMEDULLIN
20210285949 · 2021-09-16
Assignee
Inventors
Cpc classification
G01N2800/52
PHYSICS
C07K2317/24
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
G01N33/74
PHYSICS
G01N2800/56
PHYSICS
C07K16/22
CHEMISTRY; METALLURGY
C07K2317/34
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/10
CHEMISTRY; METALLURGY
A61K2039/545
HUMAN NECESSITIES
International classification
C07K16/22
CHEMISTRY; METALLURGY
Abstract
Subject matter of the present invention is a method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus, the method comprising: determining the level of pro-Adrenomedullin (SEQ ID No. 31) or fragment thereof in a sample of bodily fluid of said patient, comparing said level of pro-Adrenomedullin or fragment thereof to a pre-determined threshold or a previous level of pro-Adrenomedullin or fragment thereof, and correlating said level of pro-Adrenomedullin or fragment thereof with the risk of life-threatening deterioration or an adverse event, or correlating said level of pro-Adrenomedullin or fragment thereof with the severity, or correlating said level of pro-Adrenomedullin or fragment thereof with the success of a therapy or intervention,
wherein said pro-Adrenomedullin or fragment thereof is selected from the group consisting of PAMP (SEQ ID No. 32), MR-proADM (SEQ ID No. 33), ADM-NH2 (SEQ ID No. 20), ADM-Gly (SEQ ID No. 21) and CT-proADM (SEQ ID No. 34).
Subject matter of the present invention is an Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient in a patient infected with a Corona virus.
Claims
1. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) diagnosing or prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention or (d) therapy guidance or therapy stratification or (e) patient management in a patient infected with a Corona virus, the method comprising: determining the level of pro-Adrenomedullin (SEQ ID No. 31) or fragment thereof in a sample of bodily fluid of said patient, comparing said level of pro-Adrenomedullin or fragment thereof to a pre-determined threshold or to a previously measured level of pro-Adrenomedullin or fragment thereof, and correlating said level of pro-Adrenomedullin or fragment thereof with the risk of life-threatening deterioration or an adverse event, or correlating said level of pro-Adrenomedullin or fragment thereof with the severity, or correlating said level of pro-Adrenomedullin or fragment thereof with the success of a therapy or intervention, or correlating said level of pro-Adrenomedullin or fragment thereof with a certain therapy or intervention, or correlating said level of pro-Adrenomedullin or fragment thereof with the management of said patient, wherein said pro-Adrenomedullin or fragment thereof is selected from the group consisting of PAMP (SEQ ID No. 32), MR-proADM (SEQ ID No. 33), ADM-NH.sub.2 (SEQ ID No. 20), ADM-Gly (SEQ ID No. 21) and CT-proADM (SEQ ID No. 34).
2. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to claim 1, wherein said Corona Virus is selected from the group comprising Sars-CoV-1, Sars-CoV-2, MERS-CoV, in particular Sars-CoV-2.
3. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to claim 1, wherein said adverse event is selected from the group comprising death, organ dysfunction, shock.
4. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to claim 1, wherein said level of pro-Adrenomedullin or fragment thereof is above a pre-determined threshold.
5. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to claim 1, wherein said patient has a level of D-dimer equal or greater than 0.5 μg/ml, preferably equal or greater than 1.0 μg/ml.
6. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to claim 1, wherein the level of pro-Adrenomedullin or fragment thereof is determined by contacting said sample of bodily fluid with a capture binder that binds specifically to pro-Adrenomedullin or fragment thereof.
7. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to claim 1, wherein said determination comprises the use of a capture-binder that binds specifically to pro-Adrenomedullin or fragment thereof wherein said capture-binder may be selected from the group of antibody, antibody fragment or non-IgG scaffold.
8. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to claim 1, wherein said patient is treated with an Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold wherein said anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal and/or mid-regional part (aa 1-42) of ADM-Gly and/or ADM-NH.sub.2: TABLE-US-00056 (SEQ ID No. 23) YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA.
9. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus.
10. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to claim 9, wherein said Corona Virus is selected from the group comprising Sars-CoV-1, Sars-CoV-2, MERS-CoV, in particular Sars-CoV-2.
11. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus, wherein said patient has a level of pro-Adrenomedullin or fragment thereof in a sample of bodily fluid of said subject that is above a predetermined threshold or higher than a previously measured level of pro-Adrenomedullin when determined by a method according to claim 1.
12. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to claim 9, wherein said patient has a level of D-dimer equal or greater than 0.5 μg/ml, preferably equal or greater than 1.0 μg/ml.
13. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to claim 9, wherein said anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal (amino acid 1-21) of ADM-Gly and/or ADM-NH.sub.2: YRQSMNNFQGLRSFGCRFGTC (SEQ ID No. 14).
14. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to claim 9, wherein said antibody is a monoclonal antibody or monoclonal antibody fragment.
15. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to claim 14, wherein the complementarity determining regions (CDR's) in the heavy chain comprises the sequences: TABLE-US-00057 CDR1: SEQ ID NO: 1 GYTFSRYW CDR2: SEQ ID NO: 2 ILPGSGST CDR3: SEQ ID NO: 3 TEGYEYDGFDY and the complementarity determining regions (CDR's) in the light chain comprises the sequences: TABLE-US-00058 CDR1: SEQ ID NO: 4 QSIVYSNGNTY CDR2: RVS CDR3: SEQ ID NO: 5 FQGSHIPYT
16. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to claim 15, wherein said antibody or fragment comprises a sequence selected from the group comprising as a VH region: TABLE-US-00059 (AM-VH-C) SEQ ID NO: 6 QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGE ILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGY EYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK (AM-VH1) SEQ ID NO: 7 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGR ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK (AM-VH2-E40) SEQ ID NO: 8 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGR ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK (AM-VH3-T26-E55) SEQ ID NO: 9 QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGE ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK (AM-VH4-T26-E40-E55) SEQ ID NO: 10 QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGE ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK or a sequence that is >80% identical to each of the above depicted sequences respectively, and comprises a sequence selected from the group comprising the following sequence as a VL region: TABLE-US-00060 (AM-VL-C) SEQ ID NO: 11 DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPK LLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (AM-VL1) SEQ ID NO: 12 DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPR RLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (AM-VL2-E40) SEQ ID NO: 13 DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPR RLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC or a sequence that is >80% identical to each of the above depicted sequences.
17. Adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to claim 15, wherein said antibody or fragment comprises the following sequence as a heavy chain: TABLE-US-00061 SEQ ID NO: 35 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGE ILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK or a sequence that is >95% identical to it, and comprises the following sequence as a light chain: TABLE-US-00062 SEQ ID NO: 36 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPR LLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC or a sequence that is >95% identical to it.
18. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS).
19. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS) according to claim 18, wherein said patient has a Horowitz index below 300, in particular below 200, in particular below 100 and/or said patient is in need of mechanical ventilation.
20. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS), wherein said patient has a level of pro-Adrenomedullin or fragment thereof in a sample of bodily fluid of said subject that is above a predetermined threshold or higher than a previously measured level of pro-Adrenomedullin when determined by a method according to claim 1.
21. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS) according to claim 18, wherein the complementarity determining regions (CDR's) in the heavy chain comprises the sequences: TABLE-US-00063 CDR1: SEQ ID NO: 1 GYTFSRYW CDR2: SEQ ID NO: 2 ILPGSGST CDR3: SEQ ID NO: 3 TEGYEYDGFDY and the complementarity determining regions (CDR's) in the light chain comprises the sequences: TABLE-US-00064 CDR1: SEQ ID NO: 4 QSIVYSNGNTY CDR2: RVS CDR3: SEQ ID NO: 5 FQGSHIPYT
22. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS) according to claim 21, wherein said antibody or fragment comprises a sequence selected from the group comprising as a VH region: TABLE-US-00065 (AM-VH-C) SEQ ID NO: 6 QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGE ILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGY EYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK (AM-VH1) SEQ ID NO: 7 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGR ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK (AM-VH2-E40) SEQ ID NO: 8 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGR ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK (AM-VH3-T26-E55) SEQ ID NO: 9 QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGE ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK (AM-VH4-T26-E40-E55) SEQ ID NO: 10 QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGE ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK or a sequence that is >80% identical to each of the above depicted sequences respectively, and comprises a sequence selected from the group comprising the following sequence as a VL region: TABLE-US-00066 (AM-VL-C) SEQ ID NO: 11 DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPK LLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (AM-VL1) SEQ ID NO: 12 DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPR RLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (AM-VL2-E40) SEQ ID NO: 13 DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPR RLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC or a sequence that is >80% identical to each of the above depicted sequences.
23. Adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS) according to claim 21, wherein said antibody or fragment comprises the following sequence as a heavy chain: TABLE-US-00067 SEQ ID NO: 35 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGE ILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK or a sequence that is >95% identical to it, and comprises the following sequence as a light chain: TABLE-US-00068 SEQ ID NO: 36 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPR LLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC or a sequence that is >95% identical to it.
Description
EMBODIMENTS
[0198] 1. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) diagnosing or prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention or (d) therapy guidance or therapy stratification or (e) patient management in a patient infected with a Corona virus, the method comprising: [0199] determining the level of pro-Adrenomedullin (SEQ ID No. 31) or fragment thereof in a sample of bodily fluid of said patient, [0200] comparing said level of pro-Adrenomedullin or fragment thereof to a pre-determined threshold or to a previously measured level of pro-Adrenomedullin or fragment thereof, and [0201] correlating said level of pro-Adrenomedullin or fragment thereof with the risk of life-threatening deterioration or an adverse event, or [0202] correlating said level of pro-Adrenomedullin or fragment thereof with the severity, or [0203] correlating said level of pro-Adrenomedullin or fragment thereof with the success of a therapy or intervention, or [0204] correlating said level of pro-Adrenomedullin or fragment thereof with a certain therapy or intervention, or [0205] correlating said level of pro-Adrenomedullin or fragment thereof with the management of said patient,
[0206] wherein said pro-Adrenomedullin or fragment thereof is selected from the group consisting of PAMP (SEQ ID No. 32), MR-proADM (SEQ ID No. 33), ADM-NH.sub.2 (SEQ ID No. 20), ADM-Gly (SEQ ID No. 21) and CT-proADM (SEQ ID No. 34).
[0207] 2. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to embodiment 1, wherein said Corona Virus is selected from the group comprising Sars-CoV-1, Sars-CoV-2, MERS-CoV, in particular Sars-CoV-2.
[0208] 3. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to embodiment 1 or 2, wherein said adverse event is selected from the group comprising death, organ dysfunction, shock.
[0209] 4. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to embodiments 1 to 3, wherein said level of pro-Adrenomedullin or fragment thereof is above a pre-determined threshold.
[0210] 5. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to embodiments 1 to 4, wherein said fragment is MR-proADM (SEQ ID No. 33), and the predetermined threshold of MR-proADM in a sample of bodily fluid of said subject is between 0.5 and 2 nmol/L, preferably between 0.7 and 1.5 nmol/L, preferably between 0.8 and 1.2 nmol/L, most preferred a threshold of 1 nmol/L is applied.
[0211] 6. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to embodiments 1 to 4, wherein said fragment is ADM-NH.sub.2 (SEQ ID No. 20), and the predetermined threshold of ADM-NH.sub.2 (SEQ ID No. 20) in a sample of bodily fluid of said subject 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.
[0212] 7. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to embodiments 1 to 6, wherein said patient has a SOFA score equal or greater than 3, preferably equal or greater than 7 or said patient has a quickSOFA score equal or greater than 1, preferably equal or greater than 2.
[0213] 8. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to embodiments 1 to 7, wherein said patient has a level of D-dimer equal or greater than 0.5 μg/ml, preferably equal or greater than 1.0 μg/ml.
[0214] 9. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to embodiments 1 to 8, wherein the level of pro-Adrenomedullin or fragment thereof is determined by contacting said sample of bodily fluid with a capture binder that binds specifically to pro-Adrenomedullin or fragment thereof.
[0215] 10. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to embodiments 1 to 9, wherein said determination comprises the use of a capture-binder that binds specifically to pro-Adrenomedullin or fragment thereof wherein said capture-binder may be selected from the group of antibody, antibody fragment or non-IgG scaffold.
[0216] 11. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to embodiments 1 to 10, wherein the level of pro-Adrenomedullin or fragment thereof is determined in a bodily fluid sample of said subject and wherein said determination comprises the use of a capture-binder that binds specifically to pro-Adrenomedullin or fragment thereof wherein said capture-binder is an antibody.
[0217] 12. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to embodiments 1 to 11, wherein the level of pro-Adrenomedullin or fragment thereof is determined in a bodily fluid sample of said subject and wherein said determination comprises the use of a capture-binder that binds specifically to level of pro-Adrenomedullin or fragment thereof, wherein said capture-binder is immobilized on a surface.
[0218] 13. A method for (a) diagnosing or predicting the risk of life-threatening deterioration or an adverse event or (b) prognosing the severity or (c) predicting or monitoring the success of a therapy or intervention in a patient infected with a Corona virus according to embodiments 1 to 12, wherein said patient is treated with an Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold wherein said anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal and/or mid-regional part (aa 1-42) of ADM-Gly and/or ADM-NH.sub.2:
TABLE-US-00029 (SEQ ID No. 23) YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA.
