EA042691B1 - SPECIFIC ANTIBODIES TO HUMAN ISLAND AMYLOID POLYPEPTIDE (HIAPP) AND THEIR USE - Google Patents

Description

Field of invention

The present invention generally relates to novel molecules specifically binding to human islet amyloid polypeptide (hIAPP), also known as amylin, and/or its precursor human islet amyloid propolypeptide (proIAPP), in particular to human antibodies, as well as their fragments, derivatives and variants that recognize IAPP proteins, proIAPP, aggregated forms of IAPP, aggregated forms of proIAPP and/or fibrils of IAPP. In addition, the present invention relates to pharmaceutical and diagnostic compositions containing such binding molecules, antibodies and their mimetics, useful as a diagnostic tool for the identification of IAPP, proIAPP, aggregated IAPP, proIAPP variants and/or IAPP fibrils in plasma, as well as strategies passive vaccination for the treatment of disorders associated with aggregated IAPP, aggregated proIAPP, and IAPP fibrils, such as type 2 diabetes mellitus (T2DM) and islet rejection following clinical pancreatic islet transplantation in people with type 1 diabetes mellitus (T1D).

State of the art

Protein accumulation, modification and aggregation are pathological aspects of many metabolic diseases, including well-known neurodegenerative diseases such as Huntington's, Alzheimer's (AD) and Parkinson's (PD) diseases (Taylor et al., Science 296 (2005), 1991-1995). Abnormal protein aggregation has also been implicated in metabolic diseases such as type 2 diabetes mellitus (T2DM) and islet rejection following clinical pancreatic islet transplantation in people with type 1 diabetes mellitus (T1DM). Misfolding and aggregation of proteins lead to the development of amyloid deposits and appear to be directly related to cellular toxicity in these diseases. Islet amyloid polypeptide (IAPP or amylin), a physiological peptide co-secreted with insulin by P-cells in the pancreas, forms fibrillar aggregates in the pancreatic islets (also called islets of Langerhans) of patients with type 2 diabetes mellitus and is thought to be involved in the development of this disease (Westermark et al. (2011), Physiol. Rev. 91(3): 795-826). In addition, as previously mentioned, IAPP aggregates have been found in pancreatic islets following single islet transplantation in patients with type 1 diabetes mellitus (T1DM).

Human IAPP (hIAPP) is a 37 amino acid peptide hormone containing a disulfide bridge between cysteine residues 2 and 7 and an amidated C-terminus. Pancreatic islets are 65-80% β-cells, which produce and secrete insulin and IAPP, which are important for the regulation of blood glucose and cellular metabolism. IAPP is formed by processing the preprohormone preproIAPP, an 89 amino acid precursor produced in pancreatic β-cells.

PreproIAPP is cleaved rapidly after translation into an islet amyloid propolypeptide, a 67 amino acid peptide, which undergoes further proteolysis and post-translational modification to form hIAPP. hIAPP expression is co-regulated with insulin, as increased insulin production leads to an increase in hIAPP levels. hIAPP is released from pancreatic β-cells into the bloodstream and is involved in the regulation of glycemia by controlling gastric emptying and satiety, synergistically with insulin.

Although hIAPP acts as a regulator of cell metabolism under physiological conditions, hIAPP can aggregate and form amyloid fibrils (IAPP-amyloidosis), which is associated with β-cell deficiency, increased β-cell death, and decreased β-cell mass. There are several reasons to consider hIAPP amyloidosis as the main triggering factor in the pathogenesis of T2DM. First, hIAPP fibril deposition is found in more than 90% of patients with type 2 diabetes mellitus (Zraika et al. (2010), Diabetologia 53(6): 1046-1056). Second, hIAPP aggregation is toxic to β-cells and correlates with a decrease in insulin-producing β-cells (Butler et al. (2003), Diabetes 52(9): 23042314; Ritzel et al. (2007), Diabetes 56(1) : 65-71; Jurgens et al (2011), Am J Pathol 178(6): 2632-2640). Third, hIAPP-expressing transgenic mouse models show amyloid deposition in pancreatic islets and spontaneous development of T2DM (Janson et al. (1996), Proc. Natl. Acad. Sci. USA 93(14): 7283-7288; Hoppener et al (1999) Diabetologia 42(4): 427-434 Hull et al (2003) Diabetes 52(2): 372-379 Butler et al (2004) Diabetes 53(6): 1509-1516; Matveyenko et al. (2006), ILAR J. 47(3): 225-233; Hoppener et al. (2008), Exp Diabetes Res. 697035). They mimic human disease with β-cell dysfunction, β-cell depletion, and β-cell death comparable to those seen in tissues of T2DM patients. hIAPP expression and amyloid formation are directly correlated with β-cell apoptosis and the development of diabetes in these models, which is evidence of the contribution of human IAPP to the development of this disease. In addition, treatment that interferes with hIAPP aggregation improved the diabetic phenotype and increased the lifespan of the animal (Aitken et al. (2009), Diabetes 59(1): 161-171). hIAPP aggregation and amyloidosis are prerequisites for toxicity. Rodent non-amyloid IAPP (rIAPP), which cannot form fibrils through six amino acid substitutions, is non-toxic to β-cells. With the development of the disease, pathological aggregation of hIAPP found in human pancreatic islets can lead to

- 1 042691 β-cell function and death associated with impaired insulin secretion. In addition, compensatory increases in β-cell mass and insulin and amylin secretion to maintain normal blood glucose levels may promote formation of toxic hIAPP oligomers and deposition of hIAPP fibrils. Although the parent hIAPP oligomers are considered to be the major cytotoxic molecules, the final hIAPP fibrils may also play a role in β-cell death (Meier et al. (2006), Am. J. Physiol. Endocrinol. Metab. 291(6): E1317-1324; Haataja et al (2008), Endocr Rev 29(3): 303-316 Engel et al (2008) Proc Natl Acad Sci USA 105(16): 6033-6038). hIAPP fibrils have also been observed in isolated donor pancreatic islets and have been associated with β-cell death following clinical pancreatic islet transplantation in individuals with type 1 diabetes (Andersson et al. (2008), Exp. Diabetes Res. 562985; Udayasankar et al. (2009 ), Diabetologia 52(1): 145-153; Bohman et al (2012), Amyloid 19(2): 87-93). The exact mechanism leading to hIAPP aggregation and amyloidosis in T2DM is unknown. Insulin resistance in T2DM increases the need for insulin secretion along with the release of cellular proIAPP and hIAPP, which can cause amyloidosis, since the formation of hIAPP fibrils is concentration dependent. Another proposed mechanism is the accumulation and aggregation of the unprocessed N-terminus proIAPP caused by impaired proteolysis under conditions of insulin resistance, since partially processed proIAPP forms, in particular the 48-residue intermediate proIAPP1.48, are found in amyloid deposits (Marzban et al. (2006), Diabetes 55(8): 2192-2201). In this context, abnormal proIAPP processing may act as a trigger for hIAPP amyloidosis and increase amyloid production (Paulsson et al. (2005), Diabetes 54(7): 2117-2125; Paulsson et al. (2006), Diabetologia 49(6 ): 1237-1246; Marzban et al (2006), Diabetes 55(8): 2192-2201). Therefore, proIAPP is also considered as a suitable therapeutic target.

The clinical features of T2DM are high blood glucose levels and insulin resistance and/or insufficiency. Diabetes mellitus is a group of metabolic diseases including T1DM, T2DM and gestational diabetes. T2DM, also called adult-onset diabetes, obesity-related diabetes, and non-insulin-dependent diabetes mellitus (NIDDM), is the most common form of diabetes, accounting for approximately 90% of all cases (Gerich et al. (1998), Endocr. Rev. 19(4 ): 491-503). DM2 is characterized by a decrease in the number of functional insulin-producing β-cells. As the disease progresses, this can lead to long-term complications such as cardiovascular disease, diabetic retinopathy leading to blindness, kidney failure, frequent infections, and amputations due to poor circulation. As a consequence, T2DM is associated with reduced life expectancy. This disease affects more than 300 million people worldwide and causes more than a million deaths every year. The development of the disease is driven by both genetic determinants and environmental factors, with obesity, physical inactivity and aging considered to be the main cause (Kahn et al. (2006), Nature 444(7121): 840-846).

Current treatments for T2DM include lifestyle management (diet and exercise) and pharmacological interventions such as metformin and insulin to lower blood glucose levels by stimulating insulin release from the pancreas or enhancing insulin response. These treatments are based on symptomatic short-term improvement in diabetes. Virtually none of the available treatments have been shown to counteract hIAPP aggregation and pancreatic β-cell death. New treatment strategies using glucagon-like peptide 1 (GLP-1) analogs (Butler et al. (2009), Diabetologia 53(1): 16) and inhibitors of the GLP-1-inactivating enzyme dipeptidyl peptidase 4 (DDP4) are based on a powerful insulinotropic action of GLP-1 and its ability to enhance the proliferation of β-cells. Importantly, increased insulin release is also associated with increased amylin release. It has been experimentally shown that stimulation of insulin secretion enhances the development of islet amyloidosis in animal models, and similar effects can be expected in humans (Aston-Mourney et al. (2011), Diabetologia 54(7): 1756-1765). Thus, these treatments have the potential to exacerbate islet amyloidosis. More recent and promising strategies involve the development of anti-inflammatory drugs or antibodies that target the IL-1β pathway (Donath et al. (2008), Nat. Clin. Pract. Endocrinol. Metab. 4(5): 240-241; Ehes et al. (2009), Proc Natl Acad Sci USA 106(33): 13998-14003 Owyang et al (2010) Endocrinology 151(6): 2515-2527 Dinarello et al (2010) Curr Opin Endocrinol Diabetes Obes 17(4): 314-321 Boni-Schnetzler et al (2011), J Clin Endocrinol Metab 93(10): 4065-4074 Boni-Schnetzler et al. (2012), Br. J. Clin. Pharmacol.; Cavelti-Weder et al. (2012), Diabetes Care). Importantly, recent studies show that hIAPP specifically induces the inflammasome, an IL-1β system that activates the innate immune system (Masters et al. (2010), Nat. Immunol. 11(10): 897-904; Mandrup-Poulsen et al. (2010), Nat Immunol 11(10): 881-883), thereby supporting a therapeutic strategy directed at hIAPP aggregation.

These findings highlight the potential benefits associated with active or passive immunotherapy approaches that target hIAPP or proIAPP.

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In summary, there is an urgent need for therapeutic strategies that target aggregated hIAPP proteins, proIAPP and/or hIAPP oligomers and/or fibrils through effective and safe therapy. Passive immunization using human antibodies, evolutionarily optimized and affinity matured using the human immune system, should provide a promising new therapeutic avenue that is highly likely to have superior efficacy and safety.

The essence of the invention

The present invention makes it possible to use the hIAPP-specific immune response of healthy human subjects to isolate natural hIAPP-specific human monoclonal antibodies. In particular, the experiments carried out in accordance with the present invention have successfully allowed the isolation of hIAPP- or proIAPP-specific monoclonal antibodies from a pool of healthy human subjects or from a pool of obese patients and other groups of patients with an increased risk of developing T2DM, in whom at the time of isolation antibodies, there were no signs of DM2.

Thus, the present invention is directed to human antibodies, antigen-binding fragments and similar antigen-binding molecules capable of specifically recognizing IAPP and/or proIAPP. Unless otherwise indicated, the terms specifically recognizing IAPP or proIAPP, antibody specific for/in relation to IAPP and/or proIAPP, and antibody against IAPP and/or against proIAPP mean specifically, generally and generally antibodies to the native monomeric form of IAPP; antibodies to proIAPP, the precursor form of IAPP; antibodies specifically binding to forms of IAPP and proIAPP; antibodies that bind to aggregated, oligomeric, fibrillar or non-fibrillar variants of IAPP and/or proIAPP. The present application provides human antibodies that are selective for full-length and/or aggregated forms, eg oligomeric, fibrillar and non-fibrillar aggregated forms of IAPP and/or proIAPP.

In a particularly preferred embodiment of the present invention, the human antibody, or antigen-binding fragment thereof, exhibits the immunological binding characteristics of an antibody characterized by the VH and/or V _L variable regions shown in FIG. 1 or fig. 2.

The antigen-binding fragment of an antibody may be a single chain Fv fragment, an F(ab') fragment, an F(ab) fragment, and an F(ab') ₂ fragment, or any other antigen-binding fragment. In a specific embodiment below, the antibody or fragment thereof is a human IgG isotype antibody. Alternatively, the antibody is a chimeric human rodent antibody or a rodentized antibody, for example, a mouse antibody or a murine antibody, a rat antibody or a ratinized antibody, the rodent variants being particularly useful for diagnostic techniques and animal studies.

In addition, the present invention relates to compositions containing an antibody in accordance with the present invention or its active fragments, and to immunotherapeutic and immunodiagnostic methods in which such compositions are used in the prevention, diagnosis or treatment of disorders associated with IAPP, for example, T2DM, wherein an effective amount of the composition is administered to a patient in need thereof.

Naturally, the present invention extends to immortalized memory B lymphocytes and human B cells, respectively, which produce an antibody, or antigen-binding fragment thereof, having specific and unique characteristics as defined below.

The present invention also relates to polynucleotides encoding at least the variable region of an immunoglobulin chain of an antibody of the invention. In a preferred embodiment, said variable region comprises at least one hypervariable region (CDR) VH and/or V _L of the variable region defined in FIG. 1 or 2.

Thus, the present invention also encompasses vectors containing these polynucleotides and host cells transformed with them, as well as their use for the production of antibodies and equivalent binding molecules specific for IAPP and/or proIAPP. Means and methods for recombinant production of antibodies and their mimetics, as well as methods for screening competitive binding molecules, which may or may not be antibodies, are known in the art. At the same time, as described herein, in particular in relation to therapeutic medical use, an antibody of the present invention is a human antibody in the sense that the use of said antibody does not substantially lead to an immune response directed against such an antibody, otherwise cases observed for chimeric and even humanized antibodies.

In addition, this document describes compositions and methods that can be used to detect IAPP and/or proIAPP in samples and/or in vivo. Anti-IAPP and/or proIAPP antibodies or fragments thereof that bind IAPP and/or proIAPP can be used to screen human blood, plasma, serum, saliva, peritoneal fluid, cerebrospinal fluid (CSF) and urine for the presence of IAPP and/or proIAPP in samples, for example, using an ELISA-based assay or a surface-adapted assay. In one embodiment, the present invention relates to a method for diagnosing or monitoring the progression of an IAPP and/or proIAPP related disorder.

- 3 042691 in a subject, and this method includes determining the presence of oligomers, aggregates or fibrils of IAPP and/or proIAPP in a sample from a diagnosable subject using at least one antibody according to the present invention or a molecule that binds IAPP and/or proIAPP, having essentially the same binding specificity as any of them, where the presence of oligomers, aggregates or fibrils of IAPP and/or proIAPP is indicative of a disorder.

In addition, in one embodiment, the present invention provides the described antibodies against IAPP and/or proIAPP or fragments thereof that bind IAPP and/or proIAPP and/or molecules that bind IAPP and/or proIAPP, containing at least one CDR of an antibody according to the present invention. of the invention, for making a composition for in vivo detection (also referred to as in vivo imaging) or targeting of a therapeutic or diagnostic agent to IAPP and/or proIAPP in a human or animal body. The methods and compositions described herein can be used in disorders associated with IAPP and characterized, for example, by the occurrence of oligomeric, fibrillar and non-fibrillar aggregated forms of IAPP and/or proIAPP, for example, the diagnosis of T2DM, and can also be used to monitor disease progression and the therapeutic efficacy of the treatment provided to the subject, for example, in diagnostic methods associated with in vivo imaging. Thus, in one embodiment, an IAPP and/or proIAPP binding molecule of the present invention is provided, wherein said in vivo detection (imaging) includes positron emission tomography (PET), single photon emission tomography (SPET), near infrared optical imaging (NIR) or magnetic resonance imaging (MPT).

Thus, it is a separate object of the present invention to provide methods for the treatment, diagnosis, or prevention of diseases associated with fibrillar and/or non-fibrillar oligomeric aggregated IAPP and/or proIAPP, for example type 2 diabetes (T2DM). These methods include administering an effective concentration of the antibody or human antibody derivative to a subject wherein the antibody is targeted by IAPP and/or proIAPP.

In yet another aspect of the present invention, there is provided a peptide comprising an IAPP and/or proIAPP epitope specifically recognized by an antibody of the present invention. The specified peptide contains or consists of the amino acid sequence listed below in the Detailed Description and Examples sections or a modified sequence in which one or more amino acids have been replaced, removed and/or added. In addition, the present invention provides a method for diagnosing type 2 diabetes or risk of developing type 2 diabetes in a subject, comprising the step of determining the presence of an antibody that binds the specified peptide in a biological sample from the specified subject.

Additional embodiments of the present invention will become apparent from the following description and examples.

Brief description of the figures

Fig. 1. Amino acid and nucleotide sequences of the variable region, ie. heavy chain and light chain kappa/lambda human anti-IAPP antibodies NI-2O3.9A2 (A), NI-2O3.19H8 (B), NI203.26C11 (C), NI-203.8E3 (D), NI-203.11B12 ( E), NI-203.205F8 (F), NI-203.9B3 (G), NI-203.19F2 (H), and NI203.15C7 (I). Framework (FR) and hypervariable regions (CDRs) are indicated, with CDRs underlined. In addition, heavy (JH) and light chain (JK) J regions are indicated. Due to the cloning strategy, the amino acid sequence at the N-terminus of the heavy and light chains can potentially contain primer-driven substitutions in FR1 that do not significantly affect the biological activity of the antibody. To provide a consensus human antibody, the nucleotide and amino acid sequences of the original clone were aligned with and edited according to the appropriate human germ line variable region sequences contained in the database; see, for example, Vbase (http://vbase.mrc-cpe.cam.ac.uk/) hosted by the MRC Protein Engineering Center (Cambridge, UK). The amino acid sequence of a human antibody is indicated if the N-terminal amino acids are thought to be potentially different from the germline consensus sequence due to the PCR primer and accordingly replaced by a primer-induced corrective mutation (PIMC). PIMC-modified amino acids are highlighted in bold in the sequence.

Fig. 2. Amino acid and nucleotide sequences of the variable region, ie. heavy chain and kappa/lambda light chain human antibodies against proIAPP NI-203.1D10 (A), NI-203.2A11 (B), NI-203.10C4 (C), NI-203.20H9 (D), NI-203.26D2 ( E) and NI-203.60H3 (F). Framework (FR) and hypervariable regions (CDRs) are indicated, with CDRs underlined. In addition, the heavy (JH) and light chain (JK) J regions are indicated. Due to the cloning strategy, the amino acid sequence at the N-terminus of the heavy and light chains can potentially contain primer-driven substitutions in FR1 that do not significantly affect the biological activity of the antibody. In order to represent the consensus human antibody, the nucleotide and amino acid sequences of the original clone were aligned and adjusted according to the appropriate human germ line variable region sequences contained in the database; see, for example, Vbase (http://vbase.mrc-cpe.cam.ac.uk/) hosted by the MRC Protein Engineering Center (Cambridge,

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Great Britain). The amino acid sequence of a human antibody is indicated if the N-terminal amino acids are thought to be potentially different from the germline consensus sequence due to the PCR primer and thus substituted by primer-induced corrective mutation (PIMC). PIMC-modified amino acids are highlighted in bold in the sequence.

Fig. 3. IAPP-binding specificity of human recombinant antibodies as assessed by direct ELISA. (A) Electron micrograph of the IAPP solution (2 mg/mL) used to coat the ELISA plate. Scale bar: 1 µm. (B) Recombinant antibodies NI-203.9A2, NI-2O3.19H8, NI-203.26C11 and NI-203.8E3 showed specific binding to human IAPP (10 μg/ml). BSA (10 μg/ml) was used as a control to determine non-specific binding. Data are expressed as OD values at 450 nm.

Fig. 4. Determination of EC ₅₀ human recombinant antibodies against IAPP against IAPP and proIAPP. (A) Electron micrographs of IAPP and proIAPP (2 mg/mL) solutions used to coat the ELISA plate. Scale bar: 1 µm. (B) Plates were incubated with the indicated concentration of recombinant human antibodies NI-203.9A2, NI-2O3.19H8, NI-203.26C11 or NI-203.8E3. Antibodies NI-203.9A2, NI-2O3.19H8, NI203.26C11 and NI-203.8E3 bound to human IAPP (, 10 μg/ml) with high affinity and EC ₅₀ equal to 9, 22, 6 and 4 nm, respectively. NI-203.26d1 also bound proIAPP (□, 10 μg/ml) with an EC ₅₀ of 260 nM. The measurements were performed in duplicate and the background signal was subtracted on the BSA. Data are expressed as mean OD values at 450 nm.

Fig. 5. Antibodies of human origin against IAPP are specific to IAPP fibrils. (A) Electron micrographs of IAPP (2 mg/mL) and non-fibrillar IAPP (500 μg/mL) solutions used to coat the ELISA plate. While the IAPP solution contains fibrils, IAPP fibrils are absent from the non-fibrillar IAPP solution. Scale bar: 1 µm. (B) Plates were incubated with the indicated concentration of recombinant human antibodies NI-203.9A2, NI-2O3.19H8, NI-203.26d1, or NI-203.8E3. Antibodies NI-203.9A2, NI-2O3.19H8, NI-203.26C11 and NI-203.8E3 bind to IAPP fibrils (IAPP solution, . ) - with very low affinity, indicating specificity for IAPP fibrils. The measurements were performed in duplicate and the background signal was subtracted on the BSA. Data are expressed as mean OD values at 450 nm.

Fig. 6. IAPP-binding epitopes of recombinant human antibodies examined by pepscan analysis. (A) Pepscan images of recombinant human antibodies NI203.19H8 and NI-203.26d1 (1 μg/ml). Binding of NI-2O3.19H8 occurred at peptides 6 and 7 (line A), including amino acids 19-25 (peptide 6: 16-LVHSSNNFGA-25 SEQ ID NO: 6, peptide 7: 19SSNNFGAILS-28 SEQ ID NO: 8, consensus binding sequence: 19-SSNNFGA-25 SEQ ID NO: 4). Binding of NI-203.26C11 occurred at peptide 1 (line A) spanning amino acids 1-10 (peptide 1: 1-KCNTATCATQ-10 SEQ ID NO: 9), but not at peptide 2 spanning amino acids 4-13 (peptide 2: 4-TATCATQRLA-13 SEQ ID NO: 10). Substitution of alanine residues 2-8 in peptides 33-39 and 41 (line C) disrupted the binding of NI-203.26C11 (mutation of peptide 33: C2A, mutation of peptide 34: N3A, mutation of peptide 35: T4A, mutation of peptide 36: A5G, mutation peptide 37: A5P, peptide 38 mutation: T6A, peptide 39 mutation: C7A, peptide 41 mutation: A8P). Secondary HRP-conjugated donkey Fcy against human IgG alone from other reagents (1:20,000; secondary Ab) was used as a control. (B) Binding epitopes of various human-derived IAPP-specific antibodies identified in the indicated amino acid region of the human IAPP protein sequence. Upper panel: amino acid sequence of the full-length human IAPP (amino acids 1-37). NI: No binding epitope identified.

Fig. 7. Antibodies NI-2O3.9A2, NI-2O3.19H8 and NI-203.26C11 specifically recognize abnormal amyloid IAPP in the pancreas of patients diagnosed with type 2 diabetes mellitus (DM2). Antibodies NI-203.9A2, NI-2O3.19H8, and NI-203.26d1 show staining for T2DM pancreatic islets loaded with IAPP (amyloid) fibrils (A, B), but not T2DM pancreatic islets without IAPP fibrils (C, D) ). (A) Amyloid staining with thioflavin S (ThioS, left panel) and Congo red (CR, right panel) in the pancreatic islets of a T2DM patient. (B) Detection of IAPP fibrils in amyloid-positive pancreatic islets in T2DM using antibodies NI-203.9A2, NI-2O3.19H8, and NI-203.26C11 (brown, CR) at 50 nM (large panel and lower left inset) and 5 nM (bottom right inset). (C) Absence of amyloid in the pancreatic islets of a patient with T2DM, as determined by negative staining with thioflavin S (ThioS, left panel) and Congo red (CR, right panel). (D) Absence of staining in amyloid-negative pancreatic islets in T2DM with NI-203.9A2, NI203.19H8, and NI-203.26d1 antibodies at 50 nM. A secondary HRP-conjugated donkey anti-human antibody alone from other reagents (secondary Ab) was used as a control. Bottom insets: high magnification images of individual human pancreatic islets. Human pancreatic islets were stained with anti-insulin antibody (blue in the original sample (i.o., strong staining in this image) and stained to visualize the cell nuclei (pale blue staining i.o., weak staining in this image).

Fig. 8. Antibodies NI-203.9A2, NI-2O3.19H8, and NI-203.26C11 do not recognize physiological IAPP in human pancreatic controls. Antibodies NI-203.9A2, NI-2O3.19H8 and NI203.26C11 (50 nM) show weak staining of control human islets compared to control anti-IAPP antibody (1:100; control Ab). A secondary HRP-conjugated donkey anti-human antibody alone from other reagents (secondary Ab) was used as a control. Human pancreatic islets were stained with anti-insulin antibody (blue in original sample (i.o., strong staining in this image) and stained to visualize cell nuclei (pale blue i.o., weak staining in this image).

Fig. 9. Antibodies NI-203.9A2, NI-2O3.19H8, and NI-203.26C11 recognize abnormal IAPP fibrils in the pancreas of a diabetic cat. Detection of IAPP fibrils in pancreatic islets of a cat with T2D using antibodies NI-203.9A2, NI-2O3.19H8 and NI-203.26C11 (50 nM, brown-CR). IAPP (amyloid) fibrils were stained with Congo red (CR). A secondary HRP-conjugated donkey anti-human antibody alone from other reagents (secondary Ab) was used as a control. Lower left insets: High magnification images of individual cat pancreatic islets. Cat pancreatic islets were stained with anti-insulin antibody (blue in the original sample (i.o., strong staining in this image) and stained to visualize the cell nuclei (pale blue i.o., weak staining in this image).

