THERAPY GUIDANCE AND/OR THERAPY MONITORING FOR A TREATMENT WITH ANGIOTENSIN-RECEPTOR-AGONIST AND/OR A PRECURSOR THEREOF

Abstract

Subject matter of the present invention is an angiotensin-receptor-agonist and/or a precursor thereof for use in the treatment of a disease in a subject, ⋅wherein said disease is selected from the group comprising heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burn injuries, traumatic injuries, severe infection (microbial, viral (e.g. AIDS), parasitic diseases (e.g. Malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g. seizures, neurodegenerative diseases), autoimmune diseases, vascular diseases, hypotension and shock, and ⋅wherein said subject has an amount of DPP3 protein and/or DPP3 activity in a sample of bodily fluid that is above a predetermined threshold.

Claims

1-60. (canceled)

61. A method for the treatment of a disease selected from the group consisting of heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burn injuries, traumatic injuries, severe infection (microbial, viral (e.g. AIDS), parasitic diseases (e.g. Malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g. seizures, neurodegenerative diseases), autoimmune diseases, vascular diseases, hypotension, and shock, comprising administering to said subject an effective amount of an angiotensin-receptor-agonist and/or a precursor thereof, wherein said subject has an amount of DPP3 protein and/or DPP3 activity in a sample of bodily fluid that is above a predetermined threshold.

62. The method according to claim 61, wherein said amount of DPP3 protein and/or said DPP3 activity has been determined in a sample of bodily fluid of said subject at least once before and/or during the treatment with said Angiotensin-Receptor-Agonist and/or a precursor thereof.

63. The method according to claim 61, wherein said sample of bodily fluid of said subject is whole blood, blood plasma or blood serum.

64. The method according to claim 61, wherein said Angiotensin-Receptor-Agonist and/or a precursor thereof, in particular Angiotensin II, is administered in combination with an inhibitor of DPP3.

65. The method according to claim 64, wherein said inhibitor of DPP3 is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold.

66. The method according to claim 64, wherein said inhibitor of DPP3 is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to SEQ ID NO. 1, in particular that binds to SEQ ID NO. 2.

67. The method according to claim 64, wherein said inhibitor of DPP3 is an antibody or fragment or scaffold that is monospecific.

68. The method according to claim 61, wherein the amount of DPP3 protein and/or DPP3 activity is determined in a bodily fluid sample of said subject and comprises the steps: Contacting said sample with a capture-binder that binds specifically to full-length DPP3, separating DPP3 bound to said capture binder, adding substrate of DPP3 to said separated DPP3 and quantifying said DPP3 activity by measuring and quantifying the conversion of a substrate of DPP3 or quantifying the amount of said DPP3 protein.

69. The method according to claim 61, wherein the amount of DPP3 protein and/or DPP3 activity 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 full-length DPP3 wherein said capture-binder is an antibody.

70. A method for prognosing the risk of an adverse event in said subject, wherein said method is comprising the steps: determining the level of DPP3 in a sample of bodily fluid of said subject; comparing said level of determined DPP3 to a predetermined threshold, correlating said level of DPP3 with said risk of an adverse event in said subject, wherein an elevated level above a certain threshold is predictive for an enhanced risk of said adverse events.

71. A method for prognosing the risk of an adverse event in said subject according to claim 70, wherein said sample of bodily fluid of said subject is selected from whole blood, blood plasma and blood serum.

72. The method for prognosing the risk of an adverse event in said subject according to claim 70, wherein the amount of DPP3 protein and/or DPP3 activity is determined in a bodily fluid sample of said subject and comprises the steps: Contacting said sample with a capture-binder that binds specifically to full-length DPP3, separating DPP3 bound to said capture binder, adding substrate of DPP3 to said separated DPP3 and quantifying said DPP3 activity by measuring and quantifying the conversion of a substrate of DPP3 or quantifying the amount of said DPP3 protein.

73. The method for prognosing the risk of an adverse event in said subject according to claim 70, wherein the amount of DPP3 protein and/or DPP3 activity 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 full-length DPP3 wherein said capture-binder is an antibody.

74. A method for prognosing the risk of an organ dysfunction in said subject, wherein said method is comprising the steps: determining the level of DPP3 in a sample of bodily fluid of said subject; comparing said level of determined DPP3 to a predetermined threshold, correlating said level of DPP3 with said risk of an adverse event in said subject, wherein an elevated level above a certain threshold is predictive for an enhanced risk of said adverse events.

75. The method for prognosing the risk of an organ dysfunction in said subject according to claim 74, wherein said sample of bodily fluid of said subject is whole blood, blood plasma or blood serum.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0559] FIG. 1: Kaplan Meyer survival plots in relation to low (<68.6 ng/mL) and high (≥68.6 ng/ml) DPP3 plasma concentrations

[0560] (A) 7-Day survival of patients with sepsis/septic shock in relation to DPP3 plasma concentration (cut-off 68.6 ng/mL); (B) 7-Day survival of Patients with cardiogenic shock in relation to DPP3 plasma concentration (cut-off 68.6 ng/mL); (C) 7-day survival of patients with acute myocardial infarction in relation to DPP3 plasma concentration (cut-off 68.6 ng/mL); (D) 3-month survival of patients with dyspnea in relation to DPP3 plasma concentration; (E) 4-week survival of burned patients in relation to DPP3 plasma concentration.

[0561] FIG. 2: SDS-PAGE on a gradient gel (4-20%) of native hDPP3 purified from human erythrocyte lysate. Molecular weight marker is indicated as arrows.

[0562] FIG. 3: Experimental design—Effect of native DPP3 in an animal model.

[0563] FIG. 4: (A) DPP3 injection causes shortening fraction reduction and therefore leads to deteriorating heart function. (B) Decreased kidney function is also observed via increased renal resistive index.

[0564] FIG. 5: Association- and dissociation curve of the AK1967-DPP3 binding analysis using Octet. AK1967 loaded biosensors were dipped into a dilution series of recombinant GST-tagged human DPP3 (100, 33.3, 11.1, 3.7 nM) and association and dissociation monitored.

