Brain natriuretic peptide grafted antibody

Abstract

The present invention relates to an antibody or fragment thereof comprising at least one heterologous amino acid sequence incorporated into 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 the at least one CDR region is replaced by the at least one heterologous amino acid sequence incorporated therein. The invention also relates to such antibodies or fragments thereof for use in methods of treatment, compositions comprising such antibodies or fragments thereof, nucleic acids or mixtures of nucleic acids encoding such antibodies or fragments thereof, host cells comprising such nucleic acids or mixtures of nucleic acids, and to methods of producing such antibodies or fragments thereof.

Description

[0001] This application is a divisional application of the international application with application number PCT / EP2019 / 059101 filed on April 10, 2019 and invention name “Brain natriuretic peptide grafted antibody”. The international application entered the Chinese national phase on December 9, 2020, and its application number is 201980038639.X. Technical Field

[0002] The present invention relates to an antibody or a fragment thereof, comprising at least one heterologous amino acid sequence incorporated into at least one CDR region of the antibody or fragment thereof, wherein the 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 the at least one CDR region is replaced by the at least one heterologous amino acid sequence incorporated therein. There are at least 12 amino acid residues between: between amino acid residue HC (heavy chain) res25 according to Kabat and the first amino acid residue of BNP when the heterologous amino acid sequence is incorporated in CDRH1; between amino acid residue HC res51 according to Kabat and the first amino acid residue of BNP when the heterologous amino acid sequence is incorporated in CDRH2; between residue HC res92 according to Kabat and the first amino acid residue of BNP when the heterologous amino acid sequence is incorporated in CDRH3; between residue LC (light chain) res26 according to Kabat and the first amino acid residue of BNP when the heterologous amino acid sequence is incorporated in CDRL1; between amino acid residue LC res49 according to Kabat and the first amino acid residue of BNP when the heterologous amino acid sequence is incorporated in CDRL2; and / or between amino acid residue LC res88 according to Kabat and the first amino acid residue of BNP when the heterologous amino acid sequence is incorporated in CDRL3. In addition, there are at least 9 amino acid residues between the last amino acid residue of BNP and: amino acid residue HC res35a according to Kabat in case of inclusion of said heterologous amino acid sequence in CDRH1; amino acid residue HC res57 according to Kabat in case of inclusion of said heterologous amino acid sequence in CDRH2; amino acid residue HC res106 according to Kabat in case of inclusion of said heterologous amino acid sequence in CDRH3; amino acid residue LC res 32 according to Kabat in case of inclusion of said heterologous amino acid sequence in CDRL1; amino acid residue LC res57 according to Kabat in case of inclusion of said heterologous amino acid sequence in CDRL2; and / or amino acid residue LC res98 according to Kabat in case of inclusion of said heterologous amino acid sequence in CDRL3. The present invention also relates to such an antibody or fragment thereof for use in a method of treatment, a composition comprising such an antibody or fragment thereof, a nucleic acid or a mixture of nucleic acids encoding such an antibody or fragment thereof, a host cell comprising such a nucleic acid or such a mixture of nucleic acids, and a method of producing such an antibody or fragment thereof. Background Art

[0003] Natriuretic peptides are a family of three structurally related peptides with neurohumoral effects. Atrial natriuretic peptide (ANP) is a 28 amino acid peptide containing a central ring structure formed by a disulfide bridge between cysteine ​​residues 7 and 23. Human ANP is expressed as a 153 amino acid long pre-prohormone in atrial myocytes. Signal peptide cleavage produces a prohormone form, which is then further cleaved into mature ANP and N-terminal residues, known as NT-proANP. Similar to ANP, brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are also produced by precursor proteins and contain a central ring structure. ANP is mainly produced and released by the cardiomyocytes of the left and right atria, while BNP is mainly produced by ventricular cardiomyocytes. CNP is synthesized by vascular endothelial cells. In addition to these locations, smaller amounts of natriuretic peptides are also produced in other parts of the body, such as in the brain, kidneys and adrenal glands. Natriuretic peptides are encoded by three independent genes NPPA, NPPB and NPPC. The amino acid sequences of these three peptides are highly conserved in mammals (Potter et al., Handb Exp Pharmacol. 2009; (191): 341-66). However, significant sequence modifications of natriuretic peptides have been documented, such as truncation, amino acid exchange, and chimeric fusion (e.g., CD-NP (McKie et al., Curr Heart Fail Rep. 2010 Sep; 7 (3): 93-9)) to produce effective natriuretic peptides that activate or bind to cell receptors and can cause relevant physiological effects.

[0004] Natriuretic peptides bind to three different membrane-bound receptor types, NPR-A, NPR-B, and NPR-C, mediating their biological effects. ANP and BNP have the greatest binding affinity to NPR-A; in contrast, CNP has the greatest affinity for the NPR-B receptor. NPR-A and NPR-B contain a (microparticle) guanylate cyclase domain (pGC), whose enzymatic activity leads to an increase in (intracellular) cyclic guanosine monophosphate (cGMP). As a second messenger, cGMP regulates different cellular processes. The NPR-C receptor does not show guanylate cyclase activity and is also known as a "scavenging" receptor because it can bind natriuretic peptides, leading to their degradation by endocytosis. Additional signaling functions of the NPR-C receptor have been documented by regulating cAMP (Anand-Srivastava, Peptides. 2005 Jun; 26 (6): 1044-59).

[0005] Cardiac hormones ANP and BNP are discharged when the ventricle and atrium stretch, for example due to excessive plasma volume. They exert vasodilation effects by relaxing vascular smooth muscle, and cause blood pressure to decrease. In the kidney, ANP can cause increased urine excretion (diuresis), and increased sodium ion concentration in urine (natriuresis). ANP is considered to be a compensatory antagonist of the renin-angiotensin-aldosterone system (RAAS), which is overactivated in many cardiovascular diseases. In addition, ANP exerts other neurohumoral effects, including inhibitory effects on the sympathetic nervous system, and complex regulatory effects on baroreflexes (Woods et al., Clin Exp Pharmacol Physiol. 2004 Nov; 31 (11): 791-4). For ANP as well as BNP and CNP, anti-inflammatory, anti-hypertrophic and anti-fibrotic effects have been confirmed in animal models of different diseases (e.g. Knowles et al., 2001, J. Clin. Invest. 107:975-984; Dahrouj et al., J Pharmacol Exp Ther. 2013 Jan; 344(1):96-102; Baliga et al., Br J Pharmacol. 2014 Jul; 171(14):3463-75; Mitaka et al. Intensive Care Med Exp. 2014 Dec; 2(1):28; Werner et al., Basic Res Cardiol. 2016 Mar; 111(2):22; Kimura et al., Respir Res. 2016 Feb 19; 17:19). CNP activation of NPR-B plays an important role in bone growth (Yasoda et al., Clin. Calcium. 2009 Jul; 19(7): 1003-8) and vascular endothelial integrity (Moyes et al., J Clin Invest. 2014 Sep; 124(9): 4039-51).

[0006] The broad spectrum of physiological effects of natriuretic peptides and their receptors make them attractive targets for drug development (Lumsden et al., Curr Pharm Des. 2010; 16(37):4080-8; Buglioni et al., Annu Rev Med. 2016; 67:229-43). For example, the natriuretic cGMP system may be inhibited under various pathophysiological conditions, which may lead to hypertension, increased cell proliferation, fibrosis, inflammation, endothelial dysfunction, diabetes, metabolic syndrome, atherosclerosis, cardiac insufficiency, myocardial infarction, pulmonary hypertension, eye and kidney diseases, bone disorders, stroke and / or sexual dysfunction.

[0007] The main obstacle to the therapeutic use of natriuretic peptides is that they have a short plasma half-life in the organism, which is only a few minutes (Hunt et al., J Clin Endocrinol Metab. 1994Jun; 78(6): 1428-35; Kimura et al., EurJ Clin Pharmacol. 2007Jul; 63(7): 699-702). In addition to the endocytosis of NPR-C receptors, natriuretic peptides are also effectively proteolytically degraded by enkephalinase (NEP) and insulin degrading enzyme (IDE). The relevant short-term biological effects of the administered natriuretic peptides limit their therapeutic use mainly to acute indications. For example, in different countries, the infusion of recombinant caperitide (ANP) and nesiritide (BNP) is approved for the treatment of acute decompensated heart failure.

[0008] The treatment of chronic diseases would be greatly facilitated by providing NPR-A and NPR-B agonists with increased plasma half-life, improved proteolytic stability, and prolonged duration of action.

[0009] In recent years, several natriuretic peptide derivatives and variants have been described, such as CD-NP (McKie et al., Curr Heart Fail Rep. 2010 Sep; 7(3):93-9), ZD100 / MANP (McKie et al., Hypertension. 2010 Dec; 56(6):1152-9), PL-3994 (Edelson et al., Pulm Pharmacol Ther. 2013 Apr; 26(2):229-38), ularitide (Anker et al., Eur Heart J. 2015 Mar 21; 36(12):715-2), ANX-042 (Pan et al., Proc Natl Acad Sci USA. 2009 Jul. 7; 106(27):11282-7) and BMN-111 (Wendt et al., J. Pharmacol Exp Ther. 2015Apr; 353(1): 132-49). The half-life of CD-NP is about 18.5 minutes (Lee et al., BMC Pharmacology 2007, 7(Suppl I): P38). Other ANP and CNP derivatives are disclosed in US9,193,777 and EP2432489 A, respectively.

[0010] In addition, natriuretic peptide fusions (including Fc fusions, albumin fusions) and pegylated natriuretic peptides are also described. For example, natriuretic peptide-Fc fusions are disclosed in US2010 / 0310561, WO2008 / 154226, WO2010 / 117760, WO2006 / 107124, WO2008 / 136611 and WO2008 / 079995. Natriuretic peptide-albumin fusions are disclosed in US7,521,424 and US2014 / 0148390, and pegylated natriuretic peptides are disclosed in US2014 / 0148390.

[0011] WO2005 / 060642 records the generation of ANP and BNP peptide grafted antibody libraries obtained by inserting ANP or BNP with two random flanking amino acids at both ends into the CDRH3 region of human tetanus toxoid specific antibodies. Similarly, WO2005 / 082004 discloses the generation of ANP mimetic grafted antibody libraries obtained by replacing the entire original CDRH3 region of the 2G12 antibody with an ANP mimetic peptide with two random amino acid residues on both sides. Neither WO2005 / 060642 nor WO2005 / 082004 discloses any specific natriuretic peptide grafted antibodies, let alone functionally characterizing these antibodies.

[0012] Purpose of the Invention

[0013] In view of the prior art, an object of the present invention is to provide novel natriuretic peptide receptor agonists having increased stability in serum compared to naturally occurring wild-type natriuretic peptides. Summary of the invention

[0014] The above-mentioned objects are achieved by the teaching of the present independent claim. The inventors surprisingly found that biologically active natriuretic peptide variants with significantly increased stability in serum compared to naturally occurring wild-type natriuretic peptides can be obtained by incorporating natriuretic peptide amino acid sequences into a CDR region of an immunoglobulin molecule or a fragment thereof, despite the short length and high sequence conservation of the immunoglobulin CDR region, which has a considerable conformational inhibitory effect on the incorporation of biologically active peptides. However, the activity of natriuretic peptides incorporated into the immunoglobulin CDR region shows great variation. The inventors have found that the decisive factor for the successful incorporation of biologically active natriuretic peptide variants is the number of amino acid residues between the N-terminus and the C-terminus of the nearest neighboring CDR framework connected to the incorporated natriuretic peptide. When the N-terminal and C-terminal flanking amino acid residues between the natriuretic peptide and the adjacent CDR framework are less than a certain number, only natriuretic peptide immunoglobulin fusion constructs with no biological activity or greatly reduced biological activity are obtained. The specific linker sequences flanking the incorporated natriuretic peptides were found to be particularly conducive to achieving high peptide activity, good expression levels and / or low protein fragmentation levels.

[0015] Thus, in a first aspect, the present invention relates to an antibody or fragment thereof comprising at least one heterologous amino acid sequence incorporated into at least one CDR region of the antibody or fragment thereof, wherein the at least one heterologous amino acid sequence comprises an N-terminal linker sequence (Ntls), a natriuretic peptide and a C-terminal linker sequence (Ctls), wherein optionally at least a portion of the at least one CDR region is replaced by the heterologous amino acid sequence incorporated therein, wherein

[0016] a) at least 12 amino acid residues are present in

[0017] i) between the amino acid residue HCres25 according to Kabat (which in the heavy chain having the amino acid sequence SEQ ID NO 65 corresponds to res S25) and the first amino acid residue of the natriuretic peptide, in case said heterologous amino acid sequence is incorporated into CDRH1;

[0018] ii) in case said heterologous amino acid sequence is incorporated in CDRH2, between amino acid residue HCres51 according to Kabat (which in the heavy chain having the amino acid sequence SEQ ID NO 65 corresponds to res I51) and the first amino acid residue of the natriuretic peptide;

[0019] iii) between amino acid residue HCres92 according to Kabat (which in the heavy chain having the amino acid sequence SEQ ID NO 65 corresponds to res C96) and the first amino acid residue of the natriuretic peptide in case said heterologous amino acid sequence is incorporated into CDRH3;

[0020] iv) between the amino acid residue LCres26 according to Kabat (which in the light chain having the amino acid sequence SEQ ID NO 66 corresponds to res S25) and the first amino acid residue of the natriuretic peptide in case said heterologous amino acid sequence is incorporated in CDRL1;

[0021] v) between the amino acid residue LCres49 according to Kabat (which in the light chain having the amino acid sequence SEQ ID NO 66 corresponds to res Y51) and the first amino acid residue of the natriuretic peptide in case said heterologous amino acid sequence is incorporated in CDRL2;

[0022] and / or

[0023] vi) between the amino acid residue LCres88 according to Kabat (which in the light chain having the amino acid sequence SEQ ID NO 66 corresponds to res C90) and the first amino acid residue of the natriuretic peptide in case said heterologous amino acid sequence is incorporated in CDRL3;

[0024] And among them

[0025] b) There are at least 9 amino acid residues between the last amino acid residue of the natriuretic peptide and

[0026] i) in case said heterologous amino acid sequence is incorporated into CDRH1, amino acid residue HCres35a according to Kabat (in the heavy chain having the amino acid sequence SEQ ID NO 65, which corresponds to res M34);

[0027] ii) amino acid residue HCres57 according to Kabat (which corresponds to res T58 in the heavy chain having the amino acid sequence SEQ ID NO 65), in case said heterologous amino acid sequence is incorporated into CDRH2;

[0028] iii) in case said heterologous amino acid sequence is incorporated into CDRH3, amino acid residue HCres106 according to Kabat (which in the heavy chain having the amino acid sequence SEQ ID NO 65 corresponds to res G111);

[0029] iv) amino acid residue LCres 32 according to Kabat (which corresponds to res D34 in the light chain having the amino acid sequence SEQ ID NO 66) in case said heterologous amino acid sequence is incorporated in CDRL1;

[0030] v) amino acid residue LCres57 according to Kabat (which corresponds to res G59 in the light chain having the amino acid sequence SEQ ID NO 66) in case said heterologous amino acid sequence is included in CDRL2; and / or

[0031] vi) in case said heterologous amino acid sequence is incorporated in CDRL3, amino acid residue LCres98 according to Kabat (which in the light chain having the amino acid sequence SEQ ID NO 66 corresponds to res F102).

[0032] In other aspects, the present invention relates to the use of such antibodies or fragments thereof in methods of treatment, compositions comprising such antibodies or fragments thereof, nucleic acids or mixtures of nucleic acids encoding such antibodies or fragments thereof, host cells comprising such nucleic acids or such mixtures of nucleic acids, and methods of producing such antibodies or fragments thereof. DETAILED DESCRIPTION

[0033] The present invention may be understood more readily by reference to the following detailed description of the invention and the Examples included therein.

[0034] In a first aspect, the present invention relates to an antibody or a fragment thereof, comprising at least one heterologous amino acid sequence incorporated into at least one CDR region of the antibody or fragment thereof, wherein the at least one heterologous amino acid sequence comprises an N-terminal linker sequence (Ntls), a natriuretic peptide and a C-terminal linker sequence (Ctls), wherein optionally a portion of the at least one CDR region is replaced by at least one heterologous amino acid sequence incorporated therein.

[0035] The present inventors have found that biologically active natriuretic peptide variants with significantly increased stability in serum compared to naturally occurring wild-type natriuretic peptides can be obtained by incorporating natriuretic peptide amino acid sequences into a CDR region of an immunoglobulin molecule or fragment thereof. This discovery was completely unexpected. As is well known in the art, the shorter length and higher sequence conservation of immunoglobulin CDR regions (particularly evident in CDRL1, CDRL2, CDRL3, CDRH1 and CDRH2) have considerable conformational inhibition on the incorporation of biologically active peptides, and the surrounding immunoglobulin sequences may have an adverse effect on the expression, folding and / or biological activity of the incorporated peptides. In fact, the present inventors have found that the activity of natriuretic peptides incorporated into the immunoglobulin CDR region varies greatly, depending on the exact manner in which the natriuretic peptide grafted antibody is constructed. The decisive factor for the successful incorporation of functional (i.e., biologically active) natriuretic peptide variants has been shown to be the number of amino acid residues between the N-terminus and the CDR framework connecting the incorporated natriuretic peptide to the nearest neighbor of the incorporated natriuretic peptide. When the N-terminal and C-terminal flanking amino acid residues between the natriuretic peptide and the adjacent CDR framework connection are below a certain number, no biologically active natriuretic peptide-immunoglobulin fusion construct is obtained.

[0036] The terms "incorporated," "inserted," "integrated," "grafted," and "embedded," as well as "incorporated," "inserted," "integrated," "grafted," and "embedded" are used interchangeably herein. In the context of the present invention, these terms refer to the generation of a hybrid polynucleic acid or hybrid polypeptide by the introduction of a heterologous sequence into the original sequence of an antibody or antibody fragment. This incorporation can be performed in any manner. Typically, an antibody or fragment thereof comprising a natriuretic peptide flanked by N-terminal and C-terminal linker sequences is produced by recombinant DNA technology and expressed as described herein.

[0037] Incorporation of a natriuretic peptide flanked by N-terminal and C-terminal linker sequences into the CDR region of the original antibody or antibody fragment sequence may result in the deletion of at least a portion of the CDR region. For example, cloning of a nucleic acid sequence encoding the heterologous amino acid sequence comprising the N-terminal linker sequence, the natriuretic peptide, and the C-terminal linker sequence may be performed such that a portion of the CDR encoding sequence is replaced by the incorporated heterologous nucleic acid sequence. In specific other embodiments, the inclusion of a heterologous amino acid sequence comprising a natriuretic peptide does not result in the deletion of amino acid residues in the CDR region into which the heterologous amino acid sequence is inserted.

[0038] In the context of the present invention, the term "heterologous amino acid sequence" refers to an amino acid sequence that is not derived from an initial "empty" antibody or fragment thereof, into which the amino acid sequence is incorporated. A heterologous amino acid sequence is grafted into an antibody or fragment thereof, thereby generating a recombinant antibody molecule comprising an amino acid sequence from a different source.

[0039] The term "natriuretic peptide" refers to a peptide that can induce sodium hypernatriuresis (i.e., sodium excretion by the kidney). Natriuretic peptides include atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), Dendroaspis natriuretic peptide (DNP) and urodil. Natriuretic peptides within the meaning of the present invention may be of any origin. Natriuretic peptides include natural natriuretic peptides (such as wild-type natriuretic peptides) and mutant forms thereof, as well as homologous natriuretic peptides of different species. However, the term also includes modified natriuretic peptides, such as modified chimeric variants of different natriuretic peptides. It is known that the use of codons is different between species. Therefore, when expressing heterologous proteins in target cells, it may be necessary or at least beneficial to transform nucleic acid sequences to adapt to the codon use of target cells. Methods for designing and constructing derivatives of a given protein are well known to any person of ordinary skill in the art.