[0219] 14. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus.
[0220] 15. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to embodiment 14, wherein said Corona Virus is selected from the group comprising Sars-CoV-1, Sars-CoV-2, MERS-CoV, in particular Sars-CoV-2.
[0221] 16. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to embodiment 14 or 15, wherein said patient has a level of pro-Adrenomedullin or fragment thereof in a sample of bodily fluid of said subject that is above a predetermined threshold or higher than a previously measured level of pro-Adrenomedullin when determined by a method according to any of claims 1-12.
[0222] 17. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to embodiments 14 to 16, wherein said patient has a SOFA score equal or greater than 3, preferably equal or greater than 7 or said patient has a quickSOFA score equal or greater than 1, preferably equal or greater than 2.
[0223] 18. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to embodiments 14 to 17, wherein said patient has a level of D-dimer equal or greater than 0.5 μg/ml, preferably equal or greater than 1.0 μg/ml.
[0224] 19. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to embodiments 14 to 18, wherein said anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal (amino acid 1-21) of ADM-Gly and/or ADM-NH.sub.2:
TABLE-US-00030 (SEQ ID No. 14) YRQSMNNFQGLRSFGCRFGTC.
[0225] 20. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to embodiments 14-19, wherein said Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold exhibits a minimum binding affinity to pro-Adrenomedullin or a fragment thereof of equal or less than 10-7 M.
[0226] 21. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to embodiments 14-20, wherein said Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold wherein said antibody or fragment or scaffold blocks the bioactivity of ADM not more than 80%, preferably not more than 50%.
[0227] 22. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to embodiments 14-21, wherein said antibody is a monoclonal antibody or monoclonal antibody fragment.
[0228] 23. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to embodiment 22, wherein the complementarity determining regions (CDR's) in the heavy chain comprises the sequences:
TABLE-US-00031 CDR1: SEQ ID NO: 1 GYTFSRYW CDR2: SEQ ID NO: 2 ILPGSGST CDR3: SEQ ID NO: 3 TEGYEYDGFDY
[0229] and the complementarity determining regions (CDR's) in the light chain comprises the sequences:
TABLE-US-00032 CDR1: SEQ ID NO: 4 QSIVYSNGNTY CDR2: RVS CDR3: SEQ ID NO: 5 FQGSHIPYT
[0230] 24. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to embodiment 23, wherein said antibody or fragment comprises a sequence selected from the group comprising as a VH region:
TABLE-US-00033 (AM-VH-C) SEQ ID NO: 6 QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGE ILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGY EYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK (AM-VH1) SEQ ID NO: 7 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGR ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK (AM-VH2-E40) SEQ ID NO: 8 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGR ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK (AM-VH3-T26-E55) SEQ ID NO: 9 QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGE ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK (AM-VH4-T26-E40-E55) SEQ ID NO: 10 QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGE ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNRKPSNTKVDKRVEPK
[0231] or a sequence that is >80% identical to each of the above depicted sequences respectively, and comprises a sequence selected from the group comprising the following sequence as a VL region:
TABLE-US-00034 (AM-VL-C) SEQ ID NO: 11 DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPK LLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (AM-VL1) SEQ ID NO: 12 DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPR RLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (AM-VL2-E40) SEQ ID NO: 13 DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPR RLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC
[0232] or a sequence that is >80% identical to each of the above depicted sequences.
[0233] 25. Adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to any of embodiments 23 to 24, wherein said antibody or fragment comprises the following sequence as a heavy chain:
TABLE-US-00035 SEQ ID NO: 35 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGE ILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0234] or a sequence that is >95% identical to it,
[0235] and comprises the following sequence as a light chain:
TABLE-US-00036 SEQ ID NO: 36 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPR LLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKEIKVYAC EVTHQGLSSPVTKSFNRGEC
[0236] or a sequence that is >95% identical to it.
[0237] 26. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to any of embodiments 23 to 25, wherein said monoclonal antibody or antibody fragment is a humanized monoclonal antibody or humanized monoclonal antibody fragment.
[0238] 27. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient infected with a Corona virus according to embodiments 14-26, wherein said Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is an monoclonal antibody and is Adrecizumab and comprises the following sequence as a heavy chain:
TABLE-US-00037 SEQ ID NO: 35 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPQGLEWIGEI LPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYE YDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0239] and comprises the following sequence as a light chain:
TABLE-US-00038 SEQ ID NO: 36 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPR LLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC
[0240] or a biosimilar thereof.
[0241] 28. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS).
[0242] 29. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS) according to embodiment 28, wherein said patient has a Horowitz index below 300, in particular below 200, in particular below 100 and/or said patient is in need of mechanical ventilation.
[0243] 30. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS) according to embodiment 28 or 29, wherein said patient has a level of pro-Adrenomedullin or fragment thereof in a sample of bodily fluid of said subject that is above a predetermined threshold or higher than a previously measured level of pro-Adrenomedullin when determined by a method according to any of claims 1-12.
[0244] 31. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS) according to embodiments 28 to 30, wherein said patient has a SOFA score equal or greater than 3, preferably equal or greater than 7 or said patient has a quickSOFA score equal or greater than 1, preferably equal or greater than 2.
[0245] 32. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS) according to embodiments 28 to 31, wherein said patient has a level of D-dimer equal or greater than 0.5 μg/ml, preferably equal or greater than 1.0 μg/ml.
[0246] 33. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention i in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS) according to embodiments 28 to 32, wherein said anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal (amino acid 1-21) of ADM-Gly and/or ADM-NH2: YRQSMNNFQGLRSFGCRFGTC (SEQ ID No. 14).
[0247] 34. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS) according to embodiments 28-33, wherein said Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold exhibits a minimum binding affinity to pro-Adrenomedullin or a fragment thereof of equal or less than 10-7 M.
[0248] 35. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS) according to embodiments 28-34, wherein said Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold wherein said antibody or fragment or scaffold blocks the bioactivity of ADM not more than 80%, preferably not more than 50%.
[0249] 36. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS) according to embodiments 28-35, wherein said antibody is a monoclonal antibody or monoclonal antibody fragment.