[0565] FIG. 6: Western Blot of dilutions of blood cell lysate and detection of DPP3 with AK1967 as primary antibody.

[0566] FIG. 7: Inhibition curve of native DPP3 from blood cells with inhibitory antibody AK1967. Inhibition of DPP3 by a specific antibody is concentration dependent, with an IC.sub.50 at ˜15 ng/ml when analyzed against 15 ng/ml DPP3.

[0567] FIG. 8: Experimental setup—Effect of Procizumab in sepsis-induced heart failure.

[0568] FIG. 9: Procizumab drastically improves shortening fraction (A) and mortality rate (B) in sepsis-induced heart failure rats.

[0569] FIG. 10: Experimental design—Isoproterenol-induced cardiac stress in mice followed by Procizumab treatment (B) and control (A).

[0570] FIG. 11: Procizumab improved shortening fraction (A) and reduced the renal resistive index

[0571] (B) within 1 hour and 6 hours after administration, respectively, in isoproterenol-induced heart failure mice.

[0572] FIG. 12: Experimental setup—effect of Valsartan in healthy mice injected with DPP3.

[0573] FIG. 13: Reduction in the shortening fraction by DPP3 is rescued by the Valsartan treatment.

[0574] FIG. 14: High concentrations of DPP3 levels 24 hours after admission of septic patients were associated with worst SOFA scores.

[0575] FIG. 15: High cDPP3 plasma levels correlate with organ dysfunction in septic patients. Barplots of SOFA score in AdrenOSS1 according to the evolution of DPP3 levels during ICU stay. HH: DPP3 above median on admission and at 24 h; HL: above median on admission but below median at 24 h; LL: below median on admission and at 24 h; LH: below median on admission but above median at 24 h.

[0576] FIG. 16: High concentrations of cDPP3 levels 24 hours after admission of septic patients were associated with worst SOFA scores by organ. (A) cardiac, (B) renal, (C) respiratory, (D) liver, (E) coagulation and (F) central nervous system SOFA scores values according to dynamics levels of cDPP3 between admission and 24 h (HH: High/High, HL: High/Low, LH: Low/High, LL: Low/Low).

EXAMPLES

Example 1—Methods for the Measurement of DPP 3 Protein and DPP3 Activity

[0577] Generation of antibodies and determination DPP3 binding ability: Several murine antibodies were produced and screened by their ability of binding human DPP3 in a specific binding assay (see Table 1).

[0578] Peptides/Conjugates for Immunization:

[0579] DPP3 peptides for immunization were synthesized, see Table 1, (JPT Technologies, Berlin, Germany) with an additional N-terminal cystein (if no cystein is present within the selected DPP3-sequence) residue for conjugation of the peptides to Bovine Serum Albumin (BSA). The peptides were covalently linked to BSA by using Sulfolink-coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the manual of Perbio. Recombinant GST-hDPP3 was produced by USBio (United States Biological, Salem, Mass., USA).

[0580] Immunization of Mice, Immune Cell Fusion and Screening:

[0581] Balb/c mice were intraperitoneally (i.p.) injected with 84 μg GST-hDPP3 or 100 μg DPP3-peptide-BSA-conjugates at day 0 (emulsified in TiterMax Gold Adjuvant), 84 μg or 100 μg at day 14 (emulsified in complete Freund's adjuvant) and 42 μg or 50 μg at day 21 and 28 (in incomplete Freund's adjuvant). At day 49 the animal received an intravenous (i.v.) injection of 42 μg GST-hDPP3 or 50 μg DPP3-peptide-BSA-conjugates dissolved in saline. Three days later the mice were sacrificed and the immune cell fusion was performed.

[0582] Splenocytes from the immunized mice and cells of the myeloma cell line SP2/0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37° C. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium [RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT-Supplement]. After one week, the HAT medium was replaced with HT Medium for three passages followed by returning to the normal cell culture medium.

[0583] The cell culture supernatants were primarily screened for recombinant DPP3 binding IgG antibodies two weeks after fusion. Therefore, recombinant GST-tagged hDPP3 (USBiologicals, Salem, USA) was immobilized in 96-well plates (100 ng/well) and incubated with 50 μl cell culture supernatant per well for 2 hours at room temperature. After washing of the plate, 50 μl/well POD-rabbit anti mouse IgG was added and incubated for 1 h at RT. After a next washing step, 50 μl of a chromogen solution (3.7 mM o-phenylendiamine in citrate/hydrogen phosphate buffer, 0.012% H.sub.2O.sub.2) were added to each well, incubated for 15 minutes at RT and the chromogenic reaction stopped by the addition of 50 μl 4N sulfuric acid. Absorption was detected at 490 mm.

[0584] 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.

[0585] Mouse Monoclonal Antibody Production

[0586] Antibodies raised against GST-tagged human DPP3 or DPP3-peptides were produced via standard antibody production methods (Marx et al. 1997) and purified via Protein A. The antibody purities were ≥90% based on SDS gel electrophoresis analysis.

[0587] Characterization of Antibodies—Binding to hDPP3 and/or Immunization Peptide

[0588] To analyze the capability of DPP3/immunization peptide binding by the different antibodies and antibody clones a binding assay was performed:

[0589] a) Solid Phase

[0590] Recombinant GST-tagged hDPP3 (SEQ ID NO. 1) or a DPP3 peptide (immunization peptide, SEQ ID NO. 2) was immobilized onto a high binding microtiter plate surface (96-Well polystyrene microplates, Greiner Bio-One international AG, Austria, 1 μg/well in coupling buffer [50 mM Tris, 100 mM NaCl, pH7, 8], 1 h at RT). After blocking with 5% bovine serum albumin, the microplates were vacuum dried.

