Atrial natriuretic peptide engrafted antibodies

Abstract

This invention relates to an antibody or fragment thereof comprising at least one heterologous amino acid sequence incorporated within 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), an atrial natriuretic peptide (ANP), 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. This invention also relates to the use of such antibodies or fragments thereof in therapeutic applications, 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 such nucleic acids, and methods for preparing such antibodies or fragments thereof.

Description

[Technical Field] This invention relates to an antibody or a fragment thereof, comprising at least one heterologous amino acid sequence incorporated within at least one CDR region of the antibody or fragment thereof, wherein the at least one heterologous amino acid sequence comprises an N-terminal linker sequence (Ntls), an atrial natriuretic peptide (ANP), 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. At least 12 amino acid residues exist in the following cases: when the heteroamino acid sequence is incorporated into CDRH1, according to the Kabat amino acid residue HC (heavy chain) res25 and the first amino acid residue of ANP; when the heteroamino acid sequence is incorporated into CDRH2, according to the Kabat amino acid residue HC res51 and the first amino acid residue of ANP; when the heteroamino acid sequence is incorporated into CDRH3, according to the Kabat amino acid residue HC res92 and the first amino acid residue of ANP; when the heteroamino acid sequence is incorporated into CDRL1, according to the Kabat amino acid residue LC (light chain) res26 and the first amino acid residue of ANP; when the heteroamino acid sequence is incorporated into CDRL2, according to the Kabat amino acid residue LC res49 and the first amino acid residue of ANP; and / or when the heteroamino acid sequence is incorporated into CDRL3, according to the Kabat amino acid residue LC res49 and the first amino acid residue of ANP. res88 is the first amino acid residue of ANP. Furthermore, at least nine amino acid residues exist between the last amino acid residue of ANP and the following: HC res35a of Kabat if the heterologous amino acid sequence is incorporated into CDRH1; HC res57 of Kabat if the heterologous amino acid sequence is incorporated into CDRH2; HC res106 of Kabat if the heterologous amino acid sequence is incorporated into CDRH3; LC res32 of Kabat if the heterologous amino acid sequence is incorporated into CDRL1; LC res57 of Kabat if the heterologous amino acid sequence is incorporated into CDRL2; and / or LC res98 of Kabat if the heterologous amino acid sequence is incorporated into CDRL3. This invention also relates to the use of such antibodies or fragments thereof in therapeutic methods, 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 such nucleic acids, and methods for preparing such antibodies or fragments thereof. [Previous Technology] Natriuretic peptides (NPs) belong to a family of three structurally related peptides with neurofluidic activity. Atrial natriuretic peptide (ANP) is a 28-amino acid peptide containing a central loop structure formed by disulfide bonds between cysteine ​​residues 7 and 23. In humans, ANP is a 153-amino acid pro-hormone in atrial myocytes. The signal peptide cleaves to produce the pro-hormone form, which is then further cleaved into mature ANP and an N-terminal remnant, called 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 loop structure. ANP is primarily produced and released by cardiomyocytes in the left and right atria, while BNP is primarily produced by cardiomyocytes in the ventricles. CNP is synthesized by vascular endothelial cells. Besides these locations, natriuretic peptides are also produced in small amounts in other parts of the body, such as the brain, kidneys, and adrenal glands. Natriuretic peptides (NPs) 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). Furthermore, important sequence modifications of NPs (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)) have been described to produce potent NPs that activate or bind to cellular receptors and induce related physiological effects. Natriuretic peptides (NPs) bind to three different membrane-bound receptor types, namely NPR-A, NPR-B, and NPR-C, thereby regulating their biological effects. ANP and BNP bind to NPR-A with the highest affinity; in contrast, CNP has the highest affinity for the NPR-B receptor. NPR-A and NPR-B contain a (granular) guanylate cyclase domain (pGC), whose enzymatic activity leads to an increase in (intracellular) cyclic guanosine monophosphate (cGMP). As a second messenger, cGMP regulates various cellular processes. The NPR-C receptor does not exhibit guanylate cyclase activity and is also known as a "clearance" receptor because it binds to NPs, causing their degradation via endopoiesis. Other signaling functions of NPR-C mediated by cAMP have been described (Anand-Srivastava, Peptides. June 2005; 26(6): 1044-59). Cardiac hormones ANP and BNP are secreted during ventricular and atrial contractions, such as due to excess plasma volume. They exert vasodilatory effects and lower blood pressure through relaxation of vascular smooth muscle. In the kidneys, ANP causes urinary frequency, i.e., increased urine secretion (polyuria), and increased concentration of sodium ions in urine (sodium excretion). 