[0040] In a specific embodiment, the natriuretic peptide is selected from the wild-type natriuretic peptide of any species and the functional variant of any such wild-type natriuretic peptide. In the context of the present invention, the term "functional variant of a natriuretic peptide" or "functional natriuretic peptide variant" refers to natriuretic peptides of any origin, including natural peptides and modified peptides, which differ in amino acid sequence and / or nucleic acid sequence encoding the amino acid sequence of a given natriuretic peptide (e.g., a wild-type natriuretic peptide of a given species), but still have functional activity. In the context of the present invention, the term "functional activity" refers to the ability of a natriuretic peptide variant to perform the biological function of a naturally occurring natriuretic peptide (particularly a wild-type natriuretic peptide). Specifically, "functional activity" means that the natriuretic peptide variant is able to bind to its respective receptors. In the case of NPR-A and NPR-B ligands, "functional activity" specifically means the ability to mediate an increase in (intracellular) cyclic guanosine monophosphate (cGMP) by binding to one or both of these receptors.

[0041] In specific embodiments, the functional natriuretic peptide variant is able to perform at least about 50%, specifically at least about 60%, at least about 70%, at least about 80%, and most specifically at least about 90% of one or more biological functions of a given natriuretic peptide (e.g., a wild-type natriuretic peptide of any given species), wherein the one or more biological functions include but are not limited to binding of the natriuretic peptide to its respective receptor and / or inducing an increase in intracellular cGMP.

[0042] The functional activity of the natriuretic peptide can be measured by any method (including in vitro methods) that makes it possible to measure an increase in (intracellular) cyclic guanosine monophosphate (cGMP) or to measure changes in cGMP-regulated cellular processes, including the methods described in Examples 3 and 5. In a specific embodiment, if the EC of the (non-grafted) natriuretic peptide variant determined by the fluorometric assay described in Example 3 is 50A value below 500 nM, more specifically below 250 nM, more specifically below 150 nM, more specifically below 100 nM, more specifically below 50 nM, most specifically below 25 nM, then the (non-grafted) natriuretic peptide variant is considered to be functionally active.

[0043] This functional natriuretic peptide variant is incorporated into a CDR region of an immunoglobulin molecule or fragment thereof as described herein, producing a natriuretic peptide grafted immunoglobulin having natriuretic peptide functional activity, and the stability of the natriuretic peptide grafted immunoglobulin in serum is significantly increased compared to the non-grafted functional natriuretic peptide variant, as shown in the examples. If the natriuretic peptide grafted immunoglobulin gives a significant positive signal in any method that directly or indirectly measures the increase of (intracellular) cyclic guanosine monophosphate (cGMP) by evaluating changes in cGMP-regulated cellular processes, the natriuretic peptide grafted immunoglobulin is considered to be biologically active (i.e., functional). Specifically, the functional activity of the natriuretic peptide grafted immunoglobulin can be evaluated by the methods described in Examples 3 and 5. In the case of natriuretic peptide grafted immunoglobulins, significance is typically assessed based on: i) comparison with negative samples, such as an empty immunoglobulin scaffold, e.g., construct #209 (antibody comprising SEQ ID NO 65 and SEQ ID NO 66, TPP-5657), ii) comparison with positive samples, e.g., construct #117 (antibody comprising SEQ ID NO 67 and SEQ ID NO 66, TPP-5661), and iii) dose dependency.

[0044] Although the functional natriuretic peptide variants of the present invention may contain any number of mutations comprising additions, deletions and / or substitutions of one or more amino acids compared to the reference natriuretic peptide, the functional natriuretic peptide variants will generally retain the key features of the corresponding natriuretic peptide, such as key residues within the central loop domain. For example, the conservative residues of natriuretic peptides are described in Lincoln R. Potter et al. (HandbExp Pharmacol. 2009; (191): 341-366). Therefore, in a specific embodiment, the functional natriuretic peptide variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity with the sequence shown below:

[0045] X1CFGX2X3X4DRIX5X6X7SX8LGC

[0046] Where X1 and X5 are G or S;

[0047] X3 is R or K;

[0048] X6 is A or S; and

[0049] X2, X4, X7 and X8 can be any amino acid.

[0050] In principle, any type of natriuretic peptide can be incorporated into the CDR region of an immunoglobulin or fragment thereof as described herein. Specifically, the inventors have found that the minimum requirements for satisfactory biological activity of the grafted natriuretic peptide and the discovery of particularly suitable N-terminal and C-terminal amino acid sequences in one type of natriuretic peptide can be imparted to other types of natriuretic peptides. Without wishing to be bound by theory, it is speculated that these similar requirements for successfully embedding natriuretic peptides into immunoglobulin molecules in different natriuretic peptide types may be due to structural similarities and / or mechanisms of action in the natriuretic peptide family.

[0051] In a specific embodiment, the natriuretic peptide is selected from the group consisting of human ANP having the sequence SEQ ID NO 23, human BNP having the sequence SEQ ID NO 24, human CNP having the sequence SEQ ID NO 25, and peptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 98% sequence identity with any one of SEQ ID NOs 23 to 25. Similarly, the natriuretic peptides having sequences that deviate from the wild-type human natriuretic peptides ANP, BNP and CNP may have any natural origin, such as mutant versions of wild-type human natriuretic peptides or homologues of different species or modified natriuretic peptides. Methods for designing and constructing peptide variants are known to any one of ordinary skill in the art.

[0052] In specific such embodiments, a natriuretic peptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 98% sequence identity to any one of SEQ ID NOs 23 to 25 is a functional natriuretic peptide variant.

[0053] For a reference polynucleotide or polypeptide sequence, "percent (%) sequence identity" is defined as the percentage of nucleic acid or amino acid residues in a candidate sequence that are identical to nucleic acid or amino acid residues in a reference polynucleotide or polypeptide sequence, respectively, after alignment of the sequences and optionally introducing gaps (if necessary) to achieve maximum percentage sequence identity. Conservative substitutions are not considered part of sequence identity. In a specific embodiment, the total amount of any gaps introduced in a candidate sequence and / or reference sequence does not exceed 50%, 40%, 30%, 25%, 20%, 15% or 10% of the total amount of residues in the reference sequence. In a specific embodiment, the percentage sequence identity is determined without introducing any gaps into the candidate sequence or reference sequence (i.e., using a gapless alignment). Alignment for determining percentage amino acid sequence identity can be achieved in various ways that are fully understood by those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequence.

[0054] A natriuretic peptide having a given percentage of sequence identity with a given reference natriuretic peptide (e.g., human BNP having an amino acid sequence of SEQ ID NO 24) may contain one or more mutations including addition, deletion and / or substitution of one or more amino acids compared to the reference natriuretic peptide. According to the teachings of the present invention, the amino acids deleted, added and / or substituted may be continuous amino acids, or may be dispersed over the length of the amino acid sequence of a natriuretic peptide having a given percentage of sequence identity with a reference natriuretic peptide (e.g., human BNP having an amino acid sequence of SEQ ID NO 24). At the DNA level, a nucleic acid sequence encoding a natriuretic peptide having a given percentage of sequence identity with a given reference natriuretic peptide may differ to a greater extent due to the degeneracy of the genetic code.

[0055] According to the teachings of the present invention, any number of amino acids may be added, deleted and / or substituted as long as the specified amino acid sequence identity with the reference natriuretic peptide is maintained. In a specific embodiment, the specified amino acid sequence identity is maintained, and the natriuretic peptide variant has biological activity, i.e., a functional natriuretic peptide variant. Preferably, the reduction in the biological activity of a natriuretic peptide having a given sequence identity percentage with a given reference natriuretic peptide (e.g., human BNP with the amino acid sequence of SEQ ID NO 24) is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 25% or less than 10 compared to the reference natriuretic peptide measured in the above-mentioned assay.

[0056] The term "antibody" as used herein means an immunoglobulin molecule, in particular a dimeric immunoglobulin molecule consisting of four polypeptide chains: two heavy (H) chains and two light (L) chains, which are usually interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (referred to herein as VH) and a heavy chain constant region. The heavy chain constant region may include, for example, three domains CH1, CH2, and CH3. Each light chain consists of a light chain variable region (referred to herein as VL) and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into highly variable regions, called complementarity determining regions (CDRs), with more conservative regions, called framework regions (FRs) in between. Each VH and VL typically includes three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus, for example in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0057] As used herein, the term "complementarity determining region" (CDR; e.g., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain whose presence is necessary for antigen binding. Each variable domain typically has three CDR regions, identified as CDR1, CDR2, and CDR3. Each complementary determining region may include amino acid residues in the "complementarity determining region" defined by Kabat (e.g., about residues 24-34 (CDRL1), 50-56 (CDRL2), and 89-97 (CDRL3) in the light chain variable domain, and 31-35 (CDRH1), 50-65 (CDRH2), and 95-102 (CDRH3) in the heavy chain variable domain; (Kabat et al., Sequences of Proteins of Immulological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues from a "hypervariable loop" (e.g., about residues 26-32 (CDRL1), 50-52 (CDRL2), and 91-96 (CDRL3) in the light chain variable domain, residues 26-32 (CDRH1), 53-55 (CDRH2), and 96-101 (CDRH3) in the heavy chain variable domain (Chothia and Lesk; J Mol Biol 196:901-917 (1987)). In certain cases, the complementarity determining regions may include amino acids from both CDR regions and hypervariable loops according to the Kabat definition.

[0058] According to the amino acid sequence of its heavy chain constant domain, complete antibodies can be assigned to different "classes". There are five major classes of complete antibodies: IgA, IgD, IgE, IgG and IgM, several of which can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. In the context of the present invention, the term "antibody" includes any main class of immunoglobulin molecules (including IgG, IgE, IgM, IgD, IgA and IgY) and any subclass of immunoglobulin molecules (including IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) isolated from nature or prepared by recombinant methods, and includes all conventionally known antibodies. A preferred class of immunoglobulins used in the present invention is IgG. The term "antibody" also extends to other protein scaffolds that are capable of orienting antibody CDR inserts into an active binding conformation that is identical to that present in a native antibody, such that the observed binding of the target antigen to these chimeric proteins remains unchanged compared to the binding activity of the native antibody from which the CDRs were derived.

[0059] In the context of the present invention, the term "fragment" of an antibody / immunoglobulin refers to any portion of an antibody / immunoglobulin that contains at least one CDR region. In particular, the antibody fragments of the present invention retain the ability to increase the serum half-life of a biologically active peptide (preferably a natriuretic peptide) incorporated therein. Antibody fragments of the present invention include Fab, Fab', Fab'-SH, F(ab')2 and Fv fragments; diabodies; single domain antibodies (Dab); linear antibodies; single-chain antibody molecules (scFv); and disulfide-stabilized Fv antibody fragments (dsFv); as well as multispecific antibodies formed by antibody fragments and fragments containing VL or VH domains, which are prepared from intact immunoglobulins or by recombinant methods.

[0060] F(ab')2 or Fab can be modified to minimize or completely remove the intermolecular disulfide bond interactions between the CH1 and CL domains. The antibody fragments of the present invention can include the variable region alone or include the variable region and all or part of the following: hinge region, CH1, CH2, CH3 and CL domains. Also included are antibody fragments that include any combination of the variable region and hinge region, CH1, CH2, CH3 and CL domains.

[0061] The antibody or fragment thereof constitutes a scaffold that confers stability to the natriuretic peptide incorporated therein. For example, the serum half-life of a natriuretic peptide incorporated into the CDR region of an antibody described herein may be increased compared to a naturally occurring natriuretic peptide.

[0062] Mainly, heterologous amino acid sequences comprising natriuretic peptides can be incorporated into any immunoglobulin molecule or fragment thereof. Specifically, any species (including but not limited to humans, cattle, mice, rats, pigs, dogs, sharks, llamas (lama) and camels) and any main class and subclass immunoglobulins can be used according to the present invention. However, for therapeutic use, people or humanized antibodies may be preferred. In the context of the present invention, the term "human antibody" refers to antibodies with human immunoglobulin amino acid sequences, including antibodies separated from human immunoglobulin libraries, separated from human B cells or separated from animals of transgenic one or more human immunoglobulins, and synthetic human antibodies. In a specific embodiment, the amino acid light chain and heavy chain sequences of the variable domains are derived from human germline sequences LV 1-40 and HV 3-23, respectively (for more information, see Example 1).

[0063] In the context of the present invention, the term "humanized antibody" or "humanized antibody fragment" refers to (i) an antibody or fragment thereof derived from a non-human source (e.g., a transgenic mouse carrying a heterologous immune system), which antibody is based on human germline sequences; or (ii) a chimeric antibody or fragment thereof, in which the variable domain is derived from a non-human source and the constant domain is derived from a human source, or (iii) a CDR-grafted antibody or fragment thereof, in which the CDRs of the variable domain are from a non-human source, while one or more frameworks of the variable domain are of human origin and the constant domain (if any) is of human origin.

[0064] The antibodies or fragments thereof of the invention may be monospecific, bispecific, trispecific or of greater multispecificity.

[0065] In the context of the present invention, the term "comprises" or "comprising" means "including but not limited to". The term is intended to be open-ended to specify the presence of any claimed features, elements, integers, steps or components, but does not preclude the presence or addition of one or more other features, elements, integers, steps, components or groups thereof. Therefore, the term "comprising" encompasses the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" used throughout the application and particularly in the claims may be replaced with the term "consisting of".

[0066] In the context of the present invention, the term "approximately" or "about" means between 80% and 120%, or between 90% and 110%, including between 95% and 105%, of a given value.

[0067] In the antibodies or fragments thereof of the present invention, a) at least 12 amino acid residues are present in

[0068] i) in case a heterologous amino acid sequence is incorporated into CDRH1, between the amino acid residue HC res25 according to Kabat (which in the heavy chain having the amino acid sequence SEQ ID NO 65 corresponds to res S25) and the first amino acid residue of the natriuretic peptide;

[0069] ii) in case a heterologous amino acid sequence is incorporated into CDRH2, between amino acid residue HC res51 according to Kabat (which in the heavy chain having the amino acid sequence SEQ ID NO 65 corresponds to res I51) and the first amino acid residue of the natriuretic peptide;

[0070] iii) in case a heterologous amino acid sequence is incorporated into CDRH3, between amino acid residue HC res92 according to Kabat (which in the heavy chain having the amino acid sequence SEQ ID NO 65 corresponds to res C96) and the first amino acid residue of the natriuretic peptide;

[0071] iv) in case a heterologous amino acid sequence is incorporated into CDRL1, between the amino acid residue LC res26 according to Kabat (which in the light chain having the amino acid sequence SEQ ID NO 66 corresponds to res S25) and the first amino acid residue of the natriuretic peptide;

[0072] v) in case a heterologous amino acid sequence is incorporated into CDRL2, between the amino acid residue LC res49 according to Kabat (which in the light chain having the amino acid sequence SEQ ID NO 66 corresponds to res Y51) and the first amino acid residue of the natriuretic peptide; and / or

[0073] vi) in case a heterologous amino acid sequence is incorporated into CDRL3, between the amino acid residue LC res88 according to Kabat (which in the light chain having the amino acid sequence SEQ ID NO 66 corresponds to res C90) and the first amino acid residue of the natriuretic peptide;

[0074] and b) there are at least 9 amino acid residues between the last amino acid residue of the natriuretic peptide and

[0075] i) in case of incorporation of a heterologous amino acid sequence into CDRH1, amino acid residue HC res35a according to Kabat (in the heavy chain having the amino acid sequence SEQ ID NO 65, which corresponds to res M34);

[0076] ii) in case of incorporation of a heterologous amino acid sequence into CDRH2, amino acid residue HC res57 according to Kabat (in the heavy chain having the amino acid sequence SEQ ID NO 65, which corresponds to res T58);

[0077] iii) in case of incorporation of a heterologous amino acid sequence into CDRH3, amino acid residue HCres106 according to Kabat (which corresponds to res G111 in the heavy chain having the amino acid sequence SEQ ID NO 65);

[0078] iv) in case of incorporation of a heterologous amino acid sequence in CDRL1, amino acid residue LC res 32 according to Kabat (in the light chain having the amino acid sequence SEQ ID NO 66, which corresponds to res D34);

[0079] v) amino acid residue LCres57 according to Kabat (which corresponds to resG59 in the light chain having the amino acid sequence SEQ ID NO 66) in case of inclusion of a heterologous amino acid sequence in CDRL2; and / or

[0080] vi) In case of inclusion of a heterologous amino acid sequence in CDRL3, the amino acid residue LC res98 according to Kabat (in the light chain having the amino acid sequence SEQ ID NO 66, which corresponds to res F102).

[0081] The naming of the amino acid residues listed above refers to the amino acid position in the original immunoglobulin molecule before the inclusion of the heterologous amino acid sequence. In the context of the present invention, the amino acid residues listed above are referred to as "reference amino acids" or "reference aa". These reference amino acid residues are located at or near the CDR framework connection, but do not necessarily correspond to the standard CDR boundary definition (standard CDR boundary definition is amino acid residues S25 and W36 of CDRH1; S49 and R67 of CDRH2; K98 and W108 of CDRH3; C22 and W37 of CDRL1; Y51 and G59 of CDRL2; C90 and F102 of CDRL3. Jarasch and Skerra, Proteins 2017 Jan; 85 (1): 65-71).

[0082] The N-terminal nearest reference aa of the inserted natriuretic peptide plus the amino acid stretch between the reference aa and the first amino acid residue of the inserted natriuretic peptide is referred to herein as the "N-terminal sequence". The N-terminal sequence comprises Ntls. In a specific embodiment, the N-terminal sequence consists of Ntls plus the adjacent N-terminal reference aa.

[0083] The amino acid stretch between the last amino acid residue of the inserted natriuretic peptide and the reference aa of the C-terminus of the inserted natriuretic peptide, plus the reference aa, is referred to herein as the "C-terminal sequence". The C-terminal sequence comprises Ctls. In a specific embodiment, the C-terminal sequence consists of Ctls plus the adjacent C-terminal reference aa.

[0084] In a specific embodiment, Ntls comprises a GS linker sequence; a PN linker sequence; an amino acid sequence that is part of a human IgG antibody scaffold, or a sequence having at least 80% sequence identity therewith, in particular an amino acid sequence that is part of a fab domain scaffold of a human IgG antibody, or a sequence having at least 80% sequence identity therewith, more particularly a sequence of any one of SEQ ID NOs 1, 2 or 4, or a sequence having at least 80% sequence identity therewith; a sequence of any one of SEQ ID NOs 6, 7, 9, 11, 13, 15, 16, 17, 19 or 21; or a sequence having at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity therewith. Ntls may also comprise any combination of the amino acid sequences listed above.

[0085] In specific such embodiments, Ntls comprises a GS linker sequence; a PN linker sequence; an amino acid sequence that is part of a human IgG antibody scaffold, or a sequence having at least 80% sequence identity therewith, in particular an amino acid sequence that is part of a fab domain scaffold of a human IgG antibody, or a sequence having at least 80% sequence identity therewith, more particularly a sequence of any one of SEQ ID NOs 1, 2 or 4, or a sequence having at least 80% sequence identity therewith; a sequence of any one of SEQ ID NOs 6, 7, 9, 11, 13, 15 or 21; a sequence having at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity therewith; or any combination thereof.

[0086] In a specific embodiment, Ctls comprises a GS linker sequence; a PN linker sequence; an amino acid sequence that is part of a human IgG antibody scaffold, or a sequence having at least 80% sequence identity therewith, in particular an amino acid sequence that is part of a fab domain scaffold of a human IgG antibody, or a sequence having at least 80% sequence identity therewith, more particularly a sequence of any one of SEQ ID NOs 1, 3 or 5, or a sequence having at least 80% sequence identity therewith; a sequence of any one of SEQ ID NOs 6, 8, 10, 12, 14, 15, 17, 18, 19, 20 or 22; or a sequence having at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity therewith. Ctls may also comprise any combination of the amino acid sequences listed above.

[0087] In a specific embodiment, Ctls comprises a GS linker sequence; a PN linker sequence; an amino acid sequence that is part of a human IgG antibody scaffold, or a sequence having at least 80% sequence identity therewith, in particular an amino acid sequence that is part of a fab domain scaffold of a human IgG antibody, or a sequence having at least 80% sequence identity therewith, more particularly a sequence of any one of SEQ ID NOs 1, 3 or 5, or a sequence having at least 80% sequence identity therewith; a sequence of any one of SEQ ID NOs 6, 8, 10, 12, 14, 15, 20 or 22; a sequence having at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity therewith; or any combination thereof.