[0591] b) Labelling Procedure (Tracer)

[0592] 100 μg (100 μl) of the different antiDPP3 antibodies (detection antibody, 1 mg/ml in PBS, pH 7.4) were mixed with 10 μl acridinium NHS-ester (1 mg/ml in acetonitrile, InVent GmbH, Germany; EP 0 353 971) and incubated for 30 min at room temperature. Labelled antiDPP3 antibody was purified by gel-filtration HPLC on Shodex Protein 5 μm KW-803 (Showa Denko, Japan). The purified labeled antibody was diluted in assay buffer (50 mmol/1 potassium phosphate, 100 mmol/1 NaCl, 10 mmol/1 Na.sub.2-EDTA, 5 g/l bovine serum albumin, 1 g/l murine IgG, 1 g/l bovine IgG, 50 μmol/l amastatin, 100 μmol/l leupeptin, pH 7.4). The final concentration was approx. 5-7*10.sup.6 relative light units (RLU) of labelled compound (approx. 20 ng labeled antibody) per 200 acridinium ester chemiluminescence was measured by using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

[0593] c) hDPP3 Binding Assay

[0594] The plates were filled with 200 μl of labeled and diluted detection antibody (tracer) and incubated for 2-4 h at 2-8° C. Unbound tracer was removed by washing 4 times with 350 μl washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Well-bound chemiluminescence was measured by using the Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

[0595] Characterization of Antibodies—hDPP3-Inhibition Analysis

[0596] To analyze the capability of DPP3 inhibition by the different antibodies and antibody clones a DPP3 activity assay with known procedure (Jones et al., 1982) was performed. Recombinant GST-tagged hDPP3 was diluted in assay buffer (25 ng/ml GST-DPP3 in 50 mM Tris-HCl, pH7.5 and 100 μM ZnCl.sub.2) and 200 μl of this solution incubated with 10 μg of the respective antibody at room temperature. After 1 hour of pre-incubation, fluorogenic substrate Arg-Arg-βNA (20 μl, 2 mM) was added to the solution and the generation of free βNA over time was monitored using the Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG) at 37° C. Fluorescence of βNA is detected by exciting at 340 nm and measuring emission at 410 nm. Slopes (in RFU/min) of increasing fluorescence of the different samples are calculated. The slope of GST-hDPP3 with buffer control is appointed as 100% activity. The inhibitory ability of a possible capture-binder is defined as the decrease of GST-hDPP3 activity by incubation with said capture-binder in percent.

[0597] The following table represents a selection of obtained antibodies and their binding rate in Relative Light Units (RLU) as well as their relative inhibitory ability (%; table 1). The monoclonal antibodies raised against the below depicted DPP3 regions, were selected by their ability to bind recombinant DPP3 and/or immunization peptide, as well as by their inhibitory potential.

[0598] All antibodies raised against the GST-tagged, full length form of recombinant hDPP3 show a strong binding to immobilized GST-tagged hDPP3. Also antibodies raised against the SEQ ID NO.: 2 peptide bind to GST-hDPP3. The SEQ ID NO.: 2 antibodies also strongly bind to the immunization peptide.

TABLE-US-00001 TABLE 1 list of antibodies raised against full-length or sequences of hDPP3 and their ability to bind hDPP3 (SEQ ID NO.: 1) or immunization peptides (SEQ ID NO.: 2) in RLU, as well as the maximum inhibition or recombinant GST-hDPP3. immunization hDPP3 peptide Max. Sequence hDPP3 binding binding inhibition number Antigen/Immunogen region Clone RLU RLU of hDPP3 SEQ ID GST tagged recombinant   1-737 2552 3.023.621 0 65% NO.: 1 FL-hDPP3 2553 3.777.985 0 35% 2554 1.733.815 0 30% 2555 3.805.363 0 25% SEQ ID CETVINPETGEQIQSWYRSGE 474-493 1963   141.822         2.163.038 60% NO.: 2 1964   100.802         2.041.928 60% 1965    99.493         1.986.794 70% 1966   118.097         1.990.702 65% 1967   113.736         1.909.954 70% 1968   105.696         2.017.731 65% 1969    82.558         2.224.025 70%

[0599] The development of a luminescence immunoassay for the quantification of DPP3 protein concentrations (DPP3-LIA) as well as an enzyme capture activity assay for the quantification of DPP3 activity (DPP3-ECA) have been described recently (Rehfeld et al. 2018. JALM. in press), which is incorporated here in its entirety by reference.

Example 2—DPP3 for Prognosis of Short-Term Mortality

[0600] DPP3 concentration in plasma of a variety of diseased patients was determined using a hDPP3 immuno assay and related to the short term-mortality of the patients.

[0601] Study Cohort—Sepsis and Septic Shock

[0602] Plasma samples form 574 patients from the Adrenomedullin and Outcome in Severe Sepsis and Septic Shock (AdrenOSS) study were screened for DPP3. AdrenOSS is a prospective, observational, multinational study including 583 patients admitted to the intensive care unit with sepsis or septic shock (Hollinger et al., 2018). 292 patients were diagnosed with septic shock.

[0603] Study Cohort—Cardiogenic Shock

[0604] Plasma samples from 108 patients that were diagnosed with cardiogenic shock were screened for DPP3. Blood was drawn within 6 h from detection of cardiogenic shock. Mortality was followed for 7 days.

[0605] Study Cohort—Acute Coronary Syndrome

[0606] Plasma samples from 720 patients with acute coronary syndrome were screened for DPP3. Blood was drawn 24 hours after the onset of ChestPain. Mortality was followed for 7 days.

[0607] Study Cohort—Dyspnea:

[0608] Plasma samples from 1440 patients presenting with dyspnea (shortness of breath) were collected immediately to their entry to the emergency department of Skåne University Hospital. Patients with dyspnea may suffer from acute coronary syndrome or congestive heart failure, beside others, and have a high risk for organ failure and short-term mortality. Mortality was followed for 3 months after presentation to the emergency department.

[0609] Study Cohort—Burned Patients:

[0610] Plasma samples from 107 patients with severe burns (more than 15% of total body surface area) were screened for DPP3. Blood was drawn at admission to the hospital. Mortality was followed for 4 weeks.

[0611] hDPP3 Immunoassay:

[0612] An immune-Assay (LIA) or an activity assays (ECA) detecting the amount of human DPP3 (LIA) or the activity of human DPP3 (ECA), respectively, was used for determining the DPP3 level in patient plasma. Antibody immobilization, labelling and incubation were performed as described in Rehfeld et al. (Rehfeld et al. 2018).