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 regulation of the baroreflex (Woods et al., Clin Exp Pharmacol Physiol. 2004 Nov; 31 (11): 791-4). For ANP, BNP, and CNP, anti-inflammatory, anti-hypertrophic, and anti-fibrotic effects have been demonstrated in animal models of various diseases (e.g., Knowles et al., 2001, J. Clin. Invest. 107: 975-984; Dahrouj et al., J Pharmacol Exp Ther. Jan 2013; 344 (1): 96-102; Baliga et al., Br J Pharmacol. Jul 2014; 171 (14): 3463-75; Mitaka et al., Intensive Care Med Exp. Dec 2014; 2 (1): 28; Werner et al., Basic Res Cardiol. Mar 2016; 111 (2): 22; Kimura et al., Respir Res. Feb 19 2016; 17: 19). NPR-B activation via CNP plays an important role in bone growth (Yasoda et al., Clin. Calcium. July 2009; 19(7): 1003-8) and vascular endothelial integrity (Moyes et al., J Clin Invest. September 2014; 124(9): 4039-51). The broad range of physiological effects of natriuretic peptides and their receptors makes them attractive targets for drug research (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 can be inhibited under a variety of pathophysiological conditions, leading to hypertension, increased cell proliferation, fibrosis, inflammation, endothelial dysfunction, diabetes, metabolic syndrome, atherosclerosis, heart failure, myocardial infarction, pulmonary hypertension, eye and kidney diseases, bone diseases, stroke, and / or sexual dysfunction. The main obstacle to the therapeutic use of natriuretic peptides is their extremely short plasma half-life of only a few minutes in vivo (Hunt et al., J Clin Endocrinol Metab. June 1994; 78 (6): 1428-35; Kimura et al., Eur J Clin Pharmacol. July 2007; 63 (7): 699-702). Besides endopoiesis via NPR-C receptors, natriuretic peptides are efficiently degraded by the enzymes enkephalinase (NEP) and insulin-degrading enzyme (IDE). The short-term biological effects associated with administered natriuretic peptides limit their therapeutic use primarily to acute indications. For example, in various countries, the infusion of recombinant carperitide (ANP) and nesiritide (BNP) has been approved for the treatment of acute decompensated heart failure. Providing NPR-A and NPR-B agonists with extended plasma half-life, higher proteolytic stability, and longer duration of action will greatly promote the treatment of chronic diseases. In recent years, several diuretic natriuretic peptide derivatives and variants have been described, such as CD-NP (McKie et al., Curr Heart Fail Rep. Sep 2010; 7 (3): 93-9), ZD100 / MANP (McKie et al., Hypertension. Dec 2010; 56 (6): 1152-9), PL-3994 (Edelson et al., Pulm Pharmacol Ther. Apr 2013; 26 (2): 229-38), Ularitide (Anker et al., Eur Heart J. Mar 21 2015; 36 (12): 715-2), ANX-042 (Pan et al., Proc Natl Acad Sci USA. July 7 2009; 106 (27): 11282-7), and BMN-111. (Wendt et al., J. Pharmacol Exp Ther. April 2015; 353 (1): 132-49). The half-life of CD-NP is approximately 18.5 minutes (Lee et al., BMC Pharmacology 2007, 7 (Supplement I): P38). Other ANP and CNP derivatives are disclosed in US 9,193,777 and EP 2 432 489 A, respectively. In addition, natriuretic peptide fusions, including Fc fusions, albumin fusions, and PEGylated natriuretic peptides, have been described. Natriuretic peptide-Fc fusions are disclosed, for example, in US 2010 / 0310561, WO 2008 / 154226, WO 2010 / 117760, WO 2006 / 107124, WO 2008 / 136611, and WO 2008 / 079995. Natriuretic peptide-albumin fusions are disclosed in US 7,521,424 and US 2014 / 0148390, and PEGylated natriuretic peptides are disclosed in US 2014 / 0148390. WO 2005 / 060642 describes the generation of ANP and BNP peptide-implanted antibody libraries by inserting ANPs or BNPs with two random flanking amino acids at each end into the CDRH3 region of a human tetanus toxoid-specific antibody. Similarly, WO 2005 / 082004 discloses the generation of ANP mimic-implanted antibody libraries by replacing the entire initial CDRH3 region of a 2G12 antibody with an ANP mimic peptide flanked by two random amino acid residues on either side. Neither WO 2005 / 060642 nor WO 2005 / 082004 discloses any specific diuretic natriuretic peptide-implanted antibodies, nor does it functionally characterize such antibodies. In view of prior art, one objective of the present invention is to provide a novel natriuretic natriuretic peptide receptor agonist that has increased stability in serum compared with naturally occurring wild-type natriuretic natriuretic peptides. [Summary of the Invention] The above-described objectives are achieved through the teachings of the independent claim. Although the short length and high sequence conservation of immunoglobulin CDR regions significantly restrict the incorporation of bioactive peptides, the inventors unexpectedly discovered that a bioactive natriuretic peptide variant exhibiting significantly increased stability in serum compared to naturally occurring wild-type