BRAIN NATRIURETIC PEPTIDE ENGRAFTED ANTIBODIES

Abstract

The present invention relates to an antibody or a fragment thereof comprising at least one heterologous amino acid sequence incorporated within at least one CDR region of said antibody or fragment thereof, wherein said at least one heterologous amino acid sequence comprises an N-terminal linker sequence (Ntls), a Brain Natriuretic Peptide (BNP) and a C-terminal linker sequence (Ctls). Optionally, at least a portion of said at least one CDR region is replaced by said at least one heterologous amino acid sequence incorporated therein. The present invention further relates to such antibody or fragment thereof for use in a method for treatment, a composition comprising such antibody or fragment thereof, a nucleic acid or a mixture of nucleic acids encoding such antibody or fragment thereof, a host cell comprising such nucleic acid or such mixture of nucleic acids and to a process for producing such antibody or fragment thereof.

Claims

1. An antibody or a fragment thereof comprising at least one heterologous amino acid sequence incorporated within at least one CDR region of said antibody or fragment thereof, wherein said at least one heterologous amino acid sequence comprises an N-terminal linker sequence (Ntls), a Brain Natriuretic Peptide (BNP) and a C-terminal linker sequence (Ctls), wherein optionally at least a portion of said at least one CDR region is replaced by said at least one heterologous amino acid sequence incorporated therein, and wherein a) at least 12 amino acid residues are present between i) amino acid residue HC res25 according to Kabat and the first amino acid residue of said BNP in case of an incorporation of said heterologous amino acid sequence within CDRH1; ii) amino acid residue HC res51 according to Kabat and the first amino acid residue of said BNP in case of an incorporation of said heterologous amino acid sequence within CDRH2; iii) amino acid residue HC res92 according to Kabat and the first amino acid residue of said BNP in case of an incorporation of said heterologous amino acid sequence within CDRH3; iv) amino acid residue LC res26 according to Kabat and the first amino acid residue of said BNP in case of an incorporation of said heterologous amino acid sequence within CDRL1; v) amino acid residue LC res49 according to Kabat and the first amino acid residue of said BNP in case of an incorporation of said heterologous amino acid sequence within CDRL2; and/or vi) amino acid residue LC res88 according to Kabat and the first amino acid residue of said BNP in case of an incorporation of said heterologous amino acid sequence within CDRL3; and wherein b) at least 9 amino acid residues are present between the last amino acid residue of said BNP and i) amino acid residue HC res35a according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRH1; ii) amino acid residue HC res57 according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRH2; iii) amino acid residue HC res106 according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRH3; iv) amino acid residue LC res 32 according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRL1; v) amino acid residue LC res57 according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRL2; and/or vi) amino acid residue LC res98 according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRL3.

2. The antibody or fragment thereof according to claim 1, wherein said BNP is selected from the group consisting of human BNP having the sequence of SEQ ID NO 24 and a peptide having at least 80% sequence identity therewith.

3. The antibody or fragment thereof according to claim 1, wherein a) said Ntls comprises i) a GS linker sequence; ii) a PN linker sequence; iii) an amino acid sequence which is part of a human IgG antibody scaffold or a sequence that shares at least 80% sequence identity therewith, particularly an amino acid sequence which is part of the fab domain scaffold of a human IgG antibody or a sequence that shares at least 80% sequence identity therewith, more particularly the sequence of any one of SEQ ID NOs 1, 2 or 4 or a sequence that shares at least 80% sequence identity with any one of SEQ ID NOs 1, 2 or 4 iv) the sequence of SEQ ID NO 6 or a sequence that shares at least 60% sequence identity therewith; v) the sequence of SEQ ID NO 7 or a sequence that shares at least 60% sequence identity therewith; vi) the sequence of SEQ ID NO 9 or a sequence that shares at least 60% sequence identity therewith; vii) the sequence of SEQ ID NO 11 or a sequence that shares at least 60% sequence identity therewith; viii) the sequence of SEQ ID NO 13 or a sequence that shares at least 60% sequence identity therewith; ix) the sequence of SEQ ID NO 15 or a sequence that shares at least 60% sequence identity therewith; x) the sequence of SEQ ID NO 21 or a sequence that shares at least 60% sequence identity therewith; or xi) any combination thereof, and wherein b) said Ctls comprises i) a GS linker sequence; ii) a PN linker sequence; iii) an amino acid sequence which is part of a human IgG antibody scaffold or a sequence that shares at least 80% sequence identity therewith, particularly an amino acid sequence which is part of the fab domain scaffold of a human IgG antibody or a sequence that shares at least 80% sequence identity therewith, more particularly the sequence of any one of SEQ ID NOs 1, 3 or 5 or a sequence that shares at least 80% sequence identity with any one of SEQ ID NOs 1, 3 or 5; iv) the sequence of SEQ ID NO 6 or a sequence that shares at least 60% sequence identity therewith; v) the sequence of SEQ ID NO 8 or a sequence that shares at least 60% sequence identity therewith; vi) the sequence of SEQ ID NO 10 or a sequence that shares at least 60% sequence identity therewith; vii) the sequence of SEQ ID NO 12 or a sequence that shares at least 60% sequence identity therewith; viii) the sequence of SEQ ID NO 14 or a sequence that shares at least 60% sequence identity therewith; ix) the sequence of SEQ ID NO 15 or a sequence that shares at least 60% sequence identity therewith; x) the sequence of SEQ ID NO 20 or a sequence that shares at least 60% sequence identity therewith; xi) the sequence of SEQ ID NO 22 or a sequence that shares at least 60% sequence identity therewith; or xii) any combination thereof.

4. The antibody or fragment thereof according to claim 3, wherein i) said Ntls and said Ctls each comprise a GS linker sequence; ii) said Ntls and said Ctls each comprise a PN linker sequence; iii) said Ntls and said Ctls each comprise an amino acid sequence which is part of a human IgG antibody scaffold or a sequence that shares at least 80% sequence identity therewith, particularly an amino acid sequence which is part of the fab domain scaffold of a human IgG antibody or a sequence that shares at least 80% sequence identity therewith, more particularly said Ntls comprises the sequence of any one of SEQ ID NOs 1, 2 or 4 or a sequence that shares at least 80% sequence identity with any one of SEQ ID NOs 1, 2 or 4 and said Ctls comprises the sequence of any one of SEQ ID NOs 1, 3 or 5 or a sequence that shares at least 80% sequence identity with any one of SEQ ID NOs 1, 3 or 5; iv) said Ntls and said Ctls each comprise the sequence of SEQ ID NO 6 or a sequence that shares at least 60% sequence identity therewith; v) said Ntls comprises the sequence of SEQ ID NO 7 or a sequence that shares at least 60% sequence identity therewith and said Ctls comprises the sequence of SEQ ID NO 8 or a sequence that shares at least 60% sequence identity therewith; vi) said Ntls comprises the sequence of SEQ ID NO 9 or a sequence that shares at least 60% sequence identity therewith and said Ctls comprises the sequence of SEQ ID NO 10 or a sequence that shares at least 60% sequence identity therewith; vii) said Ntls comprises the sequence of SEQ ID NO 11 or a sequence that shares at least 60% sequence identity therewith and said Ctls comprises the sequence of SEQ ID NO 12 or a sequence that shares at least 60% sequence identity therewith; viii) said Ntls comprises the sequence of SEQ ID NO 13 or a sequence that shares at least 60% sequence identity therewith and said Ctls comprises the sequence of SEQ ID NO 14 or a sequence that shares at least 60% sequence identity therewith; ix) said Ntls and said Ctls each comprise the sequence of SEQ ID NO 15 or a sequence that shares at least 60% sequence identity therewith; x) said Ntls comprises the sequence of SEQ ID NO 9 or a sequence that shares at least 60% sequence identity therewith and said Ctls comprises the sequence of SEQ ID NO 20 or a sequence that shares at least 60% sequence identity therewith; or xi) said Ntls comprises the sequence of SEQ ID NO 21 or a sequence that shares at least 60% sequence identity therewith and said Ctls comprises the sequence of SEQ ID NO 22 or a sequence that shares at least 60% sequence identity therewith.

5. The antibody or fragment thereof according to claim 1, wherein said Ntls further comprises an anchoring element A1 at its C terminal end and/or wherein said Ctls further comprises an anchoring element A2 at its N terminal end, wherein A1 and/or A2 predominantly comprise glycine and serine residues, particularly wherein at least 60% of the amino acid residues of A1 and/or A2 are selected from glycine and serine residues.

6. The antibody or fragment thereof according to claim 1, wherein the amino acid stretch present between i) amino acid residue HC res25 according to Kabat and the first amino acid residue of the BNP in case of an incorporation of said heterologous amino acid sequence within CDRH1; ii) amino acid residue HC res51 according to Kabat and the first amino acid residue of the BNP in case of an incorporation of said heterologous amino acid sequence within CDRH2; iii) amino acid residue HC res92 according to Kabat and the first amino acid residue of the BNP in case of an incorporation of said heterologous amino acid sequence within CDRH3; iv) amino acid residue LC res26 according to Kabat and the first amino acid residue of the BNP in case of an incorporation of said heterologous amino acid sequence within CDRL1; v) amino acid residue LC res49 according to Kabat and the first amino acid residue of the BNP in case of an incorporation of said heterologous amino acid sequence within CDRL2; and/or vi) amino acid residue LC res88 according to Kabat and the first amino acid residue of the BNP in case of an incorporation of said heterologous amino acid sequence within CDRL3 comprises the sequence of any one of SEQ ID NOs 26 to 38 or a sequence having at least 80% sequence identity with any one of SEQ ID NOs 26 to 38; and wherein the amino acid stretch present between the last amino acid residue of said BNP and i) amino acid residue HC res35a according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRH1; ii) amino acid residue HC res57 according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRH2; iii) amino acid residue HC res106 according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRH3; iv) amino acid residue LC res 32 according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRL1; v) amino acid residue LC res57 according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRL2; and/or vi) amino acid residue LC res98 according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRL3 comprises the sequence of any one of SEQ ID NOs 39 to 51 or a sequence having at least 80% sequence identity with any one of SEQ ID NOs 39 to 51.

7. The antibody or fragment thereof according to claim 1, wherein said Ntls and/or said Ctls comprise(s) at least 3, 4, 5, 6, 7, 8, 9 or 10 and up to 30, 28, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 amino acid residues in total.

8. The antibody or fragment thereof according to claim 1, wherein the amino acid stretch present between i) amino acid residue HC res25 according to Kabat and amino acid residue HC res35a according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRH1; ii) amino acid residue HC res51 according to Kabat and amino acid residue HC res57 according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRH2; iii) amino acid residue HC res92 according to Kabat and amino acid residue HC res106 according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRH3; iv) amino acid residue LC res26 according to Kabat and amino acid residue LC res 32 according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRL1; v) amino acid residue LC res49 according to Kabat and amino acid residue LC res57 according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRL2; and/or vi) amino acid residue LC res88 according to Kabat and amino acid residue LC res98 according to Kabat in case of an incorporation of said heterologous amino acid sequence within CDRL3 comprises the sequence of any one of SEQ ID NOs 52 to 64 or a sequence having at least 80% sequence identity with any one of SEQ ID NOs 52 to 64.

9. The antibody or fragment thereof according to claim 1, comprising at least one further natriuretic peptide, particularly wherein said BNP and said at least one further natriuretic peptide are incorporated within at least two separate CDR regions, more particularly wherein said at least one further natriuretic peptide is selected from ANP, BNP and CNP, most particularly from ANP and CNP.

10. The antibody or fragment thereof according to claim 1, wherein said antibody or fragment thereof is a human or humanized antibody or fragment thereof, particularly wherein said antibody or fragment thereof is of the class IgG.

11. The antibody or fragment thereof according to claim 1, wherein the light chain comprises or consists of the amino acid sequence of SEQ ID NO 66 and the heavy chain comprises or consists of the amino acid sequence of any one of SEQ ID NOs 442 to 444.

12. The antibody fragment according to claim 1, wherein said antibody fragment is selected from the group consisting of Fab, Fab′, Fab′-SH, F(ab′)2, and Fv fragments; diabodies; single domain antibodies (Dabs); linear antibodies; single-chain antibody molecules (scFv); and disulfide-stabilized Fv antibody fragments (dsFv).

13. A composition comprising the antibody or fragment thereof according to claim 1 and a pharmaceutically acceptable carrier.

14. A nucleic acid or a mixture of nucleic acids encoding the antibody or fragment thereof according to claim 1.

15. A host cell comprising the nucleic acid or the mixture of nucleic acids according to claim 14.

16. A process for producing an antibody or fragment thereof, comprising culturing the host cell according to claim 15 under conditions suitable for expression of the antibody or fragment thereof.

17. The antibody or fragment thereof according to claim 1 for use in a method for treatment.

18. The antibody or fragment thereof according to claim 1 for use in the treatment of cardiovascular, renal, pulmonary, skeletal, ocular, thromboembolic or fibrotic diseases or disorders, dwarfism, achondroplasia or other cGMP-related and/or natriuretic peptide responsive disorders.

Description

SHORT DESCRIPTION OF FIGURES

[0301] FIG. 1: Mean plasma concentrations of TPP-10992 and TPP-5661 after intravenous administration of 5 mg/kg in rat.

[0302] FIG. 2: Mean plasma concentrations of TPP-12897 after intraperitoneal administration of 5 mg/kg in mice.

[0303] FIG. 3: Stability of ANP (A-C), TPP-10992 (D-F) and TPP-5661 (G-I) against proteolytic degradation. ANP, TPP-10992 and TPP-5661 activity were tested on the stable rat ANP receptor cell line directly (A, D, G), or after 4 h incubation at 37° C. with 0.6 μg/ml NEP (B, E, H) or 0.6 μg/ml IDE (C, F, I).

[0304] FIG. 4: Stability of BNP (A-C) and TPP-11155 (D-F) against proteolytic degradation. BNP and TPP-11155 activity were tested on the stable rat BNP receptor cell line directly (A, D), or after 4 h incubation at 37° C. with 0.6 μg/ml NEP (B, E) or 0.6 μg/ml IDE (C, F).

[0305] FIG. 5: Stability of CNP (A-C) and TPP-12897 (D-F) against proteolytic degradation. CNP and TPP-11155 activity were tested on the stable rat CNP receptor cell line directly (A, D), or after 4 h incubation at 37° C. with 0.6 μg/ml NEP (B, E) or 0.6 μg/ml IDE (C, F).

[0306] FIG. 6: ANP Peptide and TPP-10992 induced vasodilation dose-response curves in PE-contracted aortic rings. Concentration-response curves (0.0001-10 μM; n=3 Rats) to the ANP peptide (open circles) and TPP-10992 (closed circles) in endothelium-intact rat aortic rings contracted by phenylephrine (1 μM). Experimental values were calculated relative to the maximal changes from the contraction produced by phenylephrine in each tissue, which was taken as 100%. Potency of ANP peptide and TPP-10992 were −7.4 and −6.7 respectively (log EC.sub.50 values). Data represent the mean±S.E.M. of 2 experiments.