[0613] Results

[0614] Short-term patients' survival in Sepsis/Septic Schock was related to the DPP3 plasma concentration at admission. Patients with DPP3 plasma concentration above 68.6 ng/mL (3. Quartile) had an increased mortality risk compared to patients with DPP3 plasma concentrations below this threshold (FIG. 1A). The same relation was visible when only the septic shock patients of this cohort were analyzed for their short-term outcome in relation to DPP3 plasma concentrations. Patients with an elevated DPP3 plasma concentration had an increased mortality risk compared to patients with a low DPP3 plasma concentration. When the same cut-off is applied to patients with cardiogenic shock, also an increased risk for short-term mortality within 7 days is observed in patients with high DPP3 (FIG. 1B).

[0615] In addition, 28 day-survival of patients with acute coronary syndrome in relation to DPP3 is also increased when DPP3 is high and the respective cut-off of 68.6 ng/mL is applied (FIG. 1C).

[0616] Applying this cut-off of 68.6 ng/mL to patients that suffer from Dyspnea, a significant increased mortality risk for patients with high DPP3 is detected within a follow-up of 3 months (FIG. 1D).

[0617] Furthermore, there was an increased risk for 4-week mortality in severely burned patients that have a high DPP3 concentration above the respective cut-off off 68.6 ng/mL (FIG. 1E).

Example 3—Purification of Human Native DPP3

[0618] Human erythrocyte lysate was applied on a total of 100 ml of Sepahrose 4B resin (Sigma-Aldrich) and the flow through was collected. The resin was washed with a total of 370 mL PBS buffer, pH 7.4 and the wash fraction was combined with the collected flow through, resulting in a total volume of 2370 mL.

[0619] For the immuno-affinity purification step, 110 mg of monoclonal anti-hDPP3 mAb AK2552 were coupled to 25.5 mL of UltraLink Hydrazide Resin (Thermo Fisher Scientific) according to the manufacturer's protocol (GlycoLink Immobilization Kit, Thermo Fisher Scientific). The coupling efficiency was 98%, determined by quantification of uncoupled antibody via Bradford-technique. The resin-antibody conjugate was equilibrated with 10 bed volumes of wash-binding buffer (PBS, 0.1% TritonX-100, pH 7.4), combined with 2370 mL of cleared red blood cell lysate and incubated at 4° C. under continuous stirring for 2 h. Consequently, 100 mL of the incubation mixture was spread on ten 15 mL polypropylene columns and the flow-through was collected by centrifugation at 1000×g for 30 seconds. This step was repeated several times resulting in 2.5 mL of DPP3-loaded resin per column. Each column was washed 5 times with 10 mL of wash-binding buffer using the gravity-glow approach. DPP3 was eluted by placing each column in 15-mL falcon tube containing 2 mL of neutralization buffer (1M Tris-HCl, pH 8.0), followed by addition of 10 mL of elution buffer (100 mM Glycine-HCl, 0.1% TritonX-100, pH 3.5) per column and immediate centrifugation for 30 seconds at 1000×g. The elution step was repeated 3 times in total resulting in 360 mL of combined eluates. The pH of the neutralized eluates was 8.0.

[0620] The combined eluates were loaded on a 5 mL HiTrap Q-sephare HP column (GE Healthcare) equilibrated with IEX-buffer A1 (100 mM Glycine, 150 mM Tris, pH 8.0) using the sample pump of the Äkta Start system (GE Healthcare). After sample loading, the column was washed with five column volumes of IEX Buffer A2 (12 mM NaH.sub.2PO.sub.4, pH 7.4) to remove unbound protein. Elution of DPP3 was achieved by applying a sodium chloride gradient over 10 column volumes (50 mL) in a range of 0-1 M NaCl using IEX-buffer B (12 mM NaH.sub.2PO.sub.4, 1 M NaCl, pH 7.4). The eluates were collected in 2 mL fractions. Buffers used for ion exchange chromatography were sterile filtered using a 0.22 μM bottle-top filter.

[0621] A purification table with the respective yields and activities of each purification step is given in table 2. FIG. 2 shows an SDS-PAGE on a gradient gel (4-20%) of native hDPP3 purified from human erythrocyte lysate.

TABLE-US-00002 TABLE 2 Purification of DPP 3 from human erythrocytes DPP3 Total amount Total activity in Specific in % protein in μmol/min Yield.sup.d) activity in Purification Step (LIA).sup.a) mg.sup.b) (ECA).sup.c) in % U/mg.sup.e) factor.sup.f) Lysate 100 204160 55 100 0.00027 — IAP 80.6 71.2 46.1 84 0.65 2407 IEX 75 6.6 38.7 70 5.9 21852 .sup.a)Relative DPP3 amount was determined in all fractions using the DPP3-LIA assay. Amount of DPP3 in starting material was set to 100% and remaining DPP3 amount in purification fractions was correlated to the starting material. .sup.b)Total protein amount was determined using the method of Lowry modified by Peterson (Peterson 1977. Analytical Biochemistry 356:346-356). .sup.c)Total Arg.sub.2-βNA hydrolyzing activity in μmol of substrate converted per minute was determined using the DPP3-ECA, calibrated via β-naphtylamine (0,05-100 μM). .sup.d)Purification yield was calculated form total Arg.sub.2-βNA hydrolyzing activity. Arg.sub.2-βNA hydrolyzing activity in starting material was set to 100%. .sup.e)Specific activity is defined as μmol of substrate converted per minute and mg of total protein. .sup.f)The purification factor is the quotient of specific activities after and before each purification step.

Example 4—Effect of Native DPP3 in an Animal Model

[0622] The effect of native hDPP3 injection in healthy mice was studied by monitoring the shortening fraction and renal resistive index.