natriuretic peptides can be obtained by incorporating the amino acid sequence of a natriuretic peptide into a CDR region of an immunoglobulin molecule or fragment thereof. However, the activity of the natriuretic peptide incorporated into the immunoglobulin CDR region was confirmed to be significantly altered. The inventors found that the decisive factor for the successful incorporation of bioactive natriuretic peptide variants is the number of amino acid residues between the incorporated natriuretic peptide and the nearest adjacent CDR scaffold junction. If the number of N-terminal and C-terminal amino acid residues flanking the diuretic natriuretic peptide at the junction with the adjacent CDR framework is less than a certain value, only diuretic natriuretic peptide immunoglobulin fusion constructs with no or significantly reduced biological activity can be obtained. It has been found that specific linker sequences for flanking the incorporated diuretic natriuretic peptide are particularly beneficial for achieving high peptide activity, good expression levels, and / or low protein fragmentation levels. Therefore, in the first state, 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 at least a portion of the at least one CDR region is optionally replaced by the at least one heterologous amino acid sequence incorporated therein, and wherein a) at least 12 amino acid residues are present in the following cases: i) in the case of the heterologous amino acid sequence being incorporated into CDRH1, according to the Kabat amino acid residue HC res25 (which corresponds to res S25 in the heavy chain having the amino acid sequence of SEQ ID NO 65) and the first amino acid residue of the natriuretic peptide; ii) in the case of the heterologous amino acid sequence being incorporated into CDRH2, according to the Kabat amino acid residue HC res51 (which corresponds to res S25 in the heavy chain having the amino acid sequence of SEQ ID NO 65) and the first amino acid residue of the natriuretic peptide; iii) In the case of incorporation of the heterologous amino acid sequence into CDRH3, the first amino acid residue of the natriuretic peptide is the first amino acid residue of the natriuretic peptide, based on the amino acid residue HC res92 of Kabat (which corresponds to res C96 in the heavy chain having the amino acid sequence of SEQ ID NO 65); iv) In the case of incorporation of the heterologous amino acid sequence into CDRL1, the first amino acid residue of the natriuretic peptide is the first amino acid residue of the natriuretic peptide, based on the amino acid residue LC res26 of Kabat (which corresponds to res S25 in the light chain having the amino acid sequence of SEQ ID NO 66); v) In the case of incorporation of the heterologous amino acid sequence into CDRL2, the first amino acid residue of the natriuretic peptide is the first amino acid residue of the natriuretic peptide, based on the amino acid residue LC res49 of Kabat (which corresponds to res Y51 in the light chain having the amino acid sequence of SEQ ID NO 66); and / or vi) In the case where the heterologous amino acid sequence is incorporated into CDRL3, according to the amino acid residue LC res88 of Kabat (which corresponds to res C90 in the light chain having the amino acid sequence of SEQ ID No 66) and the amino acid residue of the first natriuretic peptide; and wherein b) there are at least 9 amino acid residues between the last amino acid residue of the natriuretic peptide and the following: i) in the case where the heterologous amino acid sequence is incorporated into CDRH1, according to the amino acid residue HC res35a of Kabat (which corresponds to res M34 in the heavy chain having the amino acid sequence of SEQ ID No 65);ii) In the case of the heteroamino acid sequence being incorporated into CDRH2, according to Kabat's amino acid residue HC res57 (in the heavy chain having the amino acid sequence of SEQ ID NO 65, this corresponds to res T58); iii) In the case of the heteroamino acid sequence being incorporated into CDRH3, according to Kabat's amino acid residue HC res106 (in the heavy chain having the amino acid sequence of SEQ ID NO 65, this corresponds to res G111); iv) In the case of the heteroamino acid sequence being incorporated into CDRL1, according to Kabat's amino acid residue LC res 32 (in the light chain having the amino acid sequence of SEQ ID NO 66, this corresponds to res D34); v) In the case of the heteroamino acid sequence being incorporated into CDRL2, according to Kabat's amino acid residue LC res57 (in the light chain having the amino acid sequence of SEQ ID NO 66, this corresponds to res T58); G59); and / or vi) in the case of the heteroamino acid sequence being incorporated into CDRL3, according to the amino acid residue LC res98 of Kabat (in the light chain having the amino acid sequence of SEQ ID NO 66, this corresponds to res F102). In other respects, the present invention relates to the use of such antibodies or fragments thereof in therapeutic treatments, 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 such nucleic acids, and methods for preparing such antibodies or fragments thereof.