[0307] FIG. 7: ANP Peptide and TPP-5661 induced vasodilation dose-response curves in PE-contracted aortic rings. Concentration-response curves (0.0001-10 μM; n=3 Rats) to the ANP peptide (open circles) and TPP-5661 (closed circles) in endothelium-intact rat aortic rings contracted by phenylephrine (1 μM). Experimental values were calculated relative to the maximal changes from the contraction produced by phenylephrine in each tissue, which was taken as 100%. Potency of ANP peptide and TPP-5661 were −7.4 and −6.5 respectively (log EC.sub.50 values). Data represent the mean±S.E.M. of 2 experiments.

[0308] FIG. 8: Hemodynamic effect of ANP in conscious rats. Rat ANP was given intraperitoneally at 0 hours. A 500 μg dose of ANP resulted in an approximately 25% drop in mean arterial blood pressure (MAP) with a duration of effect around 6-8 hours.

[0309] FIG. 9: Hemodynamic effect TPP-5661 in conscious rats. TPP-5661 was given intraperitoneally at 0 hours. A 15 mg/kg dose resulted in an approximately 20% reduction in mean arterial blood pressure (MAP) with maximum effect at 24-48 hours post application and a duration of effect greater than 6 days.

[0310] FIG. 10: Hemodynamic effect TPP-10992 in conscious rats. TPP-10992 was given intraperitoneally at 0 hours. A 30 mg/kg dose resulted in an approximately 20% reduction in mean arterial blood pressure (MAP) with maximum effect at 48 hours post application and a duration of effect greater than 6 days.

[0311] FIG. 11: Activity of BNP engrafted antibody constructs on hNPRA cells. The activity of purified compound samples on stable hNPRA-CHO kl cells was assessed by comparison to reference sample TPP-5661 and TPP-5657. Samples were tested in dilution series in quadruplets.

[0312] FIG. 12: Different human IgG isotypes provide equally suitable antibody scaffolds. Exemplary activity determination of compounds 9, 33, 65, 91, 127 and 191 IgG1 (TPP-10294, TPP-10277, TPP-10279, TPP-10282, TPP-10269 and TPP-10355, respectively), IgG2 and IgG4 isotypes. The activity of purified compound samples on stable hNPRA-CHO kl cells was assessed by comparison to reference samples compound 117 human IgG1 TPP-5661 and compound 209 human IgG1 TPP-5657. Samples were tested in dilution series in quadruplets.

[0313] FIG. 13: Equally suitable IgG antibody scaffolds originated from different species. Exemplary activity determination of compound 117 human IgG1 (TPP-5661) and compound 9 human IgG1 (TPP-10294) and their non-human IgG1 counterparts. The activity of purified compound samples on stable hNPRA-CHO kl cells was assessed by comparison to reference sample compound 209 human IgG1 (TPP-5657). Samples were tested in dilution series in quadruplets.

[0314] FIG. 14: Equally suitable human IgG antibody scaffolds originated from different germline sequences. The activity of purified compound samples on stable hNPRA-CHO kl cells was assessed by comparison to reference sample TPP-10992. Samples were tested in dilution series in quadruplets.

[0315] FIG. 15: Protective effects of TPP-12899 against LPS, IL-113 and thrombin induced endothelial barrier permeability as assessed by real-time impedance measurement.

[0316] FIG. 16: Therapeutic effects of TPP-13992 on survival (A), body weight gain (B), urinary protein/creatinine ratio (C) and left atrial weight (D); (n=8-12 (healthy control n=5), mean±SEM, One-Way ANOVA vs TPP-10155 (isotype specific control antibody).

[0317] FIG. 17: Hemodynamic assessment after Placebo, 0.1, 0.3 and 1.0 mg/kg of TPP-10992. TPP-10992 shows a dose-dependent and long-lasting (>5d) reduction in blood pressure.**p<0.01, ****p<0.0001 in comparison to placebo group using an One-way ANOVA test for repeated measurements followed by Tukey's multiple comparison test.

EXAMPLES

Example 1: Construction of Candidate TPP-5661

[0318] Candidate TPP-5661 was designed by fusion of a heterologous amino acid sequence comprising a Ntls, wild type rat ANP and a Ctls to the C-terminus of HV 3-23 (SEQ ID NO 85) by substituting the two C-terminal residues of HV 3-23 by the two N-terminal residues of the heterologous amino acid sequence and to the N-terminus of IGHJ1 (SEQ ID NO 86) by substituting the nine N-terminal residues of IGHJ1 by the 9 C-terminal residues of the heterologous amino acid sequence. The corresponding full length heavy chain sequence of SEQ ID NO 67 further comprises amino acid sequence Constant-H (SEQ ID NO 87).

[0319] Pairing of the full length heavy chain sequence of SEQ ID NO 67 harboring the inserted rat ANP (rANP) with the full length light chain sequence of SEQ ID NO 66 built by combining sequences LV 1-40 (SEQ ID NO 88), IGLJ2 (SEQ ID NO 89) and Constant-L (SEQ ID NO 90) yields the full IgG candidate TPP-5661 (see Table 1).

[0320] Shown below is the full length heavy chain sequence (SEQ ID NO 67); the incorporated heterologous amino acid sequence (Ntls-rANP-Ctls) is underlined; sequences derived from HV 3-23 and IGHJ1 are shown in bold:

TABLE-US-00003 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVS AISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTS VHQETKKYQSSPDGGSGGSLRRSSCFGGRIDRIGAQSGLGCNSFRYGSY SYTYNYEWHVDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG

[0321] The designed and synthesized antibody construct was cloned according to well-known methods in the art and confirmed by DNA sequencing using plasmid specific oligonucleotides.

Example 2: Insertion of NPs within Antibody-CDRs Results in an Increased Serum Half-Life

[0322] Determination of In Vivo Pharmacokinetic Parameters

[0323] Pharmacokinetic parameters of TPP-10992 (SEQ ID NO 76 and SEQ ID NO 66) and TPP-5661 (SEQ ID NO 67 and SEQ ID NO 66) were determined after intravenous administration of 5 mg/kg to male Wistar rats (n=3). TPP-10992 and TPP-5661 were given as a bolus injection via the tail vein. Blood samples were collected from the jugular vein via previously implanted catheters in time intervals up to 14 days (336 hours). Generated EDTA-plasma was stored at −20° C. until further analysis.

[0324] The quantification of TPP-10992 and TPP-5661 in plasma samples was performed employing an anti-human IgG ELISA (enzyme-linked immunosorbent assay) format. Pharmacokinetic parameters were calculated from plasma concentration time profiles using non-compartmental data analysis.

[0325] Mean plasma concentrations of TPP-10992 and TPP-5661 after intravenous administration over time are graphically depicted in FIG. 1.

[0326] Mean clearance and terminal half-life of TPP-10992 and TPP-5661 are summarized in Table 2 below.

TABLE-US-00004 TABLE 2 Mean clearance (CL) and terminal half-life (t.sub.1/2) of TPP-10992 and TPP-5661 after intravenous administration of 5 mg/kg in rat. TPP- Analyte TPP-5661 10992 CL [mL/h/kg] 0.62 0.27 t.sub.1/2 [h] 297 184

[0327] Determination of In Vivo Pharmacokinetic Parameters

[0328] Pharmacokinetic parameters of TPP-12897 were determined after intraperitoneal administration to female Balb/c mice (n=3). Blood samples were collected from 15 minutes up to 72 hours post application. Generated EDTA-plasma was stored at −20° C. until further analysis. The quantification of TPP-12897 in plasma samples was performed by an anti-human IgG (Immunoglobulin G) ELISA format.

[0329] Pharmacokinetic parameters were calculated from plasma concentration time profiles using non-compartmental data analysis.

[0330] Mean plasma concentrations of TPP-12897 after intraperitoneal administration over time are graphically depicted in FIG. 2.

[0331] Mean area under the curve (AUC) and terminal half-life of TPP-12897 are summarized in Table 3 below.

TABLE-US-00005 TABLE 3 Mean area under the curve (AUC) and terminal half-life (t.sub.1/2) of TPP- 12897 after intraperitoneal administration of 5 mg/kg in mice. Analyte TPP-12897 AUC [mg .Math. h/L] 7705 t.sub.1/2 [h] 194

Example 3: In Vitro, Ex Vivo and In Vivo Potency of NP Engrafted Antibodies

[0332] Activity Data of ANP Engrafted Antibodies in NPR-A Receptor Cell Line

[0333] A luminescence-based rat ANP receptor (NPR-A) cell line was generated as described previously (Wunder et al. (2013), Eur J Pharmacol. 698: 131). Accordingly, a fluorescence-based rat ANP receptor (NPR-A) cell line was generated by co-transfecting a CHO cell line, stably expressing the fluorescent calcium sensor protein GCaMP6, with plasmid constructs encoding CNGA2 (cGMP biosensor) and rat NPR-A.

[0334] ANP receptor GCaMP6 cells were cultured for one day on black, clear-bottom 384-well microtiter plates (2500 cells/well). After removal of the cell culture medium reporter cells were loaded for 20 min with Tyrode (130 mM NaCl, 5 mM KCl, 2 mM CaCl.sub.2, 20 mM HEPES, 1 mM MgCl.sub.2, 4.8 mM NaHCO.sub.3 at pH 7.4) containing a black masking dye at 37° C. and 5% CO.sub.2. IBMX (0.2 mM) was used to prevent cGMP degradation by endogenous phosphodiesterases.

[0335] Fluorescence measurements (3 min, kinetic mode) were directly started upon agonist addition. Receptor ligands were added in Tyrode containing a black masking dye and 0.1% BSA. Measurements were done on a FLIPR Tetra®.

[0336] ANP (Bachem, H-2100) stimulated concentration-dependent fluorescence signals on the NPR-A cell line with an EC.sub.50 values of 0.22 nM. TPP-5661 and TPP-10992 stimulated the rat ANP receptor reporter cell line with EC.sub.50 values of 17 nM and 180 nM, respectively. The control antibody construct TPP-5657 did not significantly stimulate the NPR-A cell line (tested up to the max. concentration of 460 nM).

[0337] To determine the sensitivity towards proteolytic degradation, the activity of receptor ligands was also characterized after 4 hours incubation with 0.6 μg/ml neutral endopeptidase (NEP, R&D Systems, 1182-ZNC) or 0.6 μg/ml insulin degrading enzyme (IDE, Merck, 407241-50UG) at 37° C.

[0338] FIG. 3 graphically depicts the stability of ANP (A-C), TPP-10992 (D-F) and TPP-5661 (G-I) against proteolytic degradation. As shown in FIG. 3, the natriuretic peptide ANP (Bachem, H-2100) showed high sensitivity towards degradation by NEP and IDE. In contrast, TPP-5661 and TPP-10992 showed high resistance to proteolytic degradation by NEP and IDE.

[0339] Activity Data of BNP Engrafted Antibodies in NPR-A Receptor Cell Line

[0340] A luminescence-based rat BNP receptor (NPR-A) cell line was generated as described previously (Wunder et al. (2013), Eur J Pharmacol. 698: 131). Accordingly, a fluorescence-based rat BNP receptor (NPR-A) cell line was generated by co-transfecting a CHO cell line, stably expressing the fluorescent calcium sensor protein GCaMP6, with plasmid constructs encoding CNGA2 (cGMP biosensor) and rat NPR-A.

[0341] BNP receptor GCaMP6 cells were cultured for one day on black, clear-bottom 384-well microtiter plates (2500 cells/well). After removal of the cell culture medium reporter cells were loaded for 20 min with Tyrode (130 mM NaCl, 5 mM KCl, 2 mM CaCl.sub.2, 20 mM HEPES, 1 mM MgCl.sub.2, 4.8 mM NaHCO.sub.3 at pH 7.4) containing a black masking dye at 37° C. and 5% CO.sub.2. IBMX (0.2 mM) was used to prevent cGMP degradation by endogenous phosphodiesterases.

[0342] Fluorescence measurements (3 min, kinetic mode) were directly started upon agonist addition. Receptor ligands were added in Tyrode containing a black masking dye and 0.1% BSA. Measurements were done on a FLIPR Tetra®.

[0343] BNP (Bachem, H-5968) stimulated concentration-dependent fluorescence signals on the NPR-A cell line with an EC.sub.50 value of 2.9 nM. TPP-9902, TPP-11153, TPP-1154, TPP-11155, TPP-11156 and TPP-11157 stimulated the rat BNP receptor reporter cell line with EC.sub.50 values of 2.3 μM, >1.9 μM, 7 nM, 12 nM, 1.2 μM and 11 nM, respectively. The control antibody construct TPP-5657 did not significantly stimulate the NPR-A cell line (tested up to the max. concentration of 460 nM).

[0344] To determine the sensitivity towards proteolytic degradation, the activity of receptor ligands was also characterized after 4 hours incubation with 0.6 μg/ml neutral endopeptidase (NEP, R&D Systems, 1182-ZNC) or 0.6 μg/ml insulin degrading enzyme (IDE, Merck, 407241-50UG) at 37° C. The natriuretic peptide BNP (Bachem, H-5968) showed high sensitivity towards degradation by NEP and IDE. In contrast, TPP-11155 and TPP-11157 showed high resistance to proteolytic degradation by NEP and IDE.

[0345] FIG. 4 graphically depicts the stability of BNP (A-C) and TPP-11155 (D-F) against proteolytic degradation.

[0346] Activity Data of CNP Engrafted Antibodies in NPR-B Receptor Cell Line

[0347] A luminescence-based rat CNP receptor (NPR-B) reporter cell line was generated and luminescence measurements were performed as described previously (Wunder et al. (2013), Eur J Pharmacol. 698: 131).

[0348] CNP receptor cells (2500 cells/well) were cultured for 1 day on opaque 384-well microtiter plates. After removal of the cell culture medium, cells were loaded for 3 h with 2.5 μg/ml coelenterazine in Ca.sup.2+-free Tyrode (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl.sub.2, 4.8 mM NaHCO.sub.3 at pH 7.4) at 37° C. and 5% CO.sub.2. Receptor ligands were added for 10 min in Ca.sup.2+-free Tyrode containing 0.1% BSA. IBMX (0.2 mM) was used to prevent cGMP degradation by endogenous phosphodiesterases Immediately before adding calcium ions (final concentration 3 mM), luminescence measurements were started by using a charge-coupled device (CCD) camera in a light tight box. Luminescence was monitored continuously for 50 s.

[0349] CNP (Bachem, H-1296) stimulated concentration-dependent luminescence signals on the rat NPR-B cell line with an EC.sub.50 value of 0.024 nM. TPP-9465, TPP-12377, TPP-12378, TPP-12897 and TPP-12899 stimulated the rat CNP receptor reporter cell line with EC.sub.50 values of 5.2 nM, 4.1 nM, 25 nM, 10 nM and 3.2 nM, respectively. TPP-12374 stimulated the rat CNP receptor reporter cell line with EC.sub.50 values of 150 nM, and TPP-12375, TPP-12376 showed only weak indication of activity.

[0350] To determine the sensitivity towards proteolytic degradation, the activity of receptor ligands was also characterized after 4 hours incubation with 0.6 μg/ml neutral endopeptidase (NEP, R&D Systems, 1182-ZNC) or 0.6 μg/ml insulin degrading enzyme (IDE, Merck, 407241-50UG) at 37° C.

[0351] In contrast to the natriuretic peptide CNP (Bachem, H-1296), TPP-12377, TPP-12897 and TPP-12899 showed high resistance to proteolytic degradation by NEP and IDE.