Fig. 10. Antibodies NI-203.9A2, NI-2O3.19H8 and NI-203.26C11 do not recognize pathological deposits of Ae in the human brain in Alzheimer's disease. No staining with NI203.9A2, NI-2O3.19H8 and NI-203.26C11 antibodies (50 nM) compared to Ap specific antibody 6E10 (1:2000; control Ab). A secondary HRP-conjugated donkey anti-human antibody alone from other reagents (secondary Ab) was used as a control. Staining was performed to visualize cell nuclei (pale blue i.o., weak staining in this image).

Fig. 11. Recombinant human/mouse chimeric antibody NI-203.9A2, NI-2O3.19H8, and NI203.26C11 binds to human IAPP with equal affinity. (A) Determination of EC ₅₀ recombinant human and mouse chimeric anti-IAPP antibodies against IAPP (, 10 μg/ml) and BSA (◊, 10 μg/ml). The plates were incubated with the indicated antibody concentrations. The measurements were performed in duplicate. Data are expressed as mean OD values at 450 nm. (B, C) EC ₅₀ values of human and mouse chimeric antibodies.

Fig. 12. Recombinant mouse chimeric antibody NI-203.9A2, NI-2O3.19H8, and NI-203.26d1 recognizes abnormal IAPP fibrils in the pancreas of patients diagnosed with type 2 diabetes mellitus (DM2). Detection of IAPP fibrils in the pancreatic islets of two human patients with T2DM (1 and 2) using chimeric antibodies NI-2O3.9A2, NI-2O3.19H8 and NI-203.26d1 at a concentration of 50 nM (brown i.o., strong dark or black staining on this image). Human pancreatic islets were stained with anti-insulin antibody (blue i.o., strong staining in this image) and stained to visualize cell nuclei (pale blue i.o., weak staining in this image).

Detailed description of the invention

In type 2 diabetes mellitus (DM2), genetic and environmental factors lead to the development of insulin resistance, followed by a compensatory increase in beta cell mass and insulin and amylin secretion (hIAPP) to maintain normal blood glucose levels. The resulting high concentrations of amylin promote the formation of toxic human islet amyloid polypeptide (hIAPP) oligomers and the deposition of hIAPP fibrils found in more than 90% of type 2 diabetic patients. The deposition of hIAPP correlates with a decrease in insulin-producing beta cells and is also thought to play a role in the death of β-cells in pancreatic islet transplants in type 1 diabetic individuals. Several human-derived antibodies from pools of healthy or obese donors with a high risk of developing type 2 diabetes but no disease have been identified and characterized in vitro, cloned and recombinantly produced using Neurimmune RTM technology, as detailed in international application WO2008 /081008 and in this application. Lead candidates are being validated in transgenic mice expressing hIAPP and fed a high-fat diet. Therapeutic efficacy is assessed by determination of beta cell mass and hIAPP-amyloid load in the pancreas, as well as plasma hIAPP levels and functional assays of glucose metabolism and insulin secretion.

Type 2 diabetes is the most common form of diabetes, accounting for about 90% of all cases. This disease affects more than 200 million people worldwide and causes more than a million deaths from diabetes each year. In Switzerland, more than 300,000 patients suffer from this disease. The prevalence of diabetes mellitus is rising sharply in both developed and developing countries due to population growth, aging, urbanization, and an increase in obesity and lack of physical activity. The global market for type 2 diabetes is US$25 billion and is projected to reach US$35 billion in 2016 at a compound annual growth rate of 6.4% between 2009 and 2016. Current therapies include dietary control and pharmacological intervention acting on various metabolic pathways to lower blood glucose levels by increasing insulin sensitivity or by stimulating insulin release from the pancreas. At the same time, none of the available treatments is able to counteract hIAPP aggregation and pancreatic beta cell death. New strategies for the treatment of type 2 diabetes include analogues of glucagon-like peptide 1 (GLP-1) and inhibitors of dipeptidyl peptidase 4 (DPP-4), an enzyme that deactivates endogenous GLP-1. These strategies are based on the potent insulinotropic action of GLP-1 and its ability to enhance beta cell proliferation. Importantly, increased insulin release is also associated with increased amylin release. It has been experimentally shown that stimulation of insulin secretion enhances the development of islet amyloidosis in animal models, and similar effects can be expected in humans. Therefore, in addition to the specific use of IAPP binding molecules according to the present invention, additionally proposed therapeutic approach may be a promising combination therapy using these molecules and the above new methods of treatment.

I. Definitions.

Unless otherwise noted, the term used herein is defined as provided in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, 2000 edition, 2003 reprint, ISBN 0198506732.

It should be noted that singular entity forms referring to one or more of the entities indicated, eg, an antibody, should be understood to represent one or more antibodies. In fact, the singular forms as well as the terms one or more and at least one may be used interchangeably herein.

Unless otherwise specified, the term IAPP is used interchangeably to refer specifically to native monomeric, oligomeric, non-fibrillar, and fibrillar forms of islet amyloid polypeptide (IAPP). The term IAPP is also used as a generic term for other IAPP conformers, such as IAPP oligomers and aggregates, such as IAPP fibrils. The term IAPP is commonly used to refer to all types and forms of IAPP in general. The term proIAPP is used interchangeably to refer specifically to the native monomeric, oligomeric, fibrillar and/or aggregated form of the islet amyloid polypeptide precursor peptide (proIAPP). Letters added before the terms IAPP or proIAPP are used to identify the organism from which a particular orthologue originates, eg hIAPP for human IAPP or mIAPP for murine IAPP.

The 37 AK amino acid sequence for human IAPP is KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY (SEQ ID NO: 1) and contains a disulfide bridge between cysteine residues 2 and 7 and an amidated C-terminus.

IAPP is formed by processing the preprohormone preproIAPP, an 89 amino acid precursor produced in pancreatic β-cells. The human preproIAPP protein sequence is as follows:

MGILKLQVFLIVLSVALNHLKATPIESHQVEKRKCNTATCATQRLANFLVHSSNNF

GAILSSTNVGSNTYGKRNAVEVLKREPLNYLPL (SEQ ID NO: 2);

the indicated sequence can also be found in the corresponding databases, for example, in the UniProt database: UniProtID: P10997 (IAPP_HUMAN).

PreproIAPP is rapidly cleaved after translation to an islet amyloid propolypeptide. The human proIAPP protein sequence is as follows:

TPIESHQVEKRKCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTYGKRNAVEV

LKREPLNYLPL (SEQ ID NO: 3);

it undergoes additional proteolysis and post-translational modifications to form hIAPP.

The amino acid sequences of wild-type human IAPP, proIAPP and preproIAPP, or recombinant sequences thereof, are represented by the above sequences according to SEQ ID NOS: 1-3.

The human anti-IAPP and proIAPP antibodies described herein specifically bind IAPP and/or proIAPP and their epitopes and bind to different conformations of IAPP and/or proIAPP and their epitopes. For example, this document describes antibodies that specifically bind pathologically aggregated forms of IAPP and/or proIAPP, for example, non-fibrillar oligomers and/or fibrillar oligomers/fibrils and/or aggregates consisting of their mixed forms.

- 7 042691

The term (pathologically) aggregated/aggregates IAPP and/or proIAPP is used interchangeably to refer specifically to the above forms. The term (pathological) aggregates or aggregates, as used herein, describes the products of accumulation or formation of clusters as a result of an erroneous/pathological interaction of IAPP and/or proIAPP with each other. These aggregates, clusters or cluster forms may be, consist essentially of, or consist of IAPP and/or proIAPP and their non-fibrillar oligomers and/or fibrillar oligomers and fibrils. As used herein, reference to an antibody that specifically binds, selectively binds, or preferentially binds (preferentially binds) IAPP and/or proIAPP refers to an antibody that does not bind other unrelated proteins. In one example, an anti-IAPP and/or proIAPP antibody described herein can bind IAPP and/or proIAPP, or an epitope thereof, and does not exhibit binding to other proteins of approximately twice background binding. An antibody that specifically binds or selectively binds an IAPP and/or proIAPP conformer refers to an antibody that does not bind all IAPP and/or proIAPP conformations, i. e. not binding at least one of the other IAPP and/or proIAPP conformers. For example, this document describes antibodies that can preferentially bind to aggregated forms of IAPP and/or proIAPP, both in vitro and in tissues derived from patients with advanced T2DM or at risk of developing T2DM. Since human antibodies against IAPP and/or proIAPP according to the present invention are isolated from a pool of healthy human subjects or from a pool of obese patients and other patient populations at increased risk of developing T2DM who did not have signs of T2DM and IAPP manifestations at the time of antibody isolation - and/or proIAPP-specific immune response, antibodies against IAPP and/or proIAPP according to the present invention may also be referred to as human autoantibodies in order to emphasize that these antibodies were actually expressed by the subjects and were not isolated from, for example, a phage library expressing human immunoglobulins, which has so far been one of the common methods for obtaining antibodies similar to human antibodies.

The term peptide is intended to include the terms polypeptide and protein (which may be used interchangeably herein at times) within their meanings. Likewise, fragments of proteins and polypeptides may also be referred to herein as peptides and referred to as peptides. However, the term peptide preferably means a polymer of amino acids containing at least 5 consecutive amino acids, preferably at least 10 consecutive amino acids, more preferably at least 15 consecutive amino acids, even more preferably at least 20 consecutive amino acids, and especially preferably at least 25 consecutive amino acids. In addition, the peptide according to the present invention usually contains no more than 100 consecutive amino acids, preferably less than 80 consecutive amino acids, and more preferably less than 50 consecutive amino acids.

Polypeptides.

As used herein, the term polypeptide is intended to encompass a single polypeptide as well as multiple polypeptides and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term polypeptide refers to any chain or chains of two or more amino acids and does not refer to products of a specific length. Thus, peptides, dipeptides, tripeptides, oligopeptides, protein, amino acid chain, or any other term used to refer to a chain or chains of two or more amino acids is included in the definition of a polypeptide, and the term polypeptide can be used in place of, or interchangeably with, any of these terms.

In addition, the term polypeptide also refers to products of post-expression modification of a polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation and modification with known protecting/blocking groups, proteolytic cleavage, or modification with unnatural amino acids. The polypeptide can be obtained from a natural biological source or produced by recombinant technologies, but not necessarily translated from a given nucleotide sequence. It can be obtained by any method, including by chemical synthesis.

The polypeptide of the invention may be 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more, or 2000 or more amino acids. Polypeptides may have a specific three-dimensional structure, although they do not necessarily have such a structure. Polypeptides with a defined three-dimensional structure are called folded, and polypeptides that do not have a defined three-dimensional structure, but can have a large number of different conformations, are called non-folded. As used herein, the term glycoprotein refers to a protein associated with at least one carbohydrate residue attached to the protein through an oxygen-containing or nitrogen-containing side chain of an amino acid residue, such as a serine or asparagine residue.

Under the selected (isolated) polypeptide or its fragment, variant or derivative

- 8 042691 nym is understood to mean a polypeptide that is not found in its natural environment. A special level of cleaning is not required. For example, an isolated polypeptide may be recovered from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered to be isolated for the purposes of the invention as native or recombinant polypeptides separated, fractionated, or partially or substantially purified by any suitable technique.

The term recombinant peptides, polypeptides or proteins refers to peptides, polypeptides or proteins produced by recombinant DNA techniques, ie. produced by microbial or mammalian cells transformed with an exogenous recombinant expression DNA construct encoding a fusion protein comprising the desired peptide. Proteins or peptides expressed in most bacterial cultures usually do not contain glycans. Proteins and polypeptides expressed in yeast may have a different glycosylation profile than polypeptides expressed in mammalian cells.

The definition of a polypeptide according to the present invention includes fragments, derivatives, analogs or variants of these polypeptides, as well as any combination thereof. The terms fragment, variant, derivative, and analog include peptides and polypeptides having an amino acid sequence sufficiently similar to that of a naturally occurring peptide. The term sufficiently similar means a first amino acid sequence containing a sufficient or minimum number of amino acid residues identical or equivalent to those of a second amino acid sequence such that the first and second amino acid sequences share a common structural domain and/or share a common functional activity. For example, amino acid sequences containing a common structural domain that is at least about 45% identical, at least about 50% identical, at least about 55% identical, at least about 60% identical, at least about 65% identical. , at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% are defined herein as reasonably similar. In a preferred embodiment, the variants are sufficiently similar to the amino acid sequence of the preferred peptides of the present invention, in particular IAPP and/or proIAPP or fragments, variants, derivatives or analogues of any of them. Such variants generally retain the functional activity of the peptides of the present invention. Variants include peptides whose amino acid sequence differs from native and wild-type peptides, respectively, by one or more amino acid deletion(s), addition(s), or substitution(s). They can be natural as well as artificially created options.

In addition, the terms fragment, variant, derivative, and analog, when referring to antibodies or antibody polypeptides of the present invention, include any polypeptide that retains at least some degree of the antigen-binding properties of the corresponding native binding molecule, antibody, or polypeptide. Fragments of the polypeptides of the present invention include proteolytic fragments as well as deletion fragments, in addition to the specific antibody fragments discussed elsewhere in this document. Antibody variants and antibody polypeptides of the present invention include the fragments described above, as well as polypeptides with amino acid sequences altered by amino acid substitutions, deletions, or insertions. Variations may occur naturally or be unnatural. Non-natural variants can be obtained using mutagenesis techniques known in the art. Polypeptide variants may contain conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of IAPP- and/or proIAPP-specific binding molecules, eg, antibodies and antibody polypeptides of the present invention, are polypeptides modified to exhibit additional properties not present in the native polypeptide. Examples include fusion proteins. Polypeptide variants may also be referred to herein as polypeptide analogs. As used herein, the term derivative of a binding molecule or fragment thereof, an antibody, or an antibody polypeptide refers to the subject polypeptide containing one or more residues chemically modified by reaction at a functional side group. In addition, derivatives include peptides containing one or more naturally occurring amino acid derivatives of the 20 standard amino acids. For example, 4-hydroxyproline can replace proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; homoserine can replace series; and ornithine can replace lysine.

- 9 042691

Determination of similarity and/or identity of molecules.

The similarity between two peptides is determined by comparing the amino acid sequence of one peptide with that of a second peptide. An amino acid of one peptide is similar to the corresponding amino acid of a second peptide if it is identical or a conservative amino acid substitution. Conservative substitutions include those described in Dayhoff, M.O., ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, D.C. (1978), and in Argos, EMBO J. 8 (1989), 779-785. For example, amino acids belonging to one of the following groups are conservative changes or substitutions: -Ala, Pro, Gly, Gln, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, Ile, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and -Asp, Glu.

The similarity between two polynucleotides is determined by comparing the nucleotide sequence of one polynucleotide with the sequence of a second polynucleotide. The nucleic acid of one polynucleotide is similar to the corresponding nucleic acid of a second polynucleotide if it is identical or if the nucleic acid is part of a coding sequence and the corresponding triplet contained in the nucleic acid encodes the same amino acid or a conservative amino acid substitution.

The determination of percent identity or similarity between two sequences is preferably carried out using the mathematical algorithm of Karlin and Altschul (1993) Proc. Natl. Acad. Sci USA 90: 5873-5877. Such an algorithm is included in the BLASTn and BLASTp programs of Altschul et al. (1990) J. Mol. Biol. 215: 403-410, available from the NCBI website (http://www.ncbi.nlm.nih.gov/blast/Blast.cge).

Percent identity or similarity is determined using standard BLASTn and BLASTp program parameters as recommended on the NCBI web page and in the BLAST Program Selection Guide for sequences of defined length and composition.

BLAST search for polynucleotides is performed using the BLASTn program. For general parameters, the Max Target Sequences field can be set to 100, the Short Requests field can be checked, the Expected Threshold field can be set to 1000, and the Word Length field can be set to 7, as recommended for short sequences (less than 20 bases). ) on the NCBI webpage. For longer sequences, the Expected Threshold field can be set to 10 and the Word Length field can be set to 11. For scoring parameters, the Match/Mismatch Score field can be set to 1-2 and the Skip Cost field can be set to linear. For filtering options and masks, the Low Complexity Regions box may be unchecked, the Species-Specific Repeats box may be unchecked, the Lookup Table Only Mask box may be checked, the DUST Filter Options box may be checked, and the Lowercase Mask box may be unchecked. In general, in this regard, you can use the Search for short almost exact matches, which provides most of the specified parameters. More information about this can be found in the BLAST Program Selection Guide posted on the NCBI web page.

Protein BLAST searches are performed using the BLASTp program. For general parameters, the Max, Target Sequence field may be set to 100, the Short Queries field may be checked, the Expected Threshold field may be set to 10, and the Word Length field may be set to 3. For scoring parameters, the Matrix field may be set to BLOSUM62 and the Skipping cost field can be set to Availability: 11 Extension: 1, the Compositional correction field can be set to Conditional compositional matrix scoring. For filtering options and masks, the Low Complexity Regions box may be unchecked, the Lookup table-only mask field may be unchecked, and the Lowercase Mask box may be unchecked.

Changes in both programs, for example, in relation to the length of the sequences to be searched, are made in accordance with the recommendations in the BLAST Program Selection Guide, published in HTML and PDF versions on the NCBI web page.

Polynucleotides:

The term polynucleotide is intended to encompass a single nucleic acid as well as multiple nucleic acids, and refers to an isolated nucleotide molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). The polynucleotide may contain a conventional phosphodiester bond or an unconventional bond (eg, an amide bond found in peptide nucleic acids (PNA)). The term nucleic acid refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide. An isolated nucleic acid or polynucleotide is understood to mean a nucleic acid, DNA or RNA molecule extracted from its native environment. For example, a recombinant polynucleotide encoding an antibody contained in a vector is considered to be isolated for the purposes of the present invention. Additional examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or largely) polynucleotides in solution. Isolated RNA molecules include in vivo RNA transcripts of the polynucleotides of the present invention.

- 10 042691 or in vitro. In addition, the selected polynucleotides or nucleic acids according to the present invention include such molecules obtained by synthesis. In addition, the polynucleotide or nucleic acid may be or may contain a regulatory element, such as a promoter, a ribosome binding site, or a transcription terminator.

As used herein, a coding region is a portion of a nucleic acid that consists of codons translated into amino acids. Although a stop codon (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered part of the coding region; however, any flanking sequences such as promoters, ribosome binding sites, transcription terminators, introns, etc. are not part of the coding region. The two or more coding regions of the present invention may be provided as a single polynucleotide construct, eg a single vector, or separate polynucleotide constructs, eg separate (different) vectors. In addition, any vector may contain a single coding region or may consist of two or more coding regions, for example, one vector may separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, the vector, polynucleotide or nucleic acid of the invention may encode heterologous coding regions, combined or not combined with a nucleic acid encoding a binding molecule, antibody or fragment, variant or derivative thereof. Heterologous coding regions include, without limitation, specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

In some embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide consists of a nucleic acid encoding a polypeptide, which may normally include a promoter and/or other transcription or translation control elements operably linked to one or more coding regions. Functional linkage is a situation where the region encoding a gene product, for example a polypeptide, is linked to one or more regulatory sequences in such a way that the expression of the gene product is under the influence or control of the regulatory sequence(s). Two DNA fragments (e.g., a polypeptide coding region and a promoter associated with it) are operably associated or operably linked if induction of promoter function results in transcription of the mRNA encoding the target gene product, and if the nature of the bond between the two DNA fragments does not interfere with the ability regulatory sequence to control the expression of the gene product or does not interfere with the transcription of template DNA. Thus, a promoter region is operably linked to a nucleic acid encoding a polypeptide if the promoter is capable of transcribing said nucleic acid. The promoter may be a cell-specific promoter that directs DNA transcription substantially only in predetermined cells. Transcription control elements other than the promoter, such as enhancers, operators, repressors, and transcription termination signals, may be operably linked to the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are described herein.

Numerous transcriptional control regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions functioning in vertebrate cells, such as but not limited to the promoter and enhancer segments of cytomegaloviruses (immediate early promoter, in combination with intron A), simian virus 40 (early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcriptional control regions include elements derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (eg, promoters induced by interferons or interleukins).

Similarly, numerous translation control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (specifically, the internal ribosome entry site or IRES, also referred to as the CITE sequence).

In other embodiments, the implementation of the polynucleotide according to the present invention is an RNA, for example, in the form of messenger RNA (mRNA).

The coding regions of the polynucleotides and nucleic acids of the present invention may be linked to additional regions encoding secretory or signal peptides that control the secretion of the polypeptide encoded by the polynucleotide of the present invention. According to the signaling hypothesis, proteins secreted by mammalian cells contain a signal peptide or secretory leader sequence that is cleaved from the mature protein after initiation of export of the growing protein chain from the rough endoplasmic reticulum. It is known to those skilled in the art that polypeptides secreted by vertebrate cells typically contain a signal peptide attached to the N-terminus of the polypeptide, which is cleaved from the full or full length polypeptide to form the secreted or mature form of the polypeptide. In some embodiments, a native signal peptide is used, such as an immunoglobulin heavy or light chain signal peptide, or a functional derivative of this sequence that retains the ability to control the secretion of a polypeptide operably linked to it. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof can be used. For example, the wild-type leader can be replaced with a human tissue plasminogen activator (TPA) or mouse β-glucuronidase leader.

The term binding molecule in the context of the present invention refers primarily to antibodies and fragments thereof, but may also refer to other non-antibody molecules that bind to IAPP and/or proIAPP, including hormones, receptors, ligands, major histocompatibility complex (MHC) molecules ), chaperones, such as, but not limited to, heat shock proteins (HSPs); Thus, for purposes of clarity only and without limiting the scope of the present invention, most of the following embodiments are discussed in relation to antibodies and antibody-like molecules, which are preferred binding molecules for the development of diagnostic and therapeutic agents.

Antibodies.

The terms antibody and immunoglobulin are used interchangeably herein. An antibody or immunoglobulin is an IAPP and/or proIAPP binding molecule that contains at least a heavy chain variable domain and typically contains at least a heavy chain and a light chain variable domain. The basic structures of vertebrate immunoglobulins are relatively well understood; see, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, ^2nd ed. 1988).

In accordance with the more detailed discussion below, the term immunoglobulin includes several broad classes of polypeptides that differ from a biochemical point of view. Those skilled in the art will recognize that heavy chains are classified into gamma, mu, alpha, delta, and epsilon (γ, μ, α, δ, ε) chains, within which there are several subclasses (eg, γ1-γ4). The nature of these chains determines the class of the antibody IgG, IgM, IgG, IgA or IgE, respectively. Subclasses (isotypes) of immunoglobulins, for example, IgG1, IgG2, IgG3 and IgG4, IgA1, etc. well studied; they are known to have functional specialization. Modified versions of each of these classes and isotypes are readily recognized by one of ordinary skill in the art in view of this description and are therefore within the scope of the present invention. All classes of immunoglobulins are expressly relevant to the present invention, however, the following discussion generally refers to IgG class immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. These four chains are usually connected by disulfide bonds in a Y-configuration, where the light chains are grouped with heavy chains, starting from the neck of the specified Y-configuration and throughout the variable region.

Light chains are classified as either kappa or lambda (κ, λ). A heavy chain of any class can be linked to a kappa or lambda light chain. In general, the light and heavy chains are covalently linked to each other, and the tail fragments of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds if the immunoglobulins are produced using hybridomas, B cells, or recombinant host cells. The heavy chain amino acid sequence is oriented from the N-terminus at the branched end of the Y-configuration to the C-terminus at the bottom of each chain.

Light and heavy chains are divided into areas of structural and functional homology. The terms constant and variable are used from a functional point of view. In this regard, it should be considered that both the light (VL) and heavy chain (VH) variable domains determine antigen recognition and specificity. Conversely, the light chain (CL) and heavy chain (CH1, CH2, or CH3) constant domains confer important biological properties such as secretion, placental barrier crossing, Fc receptor binding, complement fixation, and so on. It is accepted that the numbering of the domains of the constant region increases as they move away from the antigen-binding site or the N-terminus of the antibody. The N-terminus is the variable region and the C-terminal is the constant region; The CH3 and CL domains actually contain the C-terminus of the heavy and light chain, respectively.

As mentioned above, the variable region allows the antibody to selectively recognize and specialize

- 12 042691 to digitally bind antigen epitopes. Those. the V _L domain and the V _H domain or subset of hypervariable regions (CDRs) of an antibody collectively form a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms an antigen-binding site located at the end of each Y arm. More specifically, the antigen-binding site is defined by three CDRs on each of V _H - and V _L -peneft. Any antibody or immunoglobulin fragment that contains sufficient structure to specifically bind to an IAPP and/or proIAPP is referred to interchangeably herein as a binding fragment or an immunospecific fragment.

Natural antibodies contain six hypervariable regions, sometimes referred to as complementarity determining regions or CDRs, that are present in each antigen-binding domain and are short, non-contiguous amino acid sequences arranged in a specific manner to form the antigen-binding domain when the antibody adopts its three-dimensional configuration in an aqueous environment. The CDRs are flanked by four relatively conserved framework regions, or FRs, which exhibit less pronounced intermolecular variability. The framework regions mostly adopt the β-sheet conformation, and the CDRs form loops that connect the β-sheet structures and in some cases are included in them. Thus, the framework regions form a framework that ensures that the CDRs are positioned in the correct orientation through non-covalent interactions between the strands. The antigen-binding domain, formed in a certain way arranged CDR, defines a surface complementary to the epitope of the immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its cognate epitope. One skilled in the art can easily determine the amino acids constituting the CDRs and framework regions, respectively, for any given heavy or light chain variable region, as long as there is a precise definition; see Sequences of Proteins of Immunological Interest, Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196 (1987), 901-917, which are incorporated herein by reference in their entirety.

Where there are two or more definitions of a term that is used and/or accepted in the art, the definition of the term used herein is intended to include all such meanings, unless expressly stated otherwise. A specific example is the use of the term hypervariable region or CDR to describe non-contiguous antigen-binding sites within the variable region of heavy and light chain polypeptides. This particular area has been described by Kabat et al., U.S. Dept, of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia and Lesk, J. Mol. Biol., 196 (1987), 901-917, which are incorporated herein by reference, where definitions include overlap or subsets of amino acid residues when compared to each other. However, the application of any definition to the CDRs of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The corresponding amino acid residues included in the CDR as defined in each of the above sources, are given below in table. 1 for comparison. The exact numbers of residues included in a particular CDR depend on the sequence and size of the CDR. Those skilled in the art can readily determine which residues make up a particular hypervariable region or CDR of a human IgG subtype antibody based on the amino acid sequence of the antibody's variable region.