[0088] In a specific embodiment, the sequence contained in Ntls and / or Ctls has a sequence identity of at least 60%, specifically at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% with any one of SEQ ID NOs 1 to 22.

[0089] In the context of the present invention, the term "GS linker sequence" refers to a peptide linker that mainly comprises glycine and serine residues. Specifically, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the amino acid residues of the GS linker sequence of the present invention are selected from glycine and serine residues. The GS linker sequence of the present invention may, for example, comprise 1 to 30 amino acid residues in total. Specifically, the GS linker sequence of the present invention does not comprise more than 3, 2 or 1 amino acid residues that are not glycine or serine.

[0090] In the context of the present invention, the term "PN linker sequence" refers to a peptide linker that mainly comprises proline and asparagine residues. Specifically, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the amino acid residues of the PN linker sequence of the present invention are selected from proline and asparagine residues. The PN linker sequence of the present invention can, for example, comprise 1 to 30 amino acid residues in total. Specifically, the PN linker sequence of the present invention does not comprise more than 3, 2 or 1 amino acid residues that are not proline or asparagine. Other amino acid residues that may be present in the PN linker sequence of the present invention are, for example, lysine or glutamic acid residues.

[0091] In a specific embodiment, the linker sequence contained in Ntls and / or Ctls and selected from the following comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues: GS linker sequence; PN linker sequence; human IgG antibody scaffold linker sequence; human IgG fab domain scaffold sequence; a sequence having at least 80% sequence identity with a human IgG antibody scaffold linker sequence, a human IgG fab domain scaffold sequence or a sequence of any one of SEQ ID NOs 1 to 5; a sequence having at least 60% sequence identity with any one of SEQ ID NOs 6 to 22. The linker sequence contained in Ntls and / or Ctls may, for example, comprise 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.

[0092] In the case of a linker comprising a sequence of a human IgG antibody scaffold or a sequence having at least 80% sequence identity therewith, it may be particularly advantageous to use a sequence of a scaffold region adjacent to a CDR incorporating a heterologous amino acid sequence. For example, in the case of a heterologous amino acid sequence incorporating a CDRH2 domain, Ntls and / or Ctls may comprise a linker comprising an amino acid sequence that is a part of a framework region FRH2 or FRH3. Similarly, in the case of a heterologous amino acid sequence incorporating a CDRL2 region, Ntls and / or Ctls may comprise a linker comprising an amino acid sequence that is a part of a framework region FRH2 or FRH3.

[0093] In a specific embodiment, Ntls consists of the following: a GS linker sequence; a PN linker sequence; an amino acid sequence that is part of a human IgG antibody scaffold, or a sequence having at least 80% sequence identity therewith, in particular an amino acid sequence that is part of a fab domain scaffold of a human IgG antibody, or a sequence having at least 80% sequence identity therewith, more particularly a sequence of any one of SEQ ID NOs 1, 2 or 4, or a sequence having at least 80% sequence identity therewith; a sequence of any one of SEQ ID NOs 6, 7, 9, 11, 13, 15, 16, 17, 19 or 21; a sequence having at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity therewith; or any combination thereof.

[0094] In a specific embodiment, Ctls consists of the following: a GS linker sequence; a PN linker sequence; an amino acid sequence that is part of a human IgG antibody scaffold, or a sequence having at least 80% sequence identity therewith, in particular an amino acid sequence that is part of a fab domain scaffold of a human IgG antibody, or a sequence having at least 80% sequence identity therewith, more particularly a sequence of any one of SEQ ID NOs 1, 3 or 5, or a sequence having at least 80% sequence identity therewith; a sequence of any one of SEQ ID NOs 6, 8, 10, 12, 14, 15, 17, 18, 19, 20 or 22; a sequence having at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity therewith; or any combination thereof.

[0095] In a specific embodiment, both Ntls and Ctls comprise at least one of the above listed linker sequences or any combination thereof. In principle, any of the above listed Ntls linker sequences can be combined with any of the above listed Ctls linker sequences. In particular, any linker sequence can be combined with a GS linker. As a non-limiting example, a GS Ctls linker can be combined with an Ntls linker comprising a sequence of any one of SEQ ID NO 6, 9 or 15 or a sequence having at least 60% sequence identity with any one of SEQ ID NO 6, 9 or 15. Another non-limiting example is a GS Ntls linker combined with a Ctls linker comprising a sequence of SEQ ID NO 15 or a sequence having at least 60% sequence identity therewith.

[0096] In the case where the total length of the N-terminal and C-terminal flanking sequences is sufficient, the linker sequences listed above have been shown to be particularly advantageous for obtaining good natriuretic peptide activity. Without wishing to be bound by theory, it is believed that the connecting peptide segments listed above lead to such conformations / foldings that they contribute to a system state that is advantageous for presenting the biologically active natriuretic peptides to their respective receptors with minimal steric hindrance.

[0097] In a specific embodiment, i) Ntls comprises a GS linker sequence; a PN linker sequence; a sequence of SEQ ID NO 2, 4, 9, 11, 13 or 15; a sequence having at least 60% sequence identity with SEQ ID NO 2, 4, 9, 11, 13 or 15; or any combination thereof, and ii) Ctls comprises a GS linker sequence; a PN linker sequence; a sequence of SEQ ID NO 3, 5, 12, 14, 15 or 20; a sequence having at least 60% sequence identity with SEQ ID NO 3, 5, 12, 14, 15 or 20; or any combination thereof. These linker sequences have been shown to be particularly useful because they not only achieve high natriuretic peptide activity, but also have good expression levels in recombinant expression and are not prone to protein fragmentation during expression (see Table 9).

[0098] In a specific embodiment, Ntls and Ctls each comprise a GS linker sequence; Ntls and Ctls each comprise a PN linker sequence; Ntls and Ctls each comprise an amino acid sequence that is part of a human IgG antibody scaffold, or a sequence having at least 80% sequence identity therewith, specifically an amino acid sequence that is part of a fab domain scaffold of a human IgG antibody, or a sequence having at least 80% sequence identity therewith, more specifically Ntls comprises a sequence of any one of SEQ ID NOs 1, 2 or 4, or a sequence having at least 80% sequence identity therewith, Ctls comprises a sequence of any one of SEQ ID NOs 1, 3 or 5, or a sequence having at least 80% sequence identity therewith; Ntls and Ctls each comprise a sequence of SEQ ID NO 6, or a sequence having at least 60% sequence identity therewith; Ntls comprises a sequence of SEQ ID NO 7, or a sequence having at least 60% sequence identity therewith, Ctls comprises a sequence of SEQ ID NO 8, or a sequence having at least 60% sequence identity therewith; Ntls comprises a sequence of SEQ ID NO 9 or a sequence having at least 60% sequence identity thereto, Ctls comprises the sequence SEQ ID NO 10 or a sequence having at least 60% sequence identity thereto; Ntls comprises the sequence SEQ ID NO 11 or a sequence having at least 60% sequence identity thereto, Ctls comprises the sequence SEQ ID NO 12 or a sequence having at least 60% sequence identity thereto; Ntls comprises the sequence SEQ ID NO 13 or a sequence having at least 60% sequence identity thereto, Ctls comprises the sequence SEQ ID NO 14 or a sequence having at least 60% sequence identity thereto; Ntls and Ctls each comprise the sequence SEQ ID NO 15 or a sequence having at least 60% sequence identity thereto; Ntls comprises the sequence SEQ ID NO 16 or a sequence having at least 60% sequence identity thereto, Ctls comprises the sequence SEQ ID NO 17 or a sequence having at least 60% sequence identity thereto; Ntls comprises the sequence SEQ ID NO 17 or a sequence having at least 60% sequence identity thereto, Ctls comprises the sequence SEQ ID NO 18 or a sequence having at least 60% sequence identity thereto; Ntls and Ctls each comprise the sequence SEQ ID NO 19 or a sequence having at least 60% sequence identity thereto; Ntls comprises the sequence SEQ ID NO 9 or a sequence having at least 60% sequence identity thereto, and Ctls comprises the sequence SEQ ID NO 20 or a sequence having at least 60% sequence identity thereto;or Ntls comprises the sequence SEQ ID NO 21 or a sequence having at least 60% sequence identity thereto, and Ctls comprises the sequence SEQ ID NO 22 or a sequence having at least 60% sequence identity thereto. ;

[0099] In specific embodiments of this type, Ntls and Ctls each comprise a GS linker sequence; Ntls and Ctls each comprise a PN linker sequence; Ntls and Ctls each comprise an amino acid sequence that is part of a human IgG antibody scaffold or a sequence having at least 80% sequence identity therewith, specifically an amino acid sequence that is part of a fab domain scaffold of a human IgG antibody or a sequence having at least 80% sequence identity therewith, more specifically Ntls comprises a sequence of any one of SEQ ID NOs 1, 2 or 4 or a sequence having at least 80% sequence identity therewith, Ctls comprises a sequence of any one of SEQ ID NOs 1, 3 or 5 or a sequence having at least 80% sequence identity therewith; Ntls and Ctls each comprise a sequence of SEQ ID NO 6 or a sequence having at least 60% sequence identity therewith; Ntls comprises a sequence of SEQ ID NO 7 or a sequence having at least 60% sequence identity therewith, Ctls comprises a sequence of SEQ ID NO 8 or a sequence having at least 60% sequence identity therewith; Ntls comprises a sequence of SEQ ID NO 9 or a sequence having at least 60% sequence identity thereto, Ctls comprises the sequence SEQ ID NO 10 or a sequence having at least 60% sequence identity thereto; Ntls comprises the sequence SEQ ID NO 11 or a sequence having at least 60% sequence identity thereto, Ctls comprises the sequence SEQ ID NO 12 or a sequence having at least 60% sequence identity thereto; Ntls comprises the sequence SEQ ID NO 13 or a sequence having at least 60% sequence identity thereto, Ctls comprises the sequence SEQ ID NO 14 or a sequence having at least 60% sequence identity thereto; Ntls and Ctls each comprise the sequence SEQ ID NO 15 or a sequence having at least 60% sequence identity thereto; Ntls comprises the sequence SEQ ID NO 9 or a sequence having at least 60% sequence identity thereto, Ctls comprises the sequence SEQ ID NO 20 or a sequence having at least 60% sequence identity thereto; or Ntls comprises the sequence SEQ ID NO 21 or a sequence having at least 60% sequence identity thereto, Ctls comprises the sequence SEQ ID NO 22 or a sequence having at least 60% sequence identity thereto.

[0100] In specific embodiments of this type, Ntls and Ctls each comprise a GS linker sequence; Ntls and Ctls each comprise a PN linker sequence; Ntls comprises the sequence SEQ ID NO 2 or a sequence having at least 80% sequence identity thereto, and Ctls comprises the sequence SEQ ID NO 3 or a sequence having at least 80% sequence identity thereto; Ntls comprises the sequence SEQ ID NO 4 or a sequence having at least 80% sequence identity thereto, and Ctls comprises the sequence SEQ ID NO 5 or a sequence having at least 80% sequence identity thereto; Ntls comprises the sequence SEQ ID NO 11 or a sequence having at least 60% sequence identity thereto, and Ctls comprises the sequence SEQ ID NO 12 or a sequence having at least 60% sequence identity thereto; Ntls comprises the sequence SEQ ID NO 13 or a sequence having at least 60% sequence identity thereto, and Ctls comprises the sequence SEQ ID NO 14 or a sequence having at least 60% sequence identity thereto; Ntls and Ctls each comprise the sequence SEQ ID NO 15 or a sequence having at least 60% sequence identity thereto; or Ntls comprises the sequence SEQ ID NO 9 or a sequence having at least 60% sequence identity thereto, Ctls comprises the sequence SEQ ID NO 20 or a sequence having at least 60% sequence identity thereto. These linker combinations have been shown to be particularly useful for obtaining high natriuretic peptide activity, good expression levels in recombinant expression, and minimal or no protein fragmentation, as shown in Table 9.

[0101] In a specific embodiment, Ntls further comprises an anchoring element A1 at its C-terminal end and / or Ctls further comprises an anchoring element A2 at its N-terminal end, wherein A1 and A2 mainly comprise glycine and serine residues. In a specific embodiment, A1 and / or A2 comprise at least 1, 2, 3, 4 or 5 amino acid residues. A1 and / or A2 may comprise up to 10, 9, 8, 7, 6 or 5 amino acid residues in total. In a specific embodiment, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the amino acid residues of A1 and / or A2 are selected from glycine and serine residues. Specifically, A1 and / or A2 do not comprise more than 3, 2 or 1 amino acid residues that are not glycine or serine.

[0102] In a specific embodiment, Ntls consists of: i) an anchoring element A1 at its C-terminus and ii) a GS linker sequence; a PN linker sequence; an amino acid sequence that is part of a human IgG antibody scaffold or a sequence having at least 80% sequence identity therewith, in particular an amino acid sequence that is part of a fab domain scaffold of a human IgG antibody or a sequence having at least 80% sequence identity therewith, more particularly a sequence of any one of SEQ ID NOs 1, 2 or 4 or a sequence having at least 80% sequence identity therewith; a sequence of any one of SEQ ID NOs 6, 7, 9, 11, 13, 15, 16, 17, 19 or 21; a sequence having at least 60% sequence identity therewith; or any combination thereof.

[0103] In a specific embodiment, Ctls consists of: i) an anchoring element A2 at its N-terminus and ii) a GS linker sequence; a PN linker sequence; an amino acid sequence that is part of a human IgG antibody scaffold or a sequence having at least 80% sequence identity therewith, in particular an amino acid sequence that is part of a fab domain scaffold of a human IgG antibody or a sequence having at least 80% sequence identity therewith, more particularly a sequence of any one of SEQ ID NOs 1, 3 or 5 or a sequence having at least 80% sequence identity therewith; a sequence of any one of SEQ ID NOs 6, 8, 10, 12, 14, 15, 17, 18, 19, 20 or 22; a sequence having at least 60% sequence identity therewith; or any combination thereof.

[0104] In specific embodiments, Ntls and / or Ctls comprise at least 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues in total. Ntls and / or Ctls may, for example, comprise 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.

[0105] In a specific embodiment, the stretch of amino acids present in comprises the sequence of any one of SEQ ID NOs 26 to 38 or a sequence having at least 80%, 85%, 90%, 95% or at least 98% sequence identity to any one of SEQ ID NOs 26 to 38

[0106] i) in case said heterologous amino acid sequence is incorporated into CDRH1, between amino acid residue HCres25 according to Kabat and the first amino acid residue of the natriuretic peptide;

[0107] ii) between amino acid residue HCres51 according to Kabat and the first amino acid residue of the natriuretic peptide in case said heterologous amino acid sequence is incorporated into CDRH2;

[0108] iii) in case said heterologous amino acid sequence is incorporated into CDRH3, between amino acid residue HCres92 according to Kabat and the first amino acid residue of the natriuretic peptide;

[0109] iv) between amino acid residue LCres26 according to Kabat and the first amino acid residue of the natriuretic peptide in case said heterologous amino acid sequence is incorporated into CDRL1;

[0110] v) between amino acid residue LCres49 according to Kabat and the first amino acid residue of the natriuretic peptide in case said heterologous amino acid sequence is included in CDRL2; and / or

[0111] vi) in case said heterologous amino acid sequence is included in CDRL3, between amino acid residue LCres88 according to Kabat and the first amino acid residue of the natriuretic peptide.

[0112] In a specific embodiment, the stretch of amino acids between the last amino acid residue of the natriuretic peptide and the sequence of any one of SEQ ID NOs 39 to 51 or a sequence having at least 80%, 85%, 90%, 95% or at least 98% sequence identity with any one of SEQ ID NOs 39 to 51

[0113] i) amino acid residue HCres35a according to Kabat, in case said heterologous amino acid sequence is incorporated into CDRH1;

[0114] ii) amino acid residue HCres57 according to Kabat, in case said heterologous amino acid sequence is incorporated into CDRH2;

[0115] iii) amino acid residue HCres106 according to Kabat in case said heterologous amino acid sequence is incorporated into CDRH3;

[0116] iv) amino acid residue LCres 32 according to Kabat in case said heterologous amino acid sequence is incorporated into CDRL1;

[0117] v) amino acid residue LCres57 according to Kabat, in case said heterologous amino acid sequence is included in CDRL2; and / or

[0118] vi) amino acid residue LCres98 according to Kabat in case said heterologous amino acid sequence is incorporated into CDRL3.

[0119] In a specific embodiment, the heterologous amino acid sequence consists of Ntls, natriuretic peptides, and Ctls.

[0120] In a specific embodiment, the stretch of amino acids present in comprises the sequence of any one of SEQ ID NOs 52 to 64, or a sequence having at least 80%, 85%, 90%, 95% or at least 98% sequence identity to any one of SEQ ID NOs 52 to 64.

[0121] i) between amino acid residue HCres25 according to Kabat and amino acid residue HCres35a according to Kabat, in case said heterologous amino acid sequence is included in CDRH1;

[0122] ii) between amino acid residue HCres51 according to Kabat and amino acid residue HCres57 according to Kabat, in case said heterologous amino acid sequence is included in CDRH2;

[0123] iii) between amino acid residue HCres92 according to Kabat and amino acid residue HCres106 according to Kabat, in case said heterologous amino acid sequence is incorporated into CDRH3;

[0124] iv) between amino acid residue LCres26 according to Kabat and amino acid residue LCres 32 according to Kabat, in case said heterologous amino acid sequence is included in CDRL1;

[0125] v) between amino acid residue LCres49 according to Kabat and amino acid residue LCres57 according to Kabat, in case said heterologous amino acid sequence is included in CDRL2; and / or

[0126] vi) between amino acid residue LCres88 according to Kabat and amino acid residue LCres98 according to Kabat, in case said heterologous amino acid sequence is included in CDRL3.

[0127] In a specific embodiment, the antibody or its fragment comprises at least two natriuretic peptides. In a specific embodiment, both natriuretic peptides are contained in a heterologous amino acid sequence that also comprises Ntls and Ctls, and are incorporated into the CDR region of the antibody or its fragment as described herein. At least two natriuretic peptides can be incorporated into two corresponding CDR regions of two light chains or two heavy chains, or at least two natriuretic peptides can be incorporated into two separate CDR regions. At least two natriuretic peptides can be the same or different. In a specific embodiment of this type, the antibody or its fragment comprises at least two different natriuretic peptides, which are incorporated into at least two CDR regions.

[0128] Due to the dimeric structure of antibody molecules, a nucleic acid sequence encoding heterologous amino acid sequence (Ntls-natriuretic peptide-Ctls) is inserted into the nucleic acid of the light chain or heavy chain of the encoding immunoglobulin molecule, and usually an antibody protein carrying two natriuretic peptides located in the corresponding CDR region of two identical light chains or two identical heavy chains is produced. However, it is also contemplated that two nucleic acids encoding natriuretic peptides are inserted into two different CDR coding regions of the nucleic acid sequence encoding light chain and / or heavy chain, thereby producing an antibody molecule having four natriuretic peptides located in two corresponding CDR pairs of the dimeric antibody. Also included are dimeric immunoglobulin molecules whose light chain and / or heavy chain are different, such as dimeric antibodies carrying a single natriuretic peptide and dimeric antibodies carrying two different natriuretic peptides in two corresponding CDR regions of two light chains or two heavy chains.

[0129] In specific embodiments, the natriuretic peptide is inserted into CDRH1 and CDRH2, CDRH1 and CDRH3, CDRH2 and CDRH3, CDRH1 and CDRL1, CDRH1 and CDRL2, CDRH1 and CDRL3, CDRH2 and CDRL1, or CDRH2 and CDRL2. In specific embodiments, the antibody or fragment thereof comprises one ANP and one BNP molecule; one ANP and one CNP molecule; or one BNP and one CNP molecule.

[0130] In a specific embodiment, the natriuretic peptide contained in at least one heterologous amino acid sequence incorporated into at least one CDR region of the antibody or its fragment is BNP, and the antibody or its fragment contains at least one other natriuretic peptide. In a specific embodiment, the at least one other natriuretic peptide is also contained in a heterologous amino acid sequence, which also contains Ntls and Ctls and is incorporated into the CDR region of the antibody or its fragment. In a specific embodiment, BNP and at least one other natriuretic peptide are incorporated into two corresponding CDR regions of two light chains or two heavy chains of the antibody or its fragment. In other specific embodiments, BNP and at least one other natriuretic peptide are incorporated into at least two separate CDR regions. Specifically, the at least one other natriuretic peptide is selected from ANP, BNP and CNP, more specifically ANP and CNP.