[0352] FIG. 5 graphically depicts the stability of CNP (A-C) and TPP-12897 (D-F) against proteolytic degradation.

[0353] Activity Data of ANP Engrafted Antibodies Determined with Isolated Rat Aortic Rings

[0354] All experiments were conducted in accordance with institutional guidelines and approved by the local committee on animal experiments. Male Sprague-Dawley rats (weighing 250-300 g) were anesthetized with pentobarbital sodium (40 mg/kg i.p.), killed by decapitation, and exsanguinated. The thoracic aorta was excised and placed in ice-cold Krebs buffer of the following composition: 130 mM NaCl, 14.9 mM NaHCO.sub.3, 5.5 mM dextrose, 4.7 mM KCl, 1.18 mM KH.sub.2PO.sub.4, 1.17 mM MgSO.sub.47H.sub.2O, and 1.6 mM CaCl.sub.22H.sub.2O. The vessel was pinned in a Sylgard Petri dish filled with chilled Krebs' solution, cleaned of fat and connective tissue, and cut into ring segments of approximately 3 to 4 mm in length. Aortic rings were vertically mounted in 50-ml chambers (ADlinstruments) containing Krebs' solution at 37° C. continuously bubbled with a mixture of 95% O.sub.2 and 5% CO.sub.2. Changes in isometric force were recorded using a PowerLab data acquisition system (software Lab Chart 7.0)

[0355] After the equilibration period, aortic rings were challenged with 80 mM KCl to check tissue viability. Next, the endothelial integrity of the preparations was determined by verifying the responsiveness to acetylcholine (ACh, 1 μM) in vessels pre-contracted with Phenylephrine (PE, 1 μM). After wash-out and a period of equilibration, Phenylephrine (PE, 1 μM) was used to induce contraction, thereafter natriuretic peptides and natriuretic peptide engrafted IgGs were evaluated for vasorelaxation. Rat ANP peptide (ADH-GM-10057T, Santai Labs) was used as a reference.

[0356] As shown in FIG. 6, both ANP peptide and TPP-10992 induced dose-dependent vasodilation in PE-contracted aortic rings.

[0357] As shown in FIG. 7, both ANP peptide and TPP-5661 induced dose-dependent vasodilation in PE-contracted aortic rings.

[0358] Activity Data of ANP Engrafted Antibodies Obtained in Conscious Rats

[0359] Blood pressure and heart rate were monitored in freely moving conscious animals by radiotelemetry (Data Sciences International). Female spontaneously hypertensive rats (SHR/N Crl BR, Charles River) with a body weight of 210-300 g were used for these studies. All animals were housed in individual cages at 22-24° C. ambient temperature and maintained on a 12-hour light/dark cycle with free access to standard laboratory rat chow and water ad libitum. Telemeter (HD-10, DSI) implantation was performed a minimum of 14 days before animals were used for blood pressure measurements. Surgery was performed under aseptic conditions. After shaving the abdominal wall, a midline abdominal incision was made, and the fluid-filled sensor catheter was inserted upstream into the exposed descending aorta between the iliac bifurcation and the renal arteries. According to the DSI guidelines the tip of the telemetric catheter was located caudal to the renal arteries and secured by tissue adhesive. The transmitter body was affixed to the inner peritoneal wall before closure of the abdomen. For postsurgical protection against infections and pain a single dosage of an antibiotic (Oxytetracyclin® 10%, 60 mg/kg s.c., 0.06 ml/100 g body weight, Beta-Pharma GmbH & Co, Germany) and analgesic were injected (Rimadyl®, 4 mg/kg s.c., Pfizer, Germany). Telemetric data acquisition was performed by DSI software was predefined to sample hemodynamic data for 10 seconds repeated every 5 minutes. Data collection was started at least 2 hours before drug administration and finished after completion of measurement cycles. Data are expressed as % of basal values±SEM of at least 4 animals per group. The basal value for each animal was calculated as the average of the values measured in two hours prior to substance application (7:00-9:00 AM). Data are then expressed as averages every half hour, starting 15 minutes post application. All animals were treated with a single intraperitoneal (ip) application of test substances dissolved in phosphate buffered saline (PBS). Drug administration took place at 9:00 AM (0 hours).

[0360] The hemodynamic effect of ANP peptide is graphically depicted in FIG. 8. The hemodynamic effect of TPP-5661 is graphically depicted in FIG. 9. The hemodynamic effect of TPP-10992 is graphically depicted in FIG. 10.

Example 4: Generation of Different NP Engrafted Antibody Constructs

[0361] For constructs with a natriuretic peptide incorporation in a CDR region other than CDRH3 a presumably functionally neutral CDRH3 was designed by fusion of the three residues stretch Leu Thr Gly (IGHD7-27*01) to the C-terminus of HV 3-23 and the N-terminus of IGHJ1 (compare Example 1). The corresponding full length heavy chain sequence of SEQ ID NO 65 further comprises amino acid sequence Constant-H (SEQ ID NO 87).

[0362] Pairing of the full length heavy chain sequence of SEQ ID NO 65 without any natriuretic peptide insertion with the full length light chain sequence of SEQ ID NO 66 described in Example 1 yields the synthetic and presumably neutral IgG negative control TPP-5657.

[0363] The designed and synthesized antibody construct was cloned according to well-known methods in the art and confirmed by DNA sequencing using plasmid specific oligonucleotides.

[0364] Starting from this antibody scaffold, the following ANP engrafted antibody constructs were generated.

TABLE-US-00006 TABLE 4 Design of ANP engrafted antibody constructs SEQ ID SEQ ID NO NO # aa # aa Insertion comprised comprised N- C- Cmpd TPP Site in Ntls in Ctls term.sup.1 term.sup.2 1 TPP- CDRH1 6 2 13057 2 TPP- CDRH1 9 5 13056 3 TPP- CDRH1 12 8 13055 4 TPP- CDRH1 15 11 13054 5 TPP- CDRH1 18 10 12545 6 TPP- CDRH1 19 17 10454 7 TPP- CDRH1 19 17 10453 8 TPP- CDRH1 6 20 17 11172 9 TPP- CDRH1 23 14 10294 10 TPP- CDRH1 23 14 12547 11 TPP- CDRH1 6 24 13 11171 12 TPP- CDRH2 3 3 10841 13 TPP- CDRH2 7 7 10842 14 TPP- CDRH2 8 9 11009 15 TPP- CDRH2 10 11 11008 16 TPP- CDRH2 11 14 11018 17 TPP- CDRH2 12 8 10775 18 TPP- CDRH2 13 12 10767 19 TPP- CDRH2 9 14 9 11012 20 TPP- CDRH2 13 14 14 10 10774 21 TPP- CDRH2 14 11 11007 22 TPP- CDRH2 14 14 11179 23 TPP- CDRH2 13 14 15 10 10773 24 TPP- CDRH2 1 1 15 12 10770 25 TPP- CDRH2 15 14 10766 26 TPP- CDRH2 13 14 16 11 10772 27 TPP- CDRH2 16 11 11005 28 TPP- CDRH2 16 11 11006 29 TPP- CDRH2 16 14 11017 30 TPP- CDRH2 9 10 16 15 11169 31 TPP- CDRH2 16 15 11181 32 TPP- CDRH2 16 17 11182 33 TPP- CDRH2 9 10 17 11 10277 34 TPP- CDRH2 9 10 17 11 12553 35 TPP- CDRH2 9 10 17 11 12554 36 TPP- CDRH2 9 10 17 11 12555 37 TPP- CDRH2 9 10 17 11 12542 38 TPP- CDRH2 9 10 17 11 12543 39 TPP- CDRH2 9 10 17 11 12544 40 TPP- CDRH2 9 10 17 11 13058 41 TPP- CDRH2 9 10 17 11 13059 42 TPP- CDRH2 9 20 17 11 13060 43 TPP- CDRH2 9 20 17 11 13061 44 TPP- CDRH2 9 10 17 11 13062 45 TPP- CDRH2 9 10 17 11 13063 46 TPP- CDRH2 9 10 17 11 13064 47 TPP- CDRH2 9 10 17 11 13065 48 TPP- CDRH2 9 10 17 11 13066 49 TPP- CDRH2 9 10 17 11 12546 50 TPP- CDRH2 13 14 17 12 10452 51 TPP- CDRH2 4 5 17 12 10846 52 TPP- CDRH2 17 12 10852 53 TPP- CDRH2 6 17 13 10851 54 TPP- CDRH2 1 1 17 14 10769 55 TPP- CDRH2 17 14 10765 56 TPP- CDRH2 17 15 11180 57 TPP- CDRH2 17 17 11177 58 TPP- CDRH2 17 17 11178 59 TPP- CDRH2 17 18 17 17 11176 60 TPP- CDRH2 9 10 18 12 10278 61 TPP- CDRH2 4 5 18 13 10847 62 TPP- CDRH2 18 13 11004 63 TPP- CDRH2 2 3 18 13 10844 64 TPP- CDRH2 18 13 10853 65 TPP- CDRH2 9 10 18 13 10279 66 TPP- CDRH2 9 10 18 13 11170 67 TPP- CDRH2 9 18 13 11010 68 TPP- CDRH2 9 18 13 11011 69 TPP- CDRH2 18 14 10764 70 TPP- CDRH2 18 17 11183 71 TPP- CDRH2 16 17 18 17 11175 72 TPP- CDRH2 15 19 12 11016 73 TPP- CDRH2 15 19 14 11015 74 TPP- CDRH2 13 14 19 14 10451 75 TPP- CDRH2 1 1 19 14 10768 76 TPP- CDRH2 4 5 19 14 10848 77 TPP- CDRH2 19 14 11003 78 TPP- CDRH2 19 14 11002 79 TPP- CDRH2 2 3 19 14 10843 80 TPP- CDRH2 2 3 19 14 10845 81 TPP- CDRH2 19 14 10284 82 TPP- CDRH2 11 12 19 14 10446 83 TPP- CDRH2 19 14 10447 84 TPP- CDRH2 19 14 10854 85 TPP- CDRH2 15 19 15 11014 86 TPP- CDRH2 6 6 19 15 10849 87 TPP- CDRH2 6 19 15 10850 88 TPP- CDRH2 20 14 11013 89 TPP- CDRH2 1 1 20 15 10771 90 TPP- CDRH2 1 1 20 15 10287 91 TPP- CDRH2 20 15 10282 92 TPP- CDRH2 20 15 10285 93 TPP- CDRH2 20 15 10286 94 TPP- CDRH2 20 15 10283 95 TPP- CDRH2 9 10 20 15 11168 96 TPP- CDRH2 20 15 10857 97 TPP- CDRH2 20 15 10856 98 TPP- CDRH2 20 15 10855 99 TPP- CDRH3 10 3 10281 100 TPP- CDRH3 10 8 10280 101 TPP- CDRH3 12 10 10583 102 TPP- CDRH3 7 14 12 10582 103 TPP- CDRH3 7 8 15 18 10270 104 TPP- CDRH3 7 8 16 14 10264 105 TPP- CDRH3 16 14 10581 106 TPP- CDRH3 7 8 17 15 10263 107 TPP- CDRH3 7 8 17 18 10271 108 TPP- CDRH3 7 8 18 16 10262 109 TPP- CDRH3 7 8 18 18 10272 110 TPP- CDRH3 7 8 19 17 10261 111 TPP- CDRH3 19 17 10289 112 TPP- CDRH3 7 8 19 18 10273 113 TPP- CDRH3 7 8 20 17 10260 114 TPP- CDRH3 9 10 20 17 10275 115 TPP- CDRH3 20 18 10580 116 TPP- CDRH3 7 8 20 18 10274 117 TPP- CDRH3 7 8 20 18 5661 118 TPP- CDRH3 7 8 18 16 13226 119 TPP- CDRH3 7 8 18 16 13227 120 TPP- CDRH3 7 22 20 18 13228 121 TPP- CDRH3 7 22 20 18 13229 122 TPP- CDRH3 21 22 20 18 13230 123 TPP- CDRH3 21 22 20 18 13231 124 TPP- CDRH3 9 10 20 18 10276 125 TPP- CDRH3 20 18 10290 126 TPP- CDRH3 11 12 20 18 10445 127 TPP- CDRH3 7 8 20 18 10269 128 TPP- CDRH3 2 3 20 18 10288 129 TPP- CDRH3 7 8 20 19 10265 130 TPP- CDRH3 7 8 21 19 10268 131 TPP- CDRH3 19 22 20 10593 132 TPP- CDRH3 6 12 22 20 11174 133 TPP- CDRH3 7 8 22 20 10266 134 TPP- CDRH3 6 12 22 20 11173 135 TPP- CDRH3 11 12 22 20 10444 136 TPP- CDRH3 7 8 24 22 10267 137 TPP- CDRH3 11 12 24 22 10443 138 TPP- CDRL1 6 16 14 11163 139 TPP- CDRL1 19 13 10360 140 TPP- CDRL1 20 18 10462 141 TPP- CDRL1 21 19 10460 142 TPP- CDRL1 21 19 10461 143 TPP- CDRL1 6 6 21 19 11161 144 TPP- CDRL1 6 21 19 11162 145 TPP- CDRL1 23 14 10359 146 TPP- CDRL2 1 6 10824 147 TPP- CDRL2 5 10 10825 148 TPP- CDRL2 12 16 11019 149 TPP- CDRL2 13 17 11021 150 TPP- CDRL2 13 17 11020 151 TPP- CDRL2 14 14 10789 152 TPP- CDRL2 15 19 11022 153 TPP- CDRL2 4 5 16 15 10829 154 TPP- CDRL2 16 15 10835 155 TPP- CDRL2 16 16 10788 156 TPP- CDRL2 15 15 17 16 10571 157 TPP- CDRL2 4 5 17 16 10830 158 TPP- CDRL2 17 16 10790 159 TPP- CDRL2 19 17 16 10573 160 TPP- CDRL2 2 3 17 16 10827 161 TPP- CDRL2 19 17 16 10572 162 TPP- CDRL2 17 16 10836 163 TPP- CDRL2 6 17 17 10834 164 TPP- CDRL2 18 16 10787 165 TPP- CDRL2 4 5 18 17 10831 166 TPP- CDRL2 2 3 18 17 10828 167 TPP- CDRL2 2 3 18 17 10826 168 TPP- CDRL2 18 17 10837 169 TPP- CDRL2 19 17 10361 170 TPP- CDRL2 11 12 19 18 11023 171 TPP- CDRL2 19 18 10838 172 TPP- CDRL2 19 18 10840 173 TPP- CDRL2 19 18 10839 174 TPP- CDRL2 6 19 19 10832 175 TPP- CDRL2 6 19 19 10833 176 TPP- CDRL2 11 12 20 19 11024 177 TPP- CDRL3 2 2 10353 178 TPP- CDRL3 14 9 10780 179 TPP- CDRL3 16 10 10786 180 TPP- CDRL3 16 11 10779 181 TPP- CDRL3 16 13 10778 182 TPP- CDRL3 11 12 17 11 10783 183 TPP- CDRL3 18 12 10785 184 TPP- CDRL3 18 13 10776 185 TPP- CDRL3 18 13 10777 186 TPP- CDRL3 18 14 10784 187 TPP- CDRL3 11 12 19 13 10782 188 TPP- CDRL3 7 8 19 14 10352 189 TPP- CDRL3 19 14 10356 190 TPP- CDRL3 19 14 10354 191 TPP- CDRL3 19 14 10355 192 TPP- CDRL3 11 12 19 15 10781 193 TPP- CDRL3 7 8 20 14 10436 194 TPP- CDRL3 11 12 20 14 10440 195 TPP- CDRL3 20 14 10442 196 TPP- CDRL3 7 8 20 15 10351 197 TPP- CDRL3 7 8 20 15 10348 198 TPP- CDRL3 20 15 10358 199 TPP- CDLR3 6 12 21 15 11167 200 TPP- CDRL3 11 12 21 15 10438 201 TPP- CDRL3 11 12 21 15 10439 202 TPP- CDRL3 21 15 10441 203 TPP- CDRL3 7 8 21 16 10349 204 TPP- CDRL3 7 8 22 17 10350 205 TPP- CDRL3 11 12 23 17 10437 206 TPP- CDRL3 6 12 24 18 11166 207 TPP- CDRL3 6 6 24 20 10362 208 TPP- CDRL3 6 6 24 20 10363 209 TPP- no na n 5657 .sup.1The number of amino acid residues present between the respective N-terminal reference amino acid residue and the first amino acid of the inserted natriuretic peptide; .sup.2The number of amino acid residues present between the last amino acid of the inserted natriuretic peptide and the respective C-terminal reference amino acid residue