Table I. CDR ¹ Definitions

Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-65 52-58 VH CDR3 95-102 95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VL CDR3 89-97 91-96

The numbering of all defined CDRs in Table. I is carried out according to the numbering rule established by Kabat et al. (see below).

Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One skilled in the art can unambiguously apply this Kabat numbering system to any variable domain sequence without using any experimental data other than the sequence itself. In this document, Kabat numbering refers to the numbering system established by Kabat et al., U.S. Dept. of

- 13 042691

Health and Human Services, Sequence of Proteins of Immunological Interest (1983). Unless otherwise indicated, reference to the numbering of positions of certain amino acid residues in an antibody or antigen-binding fragment, variant or derivative thereof according to the present invention corresponds to the Kabat numbering system, which, however, is theoretical and cannot be used equally for every antibody according to the present invention. For example, depending on the position of the first CDR, the following CDRs may be shifted in any direction.

Antibodies or antigen-binding fragments, immunospecific fragments, variants or derivatives thereof according to the invention include polyclonal, monoclonal, multispecific, humanized, primatized, murinized or chimeric antibodies or human antibodies, single-chain antibodies, epitope-binding fragments, for example, Fab, Fab' and F( ab') ₂ , Fd, Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), fragments containing the V _L or V _H domain, fragments produced using a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies described herein), but are not limited to. The scFv molecules are known to those skilled in the art and are described, for example, in US Pat. , IgG2, IgG3, IgG4, IgA1 and IgA2) or a subclass of immunoglobulin molecules.

In one embodiment, an antibody of the present invention is not IgM or a pentavalent derivative thereof. In particular, in specific applications of the present invention, especially therapeutic applications, IgMs are less useful than IgGs and other divalent antibodies or corresponding binding molecules because IgMs, due to their pentavalent structure and lack of affinity maturation, often show non-specific cross-reactivity and very low affinity.

In a particularly preferred embodiment, the antibody of the present invention is not a polyclonal antibody, i. it consists essentially of one particular type of antibody rather than a mixture derived from a sample of plasma immunoglobulins.

Antibody fragments, including single chain antibodies, may contain variable region(s) alone or in combination with complete or partial hinge, CH1, CH2 and CH3 domains. In addition, the present invention includes IAPP and/or proIAPP binding fragments containing any combination of variable regions with a hinge region, CH1, CH2 and CH3 domains. Antibodies or their immunospecific fragments according to the present invention can be derived from any animal, including birds and mammals. Preferably the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken. In yet another embodiment, the variable region may be derived from cartilaginous fish (eg, sharks).

In one aspect, an antibody of the present invention is a human monoclonal antibody isolated from a human body.

Optionally, the human antibody framework is aligned and aligned with the appropriate human germ line variable region sequences contained in the database; see, for example, Vbase (http://vbase.mrc-cpe.cam.ac.uk/) hosted by the MRC Protein Engineering Center (Cambridge, UK). For example, amino acids considered potentially deviant from the true sequence of the embryonic line may arise from the PCR primer sequence lining up during cloning. Compared to engineered human-like antibodies, such as single chain antibody (scFv) fragments derived from a phage display library or xenogeneic mice, the human monoclonal antibody of the present invention is characterized by:

(i) that it is obtained using a human immune response and not animal surrogates, i.e. said antibody is produced in response to natural IAPP or proIAPP in conformations characteristic of them in the human body, (ii) in that it protects the human or is at least essential for the presence of IAPP and/or proIAPP and (iii) because Since the antibody is of human origin, the risk of cross-reactivity with self-antigens is minimized.

Thus, in accordance with the present invention, the terms human monoclonal antibody, human monoclonal autoantibody, human antibody, and the like. are used to refer to an IAPP and/or proIAPP binding molecule of human origin, i. e. isolated from a human cell, such as a B cell or hybridoma cell, or whose cDNA is directly cloned from the mRNA of a human cell, such as a human memory B cell. Human antibodies remain human antibodies even when amino acid substitutions are introduced into the antibody, for example to improve binding characteristics.

Antibodies derived from human immunoglobulin libraries or from animals transgenic for

- 14 042691 one or more human immunoglobulins not expressing endogenous immunoglobulins as described below and, for example, in US Pat.

For example, the joining of heavy and light chain antibodies like human antibodies, eg synthetic and semi-synthetic antibodies, usually isolated by phage display, does not necessarily reflect the true joining that occurs in the original human B cells. Accordingly, Fab and scFv fragments derived from recombinant expression libraries commonly used in the art can be considered artificial, with all possible inherent effects in terms of immunogenicity and stability.

In contrast, the present invention provides isolated antibodies with mature affinity of selected human subjects, characterized by the possibility of therapeutic use and tolerability in humans.

As used herein, the term rodentized antibody or rodentized immunoglobulin refers to an antibody containing one or more CDRs of a human antibody of the present invention and a human framework containing amino acid substitutions and/or deletions and/or insertions based on a rodent antibody sequence. When referring to rodents, murine and rat sequences are preferably used, and antibodies containing such sequences are referred to as murinized or ratinized, respectively. The human immunoglobulin from which the CDRs are derived is called the parent or acceptor, and the rodent antibody from which the scaffold changes are derived is called the donor. The constant regions need not be present, but if they are, they are usually substantially identical to the constant regions of the rodent antibody, ie. identical to them at least about 85-90%, preferably about 95% or more. Therefore, in some embodiments, a full-length murinized human immunoglobulin heavy or light chain comprises a mouse constant region, a human CDR, and a substantially human-specific framework that contains a series of murinized amino acid substitutions. Typically, a murinized antibody is an antibody containing a light and/or heavy chain variable murinized region. For example, a murinized antibody should not include, for example, typical chimeric antibodies, since the entire variable region of a chimeric antibody is not a mouse variable region. The modified antibody murinated during the murinization process binds to the same antigen as the original antibody from which the CDRs are derived and is generally less immunogenic in mice than the original antibody. The explanations above for murinized antibody are similarly applicable to other rodentized antibodies, such as ratinized antibodies that use rat sequences instead of mouse sequences.

As used herein, the term heavy chain portion includes amino acid sequences derived from an immunoglobulin heavy chain. The heavy chain-containing polypeptide comprises at least one of the following: a CH1 domain, a hinge (eg, upper, middle, and/or lower hinge) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, a binding polypeptide for use in the present invention may comprise a polypeptide chain containing a CH1 domain; a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain containing a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the implementation of the polypeptide according to the invention contains a polypeptide chain containing a CH3 domain. In addition, a binding polypeptide for use in the present invention may lack at least a portion of the CH2 domain (eg, all or part of the CH2 domain). As discussed above, one of ordinary skill in the art will recognize that these domains (eg, heavy chain regions) may be modified such that their amino acid sequence differs from the natural immunoglobulin molecule.

In some antibodies or antigen-binding fragments, variants or derivatives thereof described herein, the heavy chain portions of one polypeptide chain of the multimer are identical to those located on the second polypeptide chain of the multimer. Alternatively, the monomers containing the heavy chain portions of the invention are not identical. For example, each monomer may contain its own target-binding site, forming, for example, a bispecific antibody or diabody.

In yet another embodiment, the antibodies or antigen-binding fragments, variants or derivatives thereof described herein comprise a single polypeptide chain, such as scFv, and must be expressed intracellularly (intrabodies) to enable therapeutic and diagnostic use in vivo.

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The heavy chain regions of the binding polypeptide for use in the diagnostic and therapeutic methods described herein may be derived from various immunoglobulin molecules. For example, the heavy chain portion of the polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In yet another example, the heavy chain portion of the polypeptide may comprise a hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In yet another example, the heavy chain portion of the polypeptide may comprise a chimeric hinge partly derived from an IgG1 molecule and partly from an IgG4 molecule.

As used herein, the term light chain portion includes amino acid sequences derived from an immunoglobulin light chain. In a preferred embodiment, the light chain region contains at least one V _L or CL domain.

The minimum size of a peptide or polypeptide epitope for an antibody is considered to be approximately four to five amino acids. The epitopes of a peptide or polypeptide preferably contain at least seven, more preferably at least nine, and most preferably at least about 15-30 amino acids. Because the CDR can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids that make up the epitope need not be consecutive, and in some cases may not even be on the same peptide chain. In the present invention, a peptide or polypeptide epitope recognized by an antibody of the present invention comprises a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or about 15-30 consecutive or non-consecutive IAPP or proIAPP amino acids.

The terms specifically binding or specifically recognizing, which are used interchangeably herein, generally mean that a binding molecule, such as an antibody, binds to an epitope via its antigen-binding domain, and that said binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to specifically bind to an epitope if it binds to said epitope through its antigen-binding domain more easily than if it binds to a random, non-specific epitope. The term specificity is used herein to measure the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody A can be considered to have higher specificity for a given epitope than antibody B, or antibody A can be said to bind to epitope C with higher specificity than to the cognate epitope D.

The term immunological binding characteristics, or other binding characteristics of an antibody to an antigen, in all its grammatical forms, refers to the specificity, affinity, cross-reactivity, and other binding characteristics of an antibody.

By predominantly binding(s) is meant that a binding molecule, eg, an antibody, specifically binds to an epitope more readily than to a related, similar, homologous, or analogous epitope. Thus, an antibody that preferentially binds to a given epitope should be more likely to bind to said epitope than to a related epitope, even though such an antibody may cross-react with a related epitope.

In a non-limiting example, a binding molecule, such as an antibody, may be considered to preferentially bind a first epitope if it binds said first epitope with a lower dissociation constant (KD) than the antibody's KD for the second epitope. In yet another non-limiting example, an antibody may be considered to preferentially bind a first antigen if it binds said first epitope with an affinity at least an order of magnitude less than the antibody's KD for the second epitope. In yet another non-limiting example, an antibody may be considered to preferentially bind a first epitope if it binds said first epitope with an affinity at least two orders of magnitude less than the antibody's KD for the second epitope.

In yet another non-limiting example, a binding molecule, e.g., an antibody, may be considered to preferentially bind a first epitope if it binds said first epitope at a slower rate of dissociation (k(off)) than (k(off)) of an antibody for the second epitope. In yet another non-limiting example, an antibody may be considered to preferentially bind a first epitope if it binds said first epitope with an affinity at least an order of magnitude less than the k(off) of the antibody for the second epitope. In yet another non-limiting example, an antibody may be considered to preferentially bind a first epitope if it binds said first epitope with an affinity at least two orders of magnitude less than the k(off) of the antibody for the second epitope.

A binding molecule, e.g., an antibody or antigen-binding fragment, variant or derivative thereof described herein, may be referred to as binding an IAPP and/or proIAPP or a fragment, variant or specific conformation thereof with a dissociation rate (k(off)) less than or equal to 5x10 '2 with ^-1 , 10' ² with ^-1 , 5x10'3 with ^-1 or 10-3 with ^-1 . More preferably, an antibody of the present invention may be referred to as binding IAPP and/or proIAPP or a fragment, variant or specific conformation thereof with a dissociation rate (k(off)) less than or equal to 5 x ^{10 -4} s ^-1 , 10 ⁴ s ^-1 , 5x10'5 s ^-1 , or 10' ⁵ s ^-1 5x10 ^-6 s ^-1 , 10' ⁶ s ^-1 , 5x10 ^-7 s ^-1 or 10' ⁷ s ^-1 .

A binding molecule, e.g., an antibody or antigen-binding fragment, variant or derivative thereof described herein, may be referred to as binding an IAPP and/or proIAPP or a fragment, variant or specific conformation thereof with an association rate (k(on)) greater than or equal to 103 M ^-1 s ^-1 , 5x103 M ^-1 s ^-1 , 10 ⁴ M ^-1 s ^-1 or 5x10 ⁴ M ^-1 s ^-1 . More preferably, an antibody of the present invention may be referred to as binding IAPP and/or proIAPP or a fragment, variant or specific conformation thereof with an association rate (k(on)) greater than or equal to 105 M ^-1 s ^-1 , 5x105 M ^-1 s ^{- 1} , 10 ⁶ M ^-1 s ^-1 , or 5x10 ⁶ M ^-1 s ^-1 or 10 ⁷ M ^-1 s ^-1 .

A binding molecule, such as an antibody, is said to competitively inhibit the binding of a reference antibody to a given epitope if it preferentially binds to the epitope such that it blocks binding of the reference antibody to said epitope to some extent. Competitive inhibition can be determined by any method known in the art, for example, using a competitive solid-phase ELISA. An antibody can be said to competitively inhibit binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least , by 50%.

As used herein, the term affinity refers to a measure of the binding strength of individual epitopes to the CDR of a binding molecule, eg an immunoglobulin molecule; see, for example, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988) on p. 27-28. As used herein, the term avidity refers to the overall stability of the complex between an immunoglobulin population and an antigen, i. the strength of the functional association of the mixture of immunoglobulin with the antigen; see, for example, Harlow on p. 29-34. Avidity is associated both with the affinity of individual immunoglobulin molecules in a population for certain epitopes, and with the valency of immunoglobulins and antigen. For example, an interaction between a divalent monoclonal antibody and an antigen with a repetitive epitope structure, such as a polymer, will be highly avid. The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method; see, for example, Berzofsky et al., Antibody-Antigen Interactions In Fundamental Immunology, Paul, WE, Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, WH Freeman and Company New York, NY (1992 ), and the methods described herein. Common techniques for measuring the affinity of an antibody for an antigen include ELISA, RIA, and surface plasmon resonance. The measured affinity of the interaction of a particular antibody and antigen can vary under different conditions, for example, salt concentration, pH. Thus, measurements of affinity and other antigen binding parameters, eg KD, IC ₅₀ , are preferably performed using standardized antibody and antigen solutions and a standardized buffer.

In addition, binding molecules, such as antibodies or antigen-binding fragments, variants or derivatives thereof, according to the invention can be described or defined in terms of their cross-reactivity. As used herein, the term cross-reactivity (reactivity) refers to the ability of an antibody specific for one antigen to react with a second antigen; a measure of relatedness between two different antigenic substances. Thus, an antibody is cross-reactive if it binds to an epitope that is different from the epitope that induced it. The cross-reactive epitope usually contains a large number of complementary structural elements similar to those of the inducing epitope and in some cases may actually match the antibody better than the original.

For example, some antibodies have some degree of cross-reactivity in that they bind similar but not identical epitopes, e.g., epitopes with at least 95%, at least 90%, at least 85%, at least 80% , at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (calculated using methods known in the art and described herein) in relation to the reference epitope. An antibody can be said to have little or no cross-reactivity if it does not bind epitopes to less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (calculated using methods known in the art and described herein) to the reference epitope. An antibody can be considered highly specific for a particular epitope if it does not bind any analogs, orthologues, or homologues of that epitope.

In addition, binding molecules, eg antibodies or antigen-binding fragments, variants or derivatives thereof, according to the invention can be described or indicated in terms of their binding affinity for IAPP and/or proIAPP. Preferred binding affinity includes binding with a dissociation constant or K _d less than 5x10 ^-2 M, 10 ^-2 M, 5x10 ^-3 M, 10 ^-3 M, 5x10 ^-4 M, 10 ^-4 M, 5x10 ^-5 M, 10 ^-5 M,

- 17 042691

5x10 ^-6 M, 10 ^-6 M, 5x10 ^-7 M, 10 ^-7 M, 5x10 ^-8 M, 10 ^-8 M, 5x10 -9 M, 10 ^-9 M, ^{5x10 -10 M} , 10 ^-10 M, 5x10 ^-11 M, 10 ^-11

M, 5x10 ^-12 M, 10 ^-12 M, 5x10 ^-13 M, 10 ^-13 M, 5x10 ^-14 M, 10 ^-14 M, 5x10 ^-15 M or 10 ^-15 M.

As previously stated, the subunit structure and three-dimensional configuration of the constant regions of the various classes of immunoglobulins are well known. As used herein, the term VH domain includes the N-terminal immunoglobulin heavy chain variable domain, and the term CH1 domain includes the first (closest to the N-terminus) immunoglobulin heavy chain constant region domain. The CH1 domain is adjacent to the VH domain and is located N-terminally from the hinge region of the heavy chain of the immunoglobulin molecule.

As used herein, the term CH2 domain includes a region of a heavy chain molecule that extends, for example, from about residue 244 to residue 360 of an antibody according to conventional numbering schemes (residues 244-360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat EA et al., op.cit). The CH2 domain is unique in the sense that it is not related to another domain to a significant extent. Instead, two branched N-linked carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. In addition, it is well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.

As used herein, the term hinge region includes the region of the heavy chain molecule connecting the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge regions can be divided into three distinct domains: superior, middle, and inferior hinge domains; see Roux et al, J. Immunol. 161 (1998), 4083-4090.

As used herein, the term disulfide bond includes a covalent bond between two sulfur atoms. The amino acid cysteine contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In the naturally occurring IgG molecule, the CH1 and CL regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions corresponding to positions 239 and 242 according to the Kabat numbering system (positions 226 and 229 according to the EU numbering system).

In this document, the terms connected, hybrid and fused are used interchangeably. These terms refer to the combination of two or more elements or components by any means, including chemical conjugation or recombinant means. The term in-frame fusion refers to the combination of two or more open reading frames (ORFs) to form a continuous extended ORF while maintaining the correct reading frame of the original ORF when translated. Thus, a recombinant fusion protein is a single protein containing two or more segments corresponding to the polypeptides encoded by the original ORFs (these segments are not normally combined in this way in nature). Although a continuous reading frame is thus obtained throughout the combined segments, these segments can be physically or spatially separated, for example, by a linker sequence inserted without disturbing the reading frame. For example, polynucleotides encoding the CDRs of an immunoglobulin variable region may be combined without defrauding, but separated by a polynucleotide encoding at least one immunoglobulin framework or additional CDRs, provided that the hybrid CDR can be co-translated as part of a contiguous polypeptide.

The term expression as used herein refers to the process by which a gene produces a biochemical compound, such as an RNA or a polypeptide. This process includes any manifestation of the functional presence of a gene in a cell, including, without limitation, gene knockdown, as well as transient expression and stable expression. It includes, without limitation, transcription of a gene into messenger RNA (mRNA), transfer RNA (tRNA), short hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and translation of the mRNA into polypeptide(s). If the final target product is a biochemical compound, expression includes the production of said biochemical compound and all of its precursors. Expression of a gene produces a gene product. As used herein, a gene product may be a nucleic acid, such as messenger RNA, resulting from the transcription of a gene, or a polypeptide that is translated from a transcript. The gene products described herein further include nucleic acids that have undergone post-transcriptional modifications, such as polyadenylation, or polypeptides that have undergone post-translational modifications, such as methylation, glycosylation, lipid addition, association with other protein subunits, proteolytic cleavage, and the like.

As used herein, the term sample refers to any biological material obtained from a subject or patient. In one aspect, the sample may contain blood, peritoneal fluid, CSF, saliva, or urine. In other aspects, the sample may contain whole blood, blood plasma, blood serum, B cells obtained by enrichment of blood samples, and cultured cells (eg, B cells of the subject). The sample may also include a biopsy or tissue sample, including nervous tissue. In other aspects, the sample may contain whole cells and/or cell lysate. Blood samples can be obtained by methods known in the art. In one aspect, the pellet can be resuspended by vortexing at 4°C in 200 µl of buffer (20 mM Tris, pH 7.5, 0.5% Nonidet, 1 mM EDTA, 1 mM PMSF, 0.1 M NaCl, protease inhibitor IX Sigma Protease Inhibitor and IX Sigma Phosphatase Inhibitors 1 and 2) The suspension can be stored on ice for 20 minutes with occasional vortexing. After centrifugation at 15,000xg for 5 min at about 4°C, aliquots of the supernatant can be stored at about -70°C.

Diseases.

Unless otherwise indicated, the terms disorder and disease are used interchangeably herein and include any unwanted physiological changes in a subject, animal, isolated organ, tissue, or cell/cell culture.

In T2DM, genetic and environmental factors lead to the development of insulin resistance, followed by a compensatory increase in beta cell mass and secretion of insulin and amylin (IAPP) to maintain normal blood glucose levels. The resulting high concentrations of amylin promote the formation of toxic hIAPP oligomers and the deposition of hIAPP fibrils found in more than 90% of T2DM patients. Deposition of hIAPP correlates with a decrease in insulin-producing beta cells and is also thought to play a role in the death of β-cells in pancreatic islets transplanted in T1DM individuals. This application provides several human antibodies from pools of healthy or obese donors with a high risk of T2DM but no disease, cloned and produced using recombinant techniques, as detailed below.

However, in one embodiment of the invention, antibodies of the invention having binding molecules having a specificity substantially the same as any of them, polynucleotides, vectors or cells of the invention are used to prepare a pharmaceutical or diagnostic composition for prophylactic and therapeutic treatment, monitoring progression or response to treatment and/or diagnosing diseases from the group of diabetes mellitus, consisting of type 1 diabetes mellitus (T1DM), gestational diabetes, prediabetes, latent adult autoimmune diabetes (LADA; type 1.5 diabetes), and/or type 2 diabetes (SD2).

In a preferred embodiment, antibodies of the present invention having binding molecules having a specificity substantially the same as any of them, polynucleotides, vectors or cells of the present invention are used to prepare a pharmaceutical or diagnostic composition for prophylactic and therapeutic treatment, progression monitoring, or response to treatment and/or diagnosis of a group of disorders typically characterized by symptoms such as metabolic changes that precede, cause and/or are related/associated with or associated with T2DM, including diseases that cause damage to the pancreas and are therefore capable of leading to diabetes, including chronic pancreatitis, cystic fibrosis, pancreatic cancer; in diseases that increase the risk of type 2 diabetes, including Alzheimer's disease, Huntington's disease; and / or in cardiovascular diseases associated or not associated with obesity and T2DM. In one preferred embodiment, the symptoms commonly associated with the above diseases include impaired insulin sensitivity and increased secretion of insulin and/or hIAPP in the subject.

In one embodiment, the group of disorders associated with the above specific symptoms includes gestational diabetes, prediabetes (when high blood glucose does not reach the T2DM threshold or insulin resistance); metabolic syndrome in general as a risk factor for diabetes or as a condition that may exist before diabetes; islet amyloidosis in general as a risk factor for diabetes or as a condition that may exist before diabetes; obesity in general as a risk factor for diabetes or as a condition that may exist before diabetes, and/or beta cell deficiency after clinical pancreatic islet transplantation.

In addition, in a preferred embodiment of the antibodies of the present invention, binding molecules having a specificity substantially the same as any of them, polynucleotides, vectors or cells of the present invention are used to obtain a composition for detecting an altered, i. increased or decreased secretion of amylin compared to secretion of amylin in healthy subjects in the differential diagnosis of type 1 diabetes, latent autoimmune diabetes in adults (LADA; type 1.5 diabetes) compared with forms of type 2 diabetes, or disorders preceding overt type 2 diabetes, for example, gestational diabetes or prediabetes; in diseases that cause damage to the pancreas and therefore can lead to diabetes, for example, chronic pancreatitis, cystic fibrosis, pancreatic cancer; in diseases that increase the risk of type 2 diabetes, including Alzheimer's disease, Huntington's disease; and / or in cardiovascular diseases associated or not associated with obesity and T2DM.

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Disorders such as obesity and insulin resistance/hyperinsulinemia are often seen as a sign of predisposition or as a symptom of T2DM, which can lead to elevated circulating islet amyloid polypeptide (IAPP) levels ahead of overt T2DM. It has been found that amylin oligomers, fibrils and plaques (hIAPP) accumulate not only within the pancreas and kidneys, but also in the heart in patients with obesity and insulin resistance. This accumulation has been observed in association with altered cellular Ca ²⁺ homeostasis, which in turn may contribute to cardiac dysfunction in these patients.

Therefore, in one preferred embodiment, antibodies according to the present invention, binding molecules having a specificity essentially the same as any of them, polynucleotides, vectors or cells according to the present invention are used to obtain a pharmaceutical or diagnostic composition for prophylactic and therapeutic treatment, relief , monitoring progression or response to treatment, and/or diagnosing a group of disorders that occur after T2DM or are due to metabolic changes that precede and/or cause T2DM, i.e. metabolic changes that occur in the pre-diabetic state, and this group of disorders includes heart disease, strokes, diabetic retinopathy, renal failure, ketoacidosis and non-ketotic hyperosmolar coma.

More than 20 neurodegenerative disorders are known (see, for example, Table 1 on p. 511 in M. Ristow, J. Mol. Med 82 (2004), 510-529) associated with diabetes mellitus, increased insulin resistance and obesity, impaired sensitivity to insulin and excessive or impaired secretion of insulin. Therefore, in one embodiment of the invention, antibodies of the invention having binding molecules having a specificity substantially the same as any of them, polynucleotides, vectors or cells of the invention are used to prepare a pharmaceutical or diagnostic composition for prophylactic and therapeutic treatment, monitoring the progression or response to treatment and/or diagnosing neurodegenerative disorders, including Alzheimer's disease (AD), ataxia-telangiectasia (AT), Bardet-Biedl syndrome (BBS), Friedreich's ataxia (FRDA), Huntington's disease, myotonic dystrophy, narcolepsy, disease Parkinson's, Prader-Willi syndrome and Werner's syndrome.

Impaired glucose tolerance is unlikely to cause complications characteristic of diabetes mellitus, but it is associated with a higher risk of diabetes than the normal type, which may be the cause of macrovascular disease.

Insulin resistance refers to conditions with decreased sensitivity to insulin. Insulin is known to exhibit a wider range of effects, for example, in addition to affecting glucose metabolism, it also affects lipid metabolism or blood vessels and kidneys. After a decrease in insulin sensitivity, not only abnormalities in glucose metabolism can be induced, but also metabolic lipid abnormalities, such as hypertriglyceridemia, a decrease in plasma HDL levels, or hypertension, as an abnormal effect of insulin on blood vessels or kidneys.

In this document, the term diabetic (diabetic) in relation to a person in general and in this case means a random concentration of glucose in plasma or blood >200 mg/dL (>11.1 mmol/L) or fasting plasma glucose >126 mg/dL ( >7.0 mmol/L) or 2 hours after glucose loading >200 mg/dL (>11.1 mmol/L) during an oral glucose tolerance test. In addition or alternatively, the term diabetic is used for subjects who exhibit one or more of the clinical symptoms of diabetes, including increased thirst (polydipsia), frequent urination (polyuria), severe hunger, unexplained weight loss, fatigue and blurred vision, vulnerability to slow-healing ulcers and frequent infections, including infections of the bladder, vagina, and gums and/or areas of dark skin (acanthokeratoderma).