[0131] The "empty" antibody molecule consisting of two heavy chains having the sequence SEQ ID NO 65 and two light chains having the sequence SEQ ID NO 66, which does not contain a heterologous amino acid sequence comprising a natriuretic peptide, is referred to as TPP-5657. In a specific embodiment, an antibody molecule consisting of two heavy chains having the sequence SEQ ID NO 65 or a sequence having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto and two light chains having the sequence SEQ ID NO 66 or a sequence having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto is used as the initial antibody or parent antibody, and a heterologous amino acid sequence comprising a natriuretic peptide is incorporated therein. In a specific embodiment of this type, the heterologous amino acid sequence comprising a natriuretic peptide is incorporated into the CDR region of one or both heavy chains of such an antibody molecule.

[0132] In specific such embodiments, the heavy chain comprising at least one heterologous amino acid sequence incorporated into at least one of its CDR regions has the sequence of any one of SEQ ID NOs 67 to 79, or a sequence having at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs 67 to 79. In specific embodiments, the antibodies of the invention are composed of two identical heavy chains having the sequence of any one of SEQ ID NOs 67 to 79, or a sequence having at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs 67 to 79, and two identical light chains having the sequence of SEQ ID NO 66, or a sequence having at least 80%, at least 85%, at least 90% or at least 95% sequence identity thereto.

[0133] In specific other embodiments, a heterologous amino acid sequence comprising a natriuretic peptide is incorporated into the CDR region of one or both light chains of such antibody molecules. In specific such embodiments, the light chain comprising at least one heterologous amino acid sequence incorporated into at least one of its CDR regions has a sequence of any one of SEQ ID NOs 80 or 81, or a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs 80 or 81. In specific embodiments, the antibodies of the invention are composed of two identical light chains having a sequence of any one of SEQ ID NOs 80 or 81, or a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs 80 or 81, and two identical heavy chains having a sequence of SEQ ID NO 65, or a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

[0134] The following table describes the heavy and light chain composition of exemplary antibodies of the invention.

[0135] Table 1: Exemplary natriuretic peptide grafted antibody constructs

[0136]

[0137]

[0138] The antibodies or fragments thereof of the present invention include naturally occurring purified products, products of chemical synthesis steps, and products produced by recombinant technology. Based on their source, the antibodies or fragments thereof of the present invention may be glycosylated or non-glycosylated.

[0139] For example, standard recombinant DNA methods can be used to prepare and / or obtain nucleic acids encoding heavy and light chains, incorporate these nucleic acids into expression vectors, and introduce the vectors into host cells for recombinant expression (see, e.g., Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989); Ausubel, FM et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989); Goeddel, Gene Expression Technology, Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990); and Boss et al., U.S. Pat. No. 4,816,397).

[0140] Therefore, in a second aspect, the present invention relates to nucleic acids or nucleic acid mixtures encoding antibodies of the present invention or fragments thereof. These nucleic acid sequences may be optimized for mammalian expression in some cases. The DNA molecules of the present invention are not limited to the sequences disclosed herein, but also include variants thereof.

[0141] The present invention also provides a recombinant nucleic acid construct comprising one or more nucleic acid sequences of the present invention. The recombinant nucleic acid construct of the present invention may, for example, comprise a nucleic acid vector, such as a plasmid, into which a nucleic acid molecule encoding an antibody of the present invention or a fragment thereof has been inserted. It should be understood that the design of the expression vector (including the selection of regulatory sequences) is influenced by factors such as the selection of host cells, the desired protein expression level, and whether constitutive expression or inducible expression is required.

[0142] The available expression vector used for bacterial use can be constructed by inserting one or more nucleotide sequences of the present invention, suitable translation initiation and termination signals together with functional promoters into the operable reading phase. Bacterial expression vectors generally include one or more phenotypic selection markers and replication origins to ensure the maintenance of the vector and (if necessary) to provide amplification in a bacterial host. The bacterial expression vector can include elements derived from commercially available plasmids, such as known cloning vector pBR322 (ATCC37017). According to the intended use of expressed antibody or its fragment, many bacterial expression vectors can be advantageously selected. For example, if a large amount of such antibodies are needed, it may be necessary to mediate the high-level expression vector of the antibody fusion protein that is easy to purify.

[0143] The recombinant nucleic acid constructs intended for antibody expression in eukaryotic host cells may include regulatory sequences capable of regulating the expression of the open reading frame in eukaryotic cells, preferably promoters and polyadenylation signals. Promoters and polyadenylation signals that are functional in the specific cell types intended for antibody expression are preferably selected. Examples of suitable promoters include, but are not limited to, promoters from cytomegalovirus (CMV) (e.g., the strong CMV immediate early promoter), Simian virus 40 (SV40), mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) (e.g., HIV long terminal repeat (LTR) promoter), Moloney virus, Epstein-Barr virus (EBV), adenovirus (e.g., adenovirus major late promoter (ADMLP)), polyoma and Rous sarcoma virus (RSV), synthetic CAG promoters consisting of the CMV early enhancer element, the promoter, first exon and first intron of the chicken β-actin gene, and the splice acceptor of the rabbit β-globin gene; and promoters from mammalian genes such as actin, myosin, hemoglobin, muscle creatine, and metallothionein. In a specific embodiment, the eukaryotic expression cassette comprises a CMV promoter. In the context of the present invention, the term "CMV promoter" refers to a strong immediate early cytomegalovirus promoter.

[0144] Examples of suitable polyadenylation signals include, but are not limited to, the bovine growth hormone (BGH) polyadenylation site, the SV40 polyadenylation signal, and the LTR polyadenylation signal.

[0145] In addition, the recombinant nucleic acid sequence may comprise one or more enhancer sequences. The enhancer may be, for example, an enhancer of mammalian actin, myosin, hemoglobin, muscle creatine, or a viral enhancer, such as an enhancer from CMV, RSV, SV40, or EBV. For further description of viral regulatory elements and sequences thereof, see US 5,168,062 of Stinski, US 4,510,245 of Bell et al., and US 4,968,615 of Schaffner et al.

[0146] Regulatory sequences and codons are usually species-dependent, so in order to maximize protein production, regulatory sequences and codons that are effective in the species / cell type expected to be used for antibody expression are preferably selected. Those skilled in the art can produce recombinant DNA molecules that are functional in a given test species.

[0147] Mammalian recombinant expression vectors may also contain an origin of replication and a selectable marker (see, e.g., US 4,399,216, US 4,634,665, and US 5,179,017). Suitable selectable markers include genes that confer resistance to drugs such as G418, puromycin, hygromycin, blasticidin, zeocin / bleomycin, or methotrexate, or selectable markers that utilize auxotrophic factors (e.g., glutamine synthetase) on the host cell into which the vector has been introduced. For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate, the new gene confers resistance to G418, the bsd gene from Aspergillus terreus confers resistance to blasticidin, puromycin N-acetyltransferase confers resistance to puromycin, the Sh ble gene product confers resistance to bleomycin, and the Escherichia coli hygromycin resistance gene (hyg or hph) confers resistance to hygromycin. Selectable markers (such as DHFR or glutamine synthetase) can also be combined with MTX and MSX for amplification techniques.

[0148] In some embodiments, the nucleic acid sequence encoding the heavy chain and light chain is inserted into a separate vector. In other embodiments, the nucleic acid sequence encoding the heavy chain and light chain is inserted into the same vector. In addition, the nucleic acid sequence encoding the variable region of the heavy chain and / or light chain can be converted to a nucleic acid sequence encoding, for example, a full-length antibody chain, a Fab fragment or a scFv. The DNA fragment encoding VL or VH can be operably connected (so that the amino acid sequence encoded by the two DNA fragments is in the framework) to another DNA fragment encoding, for example, an antibody constant region or a flexible joint. As an example, to create a polynucleotide sequence encoding an scFv, nucleic acids encoding VH and VL can be operably linked to another fragment encoding a flexible linker so that the VH and VL sequences can be expressed as a continuous single-chain protein with the VL and VH regions connected by a flexible linker (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554). The sequences of human heavy and light chain constant regions are known in the art (see, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification.

[0149] In a specific embodiment, the nucleic acid sequence encoding the heavy chain into which the heterologous amino acid sequence comprising a natriuretic peptide is incorporated comprises the sequence of any one of SEQ ID NOs 82 or 83, or a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs 82 or 83. In specific such embodiments, the nucleic acid sequence encoding the light chain comprises the sequence of SEQ ID NO 84, or a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

[0150] In a third aspect, the present invention relates to host cells comprising nucleic acid of the present invention or nucleic acid mixtures. In the context of the present invention, the terms "host cell", "host cell line" and "host cell culture" are used interchangeably, and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of these cells. Host cells include "transformants", "converted cells", "transfectants", "transfected cells" and "transduced cells", which include cells of primary conversion / transfection / transduction and progeny derived therefrom, without considering the number of passages. The nucleic acid content of progeny may not be identical to the parental cell, and the exogenous nucleic acid included may contain mutations. This paper includes mutant progeny with the same function or biological activity as the function or biological activity screened or selected in the original transformed cell.

[0151] Transfection of the expression vector into the host cell can be performed using standard techniques, such as electroporation, nucleofection, calcium phosphate precipitation, lipofection, polycation-based transfection such as polyethyleneimine (PEI)-based transfection and DEAE-dextran transfection.

[0152] Suitable host cells include prokaryotic cells and eukaryotic cells. Examples of prokaryotic host cells are, for example, bacteria, including but not limited to Escherichia coli, Bacillus subtilis, Salmonella typhimurium and Pseudomonas, Streptomyces and Staphylococcus.

[0153] Non-limiting examples of eukaryotic host cells include yeast, insects and insect cells, plants and plant cells, transgenic animals, and mammalian cells. Suitable mammalian host cells for antibody expression include Chinese hamster ovary (CHO cells), such as CHO-K1, CHO-S, CHO-K1SV (including dhfr-CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220 and Urlaub et al., Cell. 1983 Jun; 33 (2): 405-12, used with a DHFR selective marker, such as RJ Kaufman and PA Sharp (1982) Mol. Biol. 159:601-621; and as Fan et al., Biotechnol Bioeng. 2012 Apr; 109(4): 1007-15), NS0 myeloma cells, COS cells, HEK293 cells, HKB11 cells, BHK21 cells, CAP cells, EB66 cells and SP2 cells.

[0154] Expression can also be transient or semi-stable in an expression system, such as HEK293, HEK293T, HEK293-EBNA, HEK293E, HEK293-6E, HEK293-Freestyle, HKB11, Expi293F, 293EBNALT75, CHOFreestyle, CHO-S, CHO-K1, CHO-K1SV, CHOEBNALT85, CHOS-XE, CHO-3E7 or CAP-T cells (e.g., Durocher et al., Nucleic Acids Res. 2002 Jan 15; 30(2):E9).

[0155] In a fourth aspect, the present invention relates to a method for producing an antibody or a fragment thereof, comprising culturing the host cell of the present invention. Specifically, the host cell of the present invention is cultured under conditions suitable for the expression of the antibody or a fragment thereof.

[0156] Antibody expression can be constitutive or regulated (e.g., inducible). For inducible antibody expression, host cells of the invention are typically grown to an appropriate cell density, then de-repressed / induced by an appropriate method (e.g., temperature shift or chemical induction, such as adding or removing small molecule inducers, such as tetracyclines that bind to the Tet system), and the host cells are cultured for an additional period of time.

[0157] In a specific embodiment, the method for producing an antibody of the present invention or its fragment also includes the step of recovering the antibody or its fragment from the host cell culture. The cell can be, for example, collected by centrifugation, destroyed by physical or chemical methods, and the antibody or its fragment can be further purified from the resulting crude extract. In some embodiments, the expression vector is designed so that the expressed antibody or its fragment is secreted into the culture medium of the host cell where it grows. In this case, the antibody or its fragment can be directly recovered from the culture medium using standard protein purification methods.

[0158] In a specific embodiment, the method of the present invention also includes a step of purifying and recovering the antibody or its fragment. Specifically, the antibody is purified (1) to greater than 90%, as determined, for example, by analytical chromatography or by SDS-capillary gel electrophoresis (e.g., on a Caliper LabChip GXII, GX90 or Biorad bioanalyzer device), more specifically purified to produce an antibody homogeneity of at least about 92.5%, 95%, 98% or 99%; or, the antibody is purified (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, or (3) under reducing or non-reducing conditions using Coomassie blue or preferably silver staining, by SDS-PAGE to homogeneity.

[0159] The antibodies or fragments thereof of the present invention can be recovered and purified from recombinant cell cultures by known methods, including but not limited to ammonium sulfate or ethanol precipitation, acid extraction, protein A chromatography, protein G chromatography, molecular sieve chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification (see, e.g., Colligan, Current Protocols in Immunology or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10).

[0160] In a fifth aspect, the present invention relates to a composition comprising an antibody or fragment thereof of the present invention. Specifically, the composition of the present invention is a pharmaceutical composition suitable for use in a therapeutic method, wherein the amount of the antibody or fragment thereof of the present invention contained is effective to achieve the intended purpose, i.e., to prevent or treat a specific disease state.

[0161] The composition optionally also comprises at least one pharmaceutically acceptable excipient. In the context of the present invention, the term "excipient" refers to a natural or synthetic substance formulated with the active ingredient of the drug. Suitable excipients include antiadhesives, adhesives, coatings, disintegrants, spices, pigments, lubricants, glidants, sorbents, preservatives and sweeteners. Specific examples of pharmaceutically acceptable excipients include, but are not limited to, saline, buffered saline, dextrose and water. In the context of the present invention, the term "pharmaceutically acceptable" refers to other ingredients of molecular entities and pharmaceutical compositions that are physiologically tolerable and generally do not produce adverse reactions when given to mammals (e.g., humans). The term "pharmaceutically acceptable" may also refer to approval by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopoeia or other pharmacopeias recognized for use in mammals (more specifically humans).

[0162] The compositions of the invention may also contain one or more other therapeutically active agents.

[0163] The pharmaceutical composition may be in the form of a solution, suspension, enteric-coated capsule, lyophilized powder, or any other form suitable for the intended use.

[0164] Pharmaceutical compositions for oral administration can be formulated with pharmaceutically acceptable excipients known in the art in dosages suitable for oral administration. Such excipients enable the pharmaceutical compositions to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for ingestion by patients.

[0165] Pharmaceutical preparations for oral use can be obtained by combining the active compound with a solid excipient, optionally grinding the resulting mixture, and processing the granular mixture after adding suitable auxiliaries (if necessary) to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; starch from corn, wheat, rice, potato or other plants; cellulose, such as methylcellulose, hydroxypropylmethylcellulose or sodium carboxymethylcellulose; and gums, including gum arabic and tragacanth; and proteins, such as gelatin and collagen. If necessary, disintegrants or solubilizers, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid or its salts, such as sodium alginate, can be added.

[0166] Dragee cores may be provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings for product identification or to characterize the amount of active compound, i.e., the dosage.

[0167] Pharmaceutical preparations for oral use include push-fit capsules made of gelatin, and soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient mixed with a filler or binder such as lactose or starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid such as a fatty oil, liquid paraffin or liquid polyethylene glycol with or without a stabilizer.

[0168] Pharmaceutical preparations for parenteral administration include aqueous solutions of therapeutically active agents. For injection, the pharmaceutical composition of the present invention can be prepared in aqueous solutions, preferably in physiologically compatible buffers (such as Hank's solution, Ringer's solution or physiologically buffered saline). Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. In addition, the suspension of the active agent can be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or carriers include fatty oils (such as sesame oil) or synthetic fatty acid esters (such as ethyl oleate or triglycerides) or liposomes. Optionally, the suspension may also contain a suitable stabilizer or reagent that increases the solubility of the therapeutically active agent to allow the preparation of a highly concentrated solution.

[0169] For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0170] The pharmaceutical composition of the present invention may be manufactured in a manner known in the art, eg, by means of conventional mixing, dissolving, granulating, dragee-making, suspending, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0171] The pharmaceutical composition can be provided in the form of a salt and can be formed with an acid, including but not limited to hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, etc. Salts tend to be more soluble in aqueous or other protic solvents, which are the corresponding free base forms. In other cases, a preferred preparation may be a lyophilized powder containing 1mM-50mM histidine, 0.1%-2% sucrose, 2%-7% mannitol, with a pH range of 4.5 to 7.5, which is combined with a buffer before use.

[0172] Further details of formulation and administration techniques may be found in the latest edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa.).

[0173] After preparing the pharmaceutical composition containing the antibody or fragment thereof of the present invention, it can be placed in an appropriate container and labeled for the specified condition to be treated. For the administration of the antibody or fragment of the present invention, such labeling will include the amount, frequency and method of administration.

[0174] The present invention also provides packaging (pack) and test kits containing one or more containers containing one or more components of the aforementioned compositions of the present invention. Accompanying such containers is a notification in a form prescribed by a government agency that manages the manufacture, use or sale of a drug or biological product, reflecting that the agency has approved the manufacture, use or sale of the product for human administration.

[0175] In a sixth aspect, the present invention relates to an antibody or fragment thereof of the present invention or a composition of the present invention for use in a method of treatment.

[0176] Specifically, this method of treatment involves administering a therapeutically effective amount of an antibody or fragment thereof of the present invention to a subject in need thereof. The subject may be a human or non-human animal (eg, rabbit, rat, mouse, dog, monkey or other lower primate).

[0177] In the context of the present invention, the term "therapeutically effective amount" is defined as an amount of the antibody of the present invention or its fragment sufficient to prevent or alleviate the disease symptoms of any disorder and disease mentioned herein, as a single dose or according to a multiple dose regimen, alone or in combination with other drugs. In a specific embodiment, the "therapeutically effective amount" is toxicologically tolerable. The determination of an effective dose is entirely within the capabilities of those skilled in the art. The therapeutically effective amount of a therapeutic agent generally depends to a large extent on specific patient characteristics, such as age, weight, sex and disease state, the time, frequency and route of administration, the drug combination and the nature of the disorder being treated. Common dosages of antibodies range from 0.1 micrograms to 100,000 micrograms, up to a total dose of about 10 g, depending on the route of administration.

[0178] For example, general guidance on the determination method thereof can be found in the publications of the International Conference on Harmonization and in Chapters 27 and 28 of REMINGTON'S PHARMACEUTICAL SCIENCES, pages 484-528 (18th ed., Alfonso R. Gennaro, Ed., Easton, Pa.: Mack Pub. Co., 1990). More specifically, determining the therapeutically effective amount will depend on factors such as the toxicity and efficacy of the drug, which can be determined using methods known in the art and described in the aforementioned references. In summary, the therapeutic efficacy and toxicity of a therapeutic agent can be determined in cell culture assays or animal models, such as ED50 (the dose that is therapeutically effective in 50% of the population) and LD50 (the dose that is lethal to 50% of the population), respectively. The dose ratio of ED50 to LD50 is the therapeutic index.

[0179] The antibodies or fragments thereof of the present invention are useful for treating and / or preventing cardiovascular, renal, pulmonary, skeletal, ocular, thromboembolic and fibrotic diseases and disorders, dwarfism, achondroplasia and other cGMP-related and / or natriuretic peptide-responsive disorders. Thus, in specific embodiments, the antibodies or fragments thereof are used to treat and / or prevent any one or any combination of these disorders and diseases.