[0250] 37. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS) according to embodiment 36, wherein the complementarity determining regions (CDR's) in the heavy chain comprises the sequences:
TABLE-US-00039 CDR1: SEQ ID NO: 1 GYTFSRYW CDR2: SEQ ID NO: 2 ILPGSGST CDR3: SEQ ID NO: 3 TEGYEYDGFDY
[0251] and the complementarity determining regions (CDR's) in the light chain comprises the sequences:
TABLE-US-00040 CDR1: SEQ ID NO: 4 QSIVYSNGNTY CDR2: RVS CDR3: SEQ ID NO: 5 FQGSHIPYT
[0252] 38. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS) according to embodiment 37, wherein said antibody or fragment comprises a sequence selected from the group comprising as a VH region:
TABLE-US-00041 (AM-VH-C) SEQ ID NO: 6 QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGE ILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGY EYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK (AM-VH1) SEQ ID NO: 7 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGR ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK (AM-VH2-E40) SEQ ID NO: 8 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGR ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK (AM-VH3-T26-E55) SEQ ID NO: 9 QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGE ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK (AM-VH4-T26-E40-E55) SEQ ID NO: 10 QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGE ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK
[0253] or a sequence that is >80% identical to each of the above depicted sequences respectively, and comprises a sequence selected from the group comprising the following sequence as a VL region:
TABLE-US-00042 (AM-VL-C) SEQ ID NO: 11 DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPK LLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (AM-VL1) SEQ ID NO: 12 DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPR RLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (AM-VL2-E40) SEQ ID NO: 13 DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPR RLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC
[0254] or a sequence that is >80% identical to each of the above depicted sequences.
[0255] 39. Adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS) according to any of embodiments 37 to 38, wherein said antibody or fragment comprises the following sequence as a heavy chain:
TABLE-US-00043 SEQ ID NO: 35 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGE ILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0256] or a sequence that is >95% identical to it,
[0257] and comprises the following sequence as a light chain:
TABLE-US-00044 SEQ ID NO: 36 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPR LLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC
[0258] or a sequence that is >95% identical to it.
[0259] 40. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS) according to any of embodiments 37 to 39, wherein said monoclonal antibody or antibody fragment is a humanized monoclonal antibody or humanized monoclonal antibody fragment.
[0260] 41. Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or intervention in a patient with compromised lung function and/or acute respiratory distress syndrome (ARDS) according to embodiments 28-40, wherein said Anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is an monoclonal antibody and is Adrecizumab and comprises the following sequence as a heavy chain:
TABLE-US-00045 SEQ ID NO: 35 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGE ILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0261] and comprises the following sequence as a light chain:
TABLE-US-00046 SEQ ID NO: 36 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPR LLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC
[0262] or a biosimilar thereof.
EXAMPLES
[0263] 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.
Example 1—Generation of Antibodies and Determination of their Affinity Constants
[0264] Several anti-human and anti-murine ADM antibodies were produced and their affinity constants were determined (see tables 2 and 3).
[0265] Peptides/Conjugates for Immunization:
[0266] Peptides for immunization were synthesized, see Table 2, (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.
[0267] Mouse Monoclonal Antibody Production:
[0268] A Balb/c mouse was immunized with 100 μg Peptide-BSA-Conjugate at day 0 and 14 (emulsified in 104 μl complete Freund's adjuvant) and 50 μs at day 21 and 28 (in 104 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.
[0269] 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. Horm. Metab. Res. 28: 11-15).
[0270] 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.
[0271] Human Antibody Production by Means of Phage Display:
[0272] The human naive antibody gene libraries HALT/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; Schutte et al. 2009. PLoS One 4, e6625).
[0273] 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.
[0274] Affinity Constants:
[0275] To determine the affinity of the antibodies to Adrenomedullin, the kinetics of binding of Adrenomedullin to immobilized antibody was determined by means of label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany).
[0276] 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).
[0277] 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-00047 TABLE 2 Affinity Sequence Antigen/ ADM Desig- constants Number Immunogen Region nation Kd (M) SEQ ID: 14 YRQSMNNFQ 1-21 NT-H 5.9 × 10.sup.−9 GLRSFGCRF GTC SEQ ID: 15 CTVQKLAHQ 21-32 MR-H 2 × 10.sup.−9 IYQ SEQ ID: 16 CAPRSKISP C-42-52 CT-H 1.1 × 10.sup.−9 QGY-NH2 SEQ ID: 17 YRQSMNQGSR 1-19 NT-M 3.9 × 10.sup.−9 SNGCRFGTC SEQ ID: 18 CTFQKLAHQ 19-31 MR-M 4.5 × 10.sup.−10 IYQ SEQ ID: 19 CAPRNKISPQ C-40-50 CT-M 9 × 10.sup.−9 GY-NH2
[0278] The following is a list of further obtained monoclonal antibodies:
TABLE-US-00048 TABLE 3 Max. inhibition Clone bioassay (%) Target Source number Affinity (M) (see example 2) NT-M Mouse ADM/63 5.8 × 10.sup.−9 45 NT-M Mouse ADM/364 2.2 × 10.sup.−8 48 NT-M Mouse ADM/365 3.0 × 10.sup.−8 NT-M Mouse ADM/366 1.7 × 10.sup.−8 NT-M Mouse ADM/367 1.3 × 10.sup.−8 NT-M Mouse ADM/368 1.9 × 10.sup.−8 NT-M Mouse ADM/369 2.0 × 10.sup.−8 NT-M Mouse ADM/370 1.6 × 10.sup.−8 NT-M Mouse ADM/371 2.0 × 10.sup.−8 NT-M Mouse ADM/372 2.5 × 10.sup.−8 NT-M Mouse ADM/373 1.8 × 10.sup.−8 NT-M Mouse ADM/377 1.5 × 10.sup.−8 NT-M Mouse ADM/378 2.2 × 10.sup.−8 NT-M Mouse ADM/379 1.6 × 10.sup.−8 NT-M Mouse ADM/380 1.8 × 10.sup.−8 NT-M Mouse ADM/381 2.4 × 10.sup.−8 NT-M Mouse ADM/382 1.6 × 10.sup.−8 NT-M Mouse ADM/383 1.8 × 10.sup.−8 NT-M Mouse ADM/384 1.7 × 10.sup.−8 NT-M Mouse ADM/385 1.7 × 10.sup.−8 NT-M Mouse ADM/403 1.2 × 10.sup.−8 NT-M Mouse ADM/395 1.2 × 10.sup.−8 NT-M Mouse ADM/396 3.0 × 10.sup.−8 NT-M 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 hADM Phage display ADM/B7 <1 × 10.sup.−8 hADM Phage display ADM/C7 <1 × 10.sup.−8 hADM Phage display ADM/G3 <1 × 10.sup.−8 hADM Phage display ADM/B6 <1 × 10.sup.−8 hADM Phage display ADM/B11 <1 × 10.sup.−8 hADM Phage display ADM/D8 <1 × 10.sup.−8 hADM Phage display ADM/D11 <1 × 10.sup.−8 hADM Phage display ADM/G12 <1 × 10.sup.−8
[0279] Generation of Antibody Fragments by Enzymatic Digestion:
[0280] 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.