In this document, the term non-diabetic (non-diabetic) in relation to a person in general and in this case means a fasting plasma or blood glucose level of <100 mg/dL (5.6 mmol/dL) or 2 hours after glucose load <140 mg /dl (<7.8 mmol/dl) during an oral glucose tolerance test.

In this document, the term pre-diabetic (pre-diabetic) in relation to a person in general and in this case means a fasting plasma or blood glucose level of 100-125 mg / dl (5.6-6.9 mmol / l) or 2 hours after a glucose load 140-199 mg/L (7.8-11.1 mmol/L) during an oral glucose tolerance test. Unless otherwise indicated, the terms prediabetic, prodromal, and presymptomatic are used interchangeably in this document and describe the preclinical stages of T2DM.

In addition or alternatively, a glycosylated hemoglobin (HbA1c) level can be used to diagnose diabetes in a subject. An elevated HbA1c level within or outside the 6.5% threshold is diagnosed as diabetes, ie. in general and in this case, the term diabetic is used in relation to a person, while levels from 5.7 to 6.4% indicate a high risk of developing diabetes and cardiovascular diseases and are a marker of prediabetes, or a prediabetic/presymptomatic state in a person . The term non-diabetic in relation to a person in general and in this case means that the level of HbAlc is below the threshold value of 5.7%.

Rodent Type II Diabetes Mellitus Models for Drug Development Examples of conventional animal models that spontaneously develop type II diabetes include KK-Ay mice (Nishimura M., Exp. Anim. 18, 147-157, 1969), NSY mice ( Ueda H., et al., Diabetologia 38, 503508, 1995), db/db mice (Hummel K. P., et al., Science 153, 1127-1128, 1966), ob/ob mice (Herberg L. & Kley H. K., Horm. Metab. Res. 7, 410-5, 1975) and AKITA mice (Yoshioka M., et al., Diabetes 46, 887-894, 1997).

Among these animals, KK-Ay mice and NSY mice are obese models, and db/db mice and ob/ob mice are obese models caused by abnormal leptin receptor or leptin production. On the other hand, AKITA mice are a model for diabetes caused by abnormal pancreatic β-cells.

An example of a non-obese type II diabetes animal model to date is the mouse model described in Japanese Patent Application No. 2004-65181. This mouse exhibits abnormal insulin secretion.

However, the antibodies of the present invention are preferably screened and tested in transgenic animals, e.g. hIAPP-expressing rodents, e.g., rats transgenic for human amylin in pancreatic β-cells (HIP rats), as described in Butler et al. , Diabetes 53 (2004), 1509-1516 and Matveyenko and Butler, Diabetes 55 (2006), 2106-2114. More preferably, a mouse type 2 diabetes model overexpressing human IAPP is used, as described in Matveyenko and Butler (2006), ILAR J. 47(3): 225-233, and summarized in Table 1. 2 on page 228 of the said issue. Due to the overexpression of hIAPP in these animal models, the symptoms of T2DM, such as the state of hyperglycemia, are reliably reproduced. In general, the term hyperglycemia or hyperglycemic state refers to a significantly elevated fasting plasma glucose level on two consecutive measurements taken at different time intervals and when compared to non-transgenic control littermates. Absolute values obtained from hyperglycemic animals may depend on the particular animal model used, eg h-IAPP mice (hemizygous)/ob/+ >~15-20 mM glucose; h-IAPP mice (homozygous)/FVB/N >~11 mM glucose.

Methods for testing diabetes include measuring physiological changes and analyzing the blood or plasma of diabetic (diabetic) animals compared to healthy (non-diabetic) animals. These measurements include growth dynamics, body mass index (BMI), lean mass index (LMI), food and water intake, sex differences, fasting and random blood glucose, triglycerides (TG), lipoproteins, cholesterol, liver weight and liver lipids, kidney size and function, glucose tolerance test (GTT), insulin tolerance test (GTT), blood insulin concentration, pancreatic islet cell morphology, high fat diet and calorie restriction.

As used herein, the terms occasional and non-fasted generally mean any time of the day or night, regardless of the time since the last meal.

As used herein, the term fasted generally means no caloric intake for at least 12 hours.

It should be appreciated that these definitions are current guidelines generally followed by medical practitioners as defined by the American Diabetes Association (ADA) and the German Diabetes Association (GDA). Principles may change over time and vary by region or country and depend on the group or institution (e.g. ADA, World Health Organization, NIDDK/NIH, CDC, GDA, etc.) producing the guidelines, as known to those skilled in the art in this field of technology. Physicians may also use clinical experience, the patient's medical history, and/or other information when making diagnosis and treatment decisions. Thus, these definitions may change over time in line with advances in science and medicine.

Treatment.

As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent or slow down (alleviate) an undesirable physiological change or disorder, such as the development of diabetes. Beneficial or desirable clinical outcomes include relief of symptoms, relief of disease, stabilization (i.e., no worsening) of disease state, delay or retardation of disease progression, improvement or temporary relief of disease state, and remission (partial or complete), detectable or not detectable, but are not limited to them. Treatment can also mean an increase in life expectancy compared to life expectancy without treatment. Those in need of treatment include those who already suffer from the condition or disorder, as well as those who are prone to

- 21 042691 this condition or disorder, or persons in whom the onset of this condition or disorder should be prevented.

Unless otherwise indicated, the terms drug, drug, or medicament are used interchangeably herein and refer to all:

(A) products, drugs and preparations for internal or external use, and any substance or mixture of substances intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease in humans or other animals;

(B) products, medicines and preparations (other than foodstuffs) intended to affect the structure or function of the human body or other animals; and (C) products intended to be used as a component of any product listed in (A) and (B), but not limited to.

The terms drug, medicament, or medicament include the full formula of a preparation intended for use in humans or other animals, containing one or more agents, compounds, substances, or (chemical) compositions, and, in certain other situations, other pharmaceutically inactive excipients. , for example, fillers, disintegrants, lubricating and sliding agents, binders or agents that facilitate the transport, disintegration, disaggregation, dissolution and bioavailability of a drug, drug or drug in the intended target area in the human body or other animals, for example, on the skin, in the stomach or intestines. The terms agent, compound, or substance are used interchangeably herein and refer, in a more specific context, to all pharmacologically active agents, i. agents that produce a desired biological or pharmacological effect, or have been investigated or tested for the ability to cause such a possible pharmacological effect using the methods of the present invention, but are not limited to them.

By subject or individual or animal or patient or mammal is meant any subject, more particularly a mammal, such as a human patient, for whom it is desirable to obtain a diagnosis, prognosis, prophylaxis or therapy.

pharmaceutical carriers.

Pharmaceutically acceptable carriers and routes of administration can be taken from the relevant literature known to the person skilled in the art. Pharmaceutical compositions according to the present invention can be formulated according to methods widely known in the art; see, for example, Remington: The Science and Practice of Pharmacy (2000) by the University of Sciences in Philadelphia, ISBN 0-683-306472, Vaccine Protocols. ^2nd Edition by Robinson et al., Humana Press, Totowa, New Jersey, USA, 2003; Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems. 2nd Edition by Taylor and Francis (2006), ISBN: 0-8493-1630-8. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, sterile solutions, and the like. Compositions containing such carriers can be formulated using well known conventional methods. Said pharmaceutical compositions may be administered to a subject at a suitable dose. The introduction of suitable compositions can be carried out in various ways. Examples include administration of a composition containing a pharmaceutically acceptable carrier by oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal and intracranial routes. Aerosol formulations, for example nasal sprays, include purified aqueous or other solutions of the active agent with preservatives and isotonic agents. Such compositions are preferably adjusted to a pH and isotonic state compatible with the nasal mucosa. The present invention also provides pharmaceutical compositions for oral administration, such as single domain antibody molecules (eg, nanobodies™), etc. Such oral compositions may be in the form of tablets, capsules, powder, liquid or semi-solid form. The tablet may contain a solid carrier such as gelatin or an adjuvant. Formulations for rectal or vaginal administration may be presented as suppositories with a suitable carrier; see also O'Hagan et al., Nature Reviews, Drug Discovery 2(9) (2003), 727735. Further guidance on formulations suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA , ^17th ed. (1985), and related updates. For a brief overview of drug delivery methods, see Langer, Science 249 (1990), 1527-1533.

II. Antibodies of the present invention.

The present invention relates generally to human anti-IAPP antibodies and antigen-binding fragments thereof, which preferably exhibit the immunological binding characteristics and/or biological properties of the antibodies shown in the examples. In accordance with the present invention, human monoclonal antibodies specific for IAPP and/or proIAPP were cloned from a pool of healthy human subjects.

- 22 042691

In the experiments carried out in accordance with the present invention, antibodies in the conditioned medium of human memory B cell cultures were screened in parallel for binding to aggregated oligomeric, fibrillar and/or non-fibrillar IAPP and/or proIAPP protein and bovine serum albumin (BSA). Cloning of antibodies was performed only for cultures

B cells positive for aggregated IAPP and/or proIAPP but not for BSA.

Thanks to this measure, it was possible to isolate several antibodies. Selected antibodies were further analyzed to determine the class and subclass of the light chain. Selected appropriate antibody transcripts from memory B cell cultures were then transcribed by RT-PCR, cloned, and inserted into expression vectors for recombinant production; see attached examples. Recombinant expression of human antibodies in HEK293 or CHO cells and subsequent study of their binding specificity for human IAPP and/or proIAPP protein (FIGS. 3 and 4; Example 1), and their characteristic binding to pathologically aggregated forms of these proteins (FIG. 5 Example 2) confirmed that, for the first time, human antibodies have been cloned that are highly specific for the IAPP and/or proIAPP protein and clearly recognize pathologically aggregated forms of the IAPP and/or proIAPP protein, such as IAPP fibrils. The second round of experiments confirmed the above results, as shown in FIG. 7-9 and Example 4. In addition, mouse chimeric antibodies prepared in accordance with the present invention and comprising the CDR domains of human antibodies of the present invention showed a binding affinity for human IAPPs equal to that of human antibodies, as shown in FIG. 11 and 12 and in example 6.

Thus, the present invention relates to an isolated natural human monoclonal antibody against islet amyloid polypeptide (IAPP) and its binding fragments, derivatives and variants. In one embodiment, the antibody is capable of binding human IAPP and/or proIAPP.

In one embodiment, the present invention provides an anti-IAPP and/or proIAPP antibody, or antigen-binding fragment, variant, or derivative thereof, wherein the antibody specifically binds to the same IAPP epitope as a reference antibody selected from the group consisting of NI-203.9A2, NI-2O3.19H8, NI-203.26C11, NI-203.8E3, NI-203.11B12, NI-203.205F8, NI-203.9B3, NI203.19F2, and NI-2O3.15C7. Epitope mapping revealed a sequence in human IAPP containing the amino acids 19-SSNNFGA-25 (SEQ ID NO: as a unique linear epitope recognized by the NI-2O3.19H8 antibody of the present invention, a sequence in human IAPP containing the amino acids 2-SSNNFGA-8 ( SEQ ID NO: as unique linear epitope recognized by NI-203.26d1 antibody of the present invention, sequence in human IAPP containing amino acids 10-QRLANFLVHS-19 (SEQ ID NO: 71) as unique linear epitope recognized by NI-2O3 antibody .15C7 of the present invention (See Figures 5A and 5B and Example 3) Thus, in one embodiment, an antibody of the present invention is provided, wherein said antibody specifically binds an IAPP epitope containing the amino acid sequence SSNNFGA (SEQ ID NO: 4) , CNTATCA (SEQ ID NO: 5) or QRLANFLVHS (SEQ ID NO: 71) As detailed in Example 3, epitopes of recombinant anti-IAPP antibodies NI-203.9A2, NI-203.8E3 and NI-203.19F2 have not been identified to date. , indicating that these antibodies likely bind non-linear epitopes.

In addition, in one embodiment, the present invention provides an anti-IAPP and/or proIAPP antibody, or antigen-binding fragment, variant, or derivative thereof, wherein the antibody specifically binds to the same proIAPP epitope as the reference antibody selected from the group consisting of NI antibodies. -203.1D10, NI-203.2A11, NI-2O3.1OC4, NI-203.20H9, NI-203.26D2, NI203.60H3, and NI-203.26C11.

Also, out of touch with the original experimental observations, as shown in Example 4 and FIG. 7, the anti-IAPP human monoclonal antibodies NI-203.9A2, NI-2O3.19H8, NI-2O3.26C11 and NI-203.8E3 of the present invention preferably have specific binding to pathological IAPP aggregates (fibrils in this example) rather than preferential recognition. IAPP in physiological form in the pancreas. The same is expected for the human monoclonal antibodies NI-203.19F2 and NI-2O3.15C7 against IAPP. Accordingly, the present invention provides a set of human antibodies against IAPP and/or proIAPP with a binding specificity particularly useful for diagnostic and therapeutic purposes. Thus, in one embodiment, the present invention provides antibodies capable of specifically binding pathologically aggregated forms of IAPP and/or proIAPP (see Figures 5 and 7-9 and Examples 2 and 4). See also FIG. 7 and 8 and in the description below.

In one embodiment, an antibody of the present invention exhibits the binding properties of the exemplary NI-203.9A2, NI-2O3.19H8, NI-2O3.26C11, NI-203.8E3, NI-203.19F2, and NI203.15C7 antibodies described in the examples. In addition or alternatively, the anti-IAPP and/or proIAPP antibody of the present invention preferably recognizes pathologically aggregated IAPP and/or proIAPP, e.g., IAPP fibrils, rather than physiological IAPP and/or proIAPP monomers,

- 23 042691 especially when analyzed according to examples 2-4. Thus, in one embodiment, an antibody of the present invention does not substantially recognize a physiological IAPP. The term is essentially unrecognizable, when used herein to describe the binding affinity of a molecule from the group consisting of an antibody, fragment or binding molecule thereof, to a particular target molecule, antigen and/or conformation of the target molecule and/or antigen means that a molecule of the above group binds the specified molecule, antigen and/or conformation with a binding affinity of at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times or 9 times less than the dissociation constant ( K _D ) molecules of the above group when binding another molecule, antigen and/or conformation. In a preferred embodiment, the term essentially does not recognize, when used in this application means that a molecule of the aforementioned group binds the specified molecule, antigen and/or conformation with a binding affinity of at least 10 times, 20 times, 50 times, 100 times, 1000 times or 10000 times less than the dissociation constant (K _D ) of the specified molecule of the above group when binding another molecule, antigen and/or conformation. In addition or alternatively, an anti-IAPP and/or proIAPP antibody of the present invention binds to pathogenic aggregated forms of human IAPP and/or proIAPP, in particular the forms described in Example 4. In this context, the binding characteristics may be in the range of representative antibodies NI-203.9A2, NI-2O3.19H8, NI-2O3.26C1 1 and NI-203.8E3 as shown in FIG. 4 and respectively in FIG. 5, i.e. be characterized by a half maximum effective concentration (EC ₅₀ ) of about 1 pm to 500 nM, preferably an EC ₅₀ of about 10 pM to 100 nM, most preferred EC ₅₀ of about 100 pM to 50 nM for human IAPP as shown for NI-203.19H8, or EC ₅₀ from about 100 μm to 10 nM for human IAPP as shown for NI-2O3.9A2, NI-203.26C11 and NI-203.8E3. In this context, the experimental results presented in Example 4 and shown in FIG. 4 and respectively in FIG. 5 also indicate that, in addition or alternatively, some of the anti-IAPP antibodies of the present invention do not substantially recognize proIAPP as shown for representative antibodies NI-203.9A2, NI-2O3.19H8 and NI-203.8E3. Thus, in one embodiment, an antibody of the present invention does not substantially recognize proIAPP.

In addition or alternatively, an anti-IAPP antibody of the present invention specifically binds, in addition to mature IAPP, a precursor form of IAPP, i. also proIAPP, specifically as described in Example 1. In this context, the binding characteristics may be in the range of a typical NI-203.26C11 antibody, as shown in FIG. 3 and respectively in FIG. 4, i.e. be characterized by a half maximum effective concentration (EC ₅₀ ) from about 1 pm to 500 nM, preferably EC ₅₀ from about 10 pM to 400 nM, more preferably EC ₅₀ from about 100 pM to 400 nM or from about 100 pM to 300 nM, most preferred EC ₅₀ from about 1 to 300 nM for aggregated pro-IAPP as shown for NI-203.26C11.

In addition or alternatively, an anti-IAPP antibody of the present invention specifically binds mature IAPP and does not or substantially does not bind the precursor form of IAPP, ie. proIAPP, specifically as described in Example 1. In this context, the binding characteristics may be in the range shown for typical antibodies NI-203.9A2, NI-2O3.19H8 and NI203.8E3 in FIG. 4 and respectively in FIG. 5 and above.

In one embodiment, an antibody of the present invention exhibits the binding properties of exemplary antibodies NI-203.1D10, NI-203.2A11, NI-2O3.1OC4, NI-2O3.2OH9, NI-203.26D2, and NI203.60H3 binding proIAPP preferentially over proIAPP. At the same time, by screening for antibodies specifically binding to the N-terminal flanking region of proIAPP, which is absent from IAPP, antibodies against proIAPP have been generated, as there is some evidence for a role for proIAPP, which was not N-terminally processed, rather than the full-length proIAPP peptide. in amyloid formation and cell death. Therefore, in one embodiment, an anti-proIAPP antibody of the present invention, e.g., representative antibodies NI-203.1D10, NI-203.2A11, NI203.10C4, NI-203.20H9, NI-203.26D2, and NI-2O3.60h3, does not bind or essentially does not bind the IAPP.

Some purified antibodies bind to a wide range of biomolecules, such as proteins. The skilled artisan will appreciate that the term specific is used herein to indicate that biomolecules other than IAPP and/or proIAPP proteins or fragments thereof do not substantially bind to an antigen-binding molecule, such as one of the antibodies of the present invention. In a preferred embodiment, the level of binding of the non-IAPP and/or proIAPP biomolecule results in a binding affinity of at most 20% or less, 10% or less, 5% or less, 2% or less, or 1% or less (i.e., e. at least 5, 10, 20, 50, or 100 times less) affinity for IAPP and/or proIAPP, respectively; see, for example, Example 1 and FIG. 3. In addition, the anti-IAPP and/or proIAPP antibodies of the present invention specifically bind to IAPP and/or proIAPP aggregates, as confirmed by experiments demonstrating that the antibodies of the present invention do not recognize abnormal amyloid Ae in the human brain of Alzheimer's disease and have only minimal ability to cross-link with several candidate proteins with a propensity for misfolding/aggregation, as shown for the antibody examples NI-203.9A2, NI-203.19H8, and NI-203.26C11 on paraffinized brain sections of a patient with diagnosis of Alzheimer's disease and in direct experiments based on solid-phase ELISA; see example 5 and FIG. 10. Thus, in one embodiment, an antibody of the present invention is provided that does not substantially recognize the amyloid-β ( _{A1_42} ) peptide.

In one embodiment, an anti-IAPP and/or proIAPP antibody of the present invention preferentially binds to aggregated forms of IAPP and/or proIAPP, fibrils and/or oligomers of IAPP and/or proIAPP; see the results of the direct ELISA experiments in Example 2 and the results obtained on the pancreas of patients diagnosed with T2DM and on the pancreas of diabetic cats by immunohistochemical staining described in Example 4 and shown in FIG. 7 and 9, respectively. In one embodiment, an antibody of the present invention preferentially binds aggregated forms of IAPP and/or proIAPP, wherein the aggregates comprise, consist essentially of, or consist of fibrillar forms and/or fibrillar oligomers of IAPP. In yet another embodiment, an antibody of the present invention preferentially binds aggregated forms of IAPP and/or proIAPP, wherein the aggregates comprise, consist essentially of, or consist of non-fibrillar forms and/or non-fibrillar oligomers of IAPP. In yet another embodiment, an antibody of the present invention preferentially binds aggregated forms of IAPP and/or proIAPP, wherein the aggregates comprise, consist essentially of, or consist of fibrillar or non-fibrillar forms of IAPP and/or proIAPP and/or fibrillar and non-fibrillar IAPP oligomers and/or proIAPP. In yet another embodiment, an anti-IAPP and/or proIAPP antibody of the present invention preferably binds to both native and pathologically aggregated forms of IAPP and/or proIAPP; see the experimental results presented in example 2 and 3 and obtained using direct solid-phase ELISA.

As previously mentioned, aggregates containing IAPP and/or proIAPP are also associated with amyloid deposits in the pancreatic islets of T2DM patients. Thus, in one embodiment, an antibody of the present invention may be used in the treatment of T2D.

The present invention also relates to an antibody or an antigen-binding fragment, variant or derivative thereof, wherein the antibody comprises an antigen-binding domain identical to that of an antibody selected from the group consisting of NI-2O3.9A2, NI-2O3.19H8, NI-203.26C11, NI- 203.8E3, NI203.11B12, NI-203.205F8, NI-203.9B3, NI-203.19F2, NI-2O3.15C7, NI-203.1D10, NI-203.2A11, NI-2O3.1OC4, NI-203.20H9, NI -203.26D2 and NI-203.60H3.

In addition, the present invention provides several such binding molecules, for example, antibodies and their binding fragments, the binding region, for example, the binding domain of which may contain at least one hypervariable complementarity region (CDR) of the V _H or V _L variable region, containing any from the amino acid sequences depicted in FIG. 1 or 2. The corresponding nucleotide sequences encoding the above variable regions are shown below in table. II and, accordingly, in table. III. Exemplary sets of CDRs of said V _H and/or V _L region amino acid sequences are depicted in FIG. 1 and 2. However, as discussed below, one of ordinary skill in the art is well aware that, as an addition or alternative, CDRs whose amino acid sequence differs from those shown in FIGS. 1 and respectively in FIG. 2 for one, two, three or even more amino acids in the case of CDR2 and CDR3. Thus, in one embodiment, an antibody of the present invention or an IAPP and/or proIAPP binding fragment is provided, the variable region of which contains at least one hypervariable region (CDR) shown in FIG. 1 and/or one or more CDRs containing one or more amino acid substitutions.

In one embodiment, an antibody of the present invention is one of the antibodies comprising the amino acid sequence of the VH or VL region shown in FIG. 1, or a V _H and/or V _L region containing one or more amino acid substitutions. In a preferred embodiment, the antibody of the present invention is characterized by the retention of a cognate heavy and light chain compound, as in human B cells.

In an alternative embodiment, an antibody of the present invention is an antibody, or an antigen-binding fragment, derivative, or variant thereof, that competes for binding to IAPP and/or proIAPP with at least one of the antibodies comprising the V _H or V _L region shown in FIG. 1.

The experimental results presented in Example 4 show that certain anti-IAPP and/or proIAPP antibodies of the present invention bind to pathogenic aggregated forms of human IAPP and/or proIAPP rather than to physiological forms of these proteins. Thus, in one embodiment, an antibody of the present invention recognizes IAPP and/or proIAPP aggregates rather than physiological IAPP and/or proIAPP. In addition, in one embodiment, an antibody of the present invention recognizes aggregates

- 25 042691

IAPPs containing IAPP oligomers and/or fibrils are preferred over physiological IAPPs. In another embodiment, an antibody of the present invention recognizes IAPP aggregates containing non-fibrillar IAPP and/or non-fibrillar IAPP oligomers rather than physiological IAPP.

As previously stated, some of the antibodies of the present invention have been shown to be able to bind both IAPP and its precursor, proIAPP. In addition, some of the antibodies of the present invention have been isolated from human patients due to their ability to bind proIAPP. Thus, in one embodiment, an antibody of the present invention can bind proIAPP.

Thus, in addition or alternative to the above, in one embodiment, an antibody of the present invention or an IAPP and/or proIAPP binding fragment is provided, the variable region of which contains at least one hypervariable region (CDR) shown in FIG. 2 and/or one or more CDRs containing one or more amino acid substitutions.

In one embodiment, an antibody of the present invention is one of the antibodies comprising the amino acid sequence of the VH or VL region shown in FIG. 2, or a VH and/or VL region containing one or more amino acid substitutions. In a preferred embodiment, the antibody of the present invention is characterized by the retention of a cognate heavy and light chain compound, as in human B cells.

In an alternative embodiment, an antibody of the present invention is an antibody, or antigen-binding fragment, derivative, or variant thereof, that competes for binding to IAPP and/or proIAPP with at least one of the antibodies comprising the V _H or V _L region shown in FIG. 2.

The antibody of the present invention may be a human antibody, in particular for therapeutic use. Alternatively, an antibody of the present invention is a rodent, rodentized, or chimeric human rodent antibody, preferably a mouse, murinized, or chimeric mouse-human antibody, or a rat, ratinized, or chimeric, rat-human antibody, which are particularly useful for diagnostic methods and animal research. In one embodiment, an antibody of the present invention is a chimeric human rodent antibody or a rodentized antibody.

In addition, in one embodiment, a chimeric antibody of the present invention exhibits the binding properties of the mouse chimeric examples NI-2O3.9A2, NI-2O3.19H8, and NI203.26C11 described in the examples. In addition, the mouse chimeric antibodies of the present invention bind to human IAPP fibrils with high affinity as described in Example 6. In a preferred embodiment, the binding affinity of the chimeric antibodies is close to that of their human antibody counterparts. In this context, binding characteristics may be in the range shown for typical mouse chimeric antibodies NI-203.9A2, NI-2O3.19H8, and NI-203.26d1, with _EC50 of 18.6, 23.9, and 11.5 nM, respectively, as described in Example 6 and shown in FIG. 11 and in table. C in this document. Binding to BSA was not observed.

In one embodiment, an antibody of the present invention is obtained from cultures of single or oligoclonal B cells that are cultured and culture supernatant containing antibodies produced by said B cells is screened for the presence and affinity of anti-IAPP and/or proIAPP antibodies in German The screening process includes screening for binding to native hIAPP monomers, fibrillar or non-fibrillar aggregate-like oligomers derived from a synthetic full-length hIAPP peptide with the amino acid sequence shown in SEQ ID NO: 1; and/or a separate and independent screening for binding to a synthetic peptide derived from human proIAPP (N-terminal fragment) with the amino acid sequence TPIESHQVEKRKCNTATCATQR represented by SEQ ID NO: 7.