Implementation Method

Claims

1. An antibody or a fragment thereof comprising a heterologous amino acid sequence incorporated into a CDR region of the antibody or fragment thereof, wherein the heterologous amino acid sequence comprises an N-terminal linker sequence (Ntls), an atrial natriuretic peptide (ANP), and a C-terminal linker sequence (Ctls), wherein at least a portion of the at least one CDR region is replaced by the heterologous amino acid sequence incorporated therein, and wherein a) the Ntls are present in the following ways: i) when the heterologous amino acid sequence is incorporated into CDRH1, the amino acid residue HC res25 of Kabat is present with the first amino acid residue of the ANP; ii) when the heterologous amino acid sequence is incorporated into CDRH2, the amino acid residue HC res51 of Kabat is present with the first amino acid residue of the ANP; iii) when the heterologous amino acid sequence is incorporated into CDRH3, the amino acid residue HC res92 of Kabat is present with the first amino acid residue of the ANP; iv) In the case where the heterologous amino acid sequence is incorporated into CDRL1, according to the Kabat amino acid residue LC res26 and the first amino acid residue of the ANP; v) in the case where the heterologous amino acid sequence is incorporated into CDRL2, according to the Kabat amino acid residue LC res49 and the first amino acid residue of the ANP; and / or vi) in the case where the heterologous amino acid sequence is incorporated into CDRL3, according to the Kabat amino acid residue LC res88 and the first amino acid residue of the ANP; and wherein b) the last amino acid residue of the ANP of the Ctls is present with: i) in the case where the heterologous amino acid sequence is incorporated into CDRH1, according to the Kabat amino acid residue HC res35a; ii) in the case where the heterologous amino acid sequence is incorporated into CDRH2, according to the Kabat amino acid residue HC res57; iii) In the case where the heterologous amino acid sequence is incorporated into CDRH3, according to the Kabat amino acid residue HC res106; iv) in the case where the heterologous amino acid sequence is incorporated into CDRL1, according to the Kabat amino acid residue LC res32; v) in the case where the heterologous amino acid sequence is incorporated into CDRL2, according to the Kabat amino acid residue LC res57; and / or vi) in the case where the heterologous amino acid sequence is incorporated into CDRL3, according to the Kabat amino acid residue LC res98, and wherein c) the Ntls and the Ctls contain sequences selected from the following combinations: and wherein the antibody or fragment thereof is a human or humanized antibody or fragment thereof, wherein the antibody or fragment thereof is of the IgG class, and wherein the antibody fragment is selected from the group consisting of: Fab, Fab', Fab'-SH, F(ab')2 and Fv fragments; bifunctional antibody;Single-domain antibody (Dab); linear antibody; single-chain antibody molecule (scFv); and disulfide-bonded stable Fv antibody fragment (dsFv), wherein the ANP therein is the sequence of SEQ ID NO 23.