[0281] Procedure for Fab Generation and Purification:
[0282] 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 1983.1 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. 20091 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).
[0283] Procedure for Generation and Purification of F(Ab′).sub.2 Fragments:
[0284] 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).
[0285] NT-H-Antibody Fragment Humanization:
[0286] The antibody fragment was humanized by the CDR-grafting method (Jones et al. 1986. Nature 321, 522-525). The following steps were done to achieve the humanized sequence:
[0287] Total RNA was extracted from NT-H hybridomas using the Qiagen kit. For first-round RT-PCR the 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.
[0288] The reaction set up was as follows: 5× QIAGEN OneStep RT-PCR Buffer 5.0 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.
[0289] The reaction setup was as follows: 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.
[0290] 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. After alignment with homologous human framework sequences, the resulting humanized sequence for the variable heavy chain is the following: see
[0291] Annotation for the antibody fragment sequences (SEQ ID No.: 7-13, 35 and 36): bold and underline are the CDR 1, 2, 3 chronologically arranged.
TABLE-US-00049 (AM-VH-C) SEQ ID No.: 6 QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGE ILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGY EYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNEEKPSNTKVDKRVEPK (AM-VH1) SEQ ID No.: 7 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGR ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNEEKPSNTKVDKRVEPK (AM-VH2-E40) SEQ ID No.: 8 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGR ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNEEKPSNTKVDKRVEPK (AM-VH3-T26-E55) SEQ ID No.: 9 QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGE ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNEEKPSNTKVDKRVEPK (AM-VH4-T26-E40-E55) SEQ ID No.: 10 QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGE ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNEEKPSNTKVDKRVEPK (AM-VL-C) SEQ ID No.: 11 DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPK LLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (AM-VL1) SEQ ID No.: 12 DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPR RLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (AM-VL2-E40) SEQ ID No.: 13 DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPR RLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC SEQ ID No.: 35 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGE ILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNEEKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSEEEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No.: 36 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPR LLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC
Example 2—Effect of Selected Anti-ADM-Antibodies on Anti-ADM-Bioactivity
[0292] The effect of selected ADM-antibodies on ADM-bioactivity was tested in a human recombinant Adrenomedullin receptor cAMP functional assay (Adrenomedullin Bioassay). The following materials were used: Cell line CHO-K1, Adrenomedullin receptor (CRLR+RAMP3), Receptor Accession Number Cell line (CRLR: U17473; RAMP3: AJ001016). CHO-K1 cells expressing human recombinant adrenomedullin receptor (FAST-027C) grown prior to the test in media is 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). Dose response curves were performed in parallel with the reference agonists (hADM or mADM).
[0293] Antagonist Test (96 Well):
[0294] 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.
[0295] Antibodies Testing cAMP-HTRF Assay:
[0296] 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 (SEQ ID No. 20), at the following final antibody concentrations: 100 μg/ml, 20 μg/ml, 4 μg/ml, 0.8 μg/ml, 0.16 μg/ml. The anti-m-ADM antibodies (NT-M, MR-M, CT-M) were tested for antagonist activity in human recombinant adrenomedullin receptor (FAST-027C) cAMP functional assay in the presence of 0.67 nM Mouse ADM 1-50 (SEQ ID No. 22), 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-00050 TABLE 4 Maximal inhibition of ADM-antibodies Maximal inhibition of ADM Antibody bioactivity (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 mouse IgG 0
Example 3—Stabilization of hADM by the Anti-ADM Antibody
[0297] The stabilizing effect of human ADM by human ADM antibodies was tested using a hADM immunoassay. The technology used was a sandwich coated tube luminescence immunoassay, based on Acridinium ester labelling.
[0298] 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).
[0299] 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.
[0300] 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).
[0301] 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). Tube-bound chemiluminescence was measured by using the LB 953 (Berthold, Germany).
[0302] 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.
Example 4—Sepsis Mortality
[0303] a) Early Treatment of Sepsis
[0304] 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.
[0305] 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).
[0306] 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.
[0307] Group Treatment (10 μl/g bodyweight) dose/Follow-Up: [0308] 1 NT-M, 0.2 mg/ml survival over 4 days [0309] 2 MR-M, 0.2 mg/ml survival over 4 days [0310] 3 CT-M, 0.2 mg/ml survival over 4 days [0311] 4 non-specific mouse IgG, 0.2 mg/ml survival over 4 days [0312] 5 control—PBS 10 μl/g bodyweight survival over 4 days
[0313] 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 5.
TABLE-US-00051 TABLE 5 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
[0314] 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.
[0315] b) Late Treatment of Sepsis
[0316] 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.
[0317] 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.
[0318] Group Treatment (10 μl/g bodyweight) dose/Follow-Up: [0319] 1 NT-M, FAB2 0.2 mg/ml survival over 4 days [0320] 2 control non-specific mouse IgG, 0.2 mg/ml survival over 4 days [0321] 3 vehicle:—PBS 10 μl/g bodyweight survival over 4 days
TABLE-US-00052 TABLE 6 4-day mortality 4 day mortality survival (%) PBS 0 Non-specific mouse IgG 0 NT-M FAB2 75
[0322] It can be seen from Table 6 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
[0323] 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—Bio-ADM in Patients Infected with Corona Virus (SARS-CoV-2)
[0324] Plasma samples from 12 patients that were diagnosed of being infected with Corona virus (SARS-CoV-2) were screened for bio-ADM. Bio-ADM levels were measured using an immunoassay as described in Weber et al. 2017 (Weber et al. 2017. JALM 2(2): 222-233). In addition, DPP3-levels were measured using an immunoassay (LIA) as described recently (Rehfeld et al. 2019. JALM 3(6): 943-953). The respective bio-ADM and DPP3 concentrations in individual samples are summarized in table 7.
TABLE-US-00053 TABLE 7 bio-ADM and DPP3 levels in samples from patients infected with Corona virus (SARS-CoV-2) Patient No. DPP3 (ng/ml) bio-ADM (pg/ml) 1 56 133 2 30 45 3 70 214 4 150 85 5 290 437 6 87 66 7 975 79 8 333 174 9 216 35 10 539 199 11 27 53 12 162 401 Median 156.0 109.0 mean 244.6 160.1
[0325] Bio-ADM concentrations in samples from patients infected with Corona virus (SARS-CoV-2) ranged between 35 and 437 pg/ml with a median (IQR) of 109 (56-210) pg/ml. Median plasma bio-ADM (mature ADM-NH.sub.2) in samples from (healthy) subjects was 24.7 pg/ml, the lowest value 11 pg/ml and the 99.sup.th percentile 43 pg/ml (Marino et al. 2014. Critical Care 18:R34). Bio-ADM in patients infected with Corona virus (SARS-CoV-2) were significantly elevated compared to healthy controls.