In addition or alternatively, the screening process for the presence and affinity of anti-IAPP and/or proIAPP antibodies may include the steps of an immunoreactivity (TAPIR) assay of sensitive amyloid plaque tissue, such as those described in WO2004/095031, the contents of which are incorporated herein by references made in this study by analogy with amyloid deposits in pancreatic islets. As an addition or alternative, pancreatic sections can be screened for binding to IAPP and/or proIAPP, as described by analogy in international application WO2008/081008 for sections of the brain and spinal cord.

As mentioned above, since the human monoclonal antibody of the present invention is generated during the human immune response, it must recognize epitopes that are of particular pathological significance and which may not be available or less immunogenic in immunization processes to produce, for example, mouse monoclonal antibodies and screening. in vitro phage display library, respectively. Accordingly, it is reasonable to assume that the epitope of the human anti-IAPP and/or proIAPP antibody of the present invention is unique, and no other antibody capable of binding to the epitope recognized by the human monoclonal antibody of the present invention exists; see also FIG. 5A and a figure showing the unique epitope of the NI-2O3.19H8 antibody and respectively the NI-203.26d 1 antibody of the present invention. Another indication of the uniqueness of the antibodies according to the present invention is the fact that, as indicated in example 3, the antibodies NI-2O3.9A2 and NI-203.8E3 according to the present invention bind putative conformational epitopes of IAPP aggregates, which, as indicated above, have a particular pathological value and may also not be available when producing antibodies by conventional means, such as immunization or in vitro library screening.

Thus, in one embodiment, the present invention also includes anti-IAPP antibodies and IAPP-binding molecules in general that compete with a human monoclonal antibody of the present invention for specific binding to IAPP. More specifically, the present invention relates to an antibody or antigen-binding fragment, variant or derivative thereof, wherein the antibody specifically binds to the same IAPP epitope as a reference antibody selected from the group consisting of NI-203.9A2, NI-2O3.19H8, NI- 203.26d1, NI-203.8E3, NI-203.19F2, and NI-203.15C7.

In addition, in one embodiment, the present invention also includes anti-IAPP and/or proIAPP antibodies and IAPP and/or proIAPP binding molecules in general that compete with a human monoclonal antibody of the present invention for specific binding to proIAPP. Thus, more specifically, the present invention also relates to an antibody or antigen-binding fragment, variant or derivative thereof, wherein the antibody specifically binds to the same proIAPP epitope as a reference antibody selected from the group consisting of NI203.1D10, NI-203.2A11 , NI-2O3.1OC4, NI-2O3.2OH9, NI-203.26D2, NI-2O3.6OH3 and NI-203.26C11.

In addition or alternatively, the present invention also extends to bispecific antibodies against IAPP and/or proIAPP and IAPP and/or proIAPP binding molecules in general which compete with the human monoclonal antibody of the present invention for specific binding to both IAPP and with proIAPP. Thus, more specifically, the present invention also relates to an antibody or antigen-binding fragment, variant or derivative thereof, wherein the antibody specifically binds to the same IAPP and proIAPP epitope as a typical NI-203.26C11 antibody. Thus, in light of the foregoing, in one embodiment, the present invention also provides an antibody or antigen-binding molecule that competes with an antibody of the present invention for specific binding to IAPP and/or proIAPP.

Antibody competition is determined by an assay in which the immunoglobulin of interest inhibits the specific binding of a reference antibody to a common antigen, eg, IAPP and/or proIAPP. Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (ELISA), competitive sandwich assay; see Stahli et al., Methods in Enzymology 9 (1983), 242-253; solid-phase direct biotin-avidin ELISA; see Kirkland et al., J. Immunol. 137 (1986), 3614-3619 and Cheung et al., Virology 176 (1990), 546-552; solid-phase direct analysis using labels, solid-phase direct sandwich analysis using labels; see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988); solid phase direct RIA using I ¹²⁵ as a label; see Morel et al., Molec. Immunol. 25 (1988), 7-15 and Moldenhauer et al., Scand. J. Immunol. 32 (1990), 77-82. Typically, such assays involve the use of purified IAPP and/or proIAPP and its aggregates, e.g., solid surface-bound oligomers and/or fibrils, or cells bearing any of these antigens, unlabeled test immunoglobulin, and labeled reference immunoglobulin, i.e. . a human monoclonal antibody of the present invention. Competitive inhibition is measured by determining the amount of label associated with a solid surface or cells in the presence of the test immunoglobulin. Typically, the immunoglobulin being tested is present in excess. In a preferred embodiment, the competitive binding assay is performed under the conditions described for the solid phase ELISA in the attached examples. Antibodies identified by competitive analysis (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an adjacent epitope close enough to that bound by the reference antibody to cause steric hindrance. Typically, if the competing antibody is present in excess, it will inhibit specific binding of the reference antibody to the common antigen by at least 50% or 75%. In this regard, the present invention also relates to an antibody or its antigen-binding fragment, variant or derivative, where the antibody competitively inhibits the binding of a reference antibody selected from the group consisting of NI-203.9A2, NI-2O3.19H8, NI-203.26C11, NI-203.8E3, NI-203.19F2 and NI-203.15C7, with IAPP.

In addition, the present invention also provides an antibody, or an antigen-binding fragment, variant, or derivative thereof, wherein the antibody competitively inhibits binding of a reference

- 27 042691 antibodies selected from the group consisting of NI-203.1D10, NI-203.2A11, NI-203.10C4, NI-203.20H9, NI203.26D2, NI-2O3.6OH3 and NI-203.26G11, with proIAPP.

In addition, in addition or alternatively, the present invention also provides an antibody or antigen-binding fragment, variant or derivative thereof, wherein the antibody competitively inhibits the binding of a reference antibody, e.g., a typical NI-2O3.26C11 antibody, to IAPP and/or proIAPP.

In yet another embodiment, the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain (VH) variable region, wherein at least one of the V _H -CDRs of the heavy chain variable region, or at least two of The heavy chain variable region VH-CDRs are at least 80%, 85%, 90%, or 95% identical to the amino acid sequences of the reference heavy chain VH-CDRs I, VH-CDR2, or VH-CDR3 of the antibodies described herein. Alternatively, the VH-CDR1, VH-CDR2, and VH-CDR3 V _H regions are at least 80%, 85%, 90, or 95% identical to the amino acid sequences of the reference VH-CDR1, VH-CDR2, and VH-CDR3 of the antibody heavy chain described in this document. Thus, in this embodiment, the heavy chain variable region of the invention comprises the V _H -CDR1, V _H -CDR2, and V _H -CDR3 polypeptide sequences belonging to the groups shown in FIG. 1 or 2, respectively. Although in FIG. 1 and 2 show Kabat VHCDRs, other CDR definitions such as Chothia VH-CDRs are also within the scope of the present invention and can be readily identified by one skilled in the art using the data presented in FIGS. 1 and 2.

In yet another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain (VH) variable region, wherein the VH-CDR1, VH-CDR2, and VH-CDR3 regions contain polypeptide sequences identical to the VH groups. -CDR1, VH-CDR2 and VH-CDR3 shown in FIG. 1 or 2, respectively.

In yet another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (V _H ), wherein the V _H -CDR1, V _H -CDR2, and V _H -CDR3 regions comprise the polypeptide sequences , identical to the V _H -CDR1, V _H -CDR2 and V _H -CDR3 groups shown in FIG. 1 or 2, respectively, except for one, two, three, four, five, or six amino acid substitutions in any V _H -CDR. In some embodiments, the amino acid substitutions are conservative.

In yet another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (V _L ), wherein at least one of the VL-CDRs of the light chain variable region, or at least two of The light chain variable region VL-CDRs are at least 80%, 85%, 90% or 95% identical to the amino acid sequences of the light chain VL-CDR1, VL-CDR2 or VL-CDR3 reference antibodies described herein. Alternatively, the VL-CDR1, VLCDR2, and VL-CDR3 V _L regions are at least 80%, 85, 90, or 95% identical to the amino acid sequences of the reference _VL -CDR1, VL _- CDR2, and _VL -CDR3 light chain antibodies described in this document. Thus, in this embodiment, the light chain variable region of the invention comprises the V _L -CDR1, V _L -CDR2 and V _L -CDR3 polypeptide sequences of the polypeptides shown in FIG. 1 or 2, respectively. Although in FIG. 1 and 2 show Kabat VL-CDRs, other definitions of CDRs such as Chothia VL-CDRs are also included in the present invention.

In yet another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain (VL) variable region, wherein the VL-CDR1, VL-CDR2, and VL-CDR3 regions contain polypeptide sequences identical to the VL groups. -CDR1, VL-CDR2 and VL-CDR3 shown in FIG. 1 or 2, respectively.

In yet another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (V _L ), wherein the V _L -CDR1, V _L -CDR2, and V _L -CDR3 regions comprise the polypeptide sequences , identical to the V _L -CDR1, V _L -CDR2 and V _L -CDR3 groups shown in FIG. 1 or 2, respectively, except for one, two, three, four, five, or six amino acid substitutions in any V _L CDR. In some embodiments, the amino acid substitutions are conservative.

The immunoglobulin or its cDNA coding may be further modified. Thus, a further embodiment of the present invention includes any of the steps of producing a chimeric antibody, a murinized antibody, a single chain antibody, a Fab fragment, a bispecific antibody, a hybrid antibody, a labeled antibody, or an analog of any of them. Appropriate methods are known to those skilled in the art and are described, for example, in Harlow and Lane Antibodies, A Laboratory Manual, CSH Press, Cold Spring Harbor (1988). If derivatives of these antibodies are obtained using a phage display technique, surface plasmon resonance, for example, implemented in the BIAcore system (Schier, Human Antibodies Hybridomas 7 (1996), 97-105 Malmborg, J. Immunol Methods 183 (1995), 7-13. Obtaining chimeric antibodies is described, for example, in international application WO89/09622. Methods for producing humanized antibodies are described in, for example, European application EP-A1 0239400 and international application WO90/07861. Additional sources of antibodies for use in accordance with the present invention are the so-called xenogeneic antibodies. The general principle for producing xenogeneic antibodies, eg human-like antibodies, in mice is described, for example, in international applications WO91/10741, WO94/02602, WO96/34096 and WO 96/33735. As discussed above, the antibodies of the invention may exist in various forms other than full antibodies; including, for example, in the form of Fv, Fab and F(ab) ₂ and a single chain; see, for example, international application WO88/09344. Thus, in one embodiment, an antibody of the present invention is selected from the group consisting of a single chain Fv fragment (scFv), an F(ab') fragment, an F(ab) fragment, and an F(ab') ₂ fragment. .

The antibodies of the present invention, or their corresponding immunoglobulin chain(s), may be further modified using conventional methods known in the art, e.g., deletion(s), insertion(s), substitution(s) , addition(s) and/or recombination(s) of amino acids or any other modifications known in the art, alone or in combination. Methods for making such modifications to the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to those skilled in the art; see, for example, Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994). Modifications of an antibody of the invention include chemical or enzymatic modification at one or more constituent amino acids, including side chain modifications, backbone modifications, and N- and C-terminal modifications, including acetylation, hydroxylation, methylation, amidation, and addition of carbohydrate or lipid groups, cofactors and so on. Similarly, the present invention encompasses the production of chimeric proteins containing the described antibody or any N-terminal fragment attached to a heterologous molecule, for example, an immunostimulatory ligand at the C-terminus; for relevant technical details see, for example, international application WO00/30680.

In addition, the present invention encompasses peptides, including peptides containing the binding molecules described above, for example, containing the CDR3 region of the variable region of any of these antibodies, in particular the CDR3 of the heavy chain, since it has often been observed that the CDR3 of the heavy chain (HCDR3) is a site with an increased degree of variability and predominant participation in the antigen-antibody interaction. Such peptides can be easily synthesized or obtained by recombinant means to obtain a binding agent suitable for use according to the invention. Such methods are well known to those skilled in the art. Peptides can be synthesized, for example, using commercially available automated peptide synthesizers.

Peptides can also be generated using recombinant techniques by inserting DNA expressing the peptide into an expression vector and transforming cells with the expression vector to produce the peptide.

Thus, the present invention relates to any binding molecule, for example, an antibody or a binding fragment thereof, targeted to human anti-IAPP and/or proIAPP antibodies according to the present invention and having the above-mentioned properties, i. specifically recognizing IAPP and/or proIAPP. The specificity and binding affinity of such antibodies and binding molecules can be tested using solid-phase ELISA and immunohistochemical techniques, as described herein, see, for example, examples. These characteristics of antibodies and binding molecules can also be checked by Western blotting. Preliminary results of serial experiments carried out in accordance with the present invention showed that the human anti-IAPP and/or proIAPP antibody of the present invention, in particular the antibodies NI-203.9A2, NI-2O3.19H8, NI-2O3.26C11 and NI- 203.8E3 were able to bind differently to IAPP fibrils in solid-phase ELISA. In addition, antibodies 203.9A2, NI-2O3.19H8, and NI-203.26C11 of the present invention were shown to preferentially bind to pathological structures in the human body, such as large amyloid-like deposits in pancreatic islets corresponding to pathological IAPP fibrils, according to imaging with staining with ThioS and Congo red (see Fig. 7A). Similar properties are expected to apply to the NI-203.19F2 and NI-203.15C7 antibodies.

Human antibodies NI-2O3.9A2, NI-2O3.19H8, and NI-203.26C11 showed prominent pancreatic islet staining on amyloid-positive sections, but did not show pancreatic islet staining from a T2DM patient in the absence of amyloid deposits.

- 29 042691 and from a control patient without a diagnosis of DM2 (see example 4 and Fig. 7). The antibodies of the present invention also gave positive results in the pancreas of diabetic cats with evidence of insular amyloid deposition; see fig. 9. This binding specificity to pathological forms of IAPP and/or proIAPP in human and animal tissues, in addition to the biochemical experiments shown here (see Example 2 and FIG. 5), highlights the possibility of using the antibodies of the present invention in the treatment and diagnosis of diseases. associated with the appearance of aggregated IAPPs and/or proIAPPs in the pancreas.

As an alternative to obtaining immunoglobulins directly from a culture of B cells or memory B cells, cells can be used as a source of rearranged (rearranged) heavy and light chain loci for subsequent expression and/or genetic manipulation. Rearranged antibody genes can be obtained by reverse transcription of the corresponding mRNAs to obtain cDNA. If desired, the heavy chain constant region can be exchanged for a different isotype constant region or removed entirely. Variable regions can be joined to encode single chain Fv regions. Several Fv regions can be connected to give the ability to bind to more than one target, or combinations of chimeric heavy and light chains can be used. Once the genetic material has been obtained, it is easy to construct the analogs described above that retain the ability to bind to the desired target. Methods for cloning antibody variable regions and obtaining recombinant antibodies are known to those skilled in the art and are described, for example, in Gilliland et al., Tissue Antigens 47 (1996), 1-20; Doenecke et al., Leukemia 11 (1997), 1787-1792.

After obtaining the appropriate genetic material and, if necessary, modifying it to encode an analog, coding sequences, including sequences encoding at least the heavy and light chain variable regions, can be inserted into expression systems contained in vectors that can be transfected with standard recombinant host cells. A variety of such host cells can be used; while mammalian cells are preferred for efficient processing. Typical mammalian cell lines suitable for this purpose include, but are not limited to, CHO cells, HEK293 cells, or NSO cells.

The production of antibodies or analogues is then performed by culturing the modified recombinant hosts under culture conditions suitable for growth of host cells and expression of coding sequences. The antibodies are then isolated by isolating them from the culture. Expression systems are preferably designed to incorporate signal peptides so that the resulting antibodies are released into the medium; however, intracellular production is also possible.

In accordance with the above, the present invention also relates to a polynucleotide encoding an antibody or equivalent binding molecule according to the present invention, and, in the case of an antibody, preferably at least the variable region of the immunoglobulin chain of the antibody described above. Typically, said variable region encoded by a polynucleotide contains at least one hypervariable region (CDR) of the V _H and/or V _L variable region of said antibody.

One skilled in the art will appreciate that the variable domain of an antibody containing the variable domain described above can be used to construct other polypeptides or antibodies with desired specificity and biological function. Thus, the present invention also encompasses polypeptides and antibodies containing at least one CDR of the variable domain described above and having substantially the same or similar binding properties as the antibody described in the accompanying examples. It is known to the person skilled in the art that binding affinity can be increased by amino acid substitutions in CDRs, or in hypervariable loops (Chothia and Lesk, J. Mol. Biol. 196 (1987), 901-917), which overlap with CDRs defined by by Kabat; see, for example, Riechmann, et al., Nature 332 (1988), 323-327. Thus, the present invention also relates to antibodies in which one or more of these CDRs contains one or more (preferably no more than two) amino acid substitutions. In a preferred embodiment, one or more immunoglobulin chains of an antibody of the invention contain two or all three CDRs of the variable region defined in FIG. 1 or, respectively, in FIG. 2.

Binding molecules such as antibodies or antigen-binding fragments, variants or derivatives thereof according to the invention, as known to those skilled in the art, may contain a constant region mediating one or more effector functions. For example, binding of the C1 component of complement to the constant region of an antibody can activate the complement system. Complement activation plays an important role in opsonization and lysis of pathogenic cells. Complement activation also stimulates the inflammatory response and may also be involved in the mechanism of autoimmune hypersensitivity. In addition, antibodies bind to receptors on various cells via the Fc region, wherein the Fc receptor binding site in the Fc region of an antibody binds to an Fc receptor (FcR) on the cell surface. There are several Fc receptors that are specific for different classes of antibodies, including IgG (gamma receptors), IgE (epsi

- 30 042691 lon receptors), IgA (alpha receptors) and IgM (mu receptors). The binding of an antibody to Fc receptors on the cell surface triggers a number of important and diverse biological reactions, including the uptake and destruction of antibody-coated particles, the excretion of immune complexes, the lysis of antibody-coated target cells by killer cells (so-called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.

Accordingly, some embodiments of the present invention include an antibody, or an antigen-binding fragment, variant, or derivative thereof, wherein at least a portion of one or more constant region domains are removed or otherwise modified to provide desirable biochemical characteristics, such as reduced effector functions, the ability to to non-covalent dimerization, increase in the ability to localize to the site of aggregation and deposition of IAPP and/or proIAPP, decrease in serum half-life or increase in serum half-life compared to a whole, unmodified antibody with approximately the same immunogenicity. For example, some antibodies for use in the diagnostic and therapeutic methods described herein are domain deleted antibodies that contain a polypeptide chain similar to an immunoglobulin heavy chain but lack at least a portion of one or more heavy chain domains. For example, in some antibodies, one constant region domain of the modified antibody is completely deleted, for example, the CH2 domain is completely or partially deleted. In other embodiments, certain antibodies for use in the diagnostic and therapeutic methods described herein comprise a constant region, such as an IgG heavy chain constant region modified to eliminate glycosylation, and are hereinafter referred to as aglycosylated or agly antibodies. Such agly antibodies can be obtained by enzymatic processing as well as engineering of the glycosylation consensus site(s) in the constant region. Outside of theory, it is believed that agly antibodies may have an improved in vivo safety and stability profile. Methods for obtaining aglycosylated antibodies having the desired effector function can be found, for example, in international application WO2005/018572, incorporated herein by reference in its entirety.

In certain antibodies or antigen-binding fragments, variants or derivatives thereof, described herein, the Fc region can be mutated to reduce effector function using techniques known in the art. For example, deletion or inactivation (by point mutations or other means) of the constant region domain can weaken the binding of the circulating modified antibody to the Fc receptor, thereby increasing the localization of IAPP and/or proIAPP. In other instances, constant region modifications consistent with the present invention may moderately impair complement fixation and thereby reduce the serum half-life and non-specific binding of the conjugated cytotoxin. Other constant region modifications can be used to modify disulfide bonds or oligosaccharide groups that provide enhanced localization through increased antigen specificity or antibody flexibility. The resulting physiological profile, bioavailability, and other biochemical effects of the modifications, eg, IAPP and/or proIAPP localization, biodistribution, and serum half-life, are easily measured and quantified using well known immunological techniques without undue experimentation.

The Fc region of certain antibodies or their antigen-binding fragments, variants or derivatives described herein can be mutated or exchanged for an alternative protein sequence to enhance uptake of antibodies into cells, for example, by enhancing receptor-mediated endocytosis of antibodies by Fcγ receptors, LRP or Thy1 receptors or through superantibody technology, which is said to allow antibodies to enter living cells without damaging them (Expert Opin. Biol. Ther. (2005), 237-241). For example, using techniques known in the art, fusion proteins of the binding region of an antibody and related protein ligands of cell surface receptors or sequence-specific bi- or polyspecific antibodies can be constructed to bind to IAPP and/or proIAPP as well as to the cell surface receptor. .

The Fc region of some of the antibodies or antigen-binding fragments, variants or derivatives thereof described herein can be mutated or replaced with alternative protein sequences, or the antibody can be chemically modified to enhance its ability to cross the blood-brain barrier.

Modified forms of antibodies or antigen-binding fragments, variants or derivatives thereof according to the invention can be obtained from a full-length precursor or parent antibody using techniques known in the art. Example techniques are discussed in this document in more detail. Antibodies or their antigennegative fragments, variants or derivatives according to the invention can be obtained or produced using techniques known in the art. In some embodiments, antibody molecules or

- 31 042691 fragments are obtained in a recombinant way, i.e. obtained using recombinant DNA technology. Examples of techniques for obtaining antibody molecules or fragments thereof are described in detail in other sections of this document.

Antibodies or antigen-binding fragments, variants or derivatives thereof according to the invention also include derivatives modified, for example, by covalently attaching any type of molecule to the antibody in such a way that the covalent attachment does not interfere with the specific binding of the antibody to a cognate epitope. By way of example, and not limitation, antibody derivatives include antibodies modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, modification with known protecting/blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the derivative may contain one or more non-classical amino acids.

In specific preferred embodiments, the antibodies or antigen-binding fragments, variants or derivatives thereof of the invention should not elicit an immune response detrimental to the animal being treated, eg a human. In some embodiments, binding molecules, such as antibodies or antigen-binding fragments thereof according to the invention, are derived from a patient, such as a human patient, and subsequently used to treat the same species from which they are derived, such as a human, thereby attenuating or minimizing the occurrence of harmful immune reactions.

Deimmunization can also be used to reduce the immunogenicity of an antibody. As used herein, the term deimmunization includes modifying an antibody to modify T cell epitopes; see, for example, international applications WO98/52976 and WO00/34317. For example, the VH and V _L sequences of the parent antibody are analyzed and the human T cell epitope in each V region is mapped showing the location of the epitopes in relation to hypervariable regions (CDRs) and other key sequence residues. Individual T cell epitopes from the T cell epitope map are analyzed to determine alternative amino acid substitutions with a low risk of altering the activity of the final antibody. Design a number of alternative sequences V _H and V _L containing combinations of amino acid substitutions; these sequences are then inserted into a series of binding polypeptides, eg, IAPP- and/or proIAPP-specific antibodies or immunospecific fragments thereof, for use in the diagnostic and therapeutic methods described herein, and then their functions are tested. Typically, 12-24 antibody variants are prepared and tested. The complete heavy and light chain genes containing the modified human V and So regions are then cloned into expression vectors; the resulting plasmids are introduced into cell lines to produce the whole antibody. The antibodies are then compared by appropriate biochemical and biological assays and the optimal variant is identified.

Monoclonal antibodies can be obtained by a variety of different methods known in the art, including using hybridoma, recombinant and phage display technologies, or combinations thereof. For example, monoclonal antibodies can be made using hybridoma technologies, including those known in the art and described, for example, in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas Elsevier, N.Y., 563-681 (1981), these references are incorporated herein by reference in their entirety. The term monoclonal antibody as used herein is not limited to antibodies produced by hybridoma technology. The term monoclonal antibody refers to an antibody derived from a single clone, including any clone of a eukaryotic, prokaryotic cell or phage, and not to the method by which it is obtained. Thus, the term monoclonal antibody is not limited to antibodies produced by hybridoma technology. In some embodiments, the antibodies of the present invention are derived from human B cells immortalized by transformation with Epstein-Barr virus as described herein.

In a well-known hybridoma technique (Kohler et al., Nature 256 (1975), 495), relatively short-lived, or mortal, mammalian lymphocytes, e.g., B cells, obtained from a human subject as described herein, are combined with cells from an immortal tumor line ( for example, a myeloma cell line), resulting in hybrid cells or hybridomas that are immortal and capable of producing the antibody genetically encoded by the B cell. The resulting hybrids are separated into genetically similar strains by selection, dilution and re-growth, with each individual strain containing specific genes for the formation of a single antibody. They produce antibodies that are homogeneous with respect to the desired antigen and, given their absolute genetic relationship, are called monoclonal.

- 32 042691

The hybridoma cells thus obtained are seeded and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of the non-hybrid parent myeloma cells. Those skilled in the art will appreciate that reagents, cell lines, and media for hybridoma formation, selection, and growth are available from several sources and standard protocols are well established. In general, the culture medium in which the hybridoma cells grow is tested for the production of monoclonal antibodies against the target antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells is determined using an in vitro assay, such as immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA), as described herein. Once hybridoma cells have been found to produce antibodies having the target specificity, affinity and/or activity, clones can be subcloned using the serial dilution method and grown using standard methods (see, for example, Goding, Monoclonal Antibodies:). Principles and Practice, Academic Press, pp. 59-103 (1986). In addition, it should be taken into account that monoclonal antibodies secreted by subclones can be separated from the culture medium, ascitic fluid or serum using standard purification procedures, for example, protein A gel filtration, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. .

In yet another embodiment, lymphocytes can be selected by micromanipulation and variable genes isolated. For example, peripheral blood mononuclear cells can be isolated from an immunized mammal or a naturally immune mammal, such as a human, and cultured for 7 days in vitro. Cultures can be screened for specific IgGs that meet the screening criteria. Cells can be isolated from positive wells. Individual Ig-producing B cells can be isolated using FACS or complement-mediated analysis of hemolytic plaques. Ig-producing B cells can be selected by in vitro micromanipulation and the VH and V _L genes can be amplified using, for example, RT-PCR. The VH and V _L genes can be cloned into a vector for antibody expression and transfected into cells (eg, eukaryotic or prokaryotic cells) for expression.