TABLE-US-00007 Table 4 cont.: Design of ANP engrafted antibody constructs Cmpd TPP N-terminal sequence.sup.3 C-terminal sequence.sup.4 1 TPP- SGFTFSS YAM 13057 2 TPP- SGFTFGSGSG GSGSGM 13056 3 TPP- SGFTFGSGSGSGS SGSGSGSGM 13055 4 TPP- SGFTFGSGSGSGGSGG GGSGGSGSGSGM 13054 5 TPP- SGFTFGSGSGSGSGGGSGG GSGSGGSGSGM 12545 6 TPP- SPAVVYIEILDRHPDGGSGG GSGREVPISNGSGFVVAM 10454 7 TPP- SGAVVYIEILDRHPDGGSGG GSGREVPISNGSGFVVAM 10453 8 TPP- SSSDRSALLKSKLRALLTAPR GSGREVPISNGSGFVVAM 11172 9 TPP- SGFTFGSGSGSGSGSGSPDGGSGG GSYGSGSGSGSGSGM 10294 10 TPP- SGFTFGSGSGSGSGSGSPDGGSGG GSYGSGSGSGSGSGM 12547 11 TPP- SGFTFSSDRSALLKSKLRALLTAPR GSGSGSGSGSGSGM 11171 12 TPP- ISGS GGST 10841 13 TPP- ISGSGSGS GSGSGGST 10842 14 TPP- ISGSGSGSG GSSGSGSGST 11009 15 TPP- ISGSGSGSGSG GSSGSGSGSGST 11008 16 TPP- ISGSGSGSGSGG GEKEKEKVSTAVGST 11018 17 TPP- ISGSAVVNGGSGG GKIAIGGST 10775 18 TPP- ISGPNPNKNPNPGG GSNENPNPNPGST 10767 19 TPP- ISGSVVVTSHGGSGG GGSGSGSGST 11012 20 TPP- ISGSAVVNVRGGSGG GGDKIAIGGST 10774 21 TPP- ISGSGSGSGSGSSGG GSSGSGSGSGST 11007 22 TPP- ISGLAVQIRRGGSGG GGSGRETLTLYVGST 11179 23 TPP- ISGSAVVNVRAGGSGG GGDKIAIGGST 10773 24 TPP- ISGSYAMSWVRGGSGG GSYAMSWVRQGST 10770 25 TPP- ISGPNPNKNPNPNPGG GSNPNENPNPNPGST 10766 26 TPP- ISGSAVVNVRADGGSGG GSGDKIAIGGST 10772 27 TPP- ISGSGSGSGSPDGGSGG GSSGSGSGSGST 11005 28 TPP- ISGSGSGSGSGSGGSGG GSSGSGSGSGST 11006 29 TPP- ISGSGSGSGSGSGGSGG GEKEKEKVSTAVGST 11017 30 TPP- ISGSVVVTSHQAPGSGG GSGEKKKLKSLAYGST 11169 31 TPP- ISGRYNILKIQKVGSGG GGSGEYLITYQIMGST 11181 32 TPP- ISGRQLLFCRVTLGSGG GGSGEQAYPEYLITYGST 11182 33 TPP- ISVVVTSHQAPGEGGSGG GEKKKLKSLAST 10277 34 TPP- ISGVVTSHQAPGEGGSGG GEKKKLKSLAST 12553 35 TPP- ISVVVTSHQAPGEGGSGG GEKKKLKSLGST 12554 36 TPP- ISVVVTSHQAPGEGGSGG GEKKKLKSGGST 12555 37 TPP- ISGVVTSHQAPGEGGSGG GEKKKLKSGGST 12542 38 TPP- ISGSVTSHQAPGEGGSGG GEKKKLKSGGST 12543 39 TPP- ISGSVTSHQAPGEGGSGG GEKKKGKSGGST 12544 40 TPP- ISVVVTSHQAPGSGGSGG GEKKKLKSLAST 13058 41 TPP- ISVVVTSHQAPTSGGSGG GEKKKLKSLAST 13059 42 TPP- ISVVVTSHQSPTPGGSGG GGSTPLKSLAST 13060 43 TPP- ISVVVTSHQAPGEGGSGG GGSTPLKSLAST 13061 44 TPP- ISVVVTSHQAPGEGGSGG GSTPKLKSLAST 13062 45 TPP- ISVVVTSHQSPTPGGSGG GEKKKLKSLAST 13063 46 TPP- ISVVVTSHPTPGEGGSGG GEKKKLKSLAST 13064 47 TPP- ISVVVTSHQAPSPGSTGG GEKKKLKSLAST 13065 48 TPP- ISVVVTSHQANGSGGSGG GEKKKLKSLAST 13066 49 TPP- ISVVVTSHQAPGEGGSGG GEKKKLKSLAST 12546 50 TPP- ISGSAVVNVRAPDGGSGG GSKGDKIAIGGST 10452 51 TPP- ISTSASLAITGPDGGSGG GSDRFSGSKSGST 10846 52 TPP- ISGFILPIEVYPDGGSGG GSKVRFDYDLFST 10852 53 TPP- ISSALLKSKLRALLTAPG GGSGSGSGSGSGST 10851 54 TPP- ISGSYAMSWVRQAGGSGG GSSSYAMSWVRQGST 10769 55 TPP- ISGPNPNKNPNPNPGSGG GSNPNENPNPNPGST 10765 56 TPP- IHPLQNRWALWFFKGSGG GGSGNLRLISKFDTVT 11180 57 TPP- ISGSVTIFSLATNEGSGG GGSGKTTWHRISVFGGST 11177 58 TPP- IYLEGKIDYGEYMDGSGG GGSNVRRQATTIIADNIT 11178 59 TPP- ISGSVQGIINFEQKGSGG GGSGPVKVWGSIKGGGST 11176 60 TPP- ISGVVVTSHQAPGEGGSGG GEKKKLKSLAGST 10278 61 TPP- ISGTSASLAITGPDGGSGG GSDRFSGSKSGGST 10847 62 TPP- ISGSGSGSGSGSPDGGSGG GSSGSGSGSGSGST 11004 63 TPP- ISGTYISNVNHKPDGGSGG GSNTKVDKKVEGST 10844 64 TPP- ISGGFILPIEVYPDGGSGG GSKVRFDYDLFGST 10853 65 TPP- ISGSVVVTSHQAPGGGSGG GEKKKLKSLAYGST 10279 66 TPP- ISGSVVVTSHQAPGGGSGG GEKPKPKPLAYGST 11170 67 TPP- ISGSVVVTSHQAPGGGSGG GSSGSGSGSGSGST 11010 68 TPP- ISGSVVVTSHQAPGGGSGG GSGSGSGSGSGGST 11011 69 TPP- ISGPNPNKNPNPNPGGSGG GSNPNENPNPNPGST 10764 70 TPP- ISGDIYLAINITNGEGSGG GGSGDIYLAINITNGEST 11183 71 TPP- ISGSATKAVSVLKGDGSGG GGSGVQGIINFEQKGGST 11175 72 TPP- ISGSVPKEKEKEKVSTAVGG GSGSGSGSGSGST 11016 73 TPP- ISGSVPKEKEKEKVSTAVGG GSGSGSGSGSGSGST 11015 74 TPP- ISGSSGAVVNVRAPDGGSGG GSKGDKIAIWTTGST 10451 75 TPP- ISGSYAMSWVRQAPDGGSGG GSSSYAMSWVRQGST 10768 76 TPP- ISGSTSASLAITGPDGGSGG GSDRFSGSKSGGGST 10848 77 TPP- ISGSGSGSGSGSGPDGGSGG GSGSGSGSGSGSGST 11003 78 TPP- ISGSGSGSGSGSSPDGGSGG GSYSGSGSGSGSGST 11002 79 TPP- ISTQTYISNVNHKPDGGSGG GSNTKVDKKVEPKST 10843 80 TPP- ISGSTYISNVNHKPDGGSGG GSNTKVDKKVEGGST 10845 81 TPP- ISGPNPNPNPNPNPDGGSGG GSNPNPNPNPNPGST 10284 82 TPP- ISAVQVKLELGHRPDGGSGG GSNHLRSEKLTFNST 10446 83 TPP- ISGFILPIEVYFKPDGGSGG GSPRKVRFDYDLFST 10447 84 TPP- ISGSGFILPIEVYPDGGSGG GSKVRFDYDLFGGST 10854 85 TPP- ISGSVPKEKEKEKVSTAVGG GSYGSGSGSGSGSGST 11014 86 TPP- ISDRSALLKSKLRALLTAPR GSDRSALLKSKLRAST 10849 87 TPP- ISDRSALLKSKLRALLTAPG GGSGSGSGSGSGSGST 10850 88 TPP- ISGSSDKTHTSPPSPDGGSGG GSKTHTSPPSPGGST 11013 89 TPP- ISGSYAMSWVRQASPDGGSGG GSYSSYAMSWVRGGST 10771 90 TPP- ISGSYAMSWVRQASPDGGSGG GSYSSYAMSWVRQGST 10287 91 TPP- ISGSGSGSGSGSGSPDGGSGG GSYGSGSGSGSGSGST 10282 92 TPP- ISGSPNPNPNPNPSPDGGSGG GSYPNPNPNPNPSGST 10285 93 TPP- ISGPNPNKNPNPNSPDGGSGG GSYNPNENPNPNPGST 10286 94 TPP- ISGPNPNPNPNPNSPDGGSGG GSYNPNPNPNPNPGST 10283 95 TPP- ISGSVVVTSHQAPGGSGGSGG GSGEKKKLKSLAYGST 11168 96 TPP- ISGSVVYIEILDRHPDGGSGG GSGREVPISNGSGGST 10857 97 TPP- ISGAVVYIEILDRHPDGGSGG GSGREVPISNGSGGST 10856 98 TPP- ISPAVVYIEILDRHPDGGSGG GSGREVPISNGSGFST 10855 99 TPP- CAKSPDGGSGG GSYG 10281 100 TPP- CAKSPDGGSGG GSYQHWGQG 10280 101 TPP- CAKVHQETGGSGG GSWHVQHWGQG 10583 102 TPP- CAKVHQETPDGGSGG GSYEWHVQHWGQG 10582 103 TPP- CTSVHQETKKYQSSGG GSYSYTYNYEWHVDVWGQG 10270 104 TPP- CTSVHQETSSPDGGSGG GSYSYEWHVDVWGQG 10264 105 TPP- CAKTHTSPPSPDGGSGG GSSPPSPYFQHWGQG 10581 106 TPP- CTSVHQETKSSPDGGSGG GSYSNYEWHVDVWGQG 10263 107 TPP- CTSVHQETKKYQSSPDGG GSYSYTYNYEWHVDVWGQG 10271 108 TPP- CTSVHQETKKSSPDGGSGG GSYSYNYEWHVDVWGQG 10262 109 TPP- CTSVHQETKKYQSSGGSGG GSYSYTYNYEWHVDVWGQG 10272 110 TPP- CTSVHQETKKQSSPDGGSGG GSYSYYNYEWHVDVWGQG 10261 111 TPP- CAKVHPNPNPNPNPDGGSGG GSNPNPNPNPHVDVWGQG 10289 112 TPP- CTSVHQETKKYQSSPDGGSG GSYSYTYNYEWHVDVWGQG 10273 113 TPP- CTSVHQETKKYQSSPDGGSGG GSYSYYNYEWHVDVWGQG 10260 114 TPP- CAKLTVVVTSHQAPGEGGSGG GEKKKLKSLAYFQHWGQG 10275 115 TPP- CAKSSDKTHTSPPSPDGGSGG GSKTHTSPPSPYFQHWGQG 10580 116 TPP- CTSVHQETKKYQSSPDGGSGG GSYSYTYNYEWHVDVWGQG 10274 117 TPP- CTSVHQETKKYQSSPDGGSGG GSYSYTYNYEWHVDVWGQG 5661 118 TPP- CAKVETKKYQSSPDGGSGG GSYSYTYNYEVQHWGQG 13226 119 TPP- CAKVHTKKYQSSPDGGSGG GSYSYTYNYHVQHWGQG 13227 120 TPP- CAKLTVETKKYQSSPDGGSGG GSYSYTYNYEWHVQHWGQG 13228 121 TPP- CAKLTAETKKYQSSPDGGSGG GSYSYTYNYENYFQHWGQG 13229 122 TPP- CAKGITGTKKYQSSPDGGSGG GSYSYTYNYAEYFQHWGQG 13230 123 TPP- CAKGITGTKKYQSSPDGGSGG GSYDYVWGSYAYFQHWGQG 13231 124 TPP- CAKLTSVVVTSHQAPGGGSGG GEKKKLKSLAYYFQHWGQG 10276 125 TPP- CAKVHPNPNPNPNSPDGGSGG GSYNPNPNPNPHVDVWGQG 10290 126 TPP- CAKLTQVKLELGHRPDGGSGG GSNHLRSEKLTYFQHWGQG 10445 127 TPP- CAKVHQETKKYQSSPDGGSGG GSYSYTYNYEWHVQHWGQG 10269 128 TPP- CAKTQTYISNVNHKPDGGSGG GSNTKVDKKAEYFQHWGQG 10288 129 TPP- CTSVHQETKKYQSSPDGGSGG GSYSYTTYNYEWHVDVWGQG 10265 130 TPP- CATSVHQETKKYQSSPDGGSGG GSYSYTYNYEWHVDVHWGQG 10268 131 TPP- CAKLTAEEWKKKYEKEKEKNKGS GGSGGSGGSGGAEYFQHWGQG 10593 132 TPP- CADSSDRSALLKSKLRALLTAPR GSNHLRSEKLTFNYFQHWGQG 11174 133 TPP- CTSVHQETKKYQYQSSPDGGSGG GSYSYTYTYNYEWHVDVWGQG 10266 134 TPP- CAKLTDRSALLKSKLRALLTAPR GSNHLRSEKLTFNYFQHWGQG 11173 135 TPP- CAKLTAVQVKLELGHRPDGGSGG GSNHLRSEKLTFNYFQHWGQG 10444 136 TPP- CTSVHQETKKYQSSYQSSPDGGSGG GSYSYTYSYTYNYEWHVDVWGQ 10267 G 137 TPP- CAKLTAVQVKLELGHRAQPDGGSG GSPVNHLRSEKLTFNYFQHWGQG 10443 G 138 TPP- SSSNIGSKLRALLTAPR GSGSGGSGGSGSGYD 11163 139 TPP- SGSGSGSGSGSGSPDGGSGG GSYGSGGSGSGSGD 10360 140 TPP- SSLGQIQLTIRHSSPDGGSGG GSNKLIVVVHASRNLIGYD 10462 141 TPP- SPLGQIQLTIRHSSQPDGGSGG GSRNKLIVVVHASRNLIAYD 10460 142 TPP- SSLGQIQLTIRHSSQPDGGSGG GSRNKLIVVVHASRNLIGYD 10461 143 TPP- SSSNIGSALLKSKLRALLTAPR GSSDRSALLKSKLRALLTAD 11161 144 TPP- SSSNIGSALLKSKLRALLTAPR GSGSGGSGGSGGSGSGSGYD 11162 145 TPP- SSSNIGSGSGSGSGSGSPDGGSGG GSYGSGGSGSGSGYD 10359 146 TPP- YG NSNRPSG 10824 147 TPP- YGGSGS GSGSNSNRPSG 10825 148 TPP- YGKTHTSPPSPGG GGKTHTSPPSPGNRPSG 11019 149 TPP- YGSGSGSGSGSGGG GGKTHTSPPSPSGNRPSG 11021 150 TPP- YGSKTHTSPPSPGG GGKTHTSPPSPSGNRPSG 11020 151 TPP- YGSGSGSGSGGGSGG GSGGSGSGSGNRPSG 10789 152 TPP- YGSSDKTHTSPPSPGG GGSGSGSGGSGSGSGNRPSG 11022 153 TPP- YTSASLAITGPDGGSGG GSDRFSGSKSGNRPSG 10829 154 TPP- YGFILPIEVYPDGGSGG GSKVRFDYDLFNRPSG 10835 155 TPP- YGSGSGSGSGSGGGSGG GSGSGGSGSGSGNRPSG 10788 156 TPP- YGVPKEKEKEKVSTAVGG GSAPLEVPKEKEKEKVG 10571 157 TPP- YGTSASLAITGPDGGSGG GSDRFSGSKSGGNRPSG 10830 158 TPP- YGGSGSGSGSGPDGGSGG GSGSGGSGSGSGNRPSG 10790 159 TPP- YGSGSGSGSGSGSGSGGG GSYEKEKEKNKTLKNVG 10573 160 TPP- YGTYISNVNHKPDGGSGG GSNTKVDKKVEGNRPSG 10827 161 TPP- YGAEEWKKKYEKEKEKGG GSGSGSGSGSGSGSGSG 10572 162 TPP- YGGFILPIEVYPDGGSGG GSKVRFDYDLFGNRPSG 10836 163 TPP- YGSALLKSKLRALLTAPG GSDGSGGSGSGSGNRPSG 10834 164 TPP- YGSGSGSGSGSGPDGGSGG GSGSGGSGSGSGNRPSG 10787 165 TPP- YGGTSASLAITGPDGGSGG GSDRFSGSKSGGGNRPSG 10831 166 TPP- YGGTYISNVNHKPDGGSGG GSNTKVDKKVEGGNRPSG 10828 167 TPP- YTQTYISNVNHKPDGGSGG GSNTKVDKKVEPKNRPSG 10826 168 TPP- YGGGFILPIEVYPDGGSGG GSKVRFDYDLFGGNRPSG 10837 169 TPP- YGSGSGSGSGSGSPDGGSGG GSYGSGGSGSGSGNRPSG 10361 170 TPP- YGSQVKLELGHRAPDGGSGG GSVNHLRSEKLTSGNRPSG 11023 171 TPP- YPAVVYIEILDRHPDGGSGG GSGREVPISNGSGFNRPSG 10838 172 TPP- YGGVVYIEILDRHPDGGSGG GSGREVPISNGSGGNRPSG 10840 173 TPP- YGAVVYIEILDRHPDGGSGG GSGREVPISNGSGGNRPSG 10839 174 TPP- YGSRSALLKSKLRALLTAPR GSDGSGSGGSGSGSGNRPSG 10832 175 TPP- YGSRSALLKSKLRALLTAPG GSDGSGSGGSGSGSGNRPSG 10833 176 TPP- YGSAVQVKLELGHRPDGGSGG GSNHLRSEKLTFNSGNRPSG 11024 177 TPP- CQS VVF 10353 178 TPP- CGSGSGSGPDGGSGG GSGSGSGSGF 10780 179 TPP- CQSYDILPIEPDGGSGG GSRFDYDGVVF 10786 180 TPP- CGSGSGSGSGPDGGSGG GSGSGSGSGSGF 10779 181 TPP- CGSGSGSGSGSGGGSGG GSGSGSGSGSGSGF 10778 182 TPP- CQSYDKLELGHPDGGSGG GSHLRSEKGVVF 10783 183 TPP- CQSYDILPIEVYPDGGSGG GSKVRFDYDGVVF 10785 184 TPP- CGSGSGSGSGSGPDGGSGG GSGSGSGSGSGSGF 10776 185 TPP- CGSGSGSGSGSGSDGGSGG GSGSGSGSGSGSGF 10777 186 TPP- CQSYDGFILPIEVYGGSGG GSKVRFDYDLFGVVF 10784 187 TPP- CQSYDKLELGHRAPDGGSGG GSVNHLRSEKGVVF 10782 188 TPP- CQVHQETKKYQSSPDGGSGG GSYSYTYNYEWHVVF 10352 189 TPP- CGSGSGSGSGSGSPDGGSGG GSYGSGSGSGSGSGF 10356 190 TPP- CQSYDPNPNPNPNPDGGSGG GSNPNPNPNPSGVVF 10354 191 TPP- CAAWNPNPNPNPNPNGGSGG GSNPNPNPNPNPNVF 10355 192 TPP- CQSYDQVKLELGHRAGGSGG GSVNHLRSEKLTGVVF 10781 193 TPP- CQSVHQETKKYQSSPDGGSGG GSYSYTYNYEWHVVF 10436 194 TPP- CQSYDQVKLELGHRPDGGSGG GSNHLRSEKLTGVVF 10440 195 TPP- CQSYDGFILPIEVYPDGGSGG GSKVRFDYDLFGVVF 10442 196 TPP- CTSVHQETKKYQSSPDGGSGG GSYSYTYNYEWHVDVF 10351 197 TPP- CQSVHQETKKYQSSPDGGSGG GSYSYTYNYEWHVVVF 10348 198 TPP- CGGSGSGSGSGSGSPDGGSGG GSYGSGSGSGSGSGGF 10358 199 TPP- CQCQSYDSSALLKSKLRALLTAPR GSVNHLRSEKLTGVVF 11167 200 TPP- CQSAVQVKLELGHRAPDGGSGG GSVNHLRSEKLTFNVF 10438 201 TPP- CQSYDQVKLELGHRAPDGGSGG GSVNHLRSEKLTGVVF 10439 202 TPP- CQSYDGFILPIEVYFPDGGSGG GSRKVRFDYDLFGVVF 10441 203 TPP- CQSYVHQETKKYQSSPDGGSGG GSYSYTYNYEWHVGVVF 10349 204 TPP- CQSYDVHQETKKYQSSPDGGSGG GSYSYTYNYEWHVSGVVF 10350 205 TPP- CQSYDAVQVKLELGHRAPDGGSGG GSVNHLRSEKLTFNGVVF 10437 206 TPP- CQCQSYDSSDRSALLKSKLRALLTAPR GSGGSVNHLRSEKLTGVVF 11166 207 TPP- CQSYDSSDRSALLKSKLRALLTAPR GSDRSALLKSKLRALLTAVVF 10362 208 TPP- CQSYDSSDRSALLKSKLRALLTAPE GSDRSALLKSKLRALLTAVVF 10363 209 TPP- 5657 .sup.3The N-terminal sequence corresponds to the nearest neighboring reference aa N-terminal from the inserted natriuretic peptide plus the amino acid stretch present between said reference aa and the first amino acid residue of the inserted natriuretic peptide .sup.4The C-terminal sequence corresponds the amino acid stretch present between the last amino acid residue of the inserted natriuretic peptide and the nearest neighboring reference aa C-terminal from the inserted natriuretic peptide plus and said reference aa