[0326] DPP3 concentrations ranged between 27 and 975 ng/ml with a median (IQR) of 156.0 (59.5-322.3) ng/ml. DPP3 concentrations are significantly elevated compared to healthy subjects. Samples from 5,400 normal (healthy) subjects (swedish single-center prospective population-based Study (MPP-RES)) have been measured: median (interquartile range) plasma DPP3 was 14.5 ng/ml (11.3 ng/ml-19 ng/ml).
Example 7—Change of Lung Function Under NT-ADM Antibody Treatment in Patients with Compromised Lung Function (AdrenOSS-2)
[0327] 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%.
[0328] 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=0.200).
[0329] Furthermore, different subpopulations of the cohort were analyzed. Main outcomes were 28-day mortality, change in Horovitz Index (PaO.sub.2/FiO.sub.2) (at 24 h/48 h/72 h), change in SOFA score (at 24 h/48 h/72 h) or change in respiratory SOFA score component (also based on PaO.sub.2/FiO.sub.2) (at 24 h/48 h/72 h). All p-values are 2-sided and a p-value of 0.20 should be considered significant.
[0330] A subpopulation of shock patients who met the following criteria: Horovitz-Index of <170 and mechanical ventilation at baseline (n=48) was analyzed. This group mimics critically-ill Covid-19 patients on the ICU and in need for mechanical ventilation. 28-day mortality trended to be lower in patients treated with Adrecizumab compared to placebo (p=0.37) (
[0331] A subpopulation of shock patients with ALI/ARDS which was defined via respiratory physical examination on admission (n=80) was further analyzed. The change in SOFA score was significantly lower after 24 hours (p=0.005) and 48 (p=0.025) (
[0332] Another subpopulation of shock patients was selected meeting the criterion of mechanical ventilation at baseline (n=161). 28-day mortality was significantly lower in patients treated with Adrecizumab compared to placebo (p=0.157) (
Example 8—Prognostic Value of Bio-ADM in Critically Ill Patients with COVID-19
[0333] The aim of this study was to determine if bioactive adrenomedullin (bio-ADM) can assist in the risk stratification and clinical management of critically ill COVID-19 patients.
[0334] 8.1. Study Population and Data Collection
[0335] After ethical approval (Ethical Committee of RWTH University, EK 100/20), this prospective observational study was performed between Mar. 13 and Apr. 16, 2020 at the University Hospital RWTH Aachen, Germany. All patients or their legal representatives provided written informed consent. All patients with positive SARS-CoV-2 PCR results and ICU admission were included in this study. The exclusion criteria were age <18 years old, pregnancy, and palliative care. The analysis was carried out using real time reverse transcription PCR (RT-PCR). Treatment of patients followed the standards of care in our ICU, including mechanical ventilation, veno-venous ECMO, and RRT and norepinephrine if needed. Decision on the use of veno-venous ECMO therapy was based on the recently published Extracorporeal Life Support Organization (ELSO) consensus guideline (Bartlett et al. 2020. ASAIO Journal 66: 472-474). All parameters including demographics, vital signs, laboratory values, blood gas analyses and organ support have been extracted from the patient data management system (Intellispace Critical Care and Anesthesia (ICCA) system, Philips, Netherlands).
[0336] 8.2. Bio-ADM Measurement
[0337] Blood was sampled on the day of admission and on a daily basis until day 7 for analysis of bio-ADM and standard laboratory parameters. Bio-ADM was measured in EDTA plasma with a one-step luminescence sandwich immunoassay (Weber et al. 2017. JALM 2(2): 222-233). In brief, 100 sample were incubated under agitation for one hour at room temperature with 150 μL detection antibody directed against the C-terminus of bio-ADM in a microtiter plate coated with monoclonal antibody directed against mid-regional bio-ADM. Synthetic human bio-ADM was used as calibrator. After washing, the chemiluminescence signal was measured in a microtiter plate luminescence reader (Centro LB960, Berthold Technologies, Bad Wildbad, Germany). The assay had a lower detection limit of 3 pg/mL. In a reference population of 200 healthy individuals, median (99th percentile) bio-ADM levels were 20.7 pg/mL (43 pg/mL) (Marino et al. 2014. Critical Care 18: R34).
[0338] 8.3. Statistics
[0339] Values are expressed as medians and interquartile ranges (IQR), or counts and percentages, as appropriate. Group comparisons of continuous variables were performed using Kruskal-Wallis test. Categorical data were compared using Pearson's Chi-squared Test for Count Data. Biomarker data were log-transformed. Boxplots were used to illustrate differences of bio-ADM in categorical variables. Cox proportional-hazards regression was used to analyze the effect of (log-transformed) bio-ADM on survival in univariable analyses. The assumptions of proportional hazard were tested. The predictive value of a 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. Survival curves plotted by the Kaplan-Meier method were used for illustrative purposes. 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 3.4.3 (http://www.r-project.org, library rms, Hmisc, ROCR) and Statistical Package for the Social Sciences (SPSS) version 22.0 (SPSS Inc., Chicago, Ill., USA).
[0340] 8.4. Results
[0341] In this cohort study, 53 patients with COVID-19 were consecutively included after confirmed SARS-CoV-2 infection and the need of ICU admission (n=40 male [76%], median [IQR] age 62 [57-70] years) (Table 7). Median ICU length of stay was 16 (7.5-20) days. 32 patients (60%) were discharged from ICU to normal ward prior to day 28, while 8 patients (15%) remained in the ICU and 13 patients (25%) died. Markers of systemic inflammation are shown in Table 7.