[0623] Wild type Black 6 mice (8-12 weeks, group size refer to table 3) were acclimated during 2 weeks and a baseline echocardiography was done. The mice were randomly allocated to one of the two groups and, subsequently, native DPP3 protein or PBS were injected intravenously via a retro-orbital injection with a dose of 600 μg/kg for DPP3 protein.

[0624] After DPP3 or PBS injection, cardiac function was assessed by echocardiography (Gao et al. 2011) and renal function assessed by renal resistive index (Lubas et al., 2014, Dewitte et al, 2012) at 15, 60 and 120 minutes (FIG. 3).

TABLE-US-00003 TABLE 3 list of experiment groups Group Number of Animals Treatment WT + PBS 3 PBS WT + DPP3 4 Native DPP3

[0625] Results

[0626] The mice treated with native DPP3 protein show significantly reduced shortening fraction compared to the the control group injected with PBS (FIG. 4A). The WT+DPP3 group also displays worsening renal function as observed by the renal resistive index increase (FIG. 4B).

Example 5—Development of Procizumab

[0627] Antibodies raised against SEQ ID NO. 2 were characterized in more detail (epitope mapping, binding affinities, specificity, inhibitory potential). Here the results for clone 1967 of SEQ ID NO.: 2 (AK1967; “Procizumab”) are shown as an example.

[0628] Determination of AK1967 Epitope on DPP3:

[0629] For epitope mapping of AK1967 a number of N- or C-terminally biotinylated peptides were synthesized (peptides&elephants GmbH, Hennigsdorf, Germany). These peptides include the sequence of the full immunization peptide (SEQ ID NO. 2) or fragments thereof, with stepwise removal of one amino acid from either C- or N-terminus (see table 5 for a complete list of peptides).

[0630] High binding 96 well plates were coated with 2 μg Avidin per well (Greiner Bio-One international AG, Austria) in coupling buffer (500 mM Tris-HCl, pH 7.8, 100 mM NaCl). Afterwards plates were washed and filled with specific solutions of biotinylated peptides (10 ng/well; buffer—1×PBS with 0.5% BSA)

[0631] Anti-DPP3 antibody AK1967 was labelled with a chemiluminescence label according to Example 1.

[0632] The plates were filled with 200 μl of labeled and diluted detection antibody (tracer) and incubated for 4 h at room temperature. Unbound tracer was removed by washing 4 times with 350 μl washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Well-bound chemiluminescence was measured by using the Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG). Binding of AK1967 to the respective peptides is determined by evaluation of the relative light units (RLU). Any peptide that shows a significantly higher RLU signal than the unspecific binding of AK1967 is defined as AK1967 binder. The combinatorial analysis of binding and non-binding peptides reveals the specific DPP3 epitope of AK1967.

[0633] Determination of Binding Affinities Using Octet:

[0634] The experiment was performed using Octet Red96 (ForteBio). AK1967 was captured on kinetic grade anti-humanFc (AHC) biosensors. The loaded biosensors were then dipped into a dilution series of recombinant GST-tagged human DPP3 (100, 33.3, 11.1, 3.7 nM). Association was observed for 120 seconds followed by 180 seconds of dissociation. The buffers used for the experiment are depicted in table 4. Kinetic analysis was performed using a 1:1 binding model and global fitting.

TABLE-US-00004 TABLE 4 Buffers used for Octet measurements Buffer Composition Assay Buffer PBS with 0.1% BSA, 0.02% Tween-21 Regeneration 10 mM Glycine buffer (pH 1.7) Buffer Neutralization PBS with 0.1% BSA, 0.02% Buffer Tween-21

[0635] Western Blot Analysis of Binding Specificity of AK1967:

[0636] Blood cells from human EDTA-blood were washed (3× in PBS), diluted in PBS and lysed by repeated freeze-thaw-cycles. The blood cell lysate had a total protein concentration of 250 μg/ml, and a DPP3 concentration of 10 μg/ml. Dilutions of blood cell lysate (1:40, 1:80, 1:160 and 1:320) and of purified recombinant human His-DPP3 (31.25-500 ng/ml) were subjected to SDS-PAGE and Western Blot. The blots were incubated in 1.) blocking buffer (1×PBS-T with 5% skim milk powder), 2.) primary antibody solution (AK1967 1:2.000 in blocking buffer) and 3.) HRP labelled secondary antibody (goat anti mouse IgG, 1:1.000 in blocking buffer). Bound secondary antibody was detected using the Amersham ECL Western Blotting Detection Reagent and the Amersham Imager 600 UV (both from GE Healthcare).

[0637] DPP3 Inhibition Assay:

[0638] To analyze the capability of DPP3 inhibition by AK1967 a DPP3 activity assay with known procedure (Jones et al., 1982) was performed as described in example 1. The inhibitory ability AK1967 is defined as the decrease of GST-hDPP3 activity by incubation with said antibody in percent. The resulting lowered DPP3 activities are shown in an inhibition curve in FIG. 1C.

[0639] Epitope Mapping:

[0640] The analysis of peptides that AK1967 binds to and does not bind to revealed the DPP3 sequence INPETG (SEQ ID NO.: 3) as necessary epitope for AK1967 binding (see table 5).

TABLE-US-00005 TABLE 5 Peptides used for Epitope mapping of AK1967 [00001]

[0641] Binding Affinity:

[0642] AK1967 binds with an affinity of 2.2*10.sup.−9 M to recombinant GST-hDPP3 (kinetic curves see FIG. 5).

[0643] Specificity and Inhibitory Potential:

[0644] The only protein detected with AK1967 as primary antibody in lysate of blood cells was DPP3 at 80 kDa (FIG. 6). The total protein concentration of the lysate was 250 μg/ml whereas the estimated DPP3 concentration is about 10 μg/ml. Even though there is 25 times more unspecific protein in the lysate, AK1967 binds and detects specifically DPP3 and no other unspecific binding takes place.

[0645] AK1967 inhibits 15 ng/ml DPP3 in a specific DPP3 activity assay with an IC50 of about 15 ng/ml (FIG. 7).

[0646] Chimerization/Humanization:

[0647] The monoclonal antibody AK1967 (“Procizumab”), with the ability of inhibiting DPP3 activity by 70%, was chosen as possible therapeutic antibody and was also used as template for chimerization and humanization.