[0180] Thus, the antibodies or fragments thereof of the present invention can be used in medicaments for the treatment and / or prevention of cardiovascular disorders, such as arterial and pulmonary hypertension, refractory and intractable hypertension, acute and chronic heart failure, coronary heart disease, bronchiolitis obliterans syndrome (BOS), tumor-related and oncological diseases, graft-versus-host disease, sickle cell disease, stable and unstable angina, peripheral and cardiovascular disorders, arrhythmias, atrial and ventricular arrhythmias and conduction impairment (such as atrioventricular block I-III degree (AB block I-I II)), supraventricular tachyarrhythmia, atrial fibrillation, atrial flutter, ventricular fibrillation, ventricular flutter, ventricular tachyarrhythmia, torsades de pointes, atrial and ventricular extrasystoles, AV junction extrasystoles, sick sinus syndrome, syncope, atrioventricular nodal reentrant tachycardia, Wolff-Parkinson-White syndrome, acute coronary syndrome (ACS), autoimmune heart disease (pericarditis, endocarditis, valvulitis, aortitis, cardiomyopathy), shock ( Such as cardiogenic shock, septic shock and anaphylactic shock), aneurysm, boxer cardiomyopathy (premature ventricular contraction (PVC)); can be used in drugs for the treatment and / or prevention of thromboembolic disorders and ischemia, such as myocardial ischemia, myocardial infarction, stroke, cardiac hypertrophy, transient ischemic attack, preeclampsia, inflammatory cardiovascular disorders, coronary artery and peripheral artery spasm, edema formation (such as pulmonary edema, cerebral edema, renal edema or edema caused by heart failure), peripheral circulatory disorders, reperfusion injury, arterial and venous thrombosis formation, microalbuminuria, myocardial insufficiency, endothelial dysfunction, prevention of restenosis after, for example, thrombolytic therapy, percutaneous transluminal angioplasty (PTA), transluminal coronary angioplasty (PTCA), heart transplantation and bypass surgery, and microvascular and macrovascular damage (vasculitis), increased levels of fibrinogen and low-density lipoprotein (LDL), increased concentrations of plasminogen activator inhibitor 1 (PAI-1); and in drugs for the treatment and / or prevention of erectile dysfunction and female sexual dysfunction.

[0181] In the context of the present invention, the term "heart failure" includes acute and chronic heart failure, and also includes more specific or related disease types such as acute decompensated heart failure, right heart failure, left heart failure, total heart failure, ischemic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, idiopathic cardiomyopathy, congenital heart defects, heart failure related to heart valve defects, mitral stenosis, mitral insufficiency, aortic stenosis, aortic insufficiency, tricuspid stenosis, tricuspid insufficiency, pulmonary stenosis, pulmonary insufficiency, combined heart valve defects, myocardial inflammation (myocarditis), chronic myocarditis, acute myocarditis, viral myocarditis, diabetic heart failure, alcoholic cardiomyopathy, cardiac storage disorder, diastolic heart failure and systolic heart failure, heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF) and acute exacerbation of existing chronic heart failure (exacerbated heart failure).

[0182] In addition, the antibodies or fragments thereof of the present invention can also be used to treat and / or prevent arteriosclerosis, peripheral arterial disease (PAD), impaired lipid metabolism, hypolipoproteinemia, dyslipidemia, hypertriglyceridemia, hyperlipidemia, hypercholesterolemia, abetalipoproteinemia, sitosterolemia, xanthomatosis, Tangier disease, adiposity, obesity, and combined hyperlipidemia and metabolic syndrome.

[0183] The antibodies or fragments thereof of the present invention can also be used to treat and / or prevent primary and secondary Raynaud's phenomenon, impaired microcirculation, claudication, peripheral and autonomic neuropathy, diabetic microangiopathy, diabetic retinopathy, diabetic limb ulcers, gangrene, CREST syndrome, erythromelalgia, onychomycosis, rheumatism, and to promote wound healing.

[0184] The antibodies or fragments thereof of the present invention are also suitable for the treatment of urinary disorders, such as benign prostatic syndrome (BPS), benign prostatic hyperplasia (BPH), benign prostatic hypertrophy (BPE), bladder outlet obstruction (BOO), lower urinary tract syndrome (LUTS, including feline urological syndrome (FUS)), urogenital disorders including neurogenic overactive bladder (OAB) and (IC), urinary incontinence (UI) (e.g. mixed urinary incontinence, urge urinary incontinence, stress urinary incontinence or overflow urinary incontinence (MUI, UUI, SUI, OUI)), pelvic pain, benign and malignant disorders of the male and female urogenital organs.

[0185] The antibodies or fragments thereof of the present invention are also suitable for treating and / or preventing renal disorders, in particular acute and chronic renal insufficiency and acute and chronic renal failure. In the context of the present invention, the term "renal insufficiency" includes acute and chronic manifestations of renal insufficiency, as well as potential or related renal disorders such as renal hypoperfusion, intradialytic hypotension, obstructive uropathy, glomerulopathy, glomerulonephritis, acute glomerulonephritis, glomerulosclerosis, tubulointerstitial diseases, renal disorders (such as primary and congenital renal diseases), nephritis, immune renal disorders (such as renal transplant rejection and immune complex-induced renal disorders, toxic substance-induced nephropathy, contrast agent-induced nephropathy, diabetic and non-diabetic nephropathy, pyelonephritis, renal cysts, nephrosclerosis, hypertensive nephrosclerosis and nephrotic syndrome, which Diagnostically, it can be characterized by, for example, abnormally reduced creatinine and / or water excretion, abnormally elevated blood concentrations of urea, nitrogen, potassium and / or creatinine, altered renal enzyme (e.g., glutamyl synthetase) activity, altered urine osmolality or urine volume, elevated microalbuminuria, macroalbuminuria, glomerular and arteriole lesions, tubular dilatation, hyperphosphatemia and / or the need for dialysis. The present invention also includes the use of the antibodies or fragments thereof of the present invention for the treatment and / or prevention of sequelae of renal insufficiency, such as pulmonary edema, heart failure, uremia, anemia, electrolyte disturbances (e.g., hyperkalemia, hyponatremia) and bone and carbohydrate metabolism disorders.

[0186] In addition, the antibodies or fragments thereof of the present invention are also suitable for treating and / or preventing asthmatic disorders, pulmonary arterial hypertension (PAH) and other forms of pulmonary hypertension (PH) including left heart disease, HIV, sickle cell anemia, thromboembolism (CTEPH), sarcoidosis, COPD or pulmonary fibrosis-associated pulmonary hypertension, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), acute lung injury (ALI), alpha-1-antitrypsin deficiency (AATD), pulmonary fibrosis, emphysema (e.g., smoking-induced emphysema), bronchiolitis obliterans syndrome (BOS) and cystic fibrosis (CF).

[0187] The antibodies or fragments thereof of the invention are also suitable for controlling central nervous system disorders characterized by a disorder of the NO / cGMP system. It is particularly suitable for improving perception, attention, learning or memory after cognitive impairment, such as those impairments particularly associated with the following conditions / diseases / syndromes: such as mild cognitive impairment, age-related learning and memory impairment, age-related memory loss, vascular dementia, craniocerebral trauma, stroke, dementia occurring after a stroke (post-stroke dementia), post-traumatic craniocerebral trauma, general attention disorder, attention disorder in children with learning and memory problems, Alzheimer's disease, Lewy body dementia, frontal lobe degeneration dementia (including Pick's syndrome), Parkinson's disease, progressive nuclear palsy, corticobasal degeneration dementia, amyotrophic lateral sclerosis (ALS), Huntington's disease, demyelination, multiple sclerosis, thalamic degeneration, Creutzfeld-Jacob dementia, HIV dementia, dementia schizophrenia or Korsakoff psychosis. It is also indicated for the treatment and / or prevention of central nervous system disorders, such as anxiety, stress and depressive states, CNS-related sexual dysfunction and sleep disturbances, as well as for pathological disorders controlling the intake of food, stimulants and addictive substances.

[0188] In addition, the antibody or fragment thereof of the present invention is also suitable for controlling cerebral blood flow, and is therefore an effective agent for controlling migraine. It is also suitable for preventing and controlling sequelae of cerebral infarction (stroke), such as stroke, cerebral ischemia and craniocerebral trauma. It can also be used to control pain and tinnitus.

[0189] In addition, the antibodies or fragments of the present invention have anti-inflammatory effects and can therefore be used as anti-inflammatory agents for the treatment and / or prevention of sepsis (SIRS), multiple organ failure (MODS, MOF), renal inflammatory disorders, chronic intestinal inflammation (IBD, Crohn's disease, UC), pancreatitis, peritonitis, rheumatoid disease, inflammatory skin diseases and inflammatory eye diseases.

[0190] In addition, the antibodies or fragments thereof of the present invention can also be used to treat and / or prevent autoimmune diseases.

[0191] The antibodies or fragments thereof are also suitable for the treatment and / or prevention of fibrotic disorders of internal organs such as the lungs, heart, kidneys, reproductive system, bone marrow, in particular the liver, as well as skin fibromas and fibrotic eye diseases. In the context of the present invention, the term fibrotic disorder particularly includes the following terms: liver fibrosis, cirrhosis, pulmonary fibrosis, endomyocardial fibrosis, nephropathy, glomerulonephritis, interstitial renal fibrosis, fibrotic lesions caused by diabetes, uterine fibroma, endometriosis, myelofibrosis and similar fibrotic disorders, scleroderma, morphea, keloids, hypertrophic scarring (also after surgical procedures), nevi, diabetic retinopathy, proliferative vitreoretinopathy and connective tissue disorders (e.g. sarcoidosis).

[0192] The antibodies or fragments thereof of the invention are also useful for controlling post-operative scarring, such as that resulting from glaucoma surgery.

[0193] The antibodies or fragments thereof of the present invention can also be used cosmetically for aged and keratinized skin.

[0194] Furthermore, the antibodies or fragments thereof of the present invention are useful for treating and / or preventing hepatitis, tumors, osteoporosis, glaucoma and gastroparesis.

[0195] In addition, the antibodies or fragments thereof of the present invention are suitable for treating and / or preventing ocular disorders, such as ocular diseases responsive to natriuretic peptides, retinal disorders, glaucoma including primary open-angle glaucoma (POAG), angle-closure glaucoma, congenital / developmental glaucoma, retinopathy, ocular trauma, optic neuropathy, ocular hypertension, increased intraocular pressure, diabetic retinopathy, macular degeneration (AMD), age-related eye diseases, macular edema, scleritis, uveitis, dry eyes, corneal epithelial abrasion, corneal ulcer.

[0196] In addition, the antibodies or fragments thereof of the present invention are suitable for treating bone and cartilage disorders, such as bone and cartilage diseases responsive to natriuretic peptides, arthritis, degenerative diseases of cartilage tissue, osteoarthritis, cartilage degeneration, fractures, skeletal dysplasia, achondroplasia, osteoporosis, osteogenesis imperfecta, Paget's disease of bone (PDB), metabolic bone disease, age-related bone disease, osteomyelitis, osteonecrosis, rickets, osteomalacia, growth plate injuries and diseases, joint and bone replacement related defects, Marfan syndrome, sports injuries, muscular dystrophy, Duchenne muscular dystrophy.

[0197] Therefore, in another aspect, the present invention relates to the use of the antibodies or fragments thereof according to the present invention for the treatment and / or prevention of disorders, in particular the disorders mentioned above.

[0198] In specific embodiments, the antibodies or fragments thereof of the invention can be used in methods for treating and / or preventing heart failure, angina pectoris, hypertension, pulmonary hypertension, ischemia, vascular disorders, renal insufficiency, thromboembolic disorders, fibrotic disorders, skeletal and bone disorders, ocular disorders, and arteriosclerosis.

[0199] In another aspect, the present invention relates to the use of an antibody or fragment thereof according to the present invention for the production of a medicament for the treatment and / or prevention of disorders, in particular the disorders mentioned above.

[0200] In a specific embodiment, the present invention relates to the use of the antibodies or fragments thereof of the present invention for the production of a medicament for the treatment and / or prevention of heart failure, angina pectoris, hypertension, pulmonary hypertension, ischemia, vascular disorders, renal insufficiency, thromboembolic disorders, fibrotic disorders, dementia, arteriosclerosis, skeleton and bone disorders, eye disorders, dwarfism, achondroplasia and erectile dysfunction.

[0201] In another aspect, the present invention relates to a method for the treatment and / or prevention of disorders, in particular the above-mentioned disorders, using an effective amount of at least one antibody or fragment thereof according to the present invention.

[0202] In a specific embodiment, the present invention relates to a method for treating and / or preventing heart failure, angina pectoris, hypertension, pulmonary hypertension, ischemia, vascular disorders, renal insufficiency, thromboembolic disorders, fibrotic disorders, tumors and oncological diseases, skeleton and bone disorders, eye disorders, dwarfism, achondroplasia and arteriosclerosis using an effective amount of at least one antibody of the present invention or a fragment thereof.

[0203] The antibodies or fragments thereof of the present invention may be administered as the sole agent or may be administered in combination with one or more additional therapeutic agents, and in some cases, the antibodies themselves may be modified. For example, the antibodies or fragments thereof may be conjugated to chemical entities, for example to further improve efficacy, stability and / or half-life. Specifically, the antibodies or fragments thereof of the present invention may be pegylated and / or HESylated.

[0204] Thus, in a specific embodiment, the antibodies or fragments thereof of the invention are used in a method of treatment in combination with at least one additional therapeutic agent, in particular for the purposes described above.

[0205] The present invention also provides a pharmaceutical combination comprising at least one antibody or fragment thereof of the present invention and at least one additional therapeutic agent.

[0206] In the context of the present invention, the term "pharmaceutical combination" is used in a manner known to the person skilled in the art, and this combination may be a fixed combination, a non-fixed combination or a kit of parts.

[0207] In the context of the present invention, the term "fixed combination" is used in a manner known to the person skilled in the art and is defined as a combination wherein, for example, a first active ingredient, such as one or more antibodies or fragments thereof of the present invention, and a further active ingredient are present together in one unit dose or in one single entity, such as a single dose formulation. An example of a "fixed combination" is a pharmaceutical composition wherein the first active ingredient and the further active ingredient are present as a mixture for simultaneous administration, such as in a formulation. Another example of a "fixed combination" is a pharmaceutical combination wherein the first active ingredient and the further active ingredient are present as one unit rather than as a mixture. Therefore, the present invention provides a pharmaceutical composition comprising at least one antibody or fragment thereof and at least one additional therapeutic agent, in particular for the treatment and / or prevention of the above-mentioned disorders.

[0208] In the context of the present invention, the terms "non-fixed combination" and "kit of parts" are used in a manner known to the person skilled in the art and are defined as a combination wherein the first active ingredient and the other active ingredient are present in more than one unit, for example in separate dosage preparations. An example of a non-fixed combination or a kit of parts is a combination wherein the first active ingredient and the other active ingredient are present separately.

[0209] The components of a non-fixed combination or kit-of-parts may be administered separately, sequentially, simultaneously, concurrently or chronologically staggered.

[0210] The antibody of the present invention or its fragment can be administered simultaneously with, before or after the other therapeutically active agent. In the context of the present invention, the term "simultaneously" means that the antibody of the present invention or its fragment is administered on the same day, more particularly within 12 hours, more particularly within 2 hours with at least one other therapeutically active agent.

[0211] In a specific embodiment, the administration of the antibody or fragment thereof of the present invention and at least one other therapeutic agent occurs within eight consecutive weeks, more particularly within one to six consecutive weeks. The antibody or fragment thereof of the present invention and at least one other therapeutic agent can be administered by the same route or by different routes.

[0212] The antibodies or fragments thereof of the invention can be combined, for example, with known agents of the same indication therapeutic group, such as agents for the treatment and / or prevention of diseases and conditions associated with: hypertension, heart failure, pulmonary hypertension, COPD, asthma, cystic fibrosis, achondroplasia, hyperphosphatemia, chronic kidney disease (CKD), soft tissue calcification, chronic kidney disease-related calcification, non-chronic kidney disease-related calcification, medial calcification including Moenckeberg's medial sclerosis, sclerosis), atherosclerosis, intimal calcification, CKD-associated cardiac hypertrophy, CKD-associated renal dystrophy, osteoporosis, postmenopausal osteoporosis, type II diabetes, chronic kidney disease, aging, hypophosphaturia, hyperparathyroidism, vitamin D disorders, vitamin K deficiency, vitamin K antagonist coagulants, Kawasaki disease, ACDC (arterial calcification due to CD73 deficiency), GACI (generalized arterial calcification of infancy), IBGC (idiopathic basal ganglia calcification), PXE (pseudoxanthoma elasticum), rheumatoid arthritis, Singleton-Merten syndrome, P-thalassemia, calciphylaxis, heterotrophic ossification, premature placental calcification, uterine calcification, calcified uterine fibroids, morbus fahr, aortic valve microcalcification and calcification.

[0213] Preferred examples of suitable other therapeutic agents to be combined with the antibodies or fragments thereof of the present invention:

[0214] - Organic nitrates and NO donors, such as sodium nitroprusside, nitroglycerin, isosorbide mononitrate, isosorbide dinitrate, molsidomide or SIN-1, and inhaled NO;

[0215] - compounds which inhibit the breakdown of cyclic guanosine monophosphate (cGMP), for example inhibitors of phosphodiesterase (PDE) 1, 2 and / or 5, in particular PDE 5 inhibitors, such as sildenafil, vardenafil, tadalafil, udenafil, desantafil, avanafil, mirodenafil, lodenafil or PF-00489791;

[0216] - antithrombotic agents, for example and preferably platelet aggregation inhibitors, anticoagulants or profibrinolytic substances;

[0217] - antihypertensive active ingredients, for example and preferably calcium antagonists, angiotensin AII antagonists, ACE inhibitors, NEP inhibitors, vasopeptidase inhibitors, endothelin antagonists, renin inhibitors, alpha-receptor blockers, beta-receptor blockers, mineralocorticoid receptor antagonists, rho-kinase inhibitors and diuretics;

[0218] antiarrhythmic agents, for example and preferably sodium channel blockers, beta-blockers, potassium channel blockers, calcium antagonists, If channel blockers, digitalis, vagoliytics, sympathomimetics and other antiarrhythmic drugs, such as adenosine, adenosine receptor agonists and vernakalant;

[0219] - Positive inotropic agents, such as cardiac glycosides (Dogoxin), beta-adrenergic and dopaminergic agonists, such as isoproterenol, epinephrine, norepinephrine, dopamine, or dobutamine;

[0220] - vasopressin receptor antagonists, for example and preferably conivaptan, tolvaptan, lixivaptan, mozavaptan, satavaptan, SR-121463, RWJ 676070 or BAY 86-8050 and the compounds described in WO 2010 / 105770, WO 2011 / 104322 and WO 2016 / 071212;

[0221] - active ingredients which alter lipid metabolism, for example and preferably thyroid receptor agonists, PCSK9 inhibitors, cholesterol synthesis inhibitors, for example and preferably HMG-CoA reductase inhibitors or squalene synthesis inhibitors, ACAT inhibitors, CETP inhibitors, MTP inhibitors, PPAR-alpha, PPAR-gamma and / or PPAR-delta agonists, cholesterol absorption inhibitors, lipase inhibitors, polymeric bile acid sorbents, bile acid reabsorption inhibitors and lipoprotein(a) antagonists.