TABLE-US-00054 TABLE 7 Baseline characteristics in 53 COVID-19 critically ill patients Variable all none (n = 3) mild (n = 12) Age (years) 62 [57-70] 53 [49-65] 61 [59-64] Gender male, n (%) 40 (75.5) 3 (100) 10 (83.3) Body mass index (kg/m.sup.2) 29.3 [24.9-32.6] 24.9 [24.7-28.2] 29.2 [26.3-34.9] Temperature, max (° C.) 38.1 [37.4-38.5] 38.1 [37.8-38.8] 38.1 [37.8-38.6] Heart rate (bpm) 106 [89-114] 93 [86-107] 105 [93-109] Respiratory rate (bpm) 25 [23-28] 24 [22-25] 24 [23-26] SOFA score at day of enrollment (points) 9 [7-11] 8.5 [7.75-9.25] 7 [6-9.5] Blood gas analysis (at day of enrollment) Arterial pH 7.36 [7.3-7.42] 7.47 [7.38-7.49] 7.4 [7.37-7.44] pCO.sub.2 (mmHg) 45.05 [39.25-52.02] 48 [42.05-71.3] 36.7 [33.75-41.2] pO.sub.2 (mmHg) 79 [70-91] 71 [63.5-79.5] 92 [75-104.5] SpO.sub.2 (%) 95 [94-98.25] 94 [93.5-94.5] 98 [96-99] Horowitz index (mmHg/%) 113.5 [87.5-151.25] 133 [88.5-276] 224 [167.5-275.5] Biomarker (at day of enrollment, unless stated differently) bio-ADM (pg/mL) 59.9 [37.9-101.9] 28.3 [19.9-28.4] 39.0 [29.2-54.5] bio-ADM > 70 pg/mL, n (%) 22 (41.5) 0 (0) 1 (8.3) lactate (mmol/L) 1 [0.8-1.42] 0.7 [0.5-0.95] 0.8 [0.7-0.9] IL-6 (pg/mL) 158.4 [97.42-337.4] 51.95 [34.52-69.39] 65.73 [46.88-93.52] PCT (ng/mL) 0.53 [0.13-1.89] 0.07 [0.06-0.08] 0.14 [0.11-0.25] CRP (nmol/L) 174.6 [117.4-325.62] 182.1 [182.1-182.1] 79.5 [34.25-142.43] WBC (10.sup.3/mm.sup.3) 9.3 [6.6-13] 10.4 [9.3-11.85] 6.15 [5.07-10.82] Platelets (10.sup.3/μL) 228 [198-329] 202 [200-292] 197 [139.75-235.75] Creatinine (mg/dL) 1.06 [0.76-2.18] 0.67 [0.6-0.72] 0.96 [0.76-1.21] Comorbidities Arterial hypertension, n (%) 27 (50.9) 1 (33.3) 5 (41.7) Diabetes mellitus, n (%) 13 (24.5) 0 (0) 1 (8.3) Ischemic heart disease, n (%) 10 (18.9) 0 (0) 2 (16.7) Embolism/Thrombosis, n (%) 6 (11.3) 1 (33.3) 1 (8.3) Cardiac arrhythmia, n (%) 6 (11.3) 0 (0) 1 (8.3) Cerebral vascular disease, n (%) 5 (9.4) 0 (0) 2 (16.7) COPD, n (%) 6 (11.3) 1 (33.3) 2 (16.7) Other lung diseases, n (%) 2 (3.8) 1 (33.3) 1 (8.3) Chronic kidney disease, n (%) 8 (15.1) 0 (0) 2 (16.7) Tumor disease, n (%) 4 (7.5) 0 (0) 3 (25) Smoker, n (%) 3 (5.7) 0 (0) 1 (8.3) Treatment on ICU (first 14 days, unless stated differently) ICU length of stay (days) 16 [7.5-20] 6 [4-9.5] 7.5 [3-10.5] Highest dose of Norepinephrine during 0.15 [0.06-0.29] 0.07 [0.03-0.11] 0 [0-0.09] the first 7 days (μg/kg/min) Anticoagulation, n (%) 15 (28.3) 1 (33.3) 2 (16.7) Antiplatelet, n (%) 15 (28.3) 0 (0) 4 (33.3) Antihypertensiva, n (%) 32 (60.4) 1 (33.3) 8 (66.7) Immunsupressant, n (%) 9 (17) 1 (33.3) 2 (16.7) Analgesics, n (%) 8 (15.1) 1 (33.3) 4 (33.3) Outcome Death 28 days, n (%) 13 (24.5) 1 (33.3) 0 (0) Disposition on day 28 discharged, n (%) 32 (60.4) 2 (66.7) 12 (100) on ICU post day 28, n (%) 8 (15.1) 0 (0) 0 (0) Death 28 days, n (%) 13 (24.5) 1 (33.3) 0 (0) Variable moderate (n = 13) severe (n = 25) p-value Age (years) 62 [54-67] 66 [58-72] 0.767 Gender male, n (%) 6 (46.2) 21 (84) 0.0385 Body mass index (kg/m.sup.2) 30.5 [26.7-35.2] 29.3 [24.7-31.3] 0.758 Temperature, max (° C.) 38.2 [37.0-38.5] 38.0 [37.3-38.5] 0.934 Heart rate (bpm) 91 [72-103] 112 [104-121] 0.014 Respiratory rate (bpm) 25 [22-28] 25 [23-29] 0.678 SOFA score at day of enrollment (points) 8.5 [7.75-10] 11 [9-11] 0.037 Blood gas analysis (at day of enrollment) Arterial pH 7.38 [7.33-7.43] 7.32 [7.28-7.36] 0.011 pCO.sub.2 (mmHg) 45.5 [43.2-52] 48.2 [42.1-55.4] 0.001 pO.sub.2 (mmHg) 79 [70-92] 79 [70-84] 0.345 SpO.sub.2 (%) 98 [95-100] 94 [93-97] 0.031 Horowitz index (mmHg/%) 115 [100-150] 94 [71-115] 0.002 Biomarker (at day of enrollment, unless stated differently) bio-ADM (pg/mL) 48.1 [26.9-79.8] 101.9 [67.0-201.1] <0.001 bio-ADM > 70 pg/mL, n (%) 4 (30.8) 17 (68.0) 0.002 lactate (mmol/L) 0.9 [0.7-1.5] 1.3 [1-1.7] 0.003 IL-6 (pg/mL) 211.25 [141.27-519.95] 251.5 [151.2-475.25] 0.001 PCT (ng/mL) 0.22 [0.11-0.69] 1.46 [0.66-5.06] <0.001 CRP (nmol/L) 256.3 [124.1-298.4] 251.15 [158.4-350] 0.002 WBC (10.sup.3/mm.sup.3) 8 [7.4-9.4] 10.1 [8-13.9] 0.120 Platelets (10.sup.3/μL) 237 [204-328] 263 [204-338] 0.242 Creatinine (mg/dL) 0.89 [0.6-1.08] 1.79 [1.18-3.02] 0.004 Comorbidities Arterial hypertension, n (%) 9 (69.2) 12 (48) 0.455 Diabetes mellitus, n (%) 3 (23.1) 9 (36) 0.215 Ischemic heart disease, n (%) 4 (30.8) 4 (16) 0.557 Embolism/Thrombosis, n (%) 3 (23.1) 1 (4) 0.197 Cardiac arrhythmia, n (%) 0 (0) 5 (20) 0.259 Cerebral vascular disease, n (%) 0 (0) 3 (12) 0.459 COPD, n (%) 1 (7.7) 2 (8) 0.525 Other lung diseases, n (%) 0 (0) 0 (0) 0.025 Chronic kidney disease, n (%) 3 (23.1) 3 (12) 0.708 Tumor disease, n (%) 1 (7.7) 0 (0) 0.057 Smoker, n (%) 2 (15.4) 0 (0) 0.247 Treatment on ICU (first 14 days, unless stated differently) ICU length of stay (days) 19.5 [16.5-22.75] 17.5 [15-20.75] 0.004 Highest dose of Norepinephrine during 0.15 [0.06-0.18] 0.29 [0.13-0.35] <0.001 the first 7 days (μg/kg/min) Anticoagulation, n (%) 3 (23.1) 9 (36) 0.627 Antiplatelet, n (%) 6 (46.2) 5 (20) 0.238 Antihypertensiva, n (%) 10 (76.9) 13 (52) 0.343 Immunsupressant, n (%) 4 (30.8) 2 (8) 0.289 Analgesics, n (%) 1 (7.7) 2 (8) 0.143 Outcome Death 28 days, n (%) 1 (7.7) 11 (44) 0.011 Disposition on day 28 0.001 discharged, n (%) 11 (84.6) 7 (28) on ICU post day 28, n (%) 1 (7.7) 7 (28) Death 28 days, n (%) 1 (7.7) 11 (44)
[0342] Variables are given as median [interquartile range] or number (%). ARDS, acute respiratory distress syndrome; bio-ADM, bioactive adrenomedullin; COPD, chronic obstructive pulmonary disease; CRP, C-reactive protein; ECMO, extracorporeal membrane oxygenation; FiO2, fraction of inspired oxygen; ICU, intensive care unit; IL-6, interleukin-6; pCO2, partial pressure of carbon dioxide; PCT, procalcitonin; PEEP, positive end-expiratory pressure; pO2, partial pressure of oxygen; RRT, renal replacement therapy; spO2, peripheral capillary oxygen saturation; SOFA, sequential organ failure assessment; WBC, white blood cell counts A high proportion of 38 patients (72%) presented with moderate or severe ARDS (25% moderate, 47% severe). Bio-ADM levels increased with severity of ARDS (p<0.001, bio-ADM 28.3 [19.9-28.4], 39.0 [29.2-54.5], 48.1 [26.9-79.8] and 101.9 [67.0-201.1] pg/mL compared to patients without ARDS, mild ARDS, moderate ARDS or severe ARDS, respectively) (