Alternatively, antibody-producing cell lines can be selected and cultured using techniques well known to those skilled in the art. Such techniques are described in various laboratory manuals and original publications. In this regard, techniques suitable for use in the present invention, as described below, are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991) incorporated herein by reference in its entirety, including appendices.

Antibody fragments that recognize specific epitopes can be obtained using known techniques. For example, Fab and F(ab') ₂ fragments can be generated recombinantly or by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') ₂ fragments). ). F(ab') ₂ fragments contain a variable region, a light chain constant region, and a heavy chain CH1 domain. Such fragments are sufficient for use in, for example, immunodiagnostic procedures involving the binding of immunospecific regions of immunoglobulins to detection reagents, for example, radioisotopes.

Particularly desirable is the therapeutic use of human antibodies, such as those described herein, in human patients. The human antibodies of the present invention are isolated, for example, from healthy human subjects who, due to their overweight or obesity, may be at risk of developing a metabolic disorder, such as T2DM, or from patients suffering from this disorder, but with an unusually stable the course of the disease or an unusually mild form of the disease. However, the selection of a healthy human subject with a suspected risk of developing a metabolic disorder, such as T2DM, from which antibodies can be isolated as described herein, may also be based on the presence of other risks known to increase the likelihood of developing a metabolic disorder, for example, CD2. The specified risk can be established on the basis of an examination of the individual for such risk factors associated with the development of a metabolic disorder, for example, T2DM, such as age 45 years and older; overweight or obesity; diabetes in close relatives; African American, Alaska Native, American Indian, Asian American, Hispanic/Latino, Pacific Islander, Mongoloid, or Arab ancestry; a history of gestational diabetes; the birth of at least one child weighing more than 4.5 kg; blood pressure 140/90 or higher; elevated cholesterol with, for example, a high-density lipoprotein (HDL) level below 40 mg/dl (corresponding to less than 1 mmol/l), or a triglyceride level above 200-499 mg/dl (corresponding to more than 2.35.6 mmol/l) ; sedentary lifestyle; diagnosis of Polycystic Ovary Syndrome (PCOS); diagnosis of prediabetes at previous examination - an A1C (also called HbA1c or glycohemoglobin) level of 5.7 to 6.4 percent, impaired fasting glucose (IFG), or impaired glucose tolerance (IGT); other diagnosed clinical conditions associated with insulin resistance, for example, acanthosis keratoderma; history of cardiovascular disease.

Although it is reasonable to expect that when obesity or overweight is used as an indicator of a person developing a metabolic disease, such as T2DM, protective antibodies against IAPP and/or proIAPP would be more consistently produced in obese and asymptomatic healthy subjects than in subjects those diagnosed with lower risk, for example, subjects who are not obese, but overweight or even normal weight, subjects belonging to the last two groups, can also be used as a source of human antibodies according to the present invention.

Subjects can be classified as normal weight, overweight, or obese based on measurements of the subject's height and body weight and calculation of their body mass index using the following formula

BMI=weight [kg]/(height[m]).

Based on the results, subjects are defined as having normal weight (BMI 18.5-24.9 kg/m ² ), overweight (25.0-29.9 kg/m ² ) or obese (>30 kg/m ² ) based on current World Health Organization criteria (World Health Organization (2000) Obesity: preventing and managing the global epidemic. Report of a WHO consultation. World Health Organ Tech Rep Ser 894: 1.253). Alternatively or in addition, the subject's waist circumference (WC) can also be measured based on gender-specific thresholds, defining WC as normal (<94 cm [<34.6 inches] in males and <80 cm [31.5 inches] ] in women), moderately enlarged (94-102 cm [34.6-40 inches] in men and 80-88 cm [31.5-35 inches] in women), or enlarged (>102 cm [>40 inches] in men and >88 cm [>35 inches] in women) as described in InterAct Consortium, Langenberg et al., PLoS Med. 2012 Jun;9(6): e1001230, wherein protective antibodies against IAPP and/or proIAPP should be more stable in a healthy subject with an increased waist circumference (increased WC), which corresponds to the category of obesity (maximum risk of developing T2DM); such a subject is preferably used to isolate said antibodies, however individuals with moderately elevated or normal WC can also be used to isolate anti-IAPP and/or proIAPP antibodies.

In one embodiment, an antibody of the invention comprises at least one heavy or light chain CDR of an antibody molecule. In yet another embodiment, an antibody of the invention comprises at least two CDRs from one or more antibody molecules. In yet another embodiment, an antibody of the invention comprises at least three CDRs from one or more antibody molecules. In yet another embodiment, an antibody of the invention comprises at least four CDRs from one or more antibody molecules. In yet another embodiment, an antibody of the invention comprises at least five CDRs from one or more antibody molecules. In yet another embodiment, an antibody of the invention comprises at least six CDRs from one or more antibody molecules. This document describes typical antibody molecules containing at least one CDR, which can be included in the considered antibodies.

Antibodies of the present invention can be produced by any antibody synthesis method known in the art, in particular by chemical synthesis or, preferably, by recombinant expression techniques as described herein.

In one embodiment, an antibody or antigen-binding fragment, variant or derivative thereof according to the invention comprises a synthetic constant region with one or more domains partially or completely deleted (domain-deleted antibody). In some embodiments, compatible modified antibodies include constructs or domain-deleted variants in which the CH2 domain is completely removed (ACH2 constructs). In other embodiments, the deleted domain may be replaced with a short junction peptide to provide flexibility and freedom of movement for the variable region. It will be apparent to those skilled in the art that such constructs are particularly preferred due to the regulatory effect of the CH2 domain on the rate of antibody catabolism. Domain deleted constructs can be generated using a vector encoding a human IgG1 constant domain, see, for example, international applications WO02/060955 and WO02/096948A2. The design of this vector involves the deletion of the CH2 domain and the production of a synthetic vector expressing the domain-deleted IgG1 constant region.

In some embodiments, antibodies or antigen-binding fragments, variants or derivatives thereof according to the present invention are mini-antibodies. Minibodies can be obtained using methods described in the art, see, for example, US patent 5837821 or international application WO94/09817.

In one embodiment, an antibody or antigen-binding fragment, variant or derivative thereof according to the invention comprises an immunoglobulin heavy chain with a deletion or substitution of several or even one amino acid, provided that this allows association of monomeric subunits. For example, single amino acid mutations in selected regions of the CH2 domain may be sufficient to significantly attenuate Fc binding and thus enhance IAPP and/or proIAPP localization. Similarly, it may be desirable to simply remove a portion of one or more domains of the constant region that controls the effector function (eg, complement fixation) to be regulated. Such partial deletions of the constant regions can improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the constant region domain of interest unchanged. In addition, as mentioned above, the constant regions of the described antibodies can be synthesized by mutation or substitution of one or more amino acids, which improves the profile of the resulting construct. In this regard, it is possible to disrupt the activity provided by a conserved binding site (eg, Fc binding) while maintaining essentially the configuration and immunogenic profile of the modified antibody. Some other implementation options may include the addition of one or more amino acids to the constant region to improve the desired characteristics, such as effector function or provide more pronounced attachment to the cytotoxin or carbohydrate. In such embodiments, insertion or replication of specific sequences derived from selected constant region domains may be desirable.

The present invention also provides antibodies comprising, consisting essentially of, or consisting of variants (including derivatives) of the antibody molecules (e.g., VH and/or V _L regions) described herein, wherein said antibodies or fragments thereof immunospecifically bind to IAPP and/or proIAPP. To introduce mutations into the nucleotide sequence encoding an antibody, standard techniques known to those skilled in the art can be used, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions. In a preferred embodiment, variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions , less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions compared to the VH reference region, VH-CDR1, VH-CDR2, VH-CDR3, V _L region, VL-CDR1, VL-CDR2, or VL-CDR3 . A conservative amino acid substitution is a substitution in which an amino acid residue is replaced with another amino acid residue containing a side chain with a similar charge. There are definitions in the art for families of amino acid residues containing side chains with similar charges. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine , tryptophan, histidine). In addition, mutations can be introduced randomly throughout all or part of the coding sequence, e.g., by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind IAPP and/or proIAPP) .

For example, it is possible to introduce mutations only in the framework regions or only in the CDR region of the antibody molecule. The introduced mutations can be silent or neutral missense mutations, for example, have no or little effect on the ability of the antibody to bind antigen; in fact, some of these mutations do not change the amino acid sequence at all. These types of mutations may be useful for optimizing codons or improving antibody production by hybridomas. Codon-optimized regions encoding antibodies of the present invention are described elsewhere in this document. Alternatively, non-neutral missense mutations can alter the ability of an antibody to bind an antigen. Most silent and neutral missense mutations are likely to be located in the framework regions, while most non-neutral missense mutations are likely to be located in the CDR, although this is not a requirement. One of skill in the art can design and test mutant molecules with desirable properties, such as no change in antigen-binding activity or changes in binding activity (eg, improved antigen-binding activity or change in antibody specificity). After mutagenesis, the encoded protein can be expressed by conventional methods and the functional and/or biological activity of the encoded protein (e.g., the ability to immunospecifically bind at least one IAPP and/or proIAPP epitope) can be determined using the techniques described herein or conventional modification techniques known in this field of technology.

III. Polynucleotides encoding antibodies.

A polynucleotide encoding an antibody, or an antigen-binding fragment, variant, or derivative thereof, may contain any polyribonucleotide or polydeoxyribonucleotide that may

- 35 042691 be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide encoding an antibody or an antigen-binding fragment, variant or derivative thereof may comprise single and double stranded DNA, DNA that is a mixture of single and double stranded regions, single and double stranded RNA, and RNA that is a mixture of single and double stranded regions. regions, hybrid molecules containing DNA and RNA, which may be single-stranded or, more typically, double-stranded, or a mixture of single- and double-stranded regions. In addition, a polynucleotide encoding an antibody or an antigen-binding fragment, variant or derivative thereof may contain three-stranded regions containing RNA or DNA, or both RNA and DNA. A polynucleotide encoding an antibody or antigen-binding fragment, variant or derivative thereof may also contain one or more modified bases or DNA or RNA backbone linkages modified for stability or other reasons. Modified bases include, for example, tritylated bases and non-standard bases, such as inosine. A number of changes can be made to DNA and RNA; thus, a polynucleotide encompasses chemically, enzymatically or metabolically modified forms.

An isolated polynucleotide encoding a non-naturally occurring immunoglobulin-derived polypeptide variant (e.g., an immunoglobulin heavy or light chain region) can be created by introducing one or more nucleotide substitutions, additions, or deletions to the immunoglobulin nucleotide sequence such that one or more amino acids are inserted into the encoded protein. substitutions, additions or deletions. Mutations can be introduced using standard techniques, such as site-directed mutagenesis and NDP-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues.

As is well known, RNA can be isolated from original B cells, hybridoma cells, or other transformed cells using standard techniques, such as guanidinium isothiocyanate extraction and precipitation followed by centrifugation or chromatography. If desired, mRNA can be isolated from total RNA using standard techniques such as oligo-dT-cellulose chromatography. Suitable techniques are well known in the art. In one embodiment, antibodies to the DNA encoding the light and heavy chains can be generated simultaneously or separately using reverse transcriptase and DNA polymerase according to known methods. PCR can be run using consensus constant region primers or more specific primers based on published DNA sequences and heavy and light chain amino acid sequences. As discussed above, PCR can also be used to isolate DNA clones encoding the light and heavy chains of an antibody. In this case, libraries can be screened using consensus primers or large homologous probes, such as probes for human constant regions.

DNA, typically plasmid DNA, can be isolated from cells using techniques known in the art, restricted to restriction mapping, and sequencing according to standard, well-known techniques detailed, for example, in the recombinant DNA techniques referenced above. Of course, DNA can be synthetic according to the present invention at any time during isolation or subsequent analysis.

In this context, the present invention also relates to a polynucleotide encoding at least a binding domain or variable region of an immunoglobulin chain of an antibody of the present invention. In one embodiment, the invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (V _H ), wherein at least one of the heavy chain variable region CDRs, or at least two of the heavy chain variable region VH-CDRs are at least 80%, 85%, 90% or 95% identical to the amino acid sequences of the heavy chain reference VH-CDR1, VH-CDR2 or VH-CDR3 of the antibodies described herein. Alternatively, the V _H -CDR1, V _H -CDR2, or V _H -CDR3 V _H regions are at least 80%, 85, 90, or 95% identical to the amino acid sequences of the heavy chain reference V _H -CDR1, VH-CDR2, and VH-CDR3 antibodies described herein. Thus, in this embodiment, the heavy chain variable region of the invention comprises the VH-CDR1, VH-CDR2, and VH-CDR3 polypeptide sequences of the polypeptide sequences shown in FIG. 1 or respectively contains the VH-CDR1, VH-CDR2 and VH-CDR3 polypeptide sequences of the polypeptide sequences shown in FIG. 2.

In yet another embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (V _L ), wherein at least one of the VL-CDRs of the light chain variable region, or at least two of the V _L -CDRs of the light chain variable region are at least 80%, 85%, 90%, or 95% identical to the amino acid sequences of the reference light chain VL _CDR1 , _VL -CDR2, or _VL -CDR3 light chain antibodies described herein. The alternatives

- 36 042691 V _L -CDR1, V _L -CDR2 or V _L -CDR3 regions of V _L are at least 80, 85, 90 or 95% identical to the amino acid sequences of the reference VL-CDR1, VL-CDR2 and VL-CDR3 light the antibody chains described herein. Thus, in this embodiment, the light chain variable region of the invention comprises the VL-CDR1, VL-CDR2, and VL-CDR3 polypeptide sequences of the polypeptide sequences shown in FIG. 1 or respectively contains the V _L -CDR1, V _L -CDR2 and V _L CDR3 polypeptide sequences of the polypeptide sequences shown in FIG. 2.

In another embodiment, the invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain (VH) variable region, wherein the VH-CDR1, VH-CDR2, and V _H CDR3 regions comprise the polypeptide sequences , identical to the VH-CDR1, VH-CDR2, and VHCDR3 groups shown in FIG. 1 or, respectively, identical to the VH-CDR1, VH-CDR2 and VH-CDR3 groups shown in FIG. 2.

As known in the art, sequence identity between two polypeptides or two polynucleotides is determined by comparing the amino acid or nucleotide sequence of one polypeptide or polynucleotide with the sequence of a second polypeptide or polynucleotide. As discussed herein, that a particular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to another polypeptide can be determined by using methods and computer programs/software known in the art, for example, the BESTFIT program (Wisconsin Sequence Analysis Package, version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711), but not limited to her. To find the best homology segment between two sequences, BESTFIT uses the local homology algorithm from Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489. When using BESTFIT or any other program for aligning sequences to determine if a certain sequence is, for example, 95% identical to the reference sequence in accordance with the present invention, the parameters are set, of course, in such a way as to calculate the percent identity over the entire length of the reference sequence. polypeptide sequence and that homology gaps of no more than 5% of the total number of amino acids in the reference sequence are allowed.

In a preferred embodiment of the present invention, the polynucleotide comprises, consists essentially of, or consists of a nucleic acid comprising a polynucleotide sequence of the VH or VL region of an anti-IAPP and/or proIAPP antibody, as shown in Table 1. II or III. In this regard, one skilled in the art will appreciate that polynucleotides encoding at least a light and/or heavy chain variable domain can encode the variable domain of both or only one immunoglobulin chain. Thus, in one embodiment, the polynucleotide comprises, consists essentially of, or consists of a nucleic acid comprising a polynucleotide sequence of the V _H and V _L region of an anti-IAPP and/or proIAPP antibody, as shown in Table 1. II, or the sequence of the region V _H and V _L antibodies against IAPP and/or proIAPP, as shown in table. III.

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Table II. Nucleotide sequences of the VL and VH regions of anti-IAPP antibodies

Antibody Heavy (VH) and light chain (VL/VK) variable region nucleotide sequences NI-203.9A2-V _H GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGG TCCCTGAGGCTCTCCTGTGCAGCCTCTGGATTCACGTTTAGCACCTTT GCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC TCAACTATTAGTGGTAGTGGTGATAATACATACTATGCAGACTCCCTG AAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACACTATAT CTGCAAGTGAACAGCCTGAGACCCGAGGACACGGCCGTTTATTACTGT GCGAAAAGTCCCTCGTCACTTCTGGCCACCTACTTTGACTACTGGGGC CAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 11 NI-203.9A2-V _K GAAATTGTGTTGACACAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA GACAGAGTCACCATCACTTGCCGGGCCAGTGAGAGTATTAATAGCTGG TTGGCCTGGTATCAGCAGAAACCAGGGAAAGGCCCTAAGCTCCTGATC TATAAGGCGTCTAGTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGC AGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCT GATGATTTTGCAACTTATTACTGCCAACAGCACAATAGTTATTGGACG TTCGGCCAAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 13 NI-203.19H8-V _h GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGACG TCCCTGAGACTCTCCTGTGCAGCGTCTGGGTTCACCTTCAGCAGTTAT GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGCCTGGAGTGGGTG GCAATTATATGGTATGATGGAAGTAAGGAATATTATGCAGACTCCCTG AAGGGCCGAGTCACCATCTCCAGAGACAATTCCGAGAACACTCTCTAT CTGCAACTGCACACCCTGAGAGTCGAGGACACGGCTGTGTATTTCTGT GCGAGGACAATCGCATCGGCCACCGTGGACCACGGTATGGACGTCTGG GGCCAAGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 15 NI-203.19H8-V _k GATGTTGTGATGACTCAGTCTCCTTCGTCCGTGTCTGCATCTGTAGGA GACAGAGTCACCATCACTTGTCGGGCGAGTCACGATATTAGCACCTGG TTAGCCTGGTATCAGCAGAGACCAGGGAAAGCCCCTAACCTCCTGATC TTTGGAGCATCGAGGTTGCAAAGTGGGGTCTCACCAAGGTTCAGCGGC AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCT GAAGATTTTGCAACTTACTATTGTCAACAGACTAACAATTTCCCTCCC ACCTTCGGCCAAGGGACACGACTGGAGATTAAA SEQ ID NO: 17 NI-203.26C11-V _h CAGGTGCAGCTGCAGGAGTCGGGCCCAGGATTGGTGAAGCCTTCTCAG ACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGT AATTACTACTGGACCTGGATCCGGCAGCCCGCCGGGAAGGGACTGGAG TGGATTGGGCATATCTATTCCAGTGGGACCACCAATTACAACCCCTCC CTCGAGAGTCGAGTCACCATTTCAGTAGACACGTCCAAGAACCAGTTC TCCCTGAGCCTGAACTCTGTGACCGCCGCAGACACGGCCGTTTATTAC TGTGCGAGACCACTGGCTACAGTTCCGGATGCTTTTAATATCTGGGGC CAAGGGACAATGGTCACCGTCTCTTCG SEQ ID NO: 19 NI-2O3.26C11-V _K GAAATTGTGATGACTCAGTCTCAGACTCCCTGGCTGTGTCTCTGGGC GAGAGGGCCACCATCAAGTGCAAGTCCAGCCAGAGTGTTTTATACAGC AATAAGAACTTCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCT AAATTACTCATTTACTGGGCATCTACTCGGGAATCCGGGGTCCCTGAC

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AGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAGTATTAT AGTAATCCTAACACTTTTGGCCAGGGGACCAAGGTGGAGATCAAA SEQ ID NO: 21 NI-203.8E3-V _H CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAACCTGGGTCC TCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGTAGTCAC ACTATCAGCTGGGTGCGACAGGCCCCTGGGCAAGGGCTTGAGTGGATG GGAGGGATCATCCCCATCTTTGGTACAGCAAACTACGCACAGAAGTTT CAGGACAGAGTCACGGTTACCGCGGACAAATCCACGAATACAGCCTAC ATGGAGTTGAGTAGCCTCAGACCTGAGGACACGGCCGTGTATTACTGT GCGAAGGGGGAACTGGAACCACGAATCCTCTACTACTACGGTATGGAC GTCTGGGGCCGAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 23 NI-203.8E3- _VK GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGA CAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTATACAGT GATGGAAACACCTACTTGAATTGGTTTCACCAGAGGCCAGGCCAATCT CCAAGGCGGCTAATTTATAAGGTTTCTAATCGTGACTCTGGGGTCCCA GACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGT TCAAATTGGCCAGGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 25 NI-203.11B12-V _h CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGCCTGGGGCC TCAATGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAACTAC TATTTACACTGGGTGCGACAGGCCCCTGGACAAGGACTTGAGTGGATG GGAATAATCAACCCTAGTGCTGGTAGCACAAGCTACGCACAGAAGTTC CAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTAC ATGGAACTGAGCAGCCTGAAATCTGAAGACACGGCCGTCTATTACTGT GCGAGAGATTCCGCTGGGATACAGATATGGTTCAGGGATGCTTTTGAT ATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCG SEQ ID NO:27 NI-203.11B12- _Vl CAGCCTGTGCTGACTCAGCCACCCTCTGCCTCTGCTTCCCTGGGATCC TCGGTCAAGCTCACCTGCACTCTGAACAGTGGGCACAGTAGCTACACC ATCGCATGGCATCAGCAGCAGCCAGGGAAGGCCCCTCGGTACTTGATG AAGGTTGAACATAATGGAAACTACAACAAGGGGAGCGGACTTCCTGAT CGCTTCTCAGGCTCCAGCTCTGGGGCTGACCGCTACCTCGCCATCTCC AACCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGAGACCTGGGAC ACTAGCACTAGGGTCTTCGGCGGAGGGACCAAGCTGACCGTCCTA SEQ ID NO: 29 NI-203.205F8-V _h CAGGTGCAGCTGCAGGAGTCGGGCCCCGGACTGGTGAAGCCTTCGGAG ACCCTGTCCCTCACCTGCACTGTCTCTGGTGACTCCGTCAGCAGTGGT AGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAG TGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCC CTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTC TCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTATATTCC TGTGCGAGAGTCCCCTATGGTTACGGATATAGGGGCTACGATGGGGCT TGGTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 31 NI-203.205F8- _Vk GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGG GAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTAC TTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC TATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGC AGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCT GAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACCGGTTCACT

- 39 042691

TTCGGCCCTGGGACCAAAGTGGATATCAA SEQ ID NO :33 NI-203.9B3-V _H GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGGGG TCCCTGAGACTCTCCTGTGCAGCGTCAGGATTCACCTTCAGTAGCTAT GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTG GCAGTTATCTGGTATGATGGAACTAAGAAGTACTATGCAGACTCCGTG AAGGGCCGATTCACCACCTCCAGAGACAATTCCAAGAATACGCTGTCT CTGCAAATGAACAGCCTGAGAGCCGAGGACTCGGCTGTGTATTACTGT GCGAGAGGCTTTAGCAGCAGCTGGGAGTTTGGGTTCTGGGGCCAGGGA ACCCTGGTCACCGTCTCCTCG SEQ ID NO: 35 NI-203.9B3- _Vl CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAG TCAGTCACCATCTCCTGCACTGGAACCAGCGGTTACATTTATGGTTAT AACTATGTCTCCTGGTACCAACAGCACCCCGGCAAAGCCCCCAAAGTC ATGATTTATGAGGTCACTAAGCGGCCCTCAGGGGTCCCTGATCGCTTC TCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGGCTC CAGGCTGAGGATGAGGCTGTTTATTACTGCGCCTCATATGCAGGCAGC AACAATGTAGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA SEQ ID NO: 37 NI-203.19F2-V _H GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAGGAAGCCTGGGTCC TCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCAACTTCTTGAGCTAT TCCATCAGTTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATG GGAGGGATCATCCCGATCTTTGGTACACCAAACTACGCACAGAAGTTC CAAGGAAGAGTCACAATTACGGCGGACAAATCGACGAGGACAGCCTAC ATGGAGCTGAGCAGCCTGAGATTTGATGACACGGCCGTCTATTATTGT GCGGATGCGACAAGACCGGGTACAGCAGCCTCTGGTTTCTATTACTAC GGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO:63 NI-203.19F2- _VK GAAATTGTGATGACACAGTCTCCAGACACCCTGTCTGTGTCTCCAGGT GAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAACAACAAC TTAGCCTGGTTCCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC TATGGTGCATCCACCAGGGCCACTGGTATTCCAGCCAGATTCAGTGGC AGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTACAGTCT GAAGATTTTGCAGTTTATTTCTGTCAGCAGAGTCACAATTGGCCCACT TTCGGCCCTGGGACCAAAGTGGATATCAAA SEQ ID NO:65 NI-203.15C7-V _H GAGGTGCAGCTGGTGGAGACTGGGGGAGGCGTGGTCCAGCCTGGGATG TCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTACCTAT ACTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTG TCATTTATATCATATGATGGAAGGGATAAATACTACGCAGATTCCGTG AAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACATGTTGTAT CTGCAAATGAACAGCCTGAGAGATGAGGACATGGCTGTGTATTACTGT GCGACTCTGCAAGTATGGCAACTCTACGATTACTACGGAATGGACGTC TGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO:67 NI-203.15C7-V _l CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAG AAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAAT TATGTATCTTGGTATCAGCAACTCCCAGGAACAGCCCCCAAACTCCTC ATTTATAACAGTGATAAGCGACCCTCAGGGATTCCTGACCGATTCTCT GCCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGGCTCCAG ACTGGGGACGAGGCCGATTATTACTGCGCAACATGGGATACCAGACTG AGTGCTGGGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTT SEQ ID NO:69

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Table III. Nucleotide sequences of the VH and VL region of antibodies against proIAPP