[0222] - anti-inflammatory agents, for example and preferably glucocorticoids, for example and preferably prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, beclomethasone, betamethasone, flunisolide, budesonide or fluticasone and non-steroidal anti-inflammatory agents (NSAIDs), for example and preferably acetylsalicylic acid (aspirin), ibuprofen and naproxen, 5-aminosalicylic acid derivatives, leukotriene antagonists, TNF-α-inhibitors and chemokine receptor antagonists, such as CCR1, 2 and / or 5 inhibitors;

[0223] - agents which inhibit signal transduction cascades, such as and preferably kinase inhibitors, such as and preferably tyrosine kinase and / or serine / threonine kinase inhibitors;

[0224] - agents which inhibit the degradation and modification of the extracellular matrix, for example and preferably inhibitors of matrix metalloproteinases (MMPs), for example and preferably inhibitors of chymotrypsin, stromelysine, collagenase, gelatinase and aggrecanase (preferably MMP-1, MMP-3, MMP-8, MMP-9, MMP-10, MMP-11 and MMP-13), and inhibitors of metalloelastase (MMP-12) and neutrophil elastase (HNE), for example sivelestat or DX-890;

[0225] - agents that block the binding of serotonin to its receptors, for example and preferably 5-HT2b receptor antagonists;

[0226] - antifibrotic agents, for example and preferably nintedanib, pirfenidone, adenosine A2b receptor antagonists, sphingosine-1-phosphate receptor 3 (S1P3) antagonists, autotaxin inhibitors, lysophosphatidic acid receptor 1 (LPA-1) and lysophosphatidic acid receptor 2 (LPA-2) antagonists, lysyl oxidase (LOX) inhibitors, lysyl oxidase-like 2 inhibitors, CTGF inhibitors, IL-13 antagonists, integrin antagonists, TGF-β antagonists, wnt signaling inhibitors, CCR2-antagonists;

[0227] - agents acting as bronchodilators, for example and preferably 5-HT2b receptor antagonists; β2-adrenergic agonists (short and long acting), anticholinergics and theophylline;

[0228] - agents that are antagonists of cytokines and chemokines, for example and preferably antagonists of TGF-β, CTGF, IL-1, IL-4, IL-5, IL-6, IL-8, IL-13, IL-25, IL-33, TSLP and integrins;

[0229] - organic nitrates and NO donors, for example and preferably sodium nitroprusside, nitroglycerin, isosorbide mononitrate, isosorbide dinitrate, molsidomine or SIN-1, and inhaled NO;

[0230] NO-independent, heme-dependent stimulators of soluble guanylate cyclase, for example and preferably the compounds described in WO 00 / 06568, WO 00 / 06569, WO 02 / 42301, WO 03 / 095451, WO 2011 / 147809, WO 2012 / 004258, WO 2012 / 028647 and WO 2012 / 059549;

[0231] NO-independent and heme-independent activators of soluble guanylate cyclase, for example and preferably as described in WO 01 / 19355, WO 01 / 19776, WO 01 / 19778, WO 01 / 19780, WO 02 / 070462 and WO 02 / 070510;

[0232] - agents which stimulate cGMP synthesis, such as sGC modulators, for example and preferably riociguat, cinaciguat, vericiguat;

[0233] - a prostacyclin analogue or an IP receptor agonist, for example and preferably iloprost, beraprost, treprostinil, epoprostenol or Selexipag;

[0234] - an endothelin receptor antagonist, for example and preferably bosentan, darusentan, ambrisentan or sitaxsentan;

[0235] - agents which inhibit soluble epoxide hydrolases (sEH), for example and preferably N,N′-di-cyclohexylurea, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid or 1-adamantan-1-yl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl}-urea;

[0236] - agents that interact with glucose metabolism, for example and preferably insulin, biguanides, thiazolidinediones, sulfonylureas, acarbose, DPP4 inhibitors, GLP-1 analogues or SGLT-1 inhibitors;

[0237] - a natriuretic peptide, for example and preferably atrial natriuretic peptide (ANP, carperitide), brain natriuretic peptide (BNP, nesiritide), C-type natriuretic peptide (CNP) or urodilatin;

[0238] - natriuretic peptide derivatives, for example and preferably vosoritide, cenderitide, PL3994;

[0239] - a cardiac myosin activator, for example and preferably omecamtiv mecarbil (CK-1827452);

[0240] - a calcium sensitizer, for example and preferably levosimendan;

[0241] - agents that influence cardiac energy metabolism, for example and preferably etomoxir, dichloroacetate, ranolazine or trimetazidine, full or partial adenosine A1 receptor agonists, such as GS-9667 (formerly CVT-3619), capadenoson and neladenoson;

[0242] - Agents that influence heart rate, for example and preferably ivabradine.

[0243] Antithrombotic agents are preferably understood to be compounds from the group of platelet aggregation inhibitors, anticoagulants or profibrinolytic substances.

[0244] In a specific embodiment, the antibodies of the invention or fragments thereof are administered in combination with a platelet aggregation inhibitor, for example and preferably aspirin, clopidogrel, prasugrel, ticagrelor, ticlopidine or dipyridamole.

[0245] In a specific embodiment, the antibodies or fragments thereof according to the invention are administered in combination with a thrombin inhibitor, such as for example and preferably ximelagatran, dabigatran, melagatran, bivalirudin or clexane.

[0246] In a specific embodiment, the antibodies of the invention or fragments thereof are administered in combination with a GPIIb / IIIa antagonist, for example and preferably tirofiban or abciximab.

[0247] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a factor Xa inhibitor, for example and preferably rivaroxaban, DU-176b, apixaban, betrixaban, otamixaban, fidexaban, razaxaban, letaxaban, eribaxaban, fondaparinux, idraparinux, PMD-3112, darexaban (YM-150), KFA-1982, EMD-503982, MCM-17, MLN-1021, DX 9065a, DPC 906, JTV803, SSR-126512 or SSR-128428.

[0248] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with heparin or a low molecular weight (LMW) heparin derivative.

[0249] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a vitamin K antagonist, such as for example and preferably coumarin.

[0250] Antihypertensive agents are preferably understood to be compounds from the group of calcium antagonists, angiotensin AII antagonists, ACE inhibitors, endothelin antagonists, renin inhibitors, alpha-receptor blockers, beta-receptor blockers, mineralocorticoid receptor antagonists, rho-kinase inhibitors and diuretics.

[0251] In a specific embodiment, the antibodies of the invention or fragments thereof are administered in combination with a calcium antagonist, such as for example and preferably nifedipine, amlodipine, verapamil or diltiazem.

[0252] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with an alpha-1 receptor blocker, such as, for example and preferably, prazosin.

[0253] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a beta-blocker, for example and preferably propranolol, atenolol, timolol, pindolol, alprenolol, oxprenolol, penbutolol, bupranolol, metipranolol, nadolol, mepindolol, carazolol, mesotriol, metoprolol, betaxolol, celiprolol, bisoprolol, carteolol, esmolol, labetalol, carvedolol, adalolol, landiolol, nebivolol, epanolol or bucindolol.

[0254] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with an angiotensin AII antagonist, for example and preferably losartan, candesartan, valsartan, telmisartan or embusartan, or a dual angiotensin AII antagonist / neprilysin inhibitor, for example and preferably LCZ696 (valsartan / sacubitril).

[0255] In a specific embodiment, the antibodies or fragments thereof according to the invention are administered in combination with an ACE inhibitor, such as for example and preferably enalapril, captopril, lisinopril, ramipril, delapril, fosinopril, quinapril, perindopril or fendolapril.

[0256] In a specific embodiment, the antibodies of the invention or fragments thereof are administered in combination with an endothelin antagonist, such as for example and preferably bosentan, darusentan, ambrisentan or sitaxsentan.

[0257] In a specific embodiment, the antibodies of the invention or fragments thereof are administered in combination with a renin inhibitor, for example and preferably aliskiren, SPP-600 or SPP-800.

[0258] In a specific embodiment, the antibodies of the invention or fragments thereof are administered in combination with a mineralocorticoid receptor antagonist, such as for example and preferably finerenone, spironolactone or eplerenone.

[0259] In specific embodiments, the antibodies or fragments thereof of the invention are administered in combination with loop diuretics, such as furosemide, torsemide, bumetanide, and piretanide, with potassium-sparing diuretics, such as amiloride and triamterene, with aldosterone antagonists, such as spironolactone, potassium canrenoate, and eplerenone, and with thiazide diuretics, such as hydrochlorothiazide, chlorthalidone, xipamide, and indapamide.

[0260] Lipid metabolism modifiers are preferably understood to be compounds from the group of CETP inhibitors, thyroid receptor agonists, cholesterol synthesis inhibitors such as HMG-CoA reductase inhibitors or squalene synthesis inhibitors, ACAT inhibitors, MTP inhibitors, PPAR-alpha, PPAR-gamma and / or PPAR-delta agonists, cholesterol absorption inhibitors, polymeric bile acid sorbents, bile acid reabsorption inhibitors, lipase inhibitors and lipoprotein (a) antagonists.

[0261] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a CETP inhibitor, for example and preferably dalcetrapib, anacetrapib, torcetrapib (CP-529414), JJT-705 or CETP vaccine (Avant).

[0262] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a thyroid receptor agonist, such as for example and preferably D-thyroxine, 3,5,3'-triiodothyronine (T3), CGS 23425 or acitreotide (CGS26214).

[0263] In a specific embodiment, the antibodies according to the invention or fragments thereof are administered in combination with an HMG-CoA reductase inhibitor from the class of statins, such as for example and preferably lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin or pitavastatin.

[0264] In a specific embodiment, the antibodies of the invention or fragments thereof are administered in combination with a squalene synthesis inhibitor, for example and preferably BMS-188494 or TAK-475.

[0265] In a specific embodiment, the antibodies of the invention or fragments thereof are administered in combination with an ACAT inhibitor, for example and preferably avasimibe, melinamide, pactimibe, eflucimibe or SMP-797.

[0266] In a specific embodiment, the antibodies of the invention or fragments thereof are administered in combination with an MTP inhibitor, such as for example and preferably implitapide, BMS-201038, R-103757 or JTT-130.

[0267] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a PPAR-γ agonist, for example and preferably pioglitazone or rosiglitazone.

[0268] In a specific embodiment, the antibodies of the invention or fragments thereof are administered in combination with a PPAR-delta agonist, such as for example and preferably GW 501516 or BAY 68-5042.

[0269] In a specific embodiment, the antibodies of the invention or fragments thereof are administered in combination with a cholesterol absorption inhibitor, such as for example and preferably ezetimibe, tiqueside or pamaside.

[0270] In a specific embodiment, the antibodies of the invention or fragments thereof are administered in combination with a lipase inhibitor, such as for example and preferably orlistat.

[0271] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a polymeric bile acid sorbent, such as for example and preferably cholestyramine, colestipol, colesolvam, cholesteat or colestimide.

[0272] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a bile acid reabsorption inhibitor, for example and preferably an ASBT (= IBAT) inhibitor, such as AZD-7806, S-8921, AK-105, BARI-1741, SC-435 or SC-635.

[0273] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a lipoprotein(a) antagonist, such as, for example and preferably, gemcabene calcium (CI-1027) or niacin.

[0274] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a lipoprotein(a) antagonist, such as, for example and preferably, gemcabene calcium (CI-1027) or niacin.

[0275] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a sGC modulator, for example and preferably riociguanide, cinaciguanide or veraciroxguanide.

[0276] In a specific embodiment, the antibodies of the invention or fragments thereof are administered in combination with an agent that influences glucose metabolism, such as for example and preferably insulin, sulfonylurea, acarbose, DPP4 inhibitors, GLP-1 analogs or SGLT-1 inhibitors.

[0277] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a TGFβ antagonist, such as for example and preferably pirfenidone, nintedanib or fresolimumab.

[0278] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a CCR2 antagonist, for example and preferably CCX-140.

[0279] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a TGFα antagonist, for example and preferably adalimumab.

[0280] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a galectin-3 inhibitor, for example and preferably GCS-100.

[0281] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with an Nrf-2 inhibitor, such as, for example and preferably, bardoxolone.

[0282] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a BMP-7 agonist, for example and preferably THR-184.

[0283] In a specific embodiment, the antibodies of the invention or fragments thereof are administered in combination with a NOX1 / 4 inhibitor, such as and preferably GKT-137831.

[0284] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with an agent that influences vitamin D metabolism, for example and preferably calcitriol, alfacalcidol, doxorcalciferol, maxacalcitol, paricalcitol, cholecalciferol or poncathiol.

[0285] In a specific embodiment, the antibodies of the invention or fragments thereof are administered in combination with a cytostatic agent, such as, for example and preferably, cyclophosphamide.

[0286] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with an immunosuppressant, for example and preferably cyclosporine.

[0287] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a phosphate binder, such as, for example and preferably, colestilan, sevelamer hydrochloride and sevelamer carbonate, lanthanum and lanthanum carbonate.

[0288] In a specific embodiment, an antibody or fragment thereof of the invention is administered in combination with a renal proximal tubule sodium phosphate co-transporter, such as and preferably niacin or niacinamide.

[0289] In a specific embodiment, the antibodies or fragments thereof of the present invention are administered in combination with a calcimimetic for the treatment of hyperparathyroidism.

[0290] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with an agent for the treatment of iron deficiency, such as and preferably an iron product.

[0291] In a specific embodiment, the antibody or fragment thereof of the invention is administered in combination with an agent for the treatment of hyperuricaemia, for example and preferably allopurinol or rasburicase.

[0292] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a glycoprotein hormone for the treatment of anemia, such as, for example and preferably, erythropoietin.

[0293] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a biologic for immunotherapy, such as for example and preferably abatacept, rituximab, eculizumab or belimumab.

[0294] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with vasopressin antagonists (vaptanes) for the treatment of heart failure, such as for example and preferably tolvaptan, conivaptan, lixivaptan, mozavaptan, satavaptan or relcovaptan.

[0295] In a specific embodiment, the antibodies of the invention or fragments thereof are administered in combination with a Jak inhibitor, for example and preferably ruxolitinib, tofacitinib, baricitinib, CYT387, GSK2586184, lestaurtinib, pacritinib (SB1518) or TG101348.

[0296] In a specific embodiment, the antibodies or fragments thereof of the present invention are administered in combination with a prostacyclin analog for the treatment of microthrombi.

[0297] In a specific embodiment, an antibody or fragment thereof of the invention is administered in combination with a base treatment, for example and preferably sodium bicarbonate.

[0298] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with an mTOR inhibitor, such as for example and preferably everolimus or rapamycin.

[0299] In a specific embodiment, the antibody or fragment thereof of the invention is administered in combination with an NHE3 inhibitor, for example and preferably AZD1722 or tenapanor.

[0300] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with an eNOS modulator, such as, for example and preferably, sapropterin.

[0301] In a specific embodiment, the antibodies or fragments thereof of the invention are administered in combination with a CTGF inhibitor, for example and preferably FG-3019.

[0302] One or more physiologically acceptable carriers or excipients can be used to formulate antibodies or fragments thereof for the treatment methods of the present invention in any conventional manner. Antibodies or fragments thereof of the present invention can be administered in any appropriate manner, which can be changed according to the type of disorder to be treated. Possible routes of administration include enteral (e.g., oral), parenteral (e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intracardiac, intraventricular, intrathecal, intramedullary, intralesional), intrapulmonary, and intranasal administration. In addition, antibodies or fragments thereof of the present invention can be administered by vein infusion, e.g., with a reduced dosage of antibodies or fragments thereof. Preferably, administration is by injection, most preferably by intravenous or subcutaneous injection, depending in part on whether the disease is acute or chronic. The amount to be administered will depend on multiple factors, such as individual clinical symptoms, sex, age and / or body weight, whether other drugs are administered, etc. The technician will recognize that the route of administration will vary according to the disorder to be treated or the disease.

[0303] Parenteral delivery methods include topical, intraarterial, intratumoral, intramuscular, subcutaneous, intramedullary, intrathecal, intracerebroventricular, intravenous, intraperitoneal, or intranasal administration.

[0304] In a specific embodiment, the method of treatment comprises a single or multiple administration of an antibody or fragment thereof or a pharmaceutical composition comprising the antibody or fragment thereof. The single administration dose may be the same or different. Specifically, the method of treatment comprises 1, 2, 3, 4, 5 or 6 administrations of an antibody or fragment thereof of the present invention, preferably wherein the multiple administrations occur within one to six consecutive months. The antibody or fragment thereof of the present invention may be administered, for example, every 3 to 4 days, weekly, once every two weeks or once every three weeks, depending on its half-life and clearance rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0305] Figure 1: Mean plasma concentrations of TPP-10992 and TPP-5661 in rats after intravenous administration of 5 mg / kg.

[0306] Figure 2 : Mean plasma concentration of TPP-12897 after intraperitoneal administration of 5 mg / kg to mice.

[0307] Figure 3 : Stability of ANP (AC), TPP-10992 (DF) and TPP-5661 (GI) against proteolytic degradation. The activities of ANP, TPP-10992 and TPP-5661 were tested on a stable rat ANP receptor cell line either directly (A, D, G) or after incubation for 4 h at 37°C with 0.6 μg / ml NEP (B, E, H) or 0.6 μg / ml IDE (C, F, I).

[0308] Figure 4 : Stability of BNP (AC) and TPP-11155 (DF) against proteolytic degradation. The activity of BNP and TPP-11155 was tested on a stable rat BNP receptor cell line either directly (A, D) or after incubation for 4 h at 37°C with 0.6 μg / ml NEP (B, E) or 0.6 μg / ml IDE (C, F).

[0309] Figure 5 : Stability of CNP (AC) and TPP-12897 (DF) against proteolytic degradation. The activity of CNP and TPP-11155 was tested on a stable rat CNP receptor cell line either directly (A, D) or after incubation for 4 h at 37°C with 0.6 μg / ml NEP (B, E) or 0.6 μg / ml IDE (C, F).

[0310] Figure 6 Figure : Dose-response curves of vasodilation induced by ANP peptide and TPP-10992 in PE-contracted aortic rings. Concentration-response curves of ANP peptide (open circles) and TPP-10992 (filled circles) in endothelial intact rat aortic rings contracted by phenylephrine (1 μM) (0.0001-10 μM; n=3 rats). Experimental values ​​are calculated relative to the maximum change in contraction produced by phenylephrine in each tissue (taken as 100%). The potencies of ANP peptide and TPP-10992 were -7.4 and -6.7, respectively (logEC 50 Data represent the mean ± SEM of 2 experiments.

[0311] Figure 7Figure : Dose-response curves of vasodilation induced by ANP peptide and TPP-5661 in PE-contracted aortic rings. Concentration-response curves of ANP peptide (open circles) and TPP-5661 (filled circles) in endothelial intact rat aortic rings contracted by phenylephrine (1 μM) (0.0001-10 μM; n=3 rats). Experimental values ​​are calculated relative to the maximum change in contraction produced by phenylephrine in each tissue (taken as 100%). The potencies of ANP peptide and TPP-5661 were -7.4 and -6.5, respectively (logEC 50 Data represent the mean ± SEM of 2 experiments.

[0312] Figure 8 : Hemodynamic effects of ANP in conscious rats. Rats were given ANP intraperitoneally at 0 hours. A dose of 500 μg of ANP resulted in a decrease in mean arterial blood pressure (MAP) of approximately 25%, with a duration of effect of approximately 6-8 hours.

[0313] Fig. 9 : Hemodynamic effects of TPP-5661 in conscious rats. TPP-5661 was administered intraperitoneally at 0 hours. The 15 mg / kg dose resulted in a decrease in mean arterial blood pressure (MAP) of approximately 20%, with a maximum effect at 24-48 hours after administration and a duration of effect of more than 6 days.

[0314] Fig.10 : Hemodynamic effects of TPP-10992 in conscious rats. TPP-10992 was administered intraperitoneally at 0 hours. A 30 mg / kg dose resulted in a decrease in mean arterial blood pressure (MAP) of approximately 20%, with a maximum effect at 48 hours after administration and an effect duration of more than 6 days.

[0315] Fig.11 : Activity of BNP grafted antibody constructs on hNPRA cells. The activity of purified compound samples was evaluated on stable hNPRA-CHO k1 cells by comparison with reference samples TPP-5661 and TPP-5657. Samples were tested in a dilution series in quadruplicate.

[0316] Fig.12:Different human IgG isotypes provide equally suitable antibody scaffolds. Exemplary activity assays 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 k1 cells was evaluated by comparison with reference samples compound 117 human IgG1 TPP-5661 and compound 209 human IgG1 TPP-5657. Samples were tested in a dilution series in quadruplicate.

[0317] Fig.13 : The same suitable IgG antibody scaffolds derived from different species. Exemplary activity assays of compound 117 human IgG1 (TPP-5661) and compound 9 human IgG1 (TPP-10294) and their non-human IgG1 counterparts. The activity of the purified compound samples was evaluated on stable hNPRA-CHO k1 cells by comparison with the reference sample compound 209 human IgG1 (TPP-5657). The samples were tested in a dilution series in quadruplicate.

[0318] Fig.14 : The same suitable human IgG antibody scaffolds derived from different germline sequences. The activity of the purified compound samples was evaluated on stable hNPRA-CHO k1 cells by comparison with the reference sample TPP-10992. The samples were tested in a dilution series in quadruplicate.

[0319] Fig.15 : The protective effect of TPP-12899 on LPS-, IL-1β-, and thrombin-induced endothelial barrier permeability was evaluated by real-time impedance measurement.