[0343] The majority of patients (n=44) received invasive ventilation during ICU stay (Table 7). Bio-ADM levels were significantly increased in invasively ventilated patients compared to spontaneously breathing patients (68.2 [45.5-106.6] pg/mL vs. 31.8 [18.6-48.4] pg/mL, p=0.006) (
[0344] Increased bio-ADM levels were observed in patients treated with veno-venous ECMO (n=9), compared to patients without ECMO therapy (101.9 [65.0-144.1] pg/mL vs. 53.3 [29.2-91.0] pg/mL, p=0.040) (
[0345] With respect to kidney function, there was a notable correlation between bio-ADM and serum creatinine (r=0.62, p<0.001). In line, significantly higher bio-ADM levels were found in patients receiving RRT (n=27) compared to patients without RRT (n=26) (101.9 [67.7-182.9] pg/mL vs. 40.2 [27.2-53.5] pg/mL, p<0.001) (
[0346] Bio-ADM levels were higher in non-survivors (n=13) than survivors (n=40) (107.6 [51.0-262.1] pg/mL vs. 53.3 [29.2-91.0] pg/mL, p=0.010). Notably, bio-ADM predicted 28-day mortality (C-index 0.72, 95% confidence interval [CI] 0.56-0.87, p<0.001) (
[0347] In conclusion, bio-ADM plasma levels correlate with the disease severity, need for extracorporeal organ assist, and outcome highlighting the promising value of bio-ADM in the early risk stratification and management of patients with COVID-19. Moreover, the data clearly highlight the role of endothelial dysfunction in the pathophysiology of COVID-19 and open up for future randomized trials that prospectively evaluate bio-ADM as a new objective tool for risk stratification and monitoring of patients suffering from COVID-19.
FIGURE DESCRIPTION
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[0349] Illustration of antibody formats—Fv and scFv-Variants.
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[0351] Illustration of antibody formats—heterologous fusions and bifunctional antibodies.
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[0353] Illustration of antibody formats—bivalental antibodies and bispecific antibodies.
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[0367] 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.
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[0369] This figure shows the stability of hADM in human plasma (citrate) in absence and in the presence of NT-H antibody.
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[0371] Alignment of the Fab with homologous human framework sequences.
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TABLE-US-00055 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) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGE ILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGY EYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK SEQ ID No.: 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGR ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK SEQ ID No.: 8 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGR ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPK SEQ ID No.: 9 (AM-VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYVVISWVRQAPGQGLEWMG EILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEG YEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPK SEQ ID No.: 10 (AM-VH4-T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYVVIEWVRQAPGQGLEWMG EILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEG YEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPK SEQ ID No.: 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPK LLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC SEQ ID No.: 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPR RLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC SEQ ID No.: 13 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPR RLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC 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 YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQ GY-CONH.sub.2 SEQ ID No.: 21 (Adrenomedullin 1-52-Gly (ADM 1-52-Gly): amino acids 95-147 of preproADM) YRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYG SEQ ID No.: 22 (Murine ADM 1-50) YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQLTDKDKDGMAPRNKISPQG Y-CONH.sub.2 SEQ ID No.: 23 (1-42 of human ADM): YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA SEQ ID No.: 24 (aa 43-52 of human ADM) PRSKISPQGY-NH.sub.2 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 (Proadrenomedullin N-20 terminal peptide, PAMP: amino acids 22-41 of preproADM) ARLDVASEF RKKWNKWALS R SEQ ID No.: 33 (Midregional proAdrenomedullin, MR-proADM: amino acids 45-92 of preproADM) ELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RV SEQ ID No.: 34 (C-terminal proAdrenomedullin, CT-proADM: amino acids 148-185 of preproADM) RRR RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL SEQ ID No.: 35 (heavy chain, HAM8101) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGE ILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFScSVMHEALHNHYTQKSLSLSPGK SEQ ID No.: 36 (light chain, HAM 8101) DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPR LLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC SEQ ID No.: 37 - IGHV1-69*11 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGR IIPILGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYY YYYGMDVWGQGTTVTVSS SEQ ID No. 38: - HB3 QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGE ILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGY EYDGFDYWGQGTTLTVSS