2. The antibody or fragment thereof as requested in item 1, wherein: The amino acid extension exists in the following cases: i) when the heteroamino acid sequence is incorporated into CDRH1, according to Kabat amino acid residues HC res25 and HC res35a; ii) when the heteroamino acid sequence is incorporated into CDRH2, according to Kabat amino acid residues HC res51 and HC res57; iii) when the heteroamino acid sequence is incorporated into CDRH3, according to Kabat amino acid residues HC res92 and HC res106; iv) when the heteroamino acid sequence is incorporated into CDRL1, according to Kabat amino acid residues LC res26 and LC res32; v) when the heteroamino acid sequence is incorporated into CDRL2, according to Kabat amino acid residues LC res49 and LC res57; and / or vi) In the case where the heterologous amino acid sequence is incorporated into CDRL3, according to the amino acid residues LC res88 and LC res98 of Kabat; the sequence includes any one of SEQ ID NO 52 to 64.

3. An antibody or fragment thereof as claimed in any of claims 1 to 2, wherein (i) the light chain contains the amino acid sequence of SEQ ID NO 66 and the heavy chain contains the amino acid sequence of any of SEQ ID NO 67 to 79; or (ii) the heavy chain contains the amino acid sequence of SEQ ID NO 65 and the light chain contains the amino acid sequence of any of SEQ ID NO 80 and 81.

4. An antibody or fragment thereof as claimed in any of claims 1 to 2, wherein (i) the light chain consists of the amino acid sequence of SEQ ID NO 66 and the heavy chain consists of the amino acid sequence of any of SEQ ID NO 67 to 79; or (ii) the heavy chain consists of the amino acid sequence of SEQ ID NO 65 and the light chain consists of the amino acid sequence of any of SEQ ID NO 80 and 81.

5. The antibody or fragment thereof as claimed in claim 3, wherein (i) the light chain contains the amino acid sequence of SEQ ID NO 66 and the heavy chain contains the amino acid sequence of any one of SEQ ID NO 67 to 79; or (ii) the heavy chain contains the amino acid sequence of SEQ ID NO 65 and the light chain contains the amino acid sequence of any one of SEQ ID NO 80 and 81.

6. The antibody or fragment thereof as claimed in claim 4, wherein (i) the light chain consists of the amino acid sequence of SEQ ID NO 66 and the heavy chain consists of the amino acid sequence of any one of SEQ ID NO 67 to 79; or (ii) the heavy chain consists of the amino acid sequence of SEQ ID NO 65 and the light chain consists of the amino acid sequence of any one of SEQ ID NO 80 and 81.

7. The antibody or fragment thereof as claimed in claim 5, wherein the light chain comprises the amino acid sequence of SEQ ID NO 66 and the heavy chain comprises the amino acid sequence of any one of SEQ ID NO 67 to 79.

8. The antibody or fragment thereof as claimed in claim 6, wherein the light chain consists of the amino acid sequence of SEQ ID NO 66 and the heavy chain consists of the amino acid sequences of any one of SEQ ID NO 67 to 79.

9. The antibody or fragment thereof as claimed in claim 7, wherein the light chain comprises the amino acid sequence of SEQ ID NO 66 and the heavy chain comprises the amino acid sequence of SEQ ID NO 76.

10. The antibody or fragment thereof as claimed in claim 8, wherein the light chain consists of the amino acid sequence of SEQ ID NO 66 and the heavy chain consists of the amino acid sequence of SEQ ID NO 76.

11. A composition comprising an antibody or fragment thereof as claimed in any one of claims 1 to 10 and a medically acceptable carrier.

12. A nucleic acid or a mixture of nucleic acids encoding an antibody or fragment thereof as claimed in any one of claims 1 to 10.

13. A host cell comprising nucleic acid or a mixture of nucleic acids as claimed in claim 12.

14. A method for preparing an antibody or a fragment thereof, comprising culturing a host cell as claimed in claim 13 under conditions suitable for the performance of the antibody or the fragment thereof.

15. Use of an antibody or fragment thereof as claimed in any one of claims 1 to 10 for the preparation of a pharmaceutical product for the treatment of cardiovascular, renal, lung, skeletal, ocular, thromboembolic or fibrotic diseases or conditions, dwarfism, achondroplasia or other cGMP-related and / or diuretic natriuretic peptide-responsive conditions.

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