[0320] Fig.16 : Treatment effect of TPP-13992 on survival (A), weight gain (B), urine protein / creatinine ratio (C), and left atrial weight (D); (n=8-12 (healthy controls n=5), mean±SEM, one-way ANOVA vs TPP-10155 (isotype-specific control antibody).

[0321] Fig.17 : Hemodynamic evaluation after placebo, 0.1, 0.3 and 1.0 mg / kg TPP-10992. TPP-10992 demonstrated a dose-dependent and long-lasting (>5d) reduction in blood pressure. **p<0.01, ****p<0.0001 compared with placebo, using one-way ANOVA test for repeated measures followed by Tukey's multiple comparison test.

[0322] Example

[0323] Example 1: Construction of candidate TPP-5661

[0324] Candidate TPP-5661 was designed by replacing the two C-terminal residues of HV 3-23 with a heterologous amino acid sequence comprising Ntls, wild-type rat ANP and Ctls, the heterologous amino acid sequence being fused to the C-terminus of HV 3-23 (SEQ ID NO 85), and replacing the 9 N-terminal residues of IGHJ1 with the 9 C-terminal residues of the heterologous amino acid sequence, the heterologous amino acid sequence being fused to the N-terminal residue of IGHJ1 (SEQ ID NO 86). The corresponding full-length heavy chain sequence of SEQ ID NO 67 also comprises the amino acid sequence constant H (SEQ ID NO 87).

[0325] The full-length heavy chain sequence of SEQ ID NO 67 carrying the inserted rat ANP (rANP) was paired with the full-length light chain sequence of SEQ ID NO 66 constructed by combining the sequences LV1-40 (SEQ ID NO 88), IGLJ2 ​​(SEQ ID NO 89) and constant L (SEQ ID NO 90) to generate the complete IgG candidate TPP-5661 (see Table 1).

[0326] Shown below is the full-length heavy chain sequence (SEQ ID NO 67); the included heterologous amino acid sequence (Ntls-rANP-Ctls) is underlined; the sequences derived from HV 3-23 and IGHJ1 are shown in bold:

[0327]

[0328] The designed and synthesized antibody constructs were cloned according to methods well known in the art and confirmed by DNA sequencing using plasmid-specific oligonucleotides.

[0329] Example 2: Insertion of NP into antibody-CDR results in increased serum half-life

[0330] Determination of in vivo pharmacokinetic parameters

[0331] The 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 administered as a single rapid intravenous injection via the tail vein. Blood samples were collected from the jugular vein via a previously implanted catheter at intervals of up to 14 days (336 hours). The resulting EDTA-plasma was stored at -20°C until further analysis.

[0332] TPP-10992 and TPP-5661 in plasma samples were quantified using an anti-human IgG ELISA (enzyme-linked immunosorbent assay) format. Pharmacokinetic parameters were calculated from the plasma concentration-time profiles using non-compartmental data analysis.

[0333] Figure 1 The mean plasma concentrations of TPP-10992 and TPP-5661 over time following intravenous administration are graphically depicted in FIG.

[0334] Table 2 below summarizes the mean clearance and terminal half-life of TPP-10992 and TPP-5661.

[0335] Table 2: Mean clearance (CL) and terminal half-life (t) of TPP-10992 and TPP-5661 in rats after intravenous administration of 5 mg / kg 1 / 2 ).

[0336] Analytes TPP-5661 TPP-10992 CL [mL / h / kg] 0.62 0.27 <![CDATA[t 1 / 2 ]]> [h] 297 184

[0337] Determination of in vivo pharmacokinetic parameters

[0338] The pharmacokinetic parameters of TPP-12897 were determined after intraperitoneal administration to female Balb / c mice (n=3). Blood samples were collected 15 minutes to up to 72 hours after administration. The resulting EDTA-plasma was stored at -20°C until further analysis. TPP-12897 in plasma samples was quantified using an anti-human IgG (immunoglobulin G) ELISA method.

[0339] Pharmacokinetic parameters were calculated from the plasma concentration-time profiles using non-compartmental data analysis.

[0340] Figure 2 Mean plasma concentrations of TPP-12897 over time following intraperitoneal administration are graphically depicted in FIG.

[0341] Table 3 below summarizes the mean area under the curve (AUC) and terminal half-life of TPP-12897.

[0342] Table 3: Mean area under the curve (AUC) and terminal half-life (t) of TPP-12897 after intraperitoneal administration of 5 mg / kg in mice 1 / 2 ).

[0343] Analytes TPP-12897 AUC [mg·h / L] 7705 <![CDATA[t 1 / 2 ]]> [h] 194

[0344] Example 3: In vitro, ex vivo and in vivo titers of NP-grafted antibodies

[0345] Activity data of ANP-grafted antibodies in NPR-A receptor cell lines

[0346] As previously described, a luminescent rat ANP receptor (NPR-A) cell line was generated (Wunder et al. (2013), Eur J Pharmacol. 698: 131). Therefore, a fluorescence-based rat ANP receptor (NPR-A) cell line was generated by co-transfecting a CHO cell line with plasmid constructs encoding CNGA2 (cGMP biosensor) and rat NPR-A, stably expressing the fluorescent calcium sensor protein GCaMP6.

[0347] ANP receptor GCaMP6 cells (2500 cells / well) were cultured in black transparent bottom 384-well microtiter plates for one day. After removing the cell culture medium, the reporter cells were loaded with Tyrode (130mM NaCl, 5mMKCl, 2mM CaCl2, 20mM HEPES, 1mM MgCl2, 4.8mM NaHCO3, pH 7.4) containing black masking dye for 20 minutes at 37°C and 5% CO2. IBMX (0.2mM) was used to prevent cGMP degradation by endogenous phosphodiesterase.

[0348] Fluorescence measurements were started directly after the addition of agonists (3 min, kinetic mode). Receptor ligands were added in Tyrode containing black masking dye and 0.1% BSA. Measurements are made on.

[0349] ANP (Bachem, H-2100) stimulated concentration-dependent fluorescence signals in NPR-A cell lines, EC 50 The value is 0.22nM. TPP-5661 and TPP-10992 stimulated the rat ANP receptor reporter cell line, EC 50 The values ​​were 17 nM and 180 nM. The control antibody construct TPP-5657 did not significantly stimulate the NPR-A cell line (tested up to a maximum concentration of 460 nM).

[0350] To determine the sensitivity to proteolytic degradation, the activity of receptor ligands was also characterized after incubation for 4 h at 37°C 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).

[0351] Figure 3 The stability of ANP (AC), TPP-10992 (DF), and TPP-5661 (GI) against proteolytic degradation is graphically depicted. Figure 3 As shown, natriuretic peptide ANP (Bachem, H-2100) showed high sensitivity to degradation by NEP and IDE. In contrast, TPP-5661 and TPP-10992 showed high resistance to proteolytic degradation by NEP and IDE.

[0352] Activity data of BNP-grafted antibodies in NPR-A receptor cell lines

[0353] As previously described, a luminescent rat BNP receptor (NPR-A) cell line was generated (Wunder et al. (2013), Eur J Pharmacol. 698: 131). Therefore, a fluorescence-based rat BNP receptor (NPR-A) cell line was generated by co-transfecting a CHO cell line with plasmid constructs encoding CNGA2 (cGMP biosensor) and rat NPR-A, stably expressing the fluorescent calcium sensor protein GCaMP6.

[0354] BNP receptor GCaMP6 cells (2500 cells / well) were cultured in black transparent bottom 384-well microtiter plates for one day. After removing the cell culture medium, the reporter cells were loaded with Tyrode (130mM NaCl, 5mMKCl, 2mM CaCl2, 20mM HEPES, 1mM MgCl2, 4.8mM NaHCO3, pH 7.4) containing black masking dye for 20 minutes at 37°C and 5% CO2. IBMX (0.2mM) was used to prevent cGMP degradation by endogenous phosphodiesterase.

[0355] Fluorescence measurements were started directly after the addition of agonists (3 min, kinetic mode). Receptor ligands were added in Tyrode containing black masking dye and 0.1% BSA. Measurements are made on.

[0356] BNP (Bachem, H-5968) stimulates concentration-dependent fluorescence signals in NPR-A cell lines, EC 50The value is 2.9nM. TPP-9902, TPP-11153, TPP-1154, TPP-11155, TPP-11156 and TPP-11157 stimulated the rat BNP receptor reporter cell line, EC 50 The values ​​were 2.3 μM, >1.9 μM, 7 nM, 12 nM, 1.2 μM and 11 nM. The control antibody construct TPP-5657 did not significantly stimulate the NPR-A cell line (tested up to a maximum concentration of 460 nM).

[0357] To determine the sensitivity to proteolytic degradation, the activity of the receptor ligands was also characterized after incubation for 4 hours at 37°C 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). The natriuretic peptide BNP (Bachem, H-5968) showed a higher sensitivity to degradation by NEP and IDE. In contrast, TPP-11155 and TPP-11157 showed a higher resistance to proteolytic degradation by NEP and IDE.

[0358] Figure 4 The stability of BNP (AC) and TPP-11155 (DF) against proteolytic degradation is graphically depicted.

[0359] Activity data of CNP-grafted antibodies in NPR-B receptor cell lines

[0360] A luminescence-based rat CNP receptor (NPR-B) reporter cell line was generated and luminescence measurements were performed as previously described (Wunder et al. (2013), Eur J Pharmacol. 698:131).

[0361] CNP receptor cells (2500 cells / well) were cultured in opaque 384-well microtiter plates for one day. After removing the cell culture medium, the cells were incubated with a Ca-free 2+ Cells were loaded with 2.5 μg / ml coelenterazine in Tyrode (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl2, 4.8 mM NaHCO3, pH 7.4) for 3 h. 2+ Receptor ligands were added to the Tyrode for 10 minutes. IBMX (0.2 mM) was used to prevent cGMP degradation by endogenous phosphodiesterases. Luminescence measurements were started in a light-tight box using a charge-coupled device (CCD) camera and calcium ions were immediately added (final concentration 3 mM). Luminescence was monitored for 50 seconds.

[0362] CNP (Bachem, H-1296) stimulates concentration-dependent luminescence signals on rat NPR-B cell lines, EC 50 The value is 0.024nM. TPP-9465, TPP-12377, TPP-12378, TPP-12897 and TPP-12899 stimulated the rat CNP receptor reporter cell line, EC 50 The values ​​are 5.2nM, 4.1nM, 25nM, 10nM and 3.2nM. TPP-12374 stimulates rat CNP receptor reporter cell line, EC 50 The value was 150 nM, and TPP-12375 and TPP-12376 showed only weak signs of activity.

[0363] To determine the sensitivity to proteolytic degradation, the activity of receptor ligands was also characterized after incubation for 4 h at 37°C 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).

[0364] Compared to the natriuretic peptide CNP (Bachem, H-1296), TPP-12377, TPP-12897, and TPP-12899 showed higher resistance to proteolytic degradation by NEP and IDE.

[0365] Figure 5 The stability of CNP (AC) and TPP-12897 (DF) against proteolytic degradation is graphically depicted.

[0366] Activity data of ANP-grafted antibodies measured using isolated rat aortic rings

[0367] All experiments were performed in accordance with institutional guidelines and approved by the local animal experimentation committee. Male Sprague-Dawley rats (weight 250-300 g) were anesthetized with sodium pentobarbital (40 mg / kg ip), decapitated, and exsanguinated. The thoracic aorta was removed and placed in ice-cold Krebs buffer with the following components: 130 mM NaCl, 14.9 mM NaHCO3, 5.5 mM dextrose, 4.7 mM KCl, 1.18 mM KH2PO4, 1.17 mM MgSO47H2O, and 1.6 mM CaCl22H2O. The vessels were fixed in a Sylgard dish filled with frozen Krebs solution, cleaned of fat and connective tissue, and cut into ring segments of about 3 to 4 mm in length. Aortic rings were mounted vertically in a 50 ml chamber (ADI Instruments) containing Krebs solution, bubbled continuously with a mixture of 95% O2 and 5% CO2 at 37° C. Isometric force changes were recorded using a PowerLab data acquisition system (software Lab Chart 7.0).

[0368] After the equilibration period, aortic rings were stimulated with 80 mM KCl to check tissue viability. Next, the endothelial integrity of the preparation was determined by verifying the reactivity to acetylcholine (ACh, 1 μM) in vessels pre-contracted with phenylephrine (PE, 1 μM). After a period of washout and equilibration, contraction was induced with phenylephrine (PE, 1 μM), and vasodilation was then assessed with natriuretic peptides and natriuretic peptide-grafted IgG. Rat ANP peptide (ADH-GM-10057T, Santai Labs) was used as a reference.

[0369] like Figure 6 As shown, both ANP peptide and TPP-10992 induced dose-dependent vasodilation in PE-contracted aortic rings.

[0370] like Figure 7 As shown, both ANP peptide and TPP-5661 induced dose-dependent vasodilation in PE-contracted aortic rings.

[0371] Activity data of ANP-grafted antibodies obtained in awake rats

[0372] Blood pressure and heart rate were monitored by radiotelemetry (Data Sciences International) in freely moving, conscious animals. Female spontaneously hypertensive rats (SHR / N Crl BR, Charles River) weighing 210-300 g were used for these studies. All animals were housed in individual cages at an ambient temperature of 22-24°C and maintained on a 12-h light / dark cycle with free access to standard laboratory rat chow and water. The animals were implanted with a telemeter (HD-10, DSI) for a minimum of 14 days before the animals were used for blood pressure measurements. Surgery was performed under sterile conditions. After scraping the abdominal wall, a midline abdominal incision was made and a fluid-filled sensor catheter was inserted upstream into the exposed descending aorta between the iliac artery bifurcation and the renal artery. According to the DSI guidelines, the tip of the telemetry catheter was located caudal to the renal artery and secured by tissue adhesive. The transmitter body was affixed to the inner peritoneal wall and the abdomen was then closed. For surgical protection against infection and pain, a single dose of antibiotics ( 10%, 60 mg / kg sc, 0.06 ml / 100 g body weight, Beta-Pharma GmbH & Co, Germany) and analgesics ( 4 mg / kg sc, Pfizer, Germany). Telemetry data acquisition was performed using DSI software, with a predefined repeated acquisition of sample hemodynamic data for 10 seconds every 5 minutes. Data acquisition began at least 2 hours before dosing and ended after the measurement cycle was completed. Data are expressed as % ± SEM of the basal value for at least 4 animals per group. The basal value for each animal was calculated as the average of the measurements taken two hours before administration of the substance (7:00-9:00 am). Data were then expressed as averages every half hour, starting from 15 minutes after administration. All animals were treated with a single intraperitoneal (ip) administration of the test substance dissolved in phosphate buffered saline (PBS). Dosing was performed at 9:00 am (0 hours).

[0373] Figure 8 The hemodynamic effects of ANP peptide are graphically depicted. Fig. 9 The hemodynamic effects of TPP-5661 are graphically depicted. Fig.10 The hemodynamic effects of TPP-10992 are graphically depicted.

[0374] Example 4: Generation of different NP-grafted antibody constructs

[0375] For constructs in which natriuretic peptides are incorporated into non-CDRH3 CDR regions, a CDRH3 that is presumed to be functionally neutral was designed by fusing a three-residue stretch of Leu Thr Gly (IGHD7-27*01) to the C-terminus of HV 3-23 and the N-terminus of IGHJ1 (Comparative Example 1). The corresponding full-length heavy chain sequence of SEQ ID NO 65 also includes the amino acid sequence constant H (SEQ ID NO 87).

[0376] The full length heavy chain sequence of SEQ ID NO 65 without any natriuretic peptide insert was paired with the full length light chain sequence of SEQ ID NO 66 described in Example 1 to generate a synthetic, putatively neutral IgG negative control, TPP-5657.

[0377] The designed and synthesized antibody constructs were cloned according to methods well known in the art and confirmed by DNA sequencing using plasmid-specific oligonucleotides.

[0378] Starting from this antibody scaffold, the following ANP-grafted antibody constructs were generated.

[0379] Table 4: Design of ANP-grafted antibody constructs

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389] 1 the number of amino acid residues present between each N-terminal reference amino acid residue and the first amino acid of the inserted natriuretic peptide;

[0390] 2 the number of amino acid residues present between the last amino acid of the inserted natriuretic peptide and each C-terminal reference amino acid residue;

[0391] Table 4 continued: Design of ANP-grafted antibody constructs

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401] 3 The N-terminal sequence corresponds to the reference aa of the nearest N-terminal neighbor of the inserted natriuretic peptide plus the amino acid stretch existing between the reference aa and the first amino acid residue of the inserted natriuretic peptide.

[0402] 4 The C-terminal sequence corresponds to the amino acid stretch existing between the last amino acid residue of the inserted natriuretic peptide and the reference aa nearest to the C-terminus of the inserted natriuretic peptide plus the reference aa

[0403] Furthermore, starting from the antibody scaffold TPP-5657, the following BNP-grafted human IgG1 antibody constructs were generated.

[0404] Table 5: Design of BNP-grafted antibody constructs

[0405]

[0406] 1 the number of amino acid residues present between each N-terminal reference amino acid residue and the first amino acid of the inserted natriuretic peptide;

[0407] 2 the number of amino acid residues present between the last amino acid of the inserted natriuretic peptide and each C-terminal reference amino acid residue;

[0408] 3 The corresponding ANP compound refers to an ANP-grafted antibody construct having the same integration locus and containing the same N-terminal and C-terminal sequences.

[0409] Table 5 continued: Design of BNP-grafted antibody constructs

[0410]

[0411]

[0412] 3 The N-terminal sequence corresponds to the reference aa of the nearest N-terminal neighbor of the inserted natriuretic peptide plus the amino acid stretch existing between the reference aa and the first amino acid residue of the inserted natriuretic peptide.

[0413] 4 The C-terminal sequence corresponds to the amino acid stretch existing between the last amino acid residue of the inserted natriuretic peptide and the reference aa nearest to the C-terminus of the inserted natriuretic peptide plus the reference aa

[0414]

[0415]

[0416] Example 5: In vitro activity of generated constructs

[0417] All constructs were transiently expressed in HEK293 cells according to methods well known in the art, with a targeted cell density of approximately 2x10^6 cells / ml and a total DNA concentration of approximately 1 μg / ml for the two plasmids encoding the light and heavy chains, and incubated for 5 days for expression.

[0418] Original compound sample (raw compound sample, rcs) is expressed with 0.4ml culture volume, and the supernatant of centrifugation is directly used for test.According to methods known in the art, compound concentration is assessed by IgG-Fc quantitative ELISA.In brief, the supernatant diluted 1:1500 and the human reference serum (Bethyl, RS-110-4) of 2 times dilution series starting with 400ng / ml are fixed at 37 ℃ for 1 hour in black Maxisorp 384 microtiter plates (MTP), and the microtiter plates are coated with the anti-human Fc [Sigma I2136] of 1x coating buffer (Candor, 121125) 1:440 dilution.After sealing with 100% SMART Block (Candor, 113125), the anti-human Fc-HRP [Sigma, A0170] of 1:10000 dilution is used to detect the antibody in rcs and reference samples. The dose curve of the reference samples was used to quantitatively assess the compound concentrations shown in Tables 7 and 8, "μg / ml rcs" column. All samples were administered in quadruplicate.

[0419] According to methods well known in the art, isolated compound samples (ics) were produced from 6 ml of expression culture by one-step purification with Protein-A. The acidic eluate was neutralized by adding 8% (v / v) 1M Tris / HCl (pH 9.0), quantified by absorbance at 280 nm, and normalized to a concentration of 125 nM.

[0420] Purified compound samples (pcs) were generated from at least 35 ml of expression culture by two-step purification with protein-A and subsequent SEC in PBS buffer. The values ​​shown in Tables 7 and 8, "μg / ml pcs" column, refer to the compound concentration in the expression culture supernatant determined by analytical protein A chromatography.

[0421] All activities shown in Tables 7 and 8 were measured on cells heterologously overexpressing human NPRA (hNPRA) using a cGMP quantification assay performed according to the manufacturer's instructions (cisbio; 62GM2PEH). Briefly, the assay quantifies cGMP in buffer solution or cell culture supernatant based on competition between cGMP produced by cells due to NPRA stimulation with (natriuretic peptide) samples and d2-labeled cGMP for binding to a cryptate-labeled antibody. Sample cGMP and d2-labeled cGMP compete for binding to a limited number of sites on the cryptate-labeled anti-cGMP antibody, thus The specific fluorescence signal (ie, energy transfer) is inversely proportional to the concentration of cGMP in the sample.