[0365] In addition, the following BNP engrafted human IgG1 antibody constructs were generated starting from the antibody scaffold TPP-5657.

TABLE-US-00008 TABLE 5 Design of BNP engrafted antibody constructs # aa # aa Corresp. Insertion N- C- ANP Cmpd TPP Site term.sup.1 term.sup.2 BNP Cpd.sup.3 B1 TPP- CDRH3 20 18 Hum28aa #117 9902 B2 TPP- CDRH3 22 19 Hum25aa 11153 B3 TPP- CDRH3 20 18 Rat28aa #117 11154 B4 TPP- CDRH3 17 17 Rat32aa 11155 B5 TPP- CDRH2 18 13 Hum28aa  #65 11156 B6 TPP- CDRH2 18 13 Rat28aa  #65 11157 B7 TPP- CDRH1 23 14 Hum28aa   #9 18029 B8 TPP- CDRH2  3  3 Hum28aa  #12 18031 B9 TPP- CDRH2  7  7 Hum28aa  #13 18032 B10 TPP- CDRH2 19 14 Hum28aa  #80 18033 B11 TPP- CDRH2 20 15 Hum28aa  #94 18028 B12 TPP- CDRH3 20 18 Hum28aa #122 18034 B13 TPP- CDRL3 19 14 Hum28aa #191 18030 .sup.1The number of amino acid residues present between the respective N-terminal reference amino acid residue and the first amino acid of the inserted natriuretic peptide; .sup.2The number of amino acid residues present between the last amino acid of the inserted natriuretic peptide and the respective C-terminal reference amino acid residue .sup.3Corresponding ANP Cpd. refers to an ANP engrafted antibody construct with the same integration locus and comprising the same N-terminal and C-terminal sequence

TABLE-US-00009 Table 5 cont.: Design of BNP engrafted antibody constructs Cmpd TPP N-terminal sequence.sup.3 C-terminal sequence.sup.4 B1 TPP- CTSVHQETKKYQSSPDGGSGG GSYSYTYNYEWHVDVWGQG 9902 B2 TPP- CTSVHQETKKYQSSPDGGSGGSG GGSYSYTYNYEWHVDVWGQG 11153 B3 TPP- CTSVHQETKKYQSSPDGGSGG GSYSYTYNYEWHVDVWGQG 11154 B4 TPP- CTSVHQETKKYQSSPDGG SYSYTYNYEWHVDVWGQG 11155 B5 TPP- ISGSVVVTSHQAPGGGSGG GEKKKLKSLAYGST 11156 B6 TPP- ISGSVVVTSHQAPGGGSGG GEKKKLKSLAYGST 11157 B7 TPP- SGFTFGSGSGSGSGSGSPDGGSGG GSYGSGSGSGSGSGM 18029 B8 TPP- ISGS GGST 18031 B9 TPP- ISGSGSGS GSGSGGST 18032 B10 TPP- ISGSTYISNVNHKPDGGSGG GSNTKVDKKVEGGST 18033 B11 TPP- ISGPNPNPNPNPNSPDGGSGG GSYNPNPNPNPNPGST 18028 B12 TPP- CAKGITGTKKYQSSPDGGSGG GSYSYTYNYAEYFQHWGQG 18034 B13 TPP- CAAWNPNPNPNPNPNGGSGG GSNPNPNPNPNPNVF 18030 .sup.3The N-terminal sequence corresponds to the nearest neighboring reference aa N-terminal from the inserted natriuretic peptide plus the amino acid stretch present between said reference aa and the first amino acid residue of the inserted natriuretic peptide .sup.4The C-terminal sequence corresponds the amino acid stretch present between the last amino acid residue of the inserted natriuretic peptide and the nearest neighboring reference aa C-terminal from the inserted natriuretic peptide plus and said reference aa

[0366] In addition, the following CNP engrafted human IgG1 antibody constructs were generated.

TABLE-US-00010 TABLE 6 Design of CNP engrafted antibody constructs SEQ ID NO SEQ ID NO # aa # aa % Corresp. Insertion comprised comprised N- C- μg/ml purity ANP Cmpd TPP Site in Ntls in Ctls term.sup.1 term.sup.2 pcs pcs Cpd.sup.3 C1 TPP-9465 CDRH3 7 8 21 23 69-242  97 C2 TPP-12374 CDRL3 11 12 20 20 212 C3 TPP-12375 CDRH3 7 8 21 23 6 C4 TPP-12376 CDRH3 7 8 20 22 7 C5 TPP-12377 CDRH3 8 21 23 7 C6 TPP-12378 CDRH2 9 10 19 18 3 C13 TPP-18036 CDRH1 23 14 259 100 #9 C14 TPP-18038 CDRH2 3 3 0 #12 C15 TPP-18039 CDRH2 7 7 301 100 #13 C16 TPP-18040 CDRH2 2 3 19 14 300 100 #80 C17 TPP-18035 CDRH2 20 15 243 100 #94 C18 TPP-18041 CDRH3 21 22 20 18 287 100 #122 C19 TPP-18037 CDRL3 19 14 246 100 #191 Cmpds based on TPP-12377: Difference vs. TPP-12377 C7 TPP-12895 LC_G99E C8 TPP-12896 LC_G99L 101-131 C9 TPP-12897 LC_S98D 198-304 C10 TPP-12898 LC_S98G 147-258 C11 TPP-12899 LC_A33Y 165-197  82 C12 TPP-12900 LC_A33E 109-195 .sup.1The number of amino acid residues present between the respective N-terminal reference amino acid residue and the first amino acid of the inserted natriuretic peptide; .sup.2The number of amino acid residues present between the last amino acid of the inserted natriuretic peptide and the respective C-terminal reference amino acid residue .sup.3Corresponding ANP Cpd. refers to an ANP engrafted antibody construct with the same integration locus and comprising the same N-terminal and C-terminal sequence

TABLE-US-00011 Table 6 cont.: Design of CNP engrafted antibody constructs Cmpd TPP N-terminal sequence.sup.3 C-terminal sequence.sup.4 C1 TPP- CTSVHQETKKYQSSPDGGSGGS GSGGYGSYSYTYNYEWHVDVWGQG 9465 C2 TPP- CQSYDQVKLELGHRAGGSGGS GSGGSGSVNHLRSEKLTGVVF 12374 C3 TPP- CTSVHQETKKYQSSPDGGSGGS GSGGSGSYSYTYNYEWHVDVWGQG 12375 C4 TPP- CTSVHQETKKYQSSPDGGSGG GGGSGSYSYTYNYEWHVDVWGQG 12376 C5 TPP- CTSVHQETKKYQSSPYKGANKK GSGGSGSYSYTYNYEWHVDVWGQG 12377 C6 TPP- ISGSVVVTSHQAPGGGSGGS GSGGSGEKKKLKSLAYGST 12378 .sup.3The N-terminal sequence corresponds to the nearest neighboring reference aa N-terminal from the inserted natriuretic peptide plus the amino acid stretch present between said reference aa and the first amino acid residue of the inserted natriuretic peptide of the inserted natriuretic peptide and the nearest .sup.4The C-terminal sequence corresponds the amino acid stretch present between the last amino acid residue neighboring reference aa C-terminal from the inserted natriuretic peptide plus and said reference aa

Example 5: In Vitro Activities of Generated Constructs

[0367] All constructs were expressed transiently in HEK293 cells according to well-known methods in the art, targeting a cell density of about 2×10{circumflex over ( )}6 cells/ml, a total DNA concentration of about 1 μg/ml for the two plasmids encoding the light and heavy chain and a 5 day incubation for the expression.