Antibody Heavy (VH) and light chain (VL/VK) variable region nucleotide sequences NI-203.1D10-V _H GAGGTGCAGCTGGTGCAGTCTGGCGCAGAAGTGAAGAAGCCCGGGGAGT CTCTCAGAATCTCCTGTAAGGCTTCTGGATACAGCTTCACCAACTCTTG GATCGCCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGACTACGTGGGT ATCATCTATCCTGGTGACTCTGATACCAAGTATGGCCCGTCCTTCCAAG GCCACGTCACTATCTCAGCCGACAACTTCGCCAACACCGCCTACCTGCA GTGGAGCAGCCTGAAGGCCTCCGACACCGCCATCTATTATTGTGCGAGA CGGGCAGCAGCGGCTATTAACTGGTTCGACTCCTGGGGCCAGGGAACCC TGGTCACCGTCTCCTCG SEQ ID NO: 39 NI-203.1D10-V _K GACATCCAGTTGACCCAGTCTCCACTCTCCCTGTCCGTCACCCCTGGAG AGCCGGCCTCCATCTCCTGCAGGTCTAGCCAGAGCCTCCTGCATCCTAA TGGAAACGACTATTTGGATTGGTACGTGCAGAAGCCAGGGCAGTCTCCA CAGATCGTGATCTACATGGGTTCTAATCGGGCCGCCGGGGTCCCTGACA GGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAG AGTGGAGGCTGAGGATGTTGGGACTTATTACTGCCTGCAAGCTCTACGC GGGTACACTTTTGGCCAGGGGACCAAGGTGGAAATCAAA SEQ ID NO: 41 NI-2O3.2A11-V _H CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGGGGT CCCTGAGACTCTCCTGTGTAGCGTCTGGATTCACCTTCAGCAGTTATGG CATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCA TTTGTACGGTATGATGGAAGTAATAAGTACTATGCAGACTCCGTGAAGG GCCGATTCACCATCTCCAGAGACAATTCCAAGAACTCGCTGTCTCTTCA AATGAACAGTCTGAGAACTGAAGACACGGCTGTATATTACTGCGCGAAA GAACAGGAGGACCACAAGGAAGCTTTTGACTACTGGGGCCAGGGAACCC TGGTCACCGTCTCCTCG SEQ ID NO: 43 NI-2O3.2A11-V _K GAAATTGTGATGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGG AAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGAGTTACCACCATAGC CTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGT GCATCCAGCAGGGCCACTGATATTCCCGCCAGGTTCAGTGGCAGTGGGT CTGGGACAGACTTCACTCTCACCATCAGCAGTCTGCAGTCTGAAGACTT TGCAGTTTATTACTGTCAGCAGTATAACCAGTGGCCCCTCACTTTCGGC GGAGGGACCAAGCTGGAGATCAAA SEQ ID NO: 45 NI-203.10C4-V _H GAGGTGCAGCTGGTGGAGTCTGGGGCTGAAGTGAGGAAGCCTGGGGCCT CAGTGAGGGTCTCCTGCCAGACATCTGGATACAGCGTCACCGACTACTA TCTACACTGGGTGCGACAGGCCCCTGGACAGGGCCTTGAGTGGATGGGA GTGATGAACCCGAGCAATGGAAACGTGGGCTACCCACAGAAGTTTCAGG GCCGAGTCACCATGACCGCAGACACGTCCACGGGCACAGTGTACATGGT GTTGACCGGCCTTACGGCTGGGGACACGGCCGTCTACTACTGTGCCAGA GGCGGGTCCACGCCGGGTCAGGAAGTAAGGAGTCCCCACGTCCTTGACC TCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 47 NI-203.10C4- _VK GATGTTGTGATGACTCAGTCTCCCCTCTCTCTGTCCGTCACCCCTGGAC AGCCGGCCTCCATCTCCTGCAGGTCTGATGAGAGCCTCCTGCATAGTGA TGGAAGGACCTATTTGTATTGGTATCTACAGAAGCCCGGCCAGCCTCCT CAGCTCCTGATCTATGAAGTTTCCAACCGGTTCTCGGGAGTGCCAAATA GGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACACTGAAAATCAGCCG GGTGGAGGCTGAGGATGTTGGCGTTTATTACTGCATGCAGGGTGTACAC TTTCCTCAGACGTTCGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 49

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NI-203.20H9-V _H CAGGTGCAGCTGGTGCAATCTGGGTCTGAGTTGAAGAAGCCTGGGGCCT CAGTGAAGGTCTCCTGCAAGGCTTCTGGATACATCTTCAGTAAACATGG TATCAACTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGATAGGA TGGATCAACACCAATACGGGGAACCCAACATATGCCCAGGACTTCACAG GACGATTTGTCTTCTCCTTGGACACCTCTGTCAGCACGGCATATCTGGA GATCAGCAGCCTAAAGGCTGAGGACACTGCCGTGTATTACTGTGCGAGA GAATCAGAGCCGATTTTTGGAGTTATCTATTACATGGACGTCTGGGGCA AAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 51 NI-203.20H9- _VK GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG ACAGCGTCACCATCACTTGCCGGGCAAGCCAGAGCATAAGCACTAATTT AAATTGGTATCAGAAGAAACCAGGACAAGCCCCTACGGTCTTGATCTAT GCTGCGTCCAGTTTGCAAGGTGGGGTCCCATCAAGGTTCAGGGGCCGGG GATCTGGGACATATTTCACTCTCACCATCAGCGGTCTTCAACCTGAAGA TTTTGCAACTTATTACTGTCAACACAATTACAATGATTTGTGGACGTTC GGCCAAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 53 NI-203.26D2-V _h CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGGGGT CCCTGAGACTCTCCTGTGCAGCGTCTGGGTTCACGTTCAGAACCTGTGG CATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAATGGGTGGCA TTTGTTCGGTCTGATGGAACTACTAGATATTACGCAGACTCCCTGATGG GCCGCTTCACCATCTCCAGAGACAATTCCAAGAACTCGCTGTATCTTCA AATGAACAGTCTGAGACCTGAGGACACGGCTCTTTATTACTGTGCGAGG GAAAAGGAGGATCACAGGGAAGCTTTTGACTACTGGGGCCAGGGAACCC TGGTCACCGTCTCCTCG SEQ ID NO: 55 NI-203.26D2- _VK GAAATTGTGATGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGG AAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGCGTGTTAGCACTGTAGC CTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGAT GCATCCACCAGGGCCACTGATATCCCCGCCAGGTTCAGTGGCAGTGGGT CTGGGACAGACTTCACTCTCACCATCAGCACTCTGCAATCTGAAGACTC TGCAGTTTATTACTGTCAGCAGTATAATAGGTGGCCCCTCACTTTCGGC GGAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 57 NI-203.60H3-V _H GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGCACGCCCTGGAGGCT CCCTGAGACTCTCCTGTGCAGTCGCTGGATTCACTTTCAGTGGTTATGA AATGAATTGGGTCCGCCAGGCACCAGGGAAGGGGCTGGAGTGGATTTCA TATATTAGCGGTCCTGGGGATGTGATATACTACGCAGACTCTGTGAAGG GCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTTTCTACA GATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTATTGTACGAGA GTCCCCCCTGACATCAGCTATGGATTTGATTACTGGGGCCAGGGCACCC TGGTCACCGTCTCCTCG SEQ ID NO: 59 NI-203.60H3- _VK GACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTACGAG ACAGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTT AAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCCAT GATACAGACATTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCACTG GATCTGGGACAGATTTCACTCTCACCATCAGCGGTCTGCAACCTGAAGA TTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCTACTTTT GGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 61

The present invention also includes fragments of polynucleotides according to the invention, described in other sections. In addition, the invention also provides polynucleotides encoding hybrid polynucleotides, Fab fragments and other derivatives described herein.

Polynucleotides can be obtained or produced by any method known in the art. For example, if the nucleotide sequence of an antibody is known, the polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides, such as those described in Kutmeier et al. encoding the antibody, annealing and ligation of these oligonucleotides, and then PCR amplification of the ligated oligonucleotides.

Alternatively, a polynucleotide encoding an antibody or an antigen-binding fragment, variant or derivative thereof can be derived from a nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, the nucleic acid encoding the antibody can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library derived from, or a nucleic acid , preferably polyA ⁺ RNA isolated from any tissue or cells expressing IAPP- or proIAPP-specific antibodies, e.g. hybridoma cells chosen to express the antibody) by NRPamplification using synthetic primers hybridizable at the 3' and 5' ends of the sequence , or by cloning with an oligonucleotide probe specific for the sequence of the particular gene to be detected, for example, a cDNA clone from a cDNA library encoding an antibody. The PCR-amplified nucleic acids can then be cloned into replicable cloning vectors by any method well known in the art.

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Once the nucleotide sequence and corresponding amino acid sequence of an antibody or antigen-binding fragment, variant or derivative thereof has been determined, its nucleotide sequence can be manipulated by nucleotide sequence manipulation techniques well known in the art, such as recombinant DNA techniques, site-directed mutagenesis, PCR, etc. .d. (See, for example, the procedures described in Sambrook et al., Molecular Cloning, A Laboratory Manual, ^2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1990) and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998, incorporated herein by reference in its entirety) to generate antibodies with different amino acid sequences, for example, to create amino acid substitutions, deletions and/or insertions.

IV. Expression of antibody polypeptides.

After the isolated genetic material has been manipulated to produce antibodies or antigen-binding fragments, variants or derivatives thereof according to the invention, the antibody-encoding polynucleotides are typically inserted into an expression vector for introduction into a host cell that can be used to produce the desired amount of antibody. Described herein is the recombinant expression of an antibody or fragment, derivative, or analog thereof, such as the heavy or light chain of an antibody, that binds to a target molecule. After obtaining a polynucleotide encoding an antibody molecule or an antibody heavy or light chain, or a fragment thereof (preferably containing a heavy or light chain variable domain) according to the invention, a vector for producing an antibody molecule can be obtained using recombinant DNA technology and using methods well known in this field of technology. Thus, the present document describes methods for producing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody-coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic methods, and in vivo genetic recombination. Thus, the present invention provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain or heavy or light chain variable domain thereof, operably linked to a promoter. Such vectors may contain a nucleotide sequence encoding the constant region of the antibody molecule (see, for example, international applications WO 86/05807 and WO 89/01036; and US patent No. 5122464) and an antibody variable domain that can be cloned into such a vector to express the entire heavy or light chain.

The term vector or expression vector (expression vector) is used herein to refer to vectors used in accordance with the present invention as a carrier for introducing a desired gene into a host cell and expressing it. As known to those skilled in the art, such vectors are readily selected from the group consisting of plasmids, phages, viruses, and retroviruses. In general, vectors compatible with the present invention should contain a selection marker, appropriate restriction sites to facilitate cloning of the desired gene, and be capable of entering and/or replicating in eukaryotic and prokaryotic cells. For the purposes of the present invention, numerous vector expression systems can be used. For example, one class of vectors use DNA elements derived from animal viruses, such as bovine papillomavirus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV), or SV40 virus. Other vectors involve the use of polycistronic systems with internal ribosome binding sites. In addition, it is possible to select for cells in which DNA is integrated into their chromosomes by introducing one or more markers that allow selection of transfected host cells. The marker can provide prototrophy for an auxotrophic host, resistance to biocides (eg antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be directly attached to the DNA sequences to be expressed, or introduced into the same cell by co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include signal sequences, splicing signals, as well as promoters, enhancers, and transcription termination signals.

In particularly preferred embodiments, the cloned variable region genes are inserted into an expression vector, along with heavy and light chain (preferably human) constant region genes, as discussed above. In one embodiment, this is achieved using the NEOSPLA proprietary expression vector owned by Biogen IDEC, Inc. and described in US Pat. No. 6,159,730. This vector contains a cytomegalovirus promoter/enhancer, a major mouse beta-globin promoter, an SV40 origin of replication, a bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exons 1 and 2, a dihydrofolate reductase gene, and a leader sequence. This vector has been found to achieve a very high level of antibody expression after the introduction of variable and constant region genes, transfection

- 43 042691 CHO cells followed by selection in a medium containing G418 and amplification with methotrexate. Of course, any expression vector capable of inducing expression in eukaryotic cells can be used in the present invention. Examples of suitable vectors include plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACERHCMV, pUB6/V5-His, pVAX1 and pZeoSV2 (available from Invitrogen, San Diego , California, USA), and the PCI plasmid (available from Promega, Madison, Wisconsin, USA), but are not limited to. In general, screening a large number of transformed cells to detect cells with a sufficiently high level of expression of heavy and light chains of immunoglobulins is a routine experiment that can be performed, for example, using robotic systems. Vector systems are also described in US Pat. Nos. 5,736,137 and 5,658,570, each of which is incorporated herein by reference in its entirety. This system provides high levels of expression, eg >30 pg/cell/day. Other exemplary vector systems are described, for example, in US Pat. No. 6,413,777.

In other preferred embodiments, antibodies or antigen-binding fragments, variants or derivatives of the invention may be expressed using polycistronic constructs, such as those described in US Patent Application Publication No. 2003-0157641 A1, which is incorporated herein in its entirety. In these expression systems, multiple products of the genes of interest, such as heavy and light chain antibodies, can be produced from a single polycistronic construct. The advantage of these systems is the use of internal ribosome entry site (IRES) to obtain relatively high levels of antibodies. Compatible IRES sequences are described in US Pat. No. 6,193,980, which is also incorporated herein. Those of skill in the art will appreciate that such expression systems can be used to efficiently produce the full range of antibodies described herein. Thus, in one embodiment, the present invention provides a vector comprising a polynucleotide encoding at least a binding domain or variable region of an immunoglobulin chain of an antibody, optionally in combination with a polynucleotide encoding a variable region of another immunoglobulin chain of said binding molecule.

In general, once a vector or a DNA sequence encoding an antibody monomeric subunit has been obtained, the expression vector can be introduced into an appropriate host cell. Introduction of the plasmid into the host cell is readily accomplished using standard techniques well known to those skilled in the art. These include, but are not limited to, transfection, including but not limited to lipotransfection using, for example, Fugene® or Lipofectamine, protoplast fusion, calcium phosphate precipitation, fusion of cells with DNA within them, microinjection, and infection with intact virus. As a rule, the introduction of the plasmid into the host is carried out using a standard method using co-precipitation with calcium phosphate. Host cells carrying the expression construct are grown under conditions suitable for the production of light and heavy chains and analyzed for heavy and/or light chain protein synthesis. Exemplary assay methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or fluorescence-activated cell sorter (FACS) analysis, immunohistochemistry, and the like.

The expression vector is transferred into the host cell using conventional techniques, and then the transfected cells are cultured using conventional techniques, which provides the production of antibodies for use in the methods described herein. Thus, the invention includes host cells comprising a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or at least an immunoglobulin binding domain or variable region, preferably operably linked to a heterologous promoter. In addition or alternatively, the present invention also includes a host cell containing the vector described herein above, containing a polynucleotide encoding at least the binding domain or variable region of an immunoglobulin chain of an antibody, optionally in combination with a polynucleotide encoding a variable region of another chain immunoglobulin of said binding molecule. In preferred embodiments for the expression of double chain antibodies, a single vector or vectors encoding both heavy and light chains can be co-expressed in a host cell to express the entire immunoglobulin molecule as described below.

A host cell can be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain-derived polypeptide and the second vector encoding a light chain-derived polypeptide. These two vectors may contain identical selectable markers allowing equal expression of heavy and light chain polypeptides. Alternatively, a single vector encoding both heavy and light chain polypeptides can be used. In such situations, the light chain is preferably placed before the heavy chain to avoid excess toxic free heavy chain; see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad. sci. USA 77 (1980), 2197. The heavy and light chain coding sequences may comprise cDNA or genomic DNA.

- 44 042691

As used herein, the term host cells refers to cells that carry vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene. In describing processes for isolating antibodies from recombinant hosts, the terms cell and cell culture are used interchangeably to refer to the source of antibodies, unless otherwise explicitly indicated. In other words, isolation of a polypeptide from a cell may mean isolation from centrifuged whole cells or from a cell culture containing both medium and suspended cells.

For the expression of antibody molecules for use in the methods described herein, you can use a variety of vector systems for expression in host cells. Such expression systems in host cells are vehicles that can produce and purify the coding sequences of interest, and are also cells that, when transformed or transfected with the appropriate nucleotide coding sequence, can express the antibody molecule of the invention in situ. These systems include microorganisms, eg bacteria (eg E. coli, B. subtilis), transformed with recombinant expression vectors based on bacteriophage DNA, plasmid DNA or cosmid DNA containing sequences encoding antibodies; yeast (eg Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; systems based on insect cells infected with recombinant viral expression vectors (eg, baculovirus) containing sequences encoding antibodies; plant cell systems infected with recombinant viral expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg, Ti plasmid) containing antibody-coding sequences; or systems based on mammalian cells (e.g., COS, CHO, NSO, BLK, 293, 3T3 cells) carrying recombinant expression constructs containing promoters derived from the mammalian genome (e.g., the metallothionein promoter) or from mammalian viruses (e.g., late adenovirus promoter; 7.5K vaccinia promoter), but are not limited to. Preferably, bacterial cells such as Escherichia coli are used to express the recombinant antibody molecule, and more preferably eukaryotic cells are used, especially for expression of the entire recombinant antibody molecule. For example, mammalian cells, such as Chinese Hamster Ovary (CHO) cells, in combination with a vector, such as the major intermediate promoter of human cytomegalovirus early genes, provide an efficient antibody expression system; see, for example, Foecking et al., Gene 45 (1986), 101; Cockett et al., Bio/Technology 8 (1990), 2.

The host cell line for protein expression is often derived from mammalian cells; those skilled in the art can determine specific preferred host cell lines that are best suited for expressing the desired gene product. Example host cell lines include CHO (Chinese hamster ovary cell), DG44 and DUXB11 (DHFR-negative Chinese hamster ovary cell lines), HeLa (human cervical carcinoma), CVI (monkey kidney cell line), COS (CVI derivative with T antigen). SV40), VERY, BHK (hamster kidney cells), MDCK, WI38, R1610 (Chinese hamster fibroblasts) BALBC/3T3 (mouse fibroblasts), HAK (hamster kidney cell line), SP2/O (mouse myeloma), P3X63-Ag3. 653 (mouse myeloma), but not limited to BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney cells). Particularly preferred are CHO and 293 cells. Host cell lines are generally available from commercial sources, the American Tissue Culture Collection, or from the published literature.

In addition, the host cell strain can be selected to modulate the expression of the introduced sequences or to modify and process the gene product in the specific manner desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important to the function of the protein. Different host cells have characteristic specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells can be used, which possess cellular mechanisms for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product.

Stable expression is preferred for long term high throughput production of recombinant proteins. For example, cell lines can be engineered to stably express an antibody molecule. Instead of using expression vectors containing viral origins of replication, host cells can be transformed with DNA under the control of appropriate expression control elements (eg, promoter, enhancer sequence, transcription terminators, polyadenylation sites, etc.) and a selectable marker.

- 45 042691

After introduction of foreign DNA, engineered cells can be left to grow for 1-2 days in enriched medium and then transferred to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into chromosomes and grow to form plaques, which in turn can be cloned and developed into cell lines.

This method can be successfully used to construct cell lines that stably express an antibody molecule.

A number of selection systems can be used, including herpes simplex virus thymidine kinase genes (Wigler et al., Cell 11 (1977), 223), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48 (1992) , 202) and adenine phosphoribosyl transferase (Lowy et al., Cell 22 (1980), 817) in but not limited to tk, hgprt or aprt cells, respectively. In addition, antimetabolite resistance can be used as a basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77 (1980), 357; O'Hare et al., Proc Natl Acad Sci USA 78 (1981), 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78 (1981), 2072); neo, which confers resistance to the aminoglycoside G-418 Goldspiel et al., Clinical Pharmacy 12 (1993), 488-505; Wu and Wu, Biotherapy 3 (1991), 87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32 (1993), 573-596; Mulligan, Science 260 (1993), 926-932; and Morgan and Anderson, Ann. Rev. Biochem. 62 (1993), 191-217; TIB TECH 11 (1993), 155-215; and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30 (1984), 147. Methods well known in the field of recombinant DNA technology that can be used for this purpose are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994) Colberre-Garapin et al., J Mol Biol 150:1 (1981), which are incorporated herein by reference in their entirety.

Expression levels of an antibody molecule can be increased by vector amplification, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987). If the marker in the vector system expressing the antibody is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Because the amplified region is linked to the antibody gene, antibody production will also increase; see Crouse et al., Mol. cell. Biol. 3 (1983), 257.

In vitro production allows scaling up to obtain large volumes of desired polypeptides. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include culturing in a homogeneous suspension, e.g., in an airlift or continuously stirred reactor, or culturing immobilized or encapsulated cells, e.g., in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or desired, polypeptide solutions can be purified by conventional chromatographic techniques, e.g., gel filtration, ion exchange chromatography, DEAE-cellulose chromatography, or (immuno-)affinity chromatography, e.g., after preferential biosynthesis of the synthetic hinge polypeptide, or before or after the step hydrophobic chromatography described in this document.

Genes encoding antibodies or antigen-binding fragments, variants or derivatives thereof according to the invention can also be expressed in non-mammalian cells, such as bacterial or insect or yeast or plant cells. Bacteria readily accepting nucleic acids include members of the Enterobacteriaceae family, for example strains of Escherichia coli or Salmonella; Bacillaceae, for example Bacillus subtilis; pneumococcus; Streptococcus and Haemophilus influenzae. In addition, it should be taken into account that when expressed in bacteria, heterologous polypeptides usually become part of inclusion bodies. Heterologous polypeptides should be isolated, purified and then assembled into functional molecules. If tetravalent forms of antibodies are desired, the subunits then self-assemble into tetravalent antibodies; see, for example, international application WO02/096948.

In bacterial systems, a range of expression vectors can be successfully selected depending on the intended use of the antibody molecules to be expressed. For example, if it is necessary to produce a large amount of such a protein to obtain pharmaceutical compositions of the antibody molecule, vectors that provide high levels of expression of easily purified hybrid protein products may be desirable. Such vectors include the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2 (1983), 1791) in which the antibody coding sequence can be separately ligated in-frame with the LacZ coding region such that a fusion protein is produced. ; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13 (1985), 3101-3109; Van Heeke & Schuster, J. Biol. Chem. 24 (1989), 5503-5509); and the like, but are not limited to them. In addition, to express foreign polypeptides as glutathione S-transferase (GST) fusion proteins,

- 46 042691 pGEX vectors. In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to the matrix of glutathione-agarose beads, followed by elution in the presence of free glutathione. The pGEX vector construct includes thrombin or factor Xa protease cleavage sites and the target cloned gene product can be freed from the GST fragment.

In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most widely used among eukaryotic microorganisms, although a number of other strains, such as Pichia pastoris, are also commonly available. For expression in Saccharomyces, for example, the YRp7 plasmid is used (Stinchcomb et al., Nature 282 (1979), 39; Kingsman et al., Gene 7 (1979), 141; Tschemper et al., Gene 10 (1980), 157 ). This plasmid already contains the TRP1 gene, which is a selectable marker for a mutant yeast strain lacking the ability to grow on tryptophan, such as ATCC No. 44076 or PEP4-1 (Jones, Genetics 85 (1977), 12). The presence of the trp1 defect as a characteristic of the host yeast cell genome provides conditions for efficient detection of transformation by growth in the absence of tryptophan.

In the insect cell system, Autographa californica nuclear polyhedrosis virus (AcNPV) is commonly used as a vector for the expression of foreign genes. This virus grows in the cells of Spodoptera frugiperda. The antibody coding sequence can be separately cloned into non-essential regions (eg polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (eg polyhedrin promoter).

Following recombinant expression of an antibody molecule of the invention, whole antibodies, dimers thereof, individual light and heavy chains, or other forms of an immunoglobulin of the invention may be purified according to standard procedures known in the art, including, for example, by chromatography (e.g., ion exchange, affinity, especially based on the affinity for a specific antigen compared to protein A, and size exclusion column chromatography), centrifugation, differential solubility, for example, precipitation with ammonium sulfate, or any other standard method for purifying proteins; see, for example, Scopes, Protein Purification, Springer Verlag, N.Y. (1982). In addition, a preferred method for increasing the affinity of antibodies of the invention is described in US Patent Publication 2002-0123057 A1. Thus, in one embodiment, the present invention provides a method for producing antibodies against IAPP and/or proIAPP, or immunoglobulin chains thereof, comprising:

(a) culturing the host cell as described above, said cell containing the polynucleotide or vector described above; and (b) isolating said antibody or immunoglobulin chain thereof from the culture.

In addition, in one embodiment, the present invention relates to an antibody or its immunoglobulin(s) chain(s) encoded by a polynucleotide as described above, or obtained using the specified method for obtaining antibodies against IAPP and/or proIAPP or their immunoglobulin(s) chain(s).

V. Hybrid proteins and conjugates.

In some embodiments, an antibody polypeptide contains an amino acid sequence or one or more groups not normally associated with an antibody. Typical modifications are detailed below. For example, a single chain Fv fragment of an antibody of the invention may contain a flexible linker sequence, or may be modified by the addition of a functional group (e.g., PEG, drug, toxin, or a label, such as a fluorescent, radioactive, enzymatic, NMR tag, heavy metal, and etc.).

An antibody polypeptide of the invention may comprise, consist essentially of, or consist of a fusion protein. Fusion proteins are chimeric molecules containing, for example, an immunoglobulin IAPP and/or proIAPP binding domain with at least one target binding site and at least one heterologous fragment, i.e. a fragment with which it is not normally associated in nature. The amino acid sequences may normally exist as separate proteins combined in a fusion polypeptide, or they may normally be present in the same protein but in a new order within the fusion polypeptide. Fusion proteins can be obtained, for example, by chemical synthesis, or by creating and translating a polynucleotide encoding the desired mutual arrangement of peptide regions.

The term heterologous when applied to a polynucleotide or polypeptide means that the polynucleotide or polypeptide is derived from a component that is different from the rest of the component to which it is being compared. For example, as used herein, a heterologous polypeptide combined with an antibody or antigen-binding fragment, variant or analog thereof is derived from a non-immunoglobulin polypeptide derived from an organism of the same species, or an immunoglobulin or non-immunoglobulin polypeptide from another species.

As discussed in more detail elsewhere herein, antibodies or antigen-binding fragments, variants or derivatives thereof according to the invention can then be recombinantly

- 47 042691 combine with a heterologous polypeptide at the N- and C-terminus or chemically conjugate (including covalent and non-covalent conjugation) with polypeptides or other compositions. For example, antibodies can be recombinantly combined or conjugated with molecules used as labels in a detection assay and effector molecules, such as heterologous polypeptides, drugs, radionuclides, and toxins; see, for example, international applications WO92/08495, WO91/14438, WO89/12624, US patent No. 5314995 and European patent EP 0396387.