[0648] Humanization of Murine Antibodies May be Conducted According to the Following Procedure:

[0649] For humanization of an antibody of murine origin the antibody sequence is analyzed for the structural interaction of framework regions (FR) with the complementary determining regions (CDR) and the antigen. Based on structural modelling an appropriate FR of human origin is selected and the murine CDR sequences are transplanted into the human FR. Variations in the amino acid sequence of the CDRs or FRs may be introduced to regain structural interactions, which were abolished by the species switch for the FR sequences. This recovery of structural interactions may be achieved by random approach using phage display libraries or via directed approach guided by molecular modeling (Almagro and Fransson, 2008. Humanization of antibodies. Front Biosci. 13:1619-33).

[0650] With the above context, the variable region can be connected to any subclass of constant regions (IgG, IgM, IgE. IgA), or only scaffolds, Fab fragments, Fv, Fab and F(ab)2. In example 6 and 7 below, the murine antibody variant with an IgG2a backbone was used. For chimerization and humanization a human IgG1κ backbone was used.

[0651] For epitope binding only the Complementarity Determining Regions (CDRs) are of importance. The CDRs for the heavy chain and the light chain of the murine anti-DPP3 antibody (AK1967; “Procizumab”) are shown in SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8 for the heavy chain and SEQ ID NO. 9, sequence KVS and SEQ ID NO. 10 for the light chain, respectively.

[0652] Sequencing of the anti-DPP3 antibody (AK1967; “Procizumab”) revealed an antibody heavy chain variable region (H chain) according to SEQ ID NO.: 11 and an antibody light chain variable region (L chain) according to SEQ ID NO.: 12.

Example 6—Effect of Procizumab in Sepsis-Induced Heart Failure

[0653] In this experiment, the effect of Procizumab injection in sepsis-induced heart failure rats (Rittirsch et al. 2009) was studied by monitoring the shortening fraction.

[0654] CLP Model of Septic Shock:

[0655] Male Wistar rats (2-3 months, 300 to 400 g, group size refer to table 6) from the Centre d'élevage Janvier (France) were allocated randomly to one of three groups. All the animals were anesthetized using ketamine hydrochloride (90 mg/kg) and xylazine (9 mg/kg) intraperitoneally (i.p.). For induction of polymicrobial sepsis, cecal ligation and puncture (CLP) was performed using Rittirsch's protocol with minor modification. A ventral midline incision (1.5 cm) was made to allow exteriorization of the cecum. The cecum is then ligated just below the ileocecal valve and punctured once with an 18-gauge needle. The abdominal cavity is then closed in two layers, followed by fluid resuscitation (3 ml/100 g body of weight of saline injected subcutaneously) and returning the animal to its cage. Sham animals were subjected to surgery, without getting their cecum punctured. CLP animals were randomized between placebo and therapeutic antibody.

[0656] Study Design:

[0657] The study flow is depicted in FIG. 8. After CLP or sham surgery the animals were allowed to rest for 20 hours with free access to water and food. Afterwards they were anesthetized, tracheotomy done and arterial and venous line laid. At 24 hours after CLP surgery either AK1967 or vehicle (saline) were administered with 5 mg/kg as a bolus injection followed by a 3 h infusion with 7.5 mg/kg. As a safety measure, hemodynamics were monitored invasively and continuously from t=0 till 3 h.

[0658] At t=0 (baseline) all CLP animals are in septic shock and developed a decrease in heart function (low blood pressure, low shortening fraction). At this time point Procizumab or vehicle (PBS) were injected (i.v.) and saline infusion was started. There were 1 control group and 2 CLP groups which are summarized in the table below (table 6). At the end of the experiment, the animals were euthanized, and organs harvested for subsequent analysis.

TABLE-US-00006 TABLE 6 list of experimental groups Group Number of Animals CLP Treatment Sham 7 No PBS CLP-PBS 6 Yes PBS CLP-PCZ 4 Yes PCZ

[0659] Invasive Blood Pressure:

[0660] Hemodynamic variables were obtained using the AcqKnowledge system (BIOPAC Systems, Inc., USA). It provides a fully automated blood pressure analysis system. The catheter is connected to the BIOPAC system through a pressure sensor.

[0661] For the procedure, rats were anesthetized (ketamine and xylazine). Animals were moved to the heating pad for the desired body temperature to 37-37.5° C. The temperature feedback probe was inserted into the rectum. The rats were placed on the operating table in a supine position. The trachea was opened and a catheter (16G) was inserted for an external ventilator without to damage carotid arteries and vagus nerves. The arterial catheter was inserted into the right carotid artery. The carotid artery is separate from vagus before ligation.

[0662] A central venous catheter was inserted through the left jugular vein allowing administration of PCZ or PBS.

[0663] Following surgery, the animals were allowed to rest for the stable condition prior to hemodynamic measurements. Then baseline blood pressure (BP) were recorded. During the data collection, saline infusion via arterial line was stopped.

[0664] Echocardiography:

[0665] Animals were anesthetized using ketamine hydrochloride. Chests were shaved and rats were placed in decubitus position.

[0666] For transthoracic echocardiographic (TTE) examination a commercial GE Healthcare Vivid 7 Ultra-sound System equipped with a high frequency (14-MHz) linear probe and 10-MHz cardiac probe was used. All examinations were recorded digitally and stored for subsequent off-line analysis.

[0667] Grey scale images were recorded at a depth of 2 cm. Two-dimensional examinations were initiated in a parasternal long axis view to measure the aortic annulus diameter and the pulmonary artery diameter. M-mode was also employed to measure left ventricular (LV) dimensions and assess fractional shortening (FS %). LVFS was calculated as LV end-diastolic diameter−LV end-systolic diameter/LV end-diastolic diameter and expressed in %. The time of end-diastole was therefore defined at the maximal diameter of the LV. Accordingly, end-systole was defined as the minimal diameter in the same heart cycle. All parameters were measured manually. Three heart cycles were averaged for each measurement.