[0422] Dose response curve data were analyzed using GraphPad Prism (Windows version 7.00, GraphPad Software, La Jolla California USA) and EC50 was fitted according to Y = Bottom + (Top - Bottom) / (1 + 10^((LogEC50-X)*HillSlope) with constraints imposed on bottom, top and slope (shared values ​​for all data sets for each experiment).

[0423] The activity of the original compound samples on stable hNPRA-CHO k1 cells was first evaluated by comparison with the negative control TPP-5657 and the positive samples (particularly TPP-5661). Controls and RCS were tested at two concentrations (in quadruplicate) with a relative dilution factor of 5 in order to have the fluorescence signal within the dynamic range of the assay. The assay window was defined as the difference between the inactive sample signal (maximum signal, s_max) and the high active sample signal (minimum signal, s_min), and the % activity at these two compound concentrations was calculated as 100*(s_max-s) / (s_max-s_min). The values ​​listed in Table 7, the "Active RCS" and "Stdev Active RCS" columns, represent the mean of the results at the two concentrations and their respective standard deviations. RCS signals less than half of the reference compound TPP-5661 signal (36%) were assessed as inactive (na).

[0424] The activity of several original compound samples on stable hNPRA-CHO k1 cells was re-evaluated by comparing with a 2.5-fold dilution series (8 concentrations) of the reference sample TPP-5661 starting with a 5-fold dilution. The "logEC50" fitting value as a measure of rcs activity was set relative to the corresponding value of the reference rcs TPP-5661 by calculating δ "logEC50"_compound-"logEC50"_TPP-5661; the resulting values ​​are listed in Table 7, "rel. active rcs" column. It is worth noting that the compound concentration in rcs is not taken into account, so the given value is affected by the activity and concentration of the compound of equal measure. All samples were applied in quadruplicate.

[0425] The activity of the isolated compound samples on stable hNPRA-CHO k1 cells was evaluated by EC50 determination and comparison with the reference sample TPP-5661. The samples were tested in a 2.5-fold dilution series from 80nM to 0.13nM. The logEC50 fitting value as a measure of ics activity was set relative to the corresponding value of the reference ics TPP-5661 (-8.8) by calculating delta logEC50_compound-logEC50_TPP-5661; the resulting values ​​are listed in Table 7, "rel.logEC50 ics" column. All samples were applied in quadruplicate.

[0426] The activity of purified compound samples on stable hNPRA-CHO k1 cells was evaluated in multiple experiments by EC50 determination and comparison with reference sample TPP-5661. Samples were tested in dilution series, at least in quadruplicate. The values ​​listed in the "rel.logEC50 pcs 1, 3 and 4" columns in Table 7 are from a 5-fold dilution series (≥4 concentrations) starting from 20 or 25 nM. The values ​​listed in the "rel.logEC50 pcs 2" column in Table 7 are from a 10-fold dilution series (4 concentrations) starting from 25 nM. The values ​​listed in the "rel.logEC50 pcs 5, 6, 7, 8 and 9" columns in Table 7 are from a 2.5-fold dilution series starting from 40 to 200 nM, respectively, with 8 or 12 concentrations. The logEC50 fitted value as a measure of pcs activity was set by calculating delta logEC50_compound-logEC50_TPP-5661 relative to the corresponding value of the reference TPP-5661 in the respective experiment (mean value -9.2, standard deviation 0.5).

[0427] The activity of the purified compound samples on transient hNPRA-HEK293 cells was evaluated in three experiments by EC50 determination and comparison with reference sample TPP-5661. The samples were tested in a 5-fold dilution series from 1000nM to 0.013nM. By calculating δlogEC50_compound-logEC50_TPP-5661, the logEC50 fitting value as the pcs activity measure was set relative to the corresponding value of the reference TPP-5661 in each experiment (mean value of -9.0, standard deviation of 0.4); the result values ​​are listed in Table 7, "rel.logEC50*pcs" column. All samples were applied in quadruplicate.

[0428] A summary qualitative assessment of the activity of the compounds is given in the "qualit.activity" column, and the values ​​listed in the "mean rel.logEC50" column are calculated as the mean of the given rel.logEC50 values. Compounds showing EC50 values ​​>150 times that of the reference compound TPP-5661 (i.e., mean rel.logEC50 >1.7) were qualitatively assessed as inactive ("-"); compounds showing relative logEC50 between 0.7 and 1.7 were assessed as active ("+"); compounds showing relative logEC50 <0.7 were assessed as very active ("++"); and the highest activity was observed at relative logEC below -0.7 ("+++"). Compounds that showed activity in rcs but were not confirmed as ics or pcs were marked with a uniform "y", and compounds that were assessed as inactive ("na" in the "active rcs" column) probably due to their very low expression levels (≤1 μg / ml) were also marked accordingly.

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436] No conclusive data were obtained for compounds #104 and #135. 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 therefore no activity; activity of #61 was shown after compound preparation (pcs). In most cases, 7 compounds (#7, #24, #70, #83, #90, #154, #167) showed low activity as rcs, although their expression levels were very low; activity of #24 and #90 was confirmed by compound preparation (pcs). No activity was observed in the rcs of compounds #18, #26, #29, #92, #96, #101, #104, #158, #164, and #179; however, activity was shown for compounds #18, #92, #158, and #164 when higher concentrations were used (rel. active rcs); thus, the lack of activity of #26 was confirmed.

[0437] Table 8: Expression levels and activities of brain natriuretic peptide grafted antibody constructs Not applicable (na)

[0438]

[0439] 1 the number of amino acid residues present between each N-terminal reference amino acid residue and the first amino acid inserted into the natriuretic peptide;

[0440] 2 the number of amino acid residues present between the last amino acid of the inserted natriuretic peptide and each C-terminal reference amino acid residue;

[0441] 3 The corresponding ANP Cpd refers to an ANP-grafted antibody construct with the same integration locus and containing the same N-terminal and C-terminal sequences.

[0442] A dilution series of purified compound samples were tested in quadruplicate on stable hNPRA-CHO k1 cells as described in Example 3. Fig.11 The activity of the BNP-grafted antibody constructs is graphically depicted in FIG.

[0443] Compared with TPP-1154, TPP-11155 and TPP-11157, TPP-11156, TPP-9902 and TPP-11153 showed significant activity on hNPRA cells. The opposite results were observed on rNPRA, with EC50 <20nM for TPP-1154, TPP-11155 and TPP-11157 and EC50>1μM for TPP-9902, TPP-11153 and TPP-11156 (see Example 3). This can be explained by the presence of human BNP sequences in TPP-11156, TPP-9902 and TPP-11153, while TPP-1154, TPP-11155 and TPP-11157 contain rat BNP sequences (see Table 5). Compared to all other human BNP-grafted antibody constructs, TPP-18031 and TPP-18032, which have a smaller number of additional amino acids at the N- and C-termini of BNP, were inactive on hNPRA.

[0444] Example 6: Specific linker sequences are particularly beneficial for obtaining good homogeneity and expression levels

[0445] Purity of purified compound samples was determined by capillary gel electrophoresis under reducing conditions according to the manufacturer's instructions (LabChip GX, Caliper Life Sciences) (Example 5, Table 7, "% Purity pcs" column). % Purity was calculated as the sum of the peak areas corresponding to the intact light chain and heavy chain relative to the sum of all observed peaks.

[0446] Ntls sequences comprising a GS linker sequence, a PN linker sequence or a sequence of SEQ ID NO 2, 4, 9, 11, 13 or 15, and Ctls sequences comprising a GS linker sequence, a PN linker sequence or a sequence of SEQ ID NO 3, 5, 12, 14, 15 or 20 proved to be particularly useful because they not only achieved high natriuretic peptide activity (as long as there were at least 12 amino acid residues between each N-terminal reference amino acid residue and the first amino acid of the inserted natriuretic peptide), but also achieved good expression levels (compared to the sequences used in, for example, compounds #186, #195 and #202) and (compared to the sequences used in compounds 6 and 7) lower heterogeneity (see Table 9). Very good purity was observed with a mean of 98% when using a linker sequence comprising a GS linker sequence and a linker sequence comprising a PN linker sequence. Similarly, good values ​​were observed for compounds having a linker comprising a sequence of SEQ ID NO 2, 3, 4, 5, 9, 11, 12, 13, 14, 15 and 20. It is noteworthy that Ntls with sequence SEQ ID NO 9 achieves very high purity only when combined with Ctls with sequence SEQ ID NO 20 in a compound; compounds containing a combination of Ntls with sequence SEQ ID NO 11 and Ctls with sequence SEQ ID NO 12 show very high purity only when the N-terminal side of SEQ ID NO 11 is flanked by Asp (D) instead of Thr (T) or Val (V) and when the C-terminal side of SEQ ID NO 12VNHLRSEKLT is flanked by Gly (G) instead of Tyr (Y) or Phe (F), such as in compounds #126 and #135.

[0447]

[0448]

[0449]

[0450]

[0451] Example 7: IgG1, IgG2 and IgG4 isotypes provide equally suitable antibody scaffolds

[0452] 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. A dilution series of purified compound samples were tested in quadruplicate on stable hNPRA-CHO k1 cells as described in Example 3. The activities of ANP-grafted human IgG2 and IgG4 isotype constructs (e.g., compound 9 IgG4 TPP-10992) were similar to their corresponding IgG1 isotypes, as shown in Table 3. Fig.12 Depicted graphically in .

[0453] Example 8: Human and non-human IgG provide equally suitable antibody scaffolds

[0454] Compound 9 (human IgG1 TPP-10294 and IgG4 TPP-10992) and compound 117 (human IgG1 TPP-5665) were generated as non-human IgG isotypes. A dilution series of purified compound samples were tested in quadruplicate on stable hNPRA-CHO k1 cells as described in Example 3. The activity of ANP-grafted rat and mouse isotype constructs (e.g., compound 9 rat IgG1 TPP-13992) was similar to that of their corresponding human IgG isotypes ( Fig.13 ).

[0455] Example 9: Human IgG containing different germline sequences provides equally suitable antibody scaffolds

[0456] 22 additional ANP-grafted IgG4 antibodies (compounds A to S) were constructed. In each case, ANP was incorporated into CDRH1. The heavy chains of these constructs contained different HV and CDRH3 sequences and were paired with different λ or κ light chains. Tables 10 and 11 summarize the structures of compounds A to S.

[0457] Table 10: Design of ANP-grafted antibody constructs A to S

[0458]

[0459] 1 the number of amino acid residues present between each N-terminal reference amino acid residue and the first amino acid of the inserted natriuretic peptide;

[0460] 2 the number of amino acid residues present between the last amino acid of the inserted natriuretic peptide and each C-terminal reference amino acid residue;

[0461] 3The N-terminal sequence corresponds to the reference aa of the nearest N-terminal neighbor of the inserted natriuretic peptide plus the amino acid stretch existing between the reference aa and the first amino acid residue of the inserted natriuretic peptide.

[0462] 4 The C-terminal sequence corresponds to the amino acid stretch existing between the last amino acid residue of the inserted natriuretic peptide and the reference aa nearest to the C-terminus of the inserted natriuretic peptide plus the reference aa

[0463] Table 11: Design of ANP-grafted antibody constructs A to S

[0464] 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-1 3957 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

[0465] A dilution series of purified compound samples of IgG scaffold constructs A to S containing different germline sequences were tested in quadruplicate on stable hNPRA-CHO k1 cells as described in Example 3. Fig.14 Exemplary activity data are graphically depicted in .

[0466] Example 10: CNP-grafted IgG protects against induced endothelial barrier permeability Endothelial monolayer permeability was assessed by real-time impedance measurement with the xCELLigence RTCA system using microelectrode-covered microtiter plates (E-Plate). Relative impedance changes were expressed as unitless cell index (CI) values.

[0467] Primary human pulmonary artery endothelial cells (HPAEC) were seeded at low passage numbers on collagen pre-coated E-Plates. After the tight monolayer and cell barrier with constant CI values ​​were formed, HPAEC were pretreated with the compound TPP-12899 or the respective negative control antibody construct TPP-5657 at the concentrations shown, and then the EC barrier was destroyed with the destructive agonist LPS (200 ng / ml), IL-1β (0.5 ng / ml) or thrombin (2 U / ml). CI was recorded every 10 minutes to monitor the effects on cell growth and monolayer permeability. All cell indexes were normalized at the last recording point, and then the test substance (= normalized CI) was applied.

[0468] Experiments were performed with n = 4 and 3 technical replicates per experiment. Results are presented as mean ± SEM. Data were statistically analyzed using one-way ANOVA and Sidak's multiple post hoc test; p values ​​< 0.05 were considered significant.

[0469] Fig.15 The effect on endothelial monolayer permeability expressed as Cell Index values ​​is graphically depicted. Fig.15As shown in , pretreatment of human endothelial monolayer cultures with TPP-12899 protected against induced endothelial barrier permeability in a dose-dependent manner. This was independent of the barrier disruptor administered; significant effects were observed with both fast- and strong-acting thrombin, as well as the long-acting proinflammatory stimuli LPS and IL-1β. The respective negative controls were used to show the effect.

[0470] Example 11: Long-term effects of ANP-grafted IgG in a rat model of chronic heart failure (TGR(mRenR2)27

[0471] The TGR (mRenR2) 27 rat model shows hypertension and endothelial dysfunction as well as end-organ damage. Male renin transgenic rats aged 8 weeks were used (Ganten D., Nature. 1990; 344 (6266): 541-4). In all study groups, a non-selective inhibitor of nitric oxide synthase L-NAME (Nω-nitro-L-arginine methyl ester) was administered chronically through drinking water (20 mg / l) to induce endothelial dysfunction. The rat IgG1 counterpart TPP-13992 of TPP-10294 used in Example 8 and the rat IgG1 isotype control antibody TPP-10155 were administered intraperitoneally once a week. Body weight and survival rate were assessed. From weaning, the placebo group was treated with a solvent (vehicle) (PBS) and the healthy control group was treated with captopril-food. Food and water were taken at will. The behavior and general health of the animals were observed daily. At the end of the experiment (week 14), the rats were anesthetized. The rats were then bled and the hearts removed from the chest for analysis. Urine was collected at the end of the study to determine various urine parameters such as urine protein to creatinine ratio. Fig.16 The treatment effects of TPP-13992 on survival, weight gain, urine protein / creatinine ratio, and left atrial weight are graphically depicted.

[0472] Example 12: Effects of ANP-grafted IgG on hemodynamics in healthy beagle dogs

[0473] In the primary pharmacodynamic study, the effects of TPP-10992 on cardiovascular and ECG parameters following a single subcutaneous administration were evaluated in awake, telemetered beagle dogs.

[0474] Surgically implanted telemetry device (DSI TM, USA) to measure blood pressure and heart rate, and then undergo a recovery period to allow wound closure. On the day of the study, the telemetry sensor was activated for continuous hemodynamic measurements. The transmission signal was collected by a telemetry receiver located in the animal facility. All collected data were processed by a data acquisition program and averaged over a predetermined 12-hour period. For TPP-10992, NaCl (0.9%) was used as a solvent (vehicle) and doses of 0.1 mg / kg, 0.3 mg / kg and 1.0 mg / kg body weight were administered by subcutaneous injection.

[0475] Fig.17 The results are graphically depicted in . In healthy dogs, TPP-10992 showed a dose-dependent and long-term (>5d) reduction in blood pressure, which was significant at 1.0 mg / kg sc (compared to placebo). No effect on heart rate was observed.

Claims

1. An antibody or a fragment thereof for use in treating and / or preventing hypertension, heart failure and / or kidney disease, the antibody or a fragment thereof comprising at least one heterologous amino acid sequence, wherein the at least one heterologous amino acid sequence consists of an N-terminal linker sequence (Ntls), a brain natriuretic peptide (BNP) and a C-terminal linker sequence (Ctls), wherein the amino acid sequences of the heavy chain and the light chain of the antibody are selected from the following groups: The heavy chain is SEQ ID NO 442, and the light chain is SEQ ID NO 66; The heavy chain is SEQ ID NO 443, and the light chain is SEQ ID NO 66; The heavy chain is SEQ ID NO 444, and the light chain is SEQ ID NO 66; The heavy chain is EVQLLESGGGLVQPGGSLRLSCAASGFTFGSGSGSGSGSPDGGSGGMVQGSGCFGRKMDRISSSSGLGCKVLRRGSYGSGSGSGSGMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLTGAEYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, The light chain is SEQ ID NO 66; The heavy chain is EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSTYISNVNHKPDGGSGGMVQGSGCFGRKMDRISSSSGLGCKVLRRGSNTKVDKKVEGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLTGAEYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, The light chain is SEQ ID NO 66; The heavy chain is EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGPNPNPNPNSPDGGSGGMVQGSGCFGRKMDRISSSSGLGCKVLRRGSYNPNPNPNPNPGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLTGAEYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, The light chain is SEQ ID NO 66; The heavy chain is SEQ ID NO 65, The light chain is QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCAAWNPNPNPNPNPNGGSGGMVQGSGCFGRKMDRISSSSGLGCK and The antibody fragment is selected from Fab, Fab', Fab'-SH, F(ab')2 or Fv fragment; a bivalent antibody; a single domain antibody (Dab); a linear antibody; a single chain antibody molecule (ScFv); or a disulfide bond stabilized Fv antibody fragment (dsFv).

2. Use of the antibody or fragment thereof according to claim 1, comprising at least one other natriuretic peptide.

3. Use of an antibody or fragment thereof according to claim 2, wherein the BNP and the at least one other natriuretic peptide are incorporated into at least two separate CDR regions.

4. Use of an antibody or fragment thereof according to claim 3, wherein the at least one other natriuretic peptide is selected from the group consisting of ANP, BNP and CNP.

5. Use of an antibody or fragment thereof according to claim 4, wherein the at least one other natriuretic peptide is selected from ANP and CNP.

6. Use of the antibody or fragment thereof according to any one of claims 1 to 5, wherein the antibody or fragment thereof is a human or humanized antibody or fragment thereof.

7. Use of the antibody or fragment thereof according to claim 6, wherein the antibody or fragment thereof is of the IgG class.

8. A composition for treating and / or preventing hypertension, heart failure and / or kidney disease, the composition comprising an antibody or a fragment thereof, the antibody or a fragment thereof comprising at least one heterologous amino acid sequence, wherein the at least one heterologous amino acid sequence consists of an N-terminal linker sequence (Ntls), a brain natriuretic peptide (BNP) and a C-terminal linker sequence (Ctls), wherein the amino acid sequences of the heavy chain and the light chain of the antibody are selected from the following groups: The heavy chain is SEQ ID NO 442, and the light chain is SEQ ID NO 66; The heavy chain is SEQ ID NO 443, and the light chain is SEQ ID NO 66; The heavy chain is SEQ ID NO 444, and the light chain is SEQ ID NO 66; The heavy chain is EVQLLESGGGLVQPGGSLRLSCAASGFTFGSGSGSGSGSPDGGSGGMVQGSGCFGRKMDRISSSSGLGCKVLRRGSYGSGSGSGSGMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLTGAEYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, The light chain is SEQ ID NO 66; The heavy chain is EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSTYISNVNHKPDGGSGGMVQGSGCFGRKMDRISSSSGLGCKVLRRGSNTKVDKKVEGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLTGAEYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, The light chain is SEQ ID NO 66; The heavy chain is EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGPNPNPNPNSPDGGSGGMVQGSGCFGRKMDRISSSSGLGCKVLRRGSYNPNPNPNPNPGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLTGAEYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, The light chain is SEQ ID NO 66; The heavy chain is SEQ ID NO 65, The light chain is QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCAAWNPNPNPNPNPNGGSGGMVQGSGCFGRKMDRISSSSGLGCK and The antibody fragment is selected from Fab, Fab', Fab'-SH, F(ab')2 or Fv fragment; a bivalent antibody; a single domain antibody (Dab); a linear antibody; a single chain antibody molecule (ScFv); or a disulfide bond stabilized Fv antibody fragment (dsFv).

9. Use of the composition according to claim 8, wherein the composition comprises a pharmaceutically acceptable carrier.

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