[0368] Raw compound samples (rcs) were expressed in a culture volume of 0.4 ml, and the supernatant separated by centrifugation was directly used for testing. The compound concentration was assessed by an IgG-Fc quantification ELISA according to well-known methods in the art. Briefly, 1:1500 diluted supernatant and a 2-fold dilution series of Human Reference Serum (Bethyl, RS-110-4) starting with 400 ng/ml were immobilized in black Maxisorp 384 micro titer plates (MTP) coated with anti-human Fc [Sigma 12136] in a 1:440 dilution in 1× coating buffer (Candor, 121125) for 1 h, 37° C. After blocking with 100% SMART Block (Candor, 113125) anti-human Fc-HRP [Sigma, A0170] was applied in a 1:10000 dilution for the detection of antibodies in rcs and reference samples. Dose curves of the reference sample were used for the quantitative assessment of compound concentrations shown in Tables 7 and 8, column “μg/ml rcs”. All samples were applied in quadruplets.

[0369] Isolated compound samples (ics) were generated by 1-step purification via protein-A from 6 ml expression culture and according to well-known methods in the art. Acid eluates were neutralized by addition of 8% (v/v) 1M Tris/HCl pH 9.0, quantified via absorption at 280 nm and normalized to a concentration of 125 nM.

[0370] Purified compound samples (pcs) were generated by 2-step purification via protein-A and subsequent SEC in PBS buffer from expression culture of at least 35 ml. Values shown in Tables 7 and 8, column “μg/ml pcs”, refer to the compound concentration in the expression culture supernatant determined by analytical Protein A chromatography.

[0371] All activities shown in Tables 7 and 8 were measured on cells with heterologous over expression of human NPRA (hNPRA) by use of a cGMP quantification assay conducted according to manufacturer's instructions (cisbio; 62GM2PEH). In brief, the assay quantifies cGMP in buffered solution or cell-culture supernatants based on the competition between cGMP produced by the cell as result of the NPRA stimulation through the (natriuretic peptide) sample and d2 labelled cGMP for binding to a Cryptate labelled antibody. Sample cGMP and d2 labeled cGMP compete for binding to a limited number of sites on Cryptate labeled anti-cGMP antibodies, and consequently, HTRF® specific fluorescent signal (i.e. energy transfer) is inversely proportional to the concentration of cGMP in the sample.

[0372] Dose-response curve data were analyzed with GraphPad Prism (version 7.00 for Windows, GraphPad Software, La Jolla Calif. USA) and EC50 were fitted according to Y=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((LogEC50−X)*HillSlope)) applying constraints for bottom, top and slope (shared value for all data sets of the respective experiment).

[0373] The raw compound sample activity on stable hNPRA-CHO kl cells was first assessed by comparison to the negative control TPP-5657 and to a positive sample, in particular TPP-5661. Controls and rcs were tested in quadruplets in two concentrations with a relative dilution factor of 5 aiming for a fluorescent signal (s) in the dynamic range of the assay. The assay window was defined as the difference in signal of inactive (max. signal, s_max) and highly active samples (min. signal, s_min), and for both compound concentrations the activity in % was calculated as 100*(s_max−s)/(s_max−s_min). Values listed in Table 7, columns “activity rcs” and “stdev activity rcs”, represent the average of the results for the two concentrations and the respective standard deviation. Rcs signals less than half of the signal of the reference compound TPP-5661 (36%) were assessed as not active (n.a.).

[0374] The activity of several raw compound samples on stable hNPRA-CHO kl cells was reassessed by comparison to reference sample TPP-5661 in 2.5-fold dilution series (8 concentrations) starting with a 5-fold dilution. The “log EC50” fit value as activity measure of the rcs was set in relation to the corresponding value of the reference rcs TPP-5661 by calculating the delta “log EC50”_compound−“log EC50”_TPP-5661; resulting values are listed in Table 7, column “rel. activity rcs”. Notably, the compound concentrations in the rcs was not considered, and consequently given values are influenced by compound activity and concentration in equal measure. All samples were applied in quadruplets.

[0375] The activity of isolated compound samples on stable hNPRA-CHO kl cells was assessed by EC50 determination and comparison to reference sample TPP-5661. Samples were tested in 2.5-fold dilution series from 80 nM to 0.13 nM. The log EC50 fit value as activity measure of the ics was set in relation to the corresponding value of the reference ics TPP-5661 (−8.8) by calculating the delta log EC50_compound−log EC50_TPP-5661; resulting values are listed in Table 7, column “rel. log EC50 ics”. All samples were applied in quadruplets.

[0376] The activity of purified compound samples on stable hNPRA-CHO kl cells was assessed in multiple experiments by EC50 determination and comparison to reference sample TPP-5661. Samples were tested in dilution series at least in quadruplets. Values listed in Table 7, columns “rel. log EC50 pcs 1, 3 and 4” result from 5-fold dilution series (>4 concentrations) starting with 20 or 25 nM. Values listed in Table 7, column “rel. log EC50 pcs 2” result from 10-fold dilution series (4 concentrations) starting with 25 nM. Values listed in Table 7, columns “rel. log EC50 pcs 5, 6, 7, 8 and 9” result from 2.5-fold dilution series, with 8 or 12 concentrations starting with 40 to 200 nM, respectively. The log EC50 fit value as activity measure of the pcs was set in relation to the corresponding value of the reference TPP-5661 in the respective experiment (in average −9.2, standard deviation 0.5) by calculating the delta log EC50_compound−log EC50_TPP−5661.

[0377] The activity of purified compound samples on transient hNPRA-HEK293 cells was assessed in three experiments by EC50 determination and comparison to reference sample TPP-5661. Samples were tested in 5-fold dilution series from 1000 nM to 0.013 nM. The log EC50 fit value as activity measure of the pcs was set in relation to the corresponding value of the reference TPP-5661 in the respective experiment (average of three experiments −9.0, standard deviation 0.4) by calculating the delta log EC50_compound−log EC50_TPP-5661; resulting values are listed in Table 7, columns “rel. log EC50* pcs”. All samples were applied in duplicates.

[0378] A summarized qualitative assessment of the activity of compounds is given in the column “qualit. activity”, values listed in column “average rel. log EC50” were calculated as average of given rel. log EC50 values. Compounds showing an EC50 values>50-fold of the reference compound TPP-5661, meaning average rel. log EC50>1.7, were qualitatively assessed as not active (“-”), compounds showing a relative log EC50 between 0.7 and 1.7 were assessed as active (“+”), compounds showing a relative log EC50<0.7 were assessed as very active (“++”); highest activities were observed at relative log EC50 below −0.7 (“+++”). Compounds showing an activity in rcs without confirmation as ics or pcs are marked with an unified “y”, and compounds assessed as not active (“n.a.” in column “activity rcs”) probably due to their very low expression level (<1 μg/ml, “n.e.” in column “μg/ml rcs”) are marked accordingly.