[0668] From the same parasternal long axis view, pulmonary artery flow was recorded using pulsed wave Doppler. Velocity time integral of pulmonary artery outflow was measured.

[0669] From an apical five-chamber view, mitral flow was recorded using pulsed Doppler at the level of the tip of the mitral valves.

[0670] Results:

[0671] The sepsis-induced heart failure rats treated with PBS (CLP+PBS) show reduced shortening fraction compared to the sham animals (FIG. 9A). The CLP+PBS group also displays high mortality rate (FIG. 9B). In contrast, application of Procizumab to sepsis-induced heart failure rats improves shortening fraction (FIG. 9A) and drastically reduces the mortality rate (FIG. 9B).

Example 7—Effect of Procizumab on Heart and Kidney Function

[0672] The effect of Procizumab in isoproterenol-induced heart failure in mice was studied by monitoring the shortening fraction and renal resistive index.

[0673] Isoproterenol-Induced Cardiac Stress in Mice:

[0674] Acute heart failure was induced in male mice at 3 months of age by two daily subcutaneous injections of 300 mg/kg of Isoproterenol, a non-selective β-adrenergic agonist (DL-Isoproterenol hydrochloride, Sigma Chemical Co) (ISO) for two days (Vergaro et al, 2016). The ISO dilution was performed in NaCl 0.9%. Isoproterenol-treated mice were randomly assigned to two groups (Table 7) and PBS or Procizumab (10 mg/kg) were injected intravenously after baseline echocardiography (Gao et al., 2011) and renal resistive index measurements (Lubas et al., 2014, Dewitte et al, 2012) were performed at day 3 (FIGS. 10 A and B).

[0675] Cardiac function was assessed by echocardiography (Gao et al., 2011) and by the renal resistive index (Lubas et al., 2014, Dewitte et al, 2012) at 1 hour, 6 hours and 24 hours (FIGS. 10 A and B). The group of mice that was injected with vehicle (PBS) instead of isoproterenol was subjected to no further pharmacological treatment and served as the control group (Table 7).

TABLE-US-00007 TABLE 7 list of experimental groups Group Number of Animals Treatment Sham + PBS 27 PBS HF + PBS 15 PBS HF + PCZ 20 PCZ

[0676] Results:

[0677] Application of Procizumab to isoproterenol-induced heart failure mice restores heart function within the first hour after administration (FIG. 11A). Kidney function of sick mice shows significant improvement at 6 hours post PCZ injection and is comparable to the kidney function of sham animals at 24 hours (FIG. 11B).

Example 8—Effect of Valsartan

[0678] The effect of an antagonist for the type I angiotensin II receptor (ATR1), Valsartan, in healthy mice injected with DPP3 was studied by monitoring the shortening fraction.

[0679] In this experiment, healthy Black 6 mice (8-12 weeks, group size refer to table 8) consumed water with 50 mg/kg Valsartan per day or just water (Table 8) for a period of two weeks. Subsequently, both groups received an intravenous injection of native DPP3 (600 μg/kg) and the shortening fraction was assessed according to Gao et al., 2011 at 15, 60 and 120 minutes (FIG. 12).

TABLE-US-00008 TABLE 8 list of experiment groups Group Number of Animals Treatment WT + DPP3 4 Water + PBS/DPP3 injection Val + DPP3 3 Water + Valsartan/ DPP3 injection

[0680] Results:

[0681] DPP3 injection to healthy mice lead to a significant decrease in the shortening fraction (FIG. 13). In contrast, healthy mice treated with the angiotensin II receptor antagonist, Valsartan, and then submitted to DPP3 injection, showed no signs of heart dysfunction assessed by the shortening fraction. Therefore, the cardiac function is restored by the Valsartan treatment and thus the DPP3-mediated heart dysfunction is angiotensin II mediated.

[0682] The animals that were treated with Valsartan for two weeks have been adapted to blocking of the type I angiotensin II receptor, to the subsequent inhibited angiotensin II-mediated signaling and the inhibited AngII-mediated activity of the heart function. Apparently, under Valsartan treatment, the organism switched to other ways for activating cardiac function independent of type I angiotensin II receptor signaling, as this angiotensin signaling system has been inhibited by Valsartan.

[0683] When DPP3 cleaves Ang II and thus inhibits the angiotensin II-mediated activity of the heart function, those animals that are adapted to a down-regulated angiotensin-system (Valsartan treated animals) showed no signs of heart dysfunction as assessed by the shortening fraction. In contrast thereto, animals that were not treated with the angiotensin II receptor antagonist Valsartan and were not adapted to an inhibited Ang II-mediated signaling, showed a significant decrease in the shortening fraction in response to DPP3 injection and subsequent cleavage and inactivation of AngII

[0684] This experiment clearly shows the relationship between DPP3 and angiotensin II meaning that the DPP3-induced heart dysfunction is angiotensin II-mediated.

Example 9—DPP3 and Organ Dysfunction in Sepsis

[0685] The same study as described in Example 2 (AdrenOSS-1) was used to assess the association between cDPP3, organ (e.g. cardiovascular and renal dysfunction) in patients admitted for sepsis and septic shock. The Adrenoss-1 is an European prospective, observational, multinational study (ClinicalTrials.gov NCT02393781) including 583 patients admitted to the ICU with sepsis or septic shock. The primary outcome (as described in example 2) was 28-day mortality. Secondary outcomes included organ failure defined by SOFA score, organ support with focus on vasopressor use and need for renal replacement therapy. Blood for the central laboratory was sampled within 24 hours after ICU admission and on day 2.

[0686] Median cDPP3 measured at admission in all AdrenoSS-1 patients was 45.1 ng/mL (inter quartile range 27.5-68.6). High DPP3 levels measured at admission were associated with worse metabolic parameters, renal and cardiac function and SOFA score: patients with DPP3 levels below the median had a median SOFA score (points) of 6 (IQR 4-9) compared to a median SOFA score of 8 (IQR 5-11) for patients with DPP3 levels above the median of 45.1 ng/mL (FIG. 14)

[0687] Whatever levels of cDPP3 at admission, high concentrations of cDPP3 levels 24 hours later were associated with worst SOFA scores whether global FIG. 15 or by organ (FIG. 16 A-F).

[0688] In summary these data showed that high levels of cDPP3 were associated with survival and the extent of organ dysfunction in a large international cohort septic or septic shock patients. The study found marked association between cDPP3<45.1 ng/ml at admission and short-term survival as well as the prognostic cut-off value of 45.1 pg/ml in both sepsis and septic shock. Concerning organ dysfunction, there was a positive relationship between cDPP3 and SOFA score at ICU admission. More importantly, the relationship between cPDPP3 levels and extent of organ dysfunction, seen at ICU admission, was also true during the recovery phase. Indeed, patients with high cDPP3 levels at admission who showed a decline towards normal cDPP3 values at day 2 were more likely to recover all organ function including cardiovascular, kidney, lung, liver.

TABLE-US-00009 SEQUENCES SEQ ID No. 1-hDPP3 aa 1-737 MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAAWYGGLAVLLQTSPEAPYIYALLSRLFRAQDPDQLR QHALAEGLTEEEYQAFLVYAAGVYSNMGNYKSFGDTKFVPNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMF SLEPRLRHLGLGKEGITTYFSGNCTMEDAKLAQDFLDSQNLSAYNTRLFKEVDGEGKPYYEVRLASVLGSEPSLDS EVTSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKAKAYAANSHQGQMLAQYIESFTQGSIEAHKRGSRFWIQDK GPIVESYIGFIESYRDPFGSRGEFEGFVAVVNKAMSAKFERLVASAEQLLKELPWPPTFEKDKFLTPDFTSLDVLT FAGSGIPAGINIPNYDDLRQTEGFKNVSLGNVLAVAYATQREKLTFLEEDDKDLYILWKGPSFDVQVGLHELLGHG SGKLFVQDEKGAFNFDQETVINPETGEQIQSWYRSGETWDSKFSTIASSYEECRAESVGLYLCLHPQVLEIFGFEG ADAEDVIYVNWLNMVRAGLLALEFYTPEAFNWRQAHMQARFVILRVLLEAGEGLVTITPTTGSDGRPDARVRLDRS KIRSVGKPALERFLRRLQVLKSTGDVAGGRALYEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDV QLLEYEASAAGLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA SEQ ID No. 2-hDPP3 aa 474-493 (N-Cys)-immunization peptide with additional N-terminal Cystein CETVINPETGEQIQSWYRSGE SEQ ID No. 3-hDPP3 aa 477-482-epitope of AK1967 INPETG SEQ ID No. 4-variable region of murine AK1967 in heavy chain QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMSVGWIRQPSGKGLEWLAHIWWNDNKSYNPALKSRLTISRDTS NNQVFLKIASVVTADTGTYFCARNYSYDYWGQGTTLTVSS SEQ ID No. 5-variable region of murine AK1967 in light chain DVVVTQTPLSLSVSLGDPASISCRSSRSLVHSIGSTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDF TLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK SEQ ID No. 6-CDR1 of murine AK1967 in heavy chain GFSLSTSGMS SEQ ID No. 7-CDR2 of murine AK1967 in heavy chain IWWNDNK SEQ ID No. 8-CDR 3 of murine AK1967 in heavy chain ARNYSYDY SEQ ID No. 9-CDR1 of murine AK1967 in light chain RSLVHSIGSTY CDR2 of murine AK1967 in light chain KVS SEQ ID No. 10-CDR3 of murine AK1967 in light chain SQSTHVPWT SEQ ID No. 11-humanized AK1967-heavy chain sequence (IgG1κ backbone) MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLAH IWWNDNKSYNPALKSRLTITRDTSKNQVVLTMTNMDPVDTGTYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG SEQ ID No. 12-humanized AK1967-light chain sequence (IgG1κ backbone) METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLYWYLQKPGQSPQLLIYKV SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC SEQ ID No. 13-Angiotensin II (synonyme: 5-isoleucine-angiotensin II) DRVYIHPF SEQ ID No. 14-Angiotensin II analogue (5-valine-angiotensin II) DRVYVHPF SEQ ID No. 15-Angiotensin II analogue (Asn.sup.1-Phe.sup.4) NRVFIHPF SEQ ID No. 16-Angiotensin II hexapeptide VYIHPF SEQ ID No. 17-Angiotensin II nonapeptide NRVYYVHPF SEQ ID No. 18-Angiotensin II analogue ([Asn.sup.1-Ile.sup.5-Ile.sup.8]-angiotensin II) NRVYIHPI SEQ ID No. 19-Angiotensin II analogue ([Asn.sup.1-Ile.sup.5-Ala.sup.8]-angiotensin II) NRVYIHPA SEQ ID No. 20-Angiotensin II analogue ([Asn.sup.1-diiodoTyr.sup.4-Ile.sup.5]-angiotensin II NRVYIHPF SEQ ID No. 21-Angiotensin III RVYIHPF SEQ ID No. 22-Angiotensin III analogue (Val.sup.4-angiotensin III) RVYVHPF SEQ ID No. 23-Angiotensin III analogue (Phe.sup.3-angiotensin III) RVFIHPF SEQ ID No. 24-Angiotensin III analogue ([Ile.sup.4-Ala.sup.7]-angiotensin III) RVYIHPA SEQ ID No. 25-Angiotensin III analogue (diiodoTyr.sup.3-Ile.sup.4]-angiotensin III) RVYIHPF SEQ ID No. 26-Angiotensin IV VYIHPF SEQ ID No. 27-Angiotensin IV analogue (Val.sup.3-angiotensin IV) VYVHPF SEQ ID No. 28-Angiotensin IV analogue (Phe.sup.2-angiotensin IV) VFIHPF SEQ ID No. 29-Angiotensin IV analogue ([Ile.sup.3-Ala.sup.6]-angiotensin IV) VYIHPA SEQ ID No. 30-Angiotensin IV analogue ([diiodoTyr.sup.2-Ile.sup.3-angiotensin IV) VYIHPF