TABLE-US-00012 TABLE 7 Expression levels and activities of atrial natriuretic peptide engrafted antibody constructs stdev rel. # aa # aa % acti- acti- acti- rel. rel. rel. rel. rel. rel. rel. rel. rel. rel. rel. rel. rel. qualit. average Com- N- C- μg/ml μg/ml purity vity vity vity logEC50 logEC50 logEC50 logEC50 logEC50 logEC50 logEC50 logEC50 logEC50 logEC50 logEC50* logEC50* logEC50* acti- rel pound term.sup.1 term.sup.2 rcs pcs pcs rcs rcs rcs ics pcs 1 pcs 2 pcs 3 pcs 4 pcs 5 pcs 6 pcs 7 pcs 8 pcs 9 pcs pcs pcs vity logEC50  #1 6 2 n.d. 225 93 n.d. n.a. n.a. n.a. − n.a.  #2 9 5 n.d. 179 96 n.d. n.a. n.a. n.a. − n.a.  #3 12 8 n.d. 196 97 n.d. 0,2 0,0 0,0 ++ 0,1  #4 15 10 n.d. 214 96 n.d. −0,7 −0,8 −0,8 ++ −0,8  #5 18 10 n.d. 229 93 n.d. −0,7 −0,4 −0,4 −0,5 ++ −0,5  #6 19 17 1,6 150 76 33% 18% y  #7 19 17 n.e 117 70 22% 20% y  #9 23 14 4,8 226 97 41% 27% −0,2 0,0 −0,5 −0,6 −0,4 ++ −0,3  #10 23 14 n.d. 265 96 n.d. 0,9 0,2 0,1 0,0 ++ 0,3  #12 3 3 42,0 n.d. n.d. n.a. −  #13 7 7 12,0 n.d. n.d. n.a. n.a. − n.a.  #14 8 9 14,0 n.d. n.d. n.a. n.a. − n.a.  #15 10 11 6,6 n.d. n.d. n.a. n.a. − n.a.  #16 11 14 9,8 n.d. n.d. n.a. −  #17 12 8 7,2 n.d. n.d. 53%  3% y  #18 13 12 12,0 290 98 n.a. −0,2 0,9 + 0,9  #19 14 9 9,5 n.d. n.d. 48% 11% y  #20 14 10 9,9 n.d. n.d. 64%  3% y  #21 14 11 5,8 n.d. n.d. 38% 18% 0,7 ++ 0,7  #22 14 14 23,0 n.d. n.d. 75%  9% y  #23 15 10 2,6 n.d. n.d. 60% 35% y  #24 15 12 n.e 83 52 26% 22% 0,6 ++ 0,6  #25 15 14 14,0 n.d. n.d. 25%  5% −1,0 0,8 + 0,8  #26 16 11 2,0 181 98 n.a. 1,9 −  #27 16 11 17,0 n.d. n.d. 37% 23% 1,4 + 1,4  #28 16 11 9,1 n.d. n.d. 50%  9% −0,9 0,5 ++ 0,5  #29 16 14 7,0 n.d. n.d. n.a. −  #30 16 15 35,0 n.d. n.d. 88% 13% y  #33 17 11 2,3 229 79 73% 32% −0,9 −0,1 −1,1 −0,7 −0,9 −0,5 −0,8 ++ −0,7  #34 17 11 n.d. 226 87 n.d. −0,2 ++ −0,2  #35 17 11 n.d. 215 86 n.d. −0,6 ++ −0,6  #36 17 11 n.d. 217 86 n.d. −0,1 ++ −0,1  #37 17 11 n.d. 244 86 n.d. −0,8 +++ −0,8  #38 17 11 n.d. 233 88 n.d. −0,5 ++ −0,5  #39 17 11 n.d. 208 86 n.d. −0,6 ++ −0,6  #40 17 11 n.d. 154 85 n.d. −0,5 −0,3 −0,6 ++ −0,5  #41 17 11 n.d. 106 79 n.d. n.d.  #42 17 11 n.d. 112 97 n.d. −0,6 −0,7 −0,7 ++ −0,7  #43 17 11 n.d. 141 96 n.d. −0,6 −0,8 −0,6 ++ −0,7  #44 17 11 n.d. 166 93 n.d. −0,7 −0,8 −0,8 +++ −0,8  #45 17 11 n.d. 135 90 n.d. −0,4 −0,2 −0,5 ++ −0,4  #46 17 11 n.d. 166 92 n.d. −0,5 −0,4 −0,6 ++ −0,5  #47 17 11 n.d. 123 83 n.d. −0,3 −0,3 −0,7 ++ −0,4  #49 17 11 n.d. 203 84 n.d. −0,6 −0,2 −0,1 −0,6 ++ −0,4  #50 17 12 9,0 87 97 66% 21% −1,0 0,0 −0,4 ++ −0,2  #51 17 12 6,4 n.d. n.d. 44% 39% y  #52 17 12 7,6 n.d. n.d. 26% 20% y  #53 17 13 n.d. n.d. n.d. 69%  5% y  #54 17 14 n.e 75 n.d. n.a. n.e  #55 17 14 8,8 271 95 72% 40% −0,9 −0,9 −0,4 0,2 ++ −0,4  #57 17 17 13,0 n.d. n.d. 48%  1% y  #58 17 17 33,0 n.d. n.d. 45%  9% y  #59 17 17 31,0 n.d. n.d. 87%  4% −1,1 0,0 ++ 0,0  #60 18 12 4,1 367 90 53% 31% 0,0 −0,5 ++ −0,3  #61 18 13 n.e 334 97 n.a. 0,0 −0,9 ++ −0,5  #62 18 13 11,0 n.d. n.d. 36% 14% 1,5 + 1,5  #63 18 13 9,5 239 96 70% 22% −0,1 ++ −0,1  #64 18 13 5,3 n.d. n.d. 51% 18% y  #65 18 13 1,2 253 86 69% 17% −0,5 −0,3 −1,2 −0,9 +++ −0,7  #66 18 13 38,0 n.d. n.d. 87%  4% −0,5 0,2 ++ 0,2  #67 18 13 4,8 n.d. n.d. 35% 17% −1,1 0,7 ++ 0,7  #68 18 13 9,2 n.d. n.d. 48% 13% y  #69 18 14 7,1 n.d. n.d. 66% 11% −1,0 0,3 ++ 0,3  #70 18 17 n.e n.d. n.d. 30%  2% y  #71 18 17 27,0 n.d. n.d. 89%  2% −0,5 −0,6 ++ −0,6  #72 19 12 11,0 n.d. n.d. 82%  0% −1,5 0,1 ++ 0,1  #73 19 14 9,4 n.d. n.d. 71%  0% −1,4 −0,2 ++ −0,2  #74 19 14 3,8 142 97 67%  7% −1,0 0,0 0,3 ++ 0,2  #75 19 14 4,4 50 49 95%  8% −0,4 ++ −0,4  #76 19 14 11,0 299 97 56%  2% 0,2 −0,8 ++ −0,3  #77 19 14 13,0 n.d. n.d. 38%  6% 1,1 + 1,1  #78 19 14 14,0 n.d. n.d. 55% 38% 1,0 + 1,0  #79 19 14 5,5 n.d. n.d. 67% 26% y  #80 19 14 5,4 214 95 67% 19% −0,4 −0,8 ++ −0,6  #81 19 14 6,2 n.d. n.d. 64% 10% y  #82 19 14 1,6 n.d. n.d. 57%  9% y  #83 19 14 n.e n.d. n.d. 22% 40% y  #84 19 14 11,0 n.d. n.d. 45% 30% y  #85 19 15 4,5 n.d. n.d. 74% 27% −1,2 0,9 +++ −0,9  #87 19 15 42,0 n.d. n.d. 90% 13% y  #89 20 15 n.e 85 n.d. n.a. n.e  #90 20 15 n.e 68 50 52% 18% 0,9 0,5 0,4 ++ 0,6  #91 20 15 3,4 275 99 30% 26% 0,3 0,3 −0,2 0,1 0,2 ++ 0,1  #92 20 15 2,3 n.d. n.d. n.a. −0,3 0,6 ++ 0,6  #93 20 15 4,1 286 98 7%  4% −0,8 −0,8 +++ −0,8  #94 20 15 12,0 318 98 83% 17% −0,8 −0,8 +++ −0,8  #95 20 15 38,0 n.d. n.d. 81% 16% y  #96 20 15 7,3 n.d. n.d. n.a. −  #97 20 15 8,3 n.d. n.d. 26% 17% y  #98 20 15 7,7 n.d. n.d. 40% 14% y  #99 10 3 44,0 257 100 n.a. n.a. n.a. − n.a. #100 10 8 88,0 188 99 n.a. n.a. n.a. − n.a. #101 12 10 83,0 n.d. n.d. n.a. − #102 14 12 16,0 n.d. n.d. 20%  7% y #103 15 18 7,7 101 85 50%  8% 0,7 −0,1 ++ 0,3 #104 16 14 15,0 137 98 n.a. 1,0 2,3 ? 1,7 #106 17 15 30,0 n.d. n.d. 20%  2% y #107 17 18 8,8 n.d. n.d. 50% 19% y #108 18 16 37,0 n.d. n.d. 68%  7% y #109 18 18 4,6 n.d. n.d. 62% 20% y #110 19 17 34,0 238 100 45% 25% −0,4 1,1 ++ 0,4 #111 19 17 41,0 296 98 80%  3% −0,4 0,2 ++ −0,1 #112 19 18 3,9 n.d. n.d. 31% 30% y #113 20 17 44,0 n.d. n.d. 52%  2% y #114 20 17 26,0 219 76 94%  4% 0,5 0,1 ++ 0,3 #116 20 18 9,4 247 84 71% 11% −0,3 −0,2 0,0 −0,2 0,0 −0,1 ++ −0,1 #117 20 18 12,9 238 92 36% 30% 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 ++ 0,0 #118 18 16 n.d. 191 n.d. n.d. −0,1 ++ −0,1 #119 18 16 n.d. 171 n.d. n.d. −0,2 ++ −0,2 #120 20 18 n.d. 200 n.d. n.d. −0,3 ++ −0,3 #121 20 18 n.d. 253 n.d. n.d. −0,2 ++ −0,2 #122 20 18 n.d. 115 n.d. n.d. −0,3 ++ −0,3 #123 20 18 n.d. 153 n.d. n.d. −0,1 ++ −0,1 #124 20 18 41,0 n.d. n.d. 77% 23% y #125 20 18 18,0 320 99 84% 16% −0,8 −0,1 ++ −0,5 #126 20 18 17,0 203 90 80%  2% y #127 20 18 35,0 119 92 95%  2% 0,5 −0,7 −0,5 −0,5 −0,5 ++ −0,3 #128 20 18 64,0 291 96 93%  9% y #129 20 19 28,0 n.d. n.d. 78% 12% y #130 21 19 33,0 n.d. n.d. 89%  5% y #132 22 20 7,5 n.d. n.d. 68% 20% y #133 22 20 20,0 n.d. n.d. 75% 14% y #134 22 20 9,7 n.d. n.d. 75%  3% y #135 22 20 30,0 158 88 67%  9% 0,1 3,5 ? 1,8 #136 24 22 15,0 n.d. n.d. 65% 13% y #137 24 22 61,0 n.d. n.d. 81% 23% y #139 19 13 n.e n.d. n.d. n.a. n.e #145 23 14 35,0 n.d. n.d. 73% 11% y #146 1 6 29,0 n.d. n.d. n.a. − #147 5 10 6,7 n.d. n.d. n.a. 0,8 + #151 14 14 13,0 n.d. n.d. 64% 23% −1,3 −0,4 ++ −0,4 #153 16 15 1,5 n.d. n.d. 40% 24% y #154 16 15 n.e n.d. n.d. 22% 21% y #155 16 16 7,8 n.d. n.d. 57% 17% −1,3 −0,7 ++ −0,7 #156 17 16 2,0 64 98 57% 33% −1,3 ++ #157 17 16 2,4 n.d. n.d. 23% 11% y #158 17 16 10,0 n.d. n.d. n.a. −0,6 ++ #159 17 16 4,9 n.d. n.d. 55% 22% −1,3 ++ #160 17 16 2,8 n.d. n.d. 34% 22% y #161 17 16 4,9 n.d. n.d. 34%  9% −0,7 ++ #162 17 16 n.e n.d. n.d. n.a. n.e #163 17 17 n.d. 84 85 n.d. 0,6 ++ 0,6 #164 18 16 3,0 n.d. n.d. n.a. −0,8 −0,1 ++ −0,1 #165 18 17 3,5 n.d. n.d. 53% 28% y #166 18 17 4,1 n.d. n.d. 20% 34% y #167 18 17 n.e 48 n.d. 44% 13% y #168 18 17 n.e n.d. n.d. n.a. n.e #169 19 17 2,8 78 99 20% 16% −0,1 −0,1 ++ −0,1 #170 19 18 n.e n.d. n.d. n.a. n.e #171 19 18 n.e n.d. n.d. n.a. n.e #172 19 18 n.e n.d. n.d. n.a. n.e #173 19 18 n.e n.d. n.d. n.a. n.e #174 19 19 n.d. 73 n.d. n.d. 1,3 + 1,3 #175 19 19 n.d. 105 82 n.d. 0,3 0,1 ++ 0,2 #176 20 19 n.e n.d. n.d. n.a. n.e #177 2 2 41,0 262 100 n.a. n.a. n.a. − n.a. #178 14 9 16,0 n.d. n.d. 67% 33% y #179 16 10 51,0 n.d. n.d. n.a. − #180 16 11 18,0 n.d. n.d. 37%  6% y #181 16 13 21,0 n.d. n.d. 79%  7% y #182 17 11 2,5 n.d. n.d. 73% 19% y #183 18 12 36,0 n.d. n.d. 21% 16% y #184 18 13 10,0 100 100 50%  3% y #185 18 13 17,0 81 100 72%  1% y #186 18 14 20,0 77 96 45% 22% y #187 19 13 35,0 217 95 85% 10% −1,1 +++ −1,1 #188 19 14 15,0 n.d. n.d. 77%  8% y #189 19 14 16,0 102 99 59%  4% y #190 19 14 41,0 n.d. n.d. 64% 13% y #191 19 14 29,0 248 99 94%  8% 0,1 −0,6 0,2 −0,3 ++ −0,2 #192 19 15 70,0 n.d. n.d. 90% 14% −1,0 −1,0 +++ −1,0 #193 20 14 2,7 n.d. n.d. 33% 37% y #194 20 14 32,0 98 97 75%  3% y #195 20 14 20,0 43 97 34% 12% y #196 20 15 n.e n.d. n.d. n.a. n.e #197 20 15 5,5 18 n.d. 54% 20% y #198 20 15 14,0 57 100 52% 21% y #199 21 15 7,3 n.d. n.d. 29% 12% y #200 21 15 33,0 97 n.d. 78%  7% y #201 21 15 51,0 134 97 87%  6% −0,3 0,1 −0,3 ++ #202 21 15 16,0 29 n.d. 58%  3% y #203 21 16 3,1 17 n.d. 33%  2% y −0,1 #204 22 17 5,7 20 n.d. 45% 17% y #205 23 17 34,0 n.d. n.d. 84% 19% y #206 24 18 11,0 n.d. n.d. 47% 33% y #207 24 20 12,0 115 95 59% 17% n.a. n.a. − n.a. #208 24 20 9,5 182 92 74% 11% 0,6 0,3 ++ 0,5 #209 na na 18,3 402 100 n.a. − .sup.1The number of amino acid residues present between the respective N-terminal reference amino acid residue and the first amino acid of the inserted natriuretic peptide; .sup.2The number of amino acid residues present between the last amino acid of the inserted natriuretic peptide and the respective C-terminal reference amino acid residue Not determined (n.d.), not active (n.a.), not expressed (n.e), not applicable (na)

[0379] No conclusive data for compounds #104 and #135 were obtained. 12 compounds (#54, #61, #89, #139, #162, #168, #170, #171, #172, #173, #176, #196) showed very low expression levels (<1 μg/ml in rcs) and consequently no activity; activity of #61 was shown after compound preparation (pcs). 7 compounds (#7, #24, #70, #83, #90, #154, #167) showed in most cases low activity as rcs, although their expression level was very low; the activity of #24 and #90 was confirmed by compound preparation (pcs). No activity was observed in rcs of compounds #18, #26, #29, #92, #96, #101, #104, #158, #164, #179; however, the activity of compounds #18, #92, #158, #164 was shown using higher concentrations (rel. activity rcs); the lack of activity of #26 was thereby confirmed.

TABLE-US-00013 TABLE 8 qualit. qualit. # aa # aa % activity activity Com- N- C- μg/ml purity on of corresp. pound TPP term.sup.1 term.sup.2 pcs pcs hNPRA ANP cpd..sup.3 B1 TPP-9902 20 18 304 96 ++ ++ B2 TPP-11153 22 19 202 99 + B3 TPP-11154 20 18 205 100 − ++ B4 TPP-11155 17 17 226 97 − B5 TPP-11156 18 13 115 75 ++ +++ B6 TPP-11157 18 13 125 93 − +++ B7 TPP-18029 23 14 265 99 + ++ B8 TPP-18031 3 3 260 99 − − B9 TPP-18032 7 7 262 99 − − B10 TPP-18033 19 14 310 98 + ++ B11 TPP-18028 20 15 74 99 + +++ B12 TPP-18034 20 18 315 97 ++ ++ B13 TPP-18030 19 14 304 100 + ++ #209 TPP-5657 na na 402 100 − − .sup.1The number of amino acid residues present between the respective N-terminal reference amino acid residue and the first amino acid of the inserted natriuretic peptide; .sup.2The number of amino acid residues present between the last amino acid of the inserted natriuretic peptide and the respective C-terminal reference amino acid residue .sup.3Corresponding ANP Cpd. refers to an ANP engrafted antibody construct with the same integration locus and comprising the same N-terminal and C-terminal sequence

[0380] Purified compound samples were tested as described in Example 3 in quadruplets in dilution series on stable hNPRA-CHO kl cells. The activities of BNP engrafted antibody constructs are graphically depicted in FIG. 11.

[0381] TPP-11156, TPP-9902 and TPP-11153 showed significant activity on hNPRA cells in contrast to TPP-1154, TPP-11155, and TPP-11157. Opposed results were observed on rNPRA with EC50<20 nM for TPP-1154, TPP-11155, and TPP-11157, and EC50>1 μM for TPP-9902, TPP-11153, and TPP-11156 (see Example 3). This can be explained by the presence of a human BNP sequence in TPP-11156, TPP-9902 and TPP-11153, whereas TPP-1154, TPP-11155, and TPP-11157 comprise a rat BNP sequence (see Table 5). In contrast to all other human BNP engrafted antibody constructs, TPP-18031 and TPP-18032 with low numbers of additional amino acids N- and C-terminal to BNP showed no activity on hNPRA.

Example 6: Specific Linker Sequences are Particularly Advantageous for Achieving Good Homogeneity and Expression Levels

[0382] The purity of purified compound samples (Example 5, Table 7, column “% purity pcs”) was determined by capillary Gel Electrophoresis according to manufacturer's instructions (LabChip GX, Caliper Life Sciences) under reduced conditions. The purity in % was calculated as sum of peak areas corresponding to the intact light and heavy chain relative to the sum of all peaks observed.

[0383] Ntls sequences comprising a GS linker sequence, a PN linker sequence or the sequence of SEQ ID NOs 2, 4, 9, 11, 13 or 15 and Ctls sequences comprising a GS linker sequence, a PN linker sequence or the sequence of SEQ ID NOs 3, 5, 12, 14, 15 or 20 have proven particularly useful as they not only achieve high natriuretic peptide activities (provided that at least 12 amino acid residues are present between the respective N-terminal reference amino acid residue and the first amino acid of the inserted natriuretic peptide) but also good expression levels (in contrast to e.g., sequences used in compounds #186, #195 and #202) and (in contrast to e.g., sequences used in compounds 6 and 7) a low degree of inhomogeneity (see Table 9). With linker sequences comprising a GS linker sequence as well as linker sequences comprising a PN linker sequence very good purities of 98% in average were observed. Similarly good values were observed for compounds with linkers comprising sequences of SEQ ID NOs 2, 3, 4, 5, 9, 11, 12, 13, 14, 15 and 20. Notably, the Ntls having the sequence of SEQ ID NO 9 resulted only in combination with the Ctls having the sequence of SEQ ID NO 20 in compounds with very good purity;

[0384] compounds with a combination of the Ntls having the sequence of SEQ ID NO 11 and the Ctls having the sequence of SEQ ID NO 12 showed only very good purity when SEQ ID NO 11 was flanked by Asp (D) on the N-terminal side and not by Thr (T) or Val (V) and when the SEQ ID NO 12 VNHLRSEKLT was flanked by Gly (G) on the C-terminal side but not by Tyr (Y) or Phe (F) as in compounds #126 and #135.

TABLE-US-00014 TABLE 9 Ntls and Ctls effects on antibody purity (excerpt of Table 7) Inser- SEQ ID NO SEQ ID NO % tion comprised comprised # aa # aa μg/ml purity qualit. Cpd TPP Site in Ntls in Ctls N-term.sup.1 C-term.sup.2 N-terminal seequence.sup.3 C-terminal sequence.sup.4 pcs pcs actvity   2 TPP- CDRH1 GS GS  9  5 SGFTFGSGSG GSGSGM 179  96 - 13056   3 TPP- CDRH1 GS GS 12  8 SGFTFGSGSGSGS SGSGSGSGM 196  97 ++ 13055   4 TPP- CDRH1 GS GS 15 10 SGFTFGSGSGSGGSGG GGSGGSGSGSG 214  96 ++ 13054   5 TPP- CDRH1 GS GS 18 10 SGFTFGSGSGSGSGGGSGG GSGSGGSGSGM 229  93 ++ 12545   9 TPP- CDRH1 GS GS 23 14 SGFTFGSGSGSGSGSGSPDGGSGG GSYGSGSGSGSGSGM 226  97 ++ 10294  10 TPP- CDRH1 GS GS 23 14 SGFTFGSGSGSGSGSGSPDGGSGG GSYGSGSGSGSGSGM 265  96 ++ 12547  91 TPP- CDRH2 GS GS 20 15 ISGSGSGSGSGSGSPDGGSGG GSYGSGSGSGSGSGST 275  99 ++ 10282  99 TPP- CDRH3 GS GS 10  3 CAKSPDGGSGG GSYG 257 100 - 10281 100 TPP- CDRH3 GS GS 10  8 CAKSPDGGSGG GSYQHWGQG 188  99 - 10280 169 TPP- CDRL2 GS GS 19 17 YGSGSGSGSGSGSPDGGSGG GSYGSGGSGSGSGNRPSG  78  99 ++ 10361 184 TPP- CDRL3 GS GS 18 13 CGSGSGSGSGSGPDGGSGG GSGSGSGSGSGSGF 100 100 y 10776 185 TPP- CDRL3 GS GS 18 13 CGSGSGSGSGSGSDGGSGG GSGSGSGSGSGSGF  81 100 y 10777 189 TPP- CDRL3 GS GS 19 14 CGSGSGSGSGSGSPDGGSGG GSYGSGSGSGSGSGF 102  99 y 10356 198 TPP- CDRL3 GS GS 20 15 CGGSGSGSGSGSGSPDGGSGG GSYGSGSGSGSGSGGF  57 100 y 10358  18 TPP- CDRH2 PN PN 13 12 ISGPNPNKNPNPGG GSNENPNPNPGST 290  98 + 10767  55 TPP- CDRH2 PN PN 17 14 ISGPNPNKNPNPNPGSGG GSNPNENPNPNPGST 271  95 ++ 10765  93 TPP- CDRH2 PN PN 20 15 ISGPNPNKNPNPNSPDGGSGG GSYNPNENPNPNPGST 286  98 +++ 10286    94 TPP- CDRH2 PN PN 20 15 ISGPNPNPNPNPNSPDGGSGG GSYNPNPNPNPNPGST 318  98 +++ 10283 111 TPP- CDRH3 PN PN 19 17 CAKVHPNPNPNPNPDGGSGG GSNPNPNPNPHVDVWGQG 296  98 ++ 10289 125 TPP- CDRH3 PN PN 20 18 CAKVHPNPNPNPNSPDGGSGG GSYNPNPNPNPHVDVWGQG 320  99 ++ 10290 191 TPP- CDRL3 PN PN 19 14 CAAWNPNPNPNPNPNGGSGG GSNPNPNPNPNPNVF 248  99 ++ 10355  63 TPP- CDRH2  2  3 18 13 ISGTYISNVNHKPDGGSGG GSNTKVDKKVEGST 239  96 ++ 10844  80 TPP- CDRH2  2  3 19 14 ISGSTYISNVNHKPDGGSGG GSNTKVDKKVEGGST 214  95 ++ 10845 128 TPP- CDRH3  2  3 20 18 CAKTQTYISNVNHKPDGGSGG GSNTKVDKKAEYFQHWGQG 291  96 y 10288    61 TPP- CDRH2  4  5 18 13 ISGTSASLAITGPDGGSGG GSDRFSGSKSGGST 334  97 ++ 10847    76 TPP- CDRH2  4  5 19 14 ISGSTSASLAITGPDGGSGG GSDRFSGSKSGGGST 299  97 ++ 10848 156 TPP- CDRL2 15 15 17 16 YGVPKEKEKEKVSTAVGG GSAPLEVPKEKEKEKVG  64  98 ++ 10571  42 TPP- CDRH2  9 20 17 11 ISVVVTSHQSPTPGGSGG GGSTPLKSLAST 112  97 ++ 13060  43 TPP- CDRH2  9 20 17 11 ISVVVTSHQAPGEGGSGG GGSTPLKSLAST 141  96 ++ 13061  44 TPP- CDRH2  9 20 17 11 ISVVVTSHQAPGEGGSGG GSTPKLKSLAST 166  93 +++ 13062  46 TPP- CDRH2  9 10 17 11 ISVVVTSHPTPGEGGSGG GEKKKLKSLAST 166  92 ++ 13064  45 TPP- CDRH2  9 10 17 11 ISVVVTSHQSPTPGGSGG GEKKKLKSLAST 135  90 ++ 13063  40 TPP- CDRH2  9 10 17 11 ISVVVTSHQAPGSGGSGG GEKKKLKSLAST 154  85 ++ 13058  47 TPP- CDRH2  9 10 17 11 ISVVVTSHQAPSPGSTGG GEKKKLKSLAST 123  83 ++ 13065  41 TPP- CDRH2  9 10 17 11 ISVVVTSHQAPTSGGSGG GEKKKLKSLAST 106  79 n.d. 13059 201 TPP- CDRL3 11 12 21 15 CQSYDQVKLELGHRAPDGGSGG GSVNHLRSEKLTGVVF 134  97 ++ 10439 194 TPP- CDRL3 11 12 20 14 CQSYDQVKLELGHRPDGGSGG GSNHLRSEKLTGVVF  98  97 y 10440 187 TPP- CDRL3 11 12 19 13 CQSYDKLELGHRAPDGGSGG GSVNHLRSEKGVVF 217  95 +++ 10782 126 TPP- CDRH3 11 12 20 18 CAKLTQVKLELGHRPDGGSGG GSNHLRSEKLTYFQHWGQG 203  90 y 10445 135 TPP- CDRH3 11 12 22 20 CAKLTAVQVKLELGHRPDGGSGG GSNHLRSEKLTFNYFQHWGQG 158  88 ? 10444  50 TPP- CDRH2 13 14 17 12 ISGSAVVNVRAPDGGSGG GSKGDKIAIGGST  87  97 ++ 10452  74 TPP- CDRH2 13 14 19 14 ISGSSGAVVNVRAPDGGSGG GSKGDKIAIWTTGST 142  97 ++ 10451  26 TPP- CDRH2 13 14 16 11 ISGSAVVNVRADGGSGG GSGDKIAIGGST 181  98 - 10772   6 TPP- CDRH1 19 17 SPAVVYIEILDRHPDGGSGG GSGREVPISNGSGFVVAM 150  76 y 10454   7 TPP- CDRH1 19 17 SGAVVYIEILDRHPDGGSGG GSGREVPISNGSGFVVAM 117  70 y 10453 186 TPP- CDRL3 18 14 CQSYDGFILPIEVYGGSGG GSKVRFDYDLFGVVF  77  96 y 10784 195 TPP- CDRL3 20 14 CQSYDGFILPIEVYPDGGSGG GSKVRFDYDLFGVVF  43  97 y 10442 202 TPP- CDRL3 21 15 CQSYDGFILPIEVYFPDGGSGG GSRKVRFDYDLFGVVF  29 n.d. y 10441 .sup.1The number of amino acid residues present between the respective N-terminal reference amino acid residue and the first amino acid of the inserted natriuretic peptide; .sup.2The number of amino acid residues present between the last amino acid of the inserted natriuretic peptide and the respective C-terminal reference amino acid residue .sup.3The N-terminal sequence corresponds to the nearest neighboring reference aa N-terminal from the inserted natriuretic peptide plus the amino acid stretch present between said reference aa and the first amino acid residue of the inserted natriuretic peptide .sup.4The C-terminal sequence corresponds the amino acid stretch present between the last amino acid residue of the inserted natriuretic peptide and the nearest neighboring reference aa C-terminal from the inserted natriuretic peptide plus and said reference aa

Example 7: IgG1, IgG2 and IgG4 Isotypes Provide Equally Suitable Antibody Scaffolds

[0385] Compounds 9, 33, 65, 91, 127 and 191 (human IgG1 TPP-10294, TPP-10277, TPP-10279, TPP-10282, TPP-10269 and TPP-10355, respectively) were generated as different IgG isotypes. Purified compound samples were tested as described in Example 3 in quadruplets in dilution series on stable hNPRA-CHO kl cells. The activities of ANP engrafted human IgG2 and IgG4 isotype constructs (e.g. compound 9 IgG4 TPP-10992) are similar to their corresponding IgG1 isotype as graphically depicted in FIG. 12.

Example 8: Human and Non-Human IgGs Provide Equally Suitable Antibody Scaffolds

[0386] Compound 9 (human IgG1 TPP-10294 and IgG4 TPP-10992) and compound 117 (human IgG1 TPP-5665) were generated as non-human IgG isotypes. Purified compound samples were tested as described in Example 3 in quadruplets in dilution series on stable hNPRA-CHO kl cells. ANP engrafted rat and mouse isotype constructs, e.g. compound 9 rat IgG1 TPP-13992, showed activities similar to their corresponding human IgG isotype (FIG. 13).

Example 9: Human IgGs Comprising Varying Germline Sequences Provide Equally Suitable Antibody Scaffolds

[0387] 22 additional ANP engrafted IgG4 antibodies (compounds A to S) were constructed. In each case ANP was incorporated within CDRH1. The heavy chains of these constructs comprise varying HV and CDRH3 sequences and were paired with varying lambda or kappa light chains. The structure of compounds A to S is summarized in Tables 10 and 11.

TABLE-US-00015 TABLE 10 Design of ANP engrafted antibody constructs A to S Inser- tion # aa #aa Cmpd. Site N-term.sup.1 C-term.sup.2 N-terminal sequence.sup.3 C-terminal sequence.sup.4 A-P CDRH1 23 14 SGFTFGSGSGSGSGSGSPDGGSGG GSYGSGSGSGSGSGM Q-S CDRH1 23 14 SGYSFGSGSGSGSGSGSPDGGSGG GSYGSGSGSGSGSGI .sup.1The number of amino acid residues present between the respective N-terminal reference amino acid residue and the first amino acid of the inserted natriuretic peptide; .sup.2The number of amino acid residues present between the last amino acid of the inserted natriuretic peptide and the respective C-terminal reference amino acid residue .sup.3The N-terminal sequence corresponds to the nearest neighboring reference aa N-terminal from the inserted natriuretic peptide plus the amino acid stretch present between said reference aa and the first amino acid residue of the inserted natriuretic peptide .sup.4The C-terminal sequence corresponds the amino acid stretch present between the last amino acid residue of the inserted natriuretic peptide and the nearest neighboring reference aa C-terminal from the inserted natriuretic peptide plus and said reference aa

TABLE-US-00016 TABLE 11 Design of ANP engrafted antibody constructs A to S Cmpd TPP HV CDRH3 LV/KV Purity 9 TPP-10992 HV3-23 KLTGAEYFQHW LV1-40  99 A TPP-13944 HV3-23 KLTGAEYFQHW LV2-14  98 B TPP-13945 HV3-23 KLTGAEYFQHW LV3-21  93 C TPP-13941 HV3-23 KDYGDYAEYFQHW LV1-40 100 D TPP-13956 HV3-23 KLTGAEYFQHW KV1-5  99 E TPP-13955 HV3-23 KLTGAEYFQHW KV3-20  99 F TPP-13940 HV3-23 KVLRFLEWLLYAEYFQHW LV1-40  98 G TPP-13939 HV3-23 KVQLERAEYFQHW LV1-40 100 H TPP-13943 HV3-23 KYNRNHAEYFQHW LV1-40  54 I TPP-13942 HV3-23 KYNWNDAEYFQHW LV1-40  99 J TPP-14684 HV3-23 RGATFALDW KV3-20  97 K TPP-13958 HV3-23 RGRLPDVW KV1-5  99 L TPP-13957 HV3-23 RGRLPDVW KV3-20  97 M TPP-13948 HV3-23 RGRLPDVW LV1-40  98 N TPP-14289 HV3-23 RGRLPDVW LV1-47 O TPP-13946 HV3-23 RGRLPDVW LV2-14  97 P TPP-13947 HV3-23 RGRLPDVW LV3-21  96 Q TPP-13952 HV5-51 RGRLPDVW LV2-14  99 R TPP-13953 HV5-51 RGRLPDVW LV3-21  98 S TPP-13962 HV5-51 RGRLPDVW KV1-5  99

[0388] Purified compound samples of IgG scaffold constructs A to S comprising varying germline sequences were tested as described in Example 3 in quadruplets in dilution series on stable hNPRA-CHO kl cells. Exemplary activity data are graphically depicted in FIG. 14.

Example 10: CNP Engrafted IgG Protects Against Induced Endothelial Barrier Permeability

[0389] Endothelial monolayer permeability was assayed by real-time impedance measurement with an xCELLigence RTCA system utilizing microtiter well plates covered with microelectrodes (E-Plates). Relative impedance changes are expressed as unitless Cell Index (CI) values.

[0390] Primary Human Pulmonary Artery Endothelial Cells (HPAECs) were seeded at low passages in collagen pre-coated E-Plates. After tight monolayer and cell barrier formation with constant CI values, HPAECs were pre-treated with the indicated concentrations of compound TPP-12899 or the respective negative control antibody construct TPP-5657, followed by compromising the EC barrier with the disruptive agonists LPS (200 ng/ml), IL-1I3 (0.5 ng/ml), or thrombin (2 U/ml). CI were recorded every 10 min to monitor effects on cell growth and monolayer permeability. All cell indices were normalized at the last recording point before test substance application (=normalized CI).

[0391] The experiments were performed with n=4 with 3 technical triplicates each. Results were expressed as mean±SEM. Data were statistically analyzed using one-way ANOVA followed by Sidak's multiple post-test; p-values<0.05 were considered as significant.

[0392] FIG. 15 graphically depicts the effects on endothelial monolayer permeability expressed as Cell Index values. As shown in FIG. 15, pre-treatment of human endothelial monolayer cultures with TPP-12899 protected against induced endothelial barrier permeability in a dose-dependent manner. This was independent of the applied barrier disruptive agent; both, with fast and strong acting thrombin as well as with long-lasting pro-inflammatory stimuli LPS and IL-1I3 significant effects were observed. The respective negative control showed no effect.

Example 11: Long Term Effects of ANP Engrafted IgG in a Chronic Heart Failure Rat Model (TGR(mRenR2)27

[0393] The TGR(mRenR2)27 rat model shows hypertension and endothelial dysfunction, as well as end-organ damage. Male renin-transgenic rats (Ganten D., Nature. 1990; 344(6266):541-4) at the age of 8 weeks were used. The nonselective inhibitor of nitric oxide synthetases L-NAME (Nω-Nitro-L-arginine methyl ester) was chronically administered via the drinking water (20 mg/1) in all study groups to induce endothelial dysfunction. TPP-13992, the rat IgG1 counterpart of TPP-10294 used in Example 8 and TPP-10155, a rat IgG1 isotype control antibody were administered once weekly intraperitoneally. Body weight and survival were assessed. The placebo group was treated with vehicle (PBS) and the healthy control group was treated with captopril-food from weaning on. Food and water were given ad libitum. Daily observation of the behavior and general health status of the animals was performed. At the end of the experiment (week 14), the rats were anaesthetized. The rats were then exsanguinated and the heart was removed from the thoracic cavity for analysis. Urine was collected at the end of the study to determine different urine parameters, e.g. urinary protein creatinine ratio. FIG. 16 graphically depicts the therapeutic effects of TPP-13992 on survival, body weight gain, urinary protein/creatinine ratio and left atrial weight.

Example 12: Hemodynamic Effects of ANP Engrafted IgG in Healthy Beagle Dogs

[0394] The effects of TPP-10992 on cardiovascular and ECG parameters after single subcutaneous administration were assessed in a primary pharmacodynamic study in conscious telemetered beagle dogs.

[0395] Telemetry devices (DSI™, USA) were surgically implanted to measure blood pressure as well as heart rate, followed by a recovery period to allow wound closure. On the day of the study, telemetry sensors were activated for continuous hemodynamic measurements. The transmitted signals were collected by telemetry receivers located in the animal facility. All collected data were processed by a data acquisition program and averaged over a predefined period of 12 h. As vehicle for TPP-10992 NaCl (0.9%) was used and doses of 0.1 mg/kg, 0.3 mg/kg and 1.0 mg/kg bodyweight were applied via subcutaneous injection.

[0396] The results are graphically depicted in FIG. 17. In healthy dogs, TPP-10992 showed a dose-dependent and long-lasting (>5d) reduction in blood pressure which was significant at 1.0 mg/kg s.c. (compared to placebo). No effects on heart rate were observable.