Antibodies or their antigen-binding fragments, variants or derivatives according to the invention may consist of amino acids connected to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres, and may contain amino acids other than the 20 genetically encoded amino acids. Antibodies can be modified by natural processes, such as post-translational processing, or by chemical modification methods well known in the art. Such modifications are well described in the basic texts and in more detailed monographs, as well as in the extensive research literature. Modifications can occur in any region of the antibody, including the peptide backbone, amino acid side chains, and the N- or C-terminus, or in groups such as carbohydrates. It should be appreciated that modifications of the same type may be present to the same or different extent at several sites in a given antibody. In addition, this antibody may contain modifications of many types. Antibodies can branch, for example, as a result of ubiquitination, and can be cyclic, with or without branching. Cyclic, branched and branched cyclic antibodies can be produced by natural post-translational processes or by synthetic means. Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent attachment, heme molecule covalent attachment, nucleotide or nucleotide derivative covalent attachment, lipid or lipid derivative covalent attachment, phosphatidylinositol covalent attachment, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cysteine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, PEGylation, proteolytic treatment, phosphorylation, prenylation, racemization, selenylation, transfer RNA mediated sulfation adding amino acids to the protein, such as arginylation and ubiquitination; see, for example, Proteins - Structure And Molecular Properties, T. E. Creighton, W.H. Freeman and Company, New York ^2nd Ed., (1993); Posttranslational Covalent Modification Of Proteins, B. C Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182 (1990), 626-646; Rattan et al., Ann. NY Acad. sci. 663 (1992), 48-62).

In addition, the present invention provides fusion proteins comprising an antibody or antigen-binding fragment, variant or derivative thereof and a heterologous polypeptide. In one embodiment, a fusion protein of the invention comprises, consists essentially of, or consists of a polypeptide comprising the amino acid sequence of one or more VH regions of an antibody of the invention, or the amino acid sequence of one or more VL regions of an antibody of the invention, or fragments and variants thereof, and the sequence heterologous polypeptide. In yet another embodiment, a fusion protein for use in the diagnostic and therapeutic methods described herein comprises, consists essentially of, or consists of a polypeptide comprising the amino acid sequence of one, two, three of the VH-CDRs of an antibody or fragments, variants, or derivatives, or the amino acid sequence of one, two, three of the V _l -CDR antibodies or its fragments, variants or derivatives, and the sequence of the heterologous polypeptide. In one embodiment, the fusion protein comprises a polypeptide comprising the VH-CDR3 amino acid sequence of an antibody of the present invention, or a fragment, derivative, or variant thereof, and a heterologous polypeptide sequence, said fusion protein specifically binding to IAPP and/or proIAPP. In another embodiment, the fusion protein comprises a polypeptide comprising the amino acid sequence of at least one VH region of an antibody of the invention and the amino acid sequence of at least one VL region of an antibody of the invention or fragments, derivatives or variants thereof, and the sequence of a heterologous polypeptide. In a preferred embodiment, the V _H and V L regions of the fusion protein correspond to a single source antibody (or scFv or Fab fragment) that specifically binds IAPP and/or proIAPP. In yet another embodiment, a fusion protein for use in the diagnostic and therapeutic methods described herein comprises a polypeptide comprising the amino acid sequence of one, two, three or more of the VH-CDRs of the antibody and the amino acid sequence of one, two, three or more of the VL -CDR of the antibody or fragments and variants thereof, and the sequence of the heterologous polypeptide. Preferably two, three, four, five, six or more VH-CDRs or VL-CDRs correspond to a single source antibody (or scFv or Fab fragment) of the invention. The present invention also encompasses nucleic acid molecules encoding these fusion proteins.

Examples of fusion proteins described in the literature include T-cell receptor fusions

- 48 042691 (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84 (1987), 2936-2940; CD4 (Capon et al., Nature 337 (1989), 525531; Traunecker et al., Nature 339 ( 1989), 68-70; Zettmeissl et al., DNA Cell Biol. USA 9 (1990), 347-353; and Byrn et al., Nature 344 (1990), 667-670); L-selectin (homing receptor) (Watson et al., J. Cell. Biol. 110 (1990), 2221-2229; and Watson et al., Nature 349 (1991), 164-167); CD44 (Aruffo et al., Cell 61 (1990) , 13031313); CD28 and B7 (Linsley et al., J. Exp. Med. 173 (1991), 721-730); CTLA-4 (Lisley et al., J. Exp. Med. 174 (1991), 561 -569); CD22 (Stamenkovic et al., Cell 66 (1991), 1133-1144); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88 (1991), 10535-10539; Lesslauer et al., Eur. J. Immunol. 27 (1991), 2883-2886, and Peppel et al., J. Exp. Med. 174 (1991), 1483-1489 (1991), and IgE receptor (Ridgway and Gorman , J. Cell Biol 115 (1991), Abstract No. 1448).

As discussed elsewhere herein, antibodies or antigen-binding fragments, variants or derivatives thereof of the invention may be combined with heterologous polypeptides to increase the in vivo half-life of the polypeptides or for use in immunoassays using methods known in the art. For example, in one embodiment, antibodies of the invention can be conjugated with PEG to increase their in vivo half-life; see, for example, Leong et al., Cytokine 16 (2001), 106-119; Adv. in Drug Deliv. Rev. 54 (2002), 531; or Weir et al., Biochem. soc. Transactions 30 (2002), 512.

In addition, antibodies or their antigen-binding fragments, variants or derivatives according to the invention can be combined with marker sequences, such as a peptide, to facilitate their purification or detection. In preferred embodiments, the marker amino acid sequence is a hexahistidine peptide (HIS), such as the marker provided in the pQE vector (QIAGEN Inc., 9259 Eaton Avenue, Chatsworth, CA, USA 91311), among other markers, many of which are available for purchase. As described in Gentz et al., Proc. Natl. Acad. sci. USA 86 (1989), 821-824, for example, hexahistidine provides a convenient purification of the fusion protein. Other peptide markers useful for purification include, but are not limited to, the HA marker, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37 (1984), 767), GST, c-myc, and the flag marker. ; see, for example, a review of epitope marking techniques in Bill Brizzard, BioTechniques 44 (2008) 693-695 and a list of the most common epitope markers according to the present invention in table. 1 on page 694 of said publication, the substance of which is expressly incorporated herein by reference.

Fusion proteins can be obtained using methods well known in the art; see, for example, US Pat. The DNA encoding the fusion protein is then transferred by transfection into a host cell for expression, which is carried out as described above.

The antibodies of the present invention can be used in unconjugated form or conjugated to at least one of a variety of molecules, for example, to enhance the therapeutic properties of the molecule, to facilitate target detection, or to image or treat a patient. Antibodies or their antigen-binding fragments, variants or derivatives according to the invention can be labeled or conjugated before or after purification, when it is carried out. In particular, antibodies or antigen-binding fragments, variants or derivatives thereof according to the invention can be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents or PEGs.

Conjugates, which are immunotoxins containing conventional antibodies, are widely described in the art. Toxins can be attached to antibodies using conventional attachment techniques, or immunotoxins containing the toxin protein moiety can be produced as fusion proteins. Antibodies according to the present invention can be used appropriately to obtain such immunotoxins. An example of such immunotoxins are those described by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and Fanger, Immunol. Today 12 (1991), 51-54.

Those skilled in the art will appreciate that the conjugates can also be assembled using various methods depending on the selected conjugate agent. For example, biotin conjugates are prepared, for example, by reacting an IAPP and/or proIAPP binding polypeptide with an activated biotin ester, eg biotin-N-hydroxysuccinimide ester. Similarly, fluorescent marker conjugates can be prepared in the presence of a binding agent, such as those listed herein, or by reaction with an isothiocyanate, preferably fluorescein isothiocyanate. Conjugates of antibodies or their antigennegative fragments, variants or derivatives according to the present invention receive a similar way.

The present invention also encompasses antibodies or antigen-binding fragments, variants or derivatives thereof according to the invention conjugated to a diagnostic or therapeutic agent. Antibodies can be used for diagnostic purposes, for example, to demonstrate the presence of a metabolic disease, such as T2DM, to indicate the risk of metabolic disease.

- 49 042691 disease, to monitor the development or progression of a metabolic disease, i.e. a disease in which the appearance of or which is associated with an aggregated IAPP and/or proIAPP, as part of a clinical analysis procedure to determine, for example, the effectiveness of a particular treatment and/or prophylaxis regimen, is observed. Thus, in one embodiment, the present invention relates to an antibody provided with a detectable label. In addition, in one embodiment, the present invention relates to an antibody attached to a medicinal substance. Detection can be facilitated by attaching an antibody or antigen-binding fragment, variant or derivative thereof to the substance to be detected. The substance or label to be detected can generally be an enzyme; a heavy metal, preferably gold; a dye, preferably a fluorescent or luminescent dye; or radioactive label. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals for use with various types of positron emission tomography, and non-radioactive paramagnetic metal ions; see, for example, US Pat. No. 4,741,900 for metal ions that can be conjugated with antibodies for diagnostic use in accordance with the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic complex groups include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin; and examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, 'In or ⁹⁹ Tc. Thus, in one embodiment, the present invention provides an antibody labeled with a detectable label, wherein the detectable label is selected from the group consisting of an enzyme, a radioisotope, a fluorophore, and a heavy metal.

An antibody or antigen-binding fragment, variant or derivative thereof can also be labeled with a detectable label by attaching it to a chemiluminescent compound. The presence of the chemiluminescently labeled antibody is then determined by detecting the luminescence that occurs during the chemical reaction. Examples of particularly suitable chemiluminescent labels are luminol, isoluminol, acridinium theromatic ester, imidazole, salts and acridinium oxalate ester.

One method that allows an antibody or its antigen-binding fragment, variant or derivative to be labeled with a detectable label is its binding to an enzyme and the use of the binding product in an enzyme immunoassay (ELISA) (Voller, A., The Enzyme Linked Immunosorbent Assay (ELISA) Microbiological Associates Quarterly Publication, Walkersville, Md., Diagnostic Horizons 2 (1978), 1-7); Voller et al., J. Clin. Pathol. 31 (1978), 507-520; Butler, Meth. Enzymol. 73 (1981), 482-523; Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla. (1980); Ishikawa, E. et al. (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo (1981). The antibody-bound enzyme must react with an appropriate substrate, preferably a chromogenic substrate, to form a chemical group that can be detected (detected), for example, spectrophotometrically, fluorimetrically, or visually. Enzymes that can be used to label an antibody include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid glucose isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, catalase, but not limited to glucose-6phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In addition, detection can be performed by colorimetric methods using the chromogenic substrate of the enzyme. Detection can also be performed by visually comparing the severity of the enzymatic reaction of the substrate compared to similarly prepared standards.

Detection can also be performed using any other type of immunoassay. For example, when an antibody or an antigen-binding fragment, variant or derivative thereof is radioactively labeled, the antibodies can be detected by radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society , (March, 1986)) incorporated herein by reference). A radioactive isotope can be detected using, among other things, a gamma counter, a scintillation counter, or autoradiography.

An antibody or antigen-binding fragment, variant or derivative thereof can also be labeled with fluorescent metals such as ¹⁵² Eu or other lanthanide metals. These metals can be attached to the antibody with a metal chelating group, such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Techniques for conjugating various molecules to an antibody or antigen-binding fragment, variant or derivative thereof are well known, see for example Arnon et al., Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et

- 50 042691 al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., Antibodies For Drug Delivery, in Controlled Drug Delivery ( ^2nd Ed.), Robinson et al. (eds.), Marcel Dekker, Inc. , pp. 623-53 (1987); Thorpe, Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review, in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 ( 1985); Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates, Immunol Rev. 62 (1982), 119-158.

As already mentioned, in some embodiments, a group can be conjugated to improve the stability and potency of a binding molecule, eg, a binding polypeptide, eg, an antibody, or an immunospecific fragment thereof. For example, in one embodiment, binding molecules of the invention can be conjugated with PEG to increase their in vivo half-life. Leong et al., Cytokine 16 (2001), 106; Adv. in Drug Deliv. Rev. 54 (2002), 531; or Weir et al., Biochem. soc. Transactions 30 (2002), 512.

VI. Compositions and methods of application.

The present invention relates to compositions containing the aforementioned IAPP and/or proIAPP binding molecules, for example, an antibody or antigen binding fragment according to the present invention or a derivative or variant thereof, or a polynucleotide, vector or cell according to the invention, as described above. In one embodiment, the composition according to the present invention is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition according to the present invention may contain additional agents, such as interleukins and interferons, depending on the intended use of the pharmaceutical composition. For use in the treatment of a metabolic disease that produces or is associated with aggregated IAPP and/or proIAPP, such as T2D, the additional agent may be selected from the group consisting of small organic molecules, anti-IAPP and/or proIAPP antibodies, and combinations thereof. Thus, in a particularly preferred embodiment, the present invention relates to the use of an IAPP and/or proIAPP binding molecule, e.g., an antibody or antigen-binding fragment thereof according to the present invention, or a binding molecule having substantially the same binding specificity as any of them, a polynucleotide, a vector or a cell according to the present invention for the preparation of a pharmaceutical or diagnostic composition for the prophylactic and therapeutic treatment of a metabolic disease, for monitoring the progression of a metabolic disease or response to treatment of a metabolic disease in a subject, or for determining the risk of developing a metabolic disease in a subject.

Thus, in one embodiment, the present invention relates to a method of treating a metabolic disorder characterized by abnormal accumulation and/or deposition of IAPP or proIAPP in the islets of Langerhans, comprising administering to a subject in need thereof a therapeutically effective amount of any of the IAPP- and/or proIAPP-binding molecules, antibodies, polynucleotides, vectors or cells according to the present invention. The term metabolic disorder includes a group of disorders typically characterized by symptoms such as metabolic changes that precede, cause, and/or are related to/associated with or associated with T2DM, including diseases that cause damage to the pancreas and are therefore capable of leading to diabetes, including chronic pancreatitis, cystic fibrosis , pancreas cancer; in diseases that increase the risk of type 2 diabetes, including Alzheimer's disease, Huntington's disease; in cardiovascular diseases associated or not associated with obesity and DM2; and/or CD2 itself, but is not limited to them.

A particular advantage of the therapeutic approach of the present invention is that the antibodies of the present invention are derived from B-cells or memory B-cells of healthy human subjects with no evidence of a disease in which there is or is associated with aggregated IAPP and/or proIAPP, e.g. , T2D, and thus, with some probability, may prevent the clinical manifestation of disease associated with aggregated IAPP and/or proIAPP, or reduce the risk of clinical manifestation of the disease, or delay the onset or progression of the clinical manifestation of the disease. As a rule, antibodies according to the present invention have already successfully undergone somatic maturation, i. optimizing the selectivity and efficiency of binding to a high affinity IAPP and/or proIAPP target molecule due to the somatic variability of antibody variable regions.

The knowledge that such cells are not activated by related or other physiological proteins or cellular structures in vivo, for example, in the human body, in the sense of autoimmune or allergic reactions, is also of great medical importance, since it means a significant increase in the chances of successful survival throughout all phases of clinical trials. It can be said that their tolerability, efficacy and acceptability have already been demonstrated prior to the preclinical and clinical development of a prophylactic or therapeutic antibody in at least one human subject. Thus, human anti-IAPP antibodies can be expected

- 51 042691 and / or proIAPP according to the present invention, their effectiveness as a therapeutic agent in relation to the target structure and the reduced likelihood of associated side effects significantly increase the clinical success rate.

The present invention also provides a pharmaceutical and diagnostic package or kit, respectively, consisting of one or more containers filled with one or more of the components described above, for example, an antibody against IAPP and/or proIAPP, its binding fragment, derivative or variant, polynucleotide , vector or cells according to the present invention. Such container(s) may be accompanied by a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals and biological products, indicating the agency's approval of the manufacture, use, or sale for medical use. As an add-on or alternative, the kit includes reagents and/or instructions for use in appropriate diagnostic tests. The composition, for example the kit according to the present invention, is of course particularly suitable for the risk assessment, diagnosis, prevention and treatment of a disorder accompanied by the presence of aggregated IAPP and/or proIAPP, and is particularly useful for the treatment of disorders typically characterized by symptoms such as metabolic changes preceding, causing and/or associated/associated with or associated with T2DM, including diseases that cause damage to the pancreas and therefore can lead to diabetes, including, for example, chronic pancreatitis, cystic fibrosis, pancreatic cancer; in diseases that increase the risk of type 2 diabetes, including Alzheimer's disease, Huntington's disease; in cardiovascular diseases associated or not associated with obesity and DM2; and/or CD2 itself.

Pharmaceutical compositions according to the present invention can be formulated according to methods widely known in the art; see, for example, Remington: The Science and Practice of Pharmacy (2000) by the University of Sciences in Philadelphia, ISBN 0-683-306472. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, sterile solutions, and the like. Compositions containing such carriers can be formulated using well known conventional methods. Said pharmaceutical compositions may be administered to a subject at a suitable dose. The introduction of suitable compositions can be carried out in various ways, for example, by intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, topical or intradermal administration, or delivery to the spinal cord or brain. Aerosol formulations, for example nasal sprays, include purified aqueous or other solutions of the active agent with preservatives and isotonic agents. Such compositions are preferably adjusted to a pH and isotonic state compatible with the nasal mucosa. Formulations for rectal or vaginal administration may be presented as a suppository with a suitable carrier.

The dosage regimen can be determined by the attending physician, taking into account clinical factors. As is known in the medical field, dosages for any patient depend on many factors, including patient size, body surface area, age, particular compound to be administered, sex, time and route of administration, general health, and other concomitantly administered drugs. A typical dose may be, for example, from 0.001 to 1000 μg (or the appropriate amount of nucleic acid to express or inhibit expression in this range); however, doses below or above this typical range are acceptable, especially considering the above factors. Typically, the dosage may vary, for example, from about 0.0001 to 100 mg/kg, more often from 0.01 to 5 mg/kg (for example, 0.02, 0.25, 0.5, 0.75, 1 , 2 mg/kg, etc.) body weight. For example, the dosage may be 1 mg/kg body weight or 10 mg/kg body weight, or 1 to 10 mg/kg, preferably at least 1 mg/kg. Doses between the above ranges are also within the scope of the present invention. Subjects can be administered such doses daily, every other day or several days, weekly, or on any other schedule determined through empirical analysis. Typical treatment involves the introduction of a series of dosages over a long period of time, for example, at least six months. Additional typical treatment regimens involve the introduction of once every two weeks or once a month or every 3-6 months. Typical dosing regimens include administration of 1-10 mg/kg or 15 mg/kg every day, 30 mg/kg every other day or 60 mg/kg once a week. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered is within the indicated range. Progress can be monitored through periodic evaluations. Preparations for parenteral administration include sterile aqueous and non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffer media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements (eg, Ringer's dextrose supplements), and the like. In addition, preservatives and other additives may be present, such as antimicrobials, antioxidants, chelating agents and inert gases, and the like. In addition, the pharmaceutical composition according to the invention may contain additional agents, such as dopamine or psychopharmacological agents, depending on the intended use of the pharmaceutical composition.

In addition, in a preferred embodiment of the present invention, the pharmaceutical composition may be formulated as a vaccine, for example, if the pharmaceutical composition of the invention comprises an anti-IAPP and/or proIAPP antibody or a passive immunization binding fragment, derivative or variant thereof. As mentioned in the Background section, there are several data sets showing that aggregated IAPP and/or proIAPP variants are the main trigger of T2DM pathogenesis (Zraika et al. (2010), Diabetologia 53(6): 1046-1056; Westermark et al (2011), Physiol Rev 91(3): 795-826, Jurgens et al (2011), Am J Pathol 178(6): 2632-2640, Hoppener et al (2008), Exp. Diabetes Res. 697035) and treatment that interferes with hIAPP aggregation improved the diabetic phenotype and increased the lifespan of the animal (Aitken et al. (2009), Diabetes 59(1): 161-171). Accordingly, it is reasonable to expect that passive immunization with a human anti-IAPP and/or proIAPP antibody and equivalent IAPP or proIAPP binding molecules of the present invention would avoid some of the undesirable effects of an active immunization therapeutic approach, as noted in the Background section. Thus, the present antibodies against IAPP and/or proIAPP and their equivalents according to the present invention should be particularly useful as a vaccine for the prevention or amelioration of diseases in which the presence or which is observed to be present or caused by aggregated IAPP and/or proIAPP, for example, metabolic changes preceding causing and/or associated/associated with or associated with T2DM, including diseases that cause damage to the pancreas and therefore can lead to diabetes, including chronic pancreatitis, cystic fibrosis, pancreatic cancer; in diseases that increase the risk of type 2 diabetes, including Alzheimer's disease, Huntington's disease; in cardiovascular diseases associated or not associated with obesity and DM2; and/or CD2 itself.

In one embodiment, it may be advantageous to use recombinant bispecific or multispecific antibody constructs of the present invention. For reference, see Fischer and Leger, Pathobiology 74 (2007), 3-14. One binding arm of such a bispecific molecule can be targeted by IAPP and the other arm by another component involved in the pathogenesis of diabetes mellitus, for example, proIAPP (in addition to the antibodies of the present invention, which are bispecific against IAPP and proIAPP, as described above for the typical NI203 antibody. 26C11). Either the second binding arm of such a bispecific molecule can bind to another component involved in the pathogenesis of diabetes mellitus, such as IL-1b and IL-6, or block the sodium and glucose co-transporter-2 (SGLT2) or CD33; this intervention is believed to promote regression of diabetes in NOD mice by inducing adaptive regulatory T cells (Ablamunits et al., Diabetes 61 (2012), 145-154; Belghith et al., Nat Med 9 (2003), 1202-1208 ).

In one embodiment, it may be advantageous to use recombinant Fab (rFab) and single chain fragments (scFv) of the antibody of the present invention, which can more easily penetrate the cell membrane. For example, in an article by Robert et al., Protein Eng. Des. Sel. (2008) Oct 16; S1741-0134 published online previously describes the use of chimeric recombinant Fabs (rFab) and single chain fragments (scFv) of the WO-2 monoclonal antibody that recognizes an epitope in the N-terminal region of Ae. The constructed fragments could:

(i) prevent the formation of amyloid fibrils, (ii) disaggregate the formed Ae1 fibrils. ₄ 2, and (iii) inhibit APi-42 oligomer mediated neurotoxicity in vitro as effectively as the entire IgG molecule.

The purported benefits of using small Fab- and scFv-engineered antibody formats devoid of effector function include more efficient penetration of the blood-brain barrier and minimization of the risk of triggering inflammatory side reactions. In addition to retaining the binding specificity of full-length antibodies, scFvs and single-domain antibodies can be expressed as single genes and intracellularly in mammalian cells as intrabodies, with the possibility of changing the folding, interaction, modification, or intracellular localization of their targets; see, for example, Miller and Messer, Molecular Therapy 12 (2005), 394-401.

According to another approach, Muller et al., Expert Opin. Biol. Ther. (2005), 237-241, described a technological platform, the so-called superantibody technology, which is believed to allow antibodies to enter living cells without harming them. Such cell-penetrating antibodies open up new diagnostic and therapeutic possibilities. The term TransMab (TransMAT) has been coined for these antibodies.

In a further embodiment, the co-administration or sequential administration of other antibodies used to treat a disease associated with the occurrence of aggregated IAPPs and/or proIAPPs may be desirable. In one embodiment, the implementation of additional antibody is part of the pharmaceutical composition according to the present invention. Examples of antibodies that can be used to treat a subject include, but are not limited to, anti-CD33, SGLT2, IL-6, and IL-1 antibodies.

In an additional embodiment, the co-administration or sequential administration of other agents used to treat a disease associated with aggregated IAPP and/or proIAPP and/or diabetes may be desirable. In one embodiment, the implementation of the additional agent is part of the pharmaceutical composition according to the present invention. Examples of agents that can be used to treat a subject include: insulin and insulin analogs; modulators of the insulin signaling pathway, e.g. protein tyrosine phosphatase inhibitors (PTPases), macromolecular mimetic compounds and inhibitors of glutamine-fructose-6-phosphate amidotransferase (GFAT), DPP-IV inhibitors, agents affecting unregulated production of glucose in the liver, e.g. glucose-6-phosphatase (G6Pase) inhibitors, fructose-1,6-bisphosphatase (F-1,6-BPase) inhibitors, glycogen phosphorylase (GP) inhibitors, glucagon receptor antagonists, phosphoenolpyruvate carboxykinase (PEPCK) inhibitors, pyruvate dehydrogenase kinase (PDHK) inhibitors , insulin sensitivity enhancers, insulin secretion enhancers, α-glucosidase inhibitors, gastric emptying inhibitors; glucagon-like peptide-1 (GLP-1) receptor agonists; sulfonylurea agents; biguanide agents, eg metformin; alpha-glucosidase inhibitors; peroxisome proliferator-activated receptor (PPAR) agonists; meglitinide agents; dipeptidyl peptidase (DPP) inhibitors IV; inhibitors of PDE1, PDE5, PDE9, PDE10 or PDE11 (PDE=phosphodiesterase); amylin agonists (eg pramlintide and other amylin analogs); cinnamon; glucagon receptor antagonists; glycogen phosphorylase inhibitors; fructose-1,6-bisphosphate inhibitors; cannabinoid receptor (CB1) antagonists; anti-obesity drugs, for example, appetite suppressants, satiety-enhancing agents, and drugs that increase energy expenditure; anti-inflammatory agents, or any combination thereof, but are not limited to. Examples of agents that can be used to treat or prevent islet rejection after clinical pancreatic islet transplantation include agents in the group containing sirolimus (rapamycin), calcineurin inhibitors (eg, tacrolimus), cyclosporine, mycophenolate mofetil, FTY-720, cyclosporine, corticosteroids, and anti-IL-2 receptor monoclonal antibodies (eg, daclizumab), glucagon-like peptide-1 (GLP-1) receptor agonists (see, for example, Noguchi et al., Acta Med. Okayama, 60 (2006), and international application WO2012088157), but are not limited to them. Thus, in one embodiment, a composition is provided further comprising an agent useful in the treatment of type 2 diabetes mellitus (T2DM) and/or in the treatment or prevention of islet rejection following clinical pancreatic islet transplantation. Examples of other agents that can be used in conjunction with the pharmaceutical composition according to the present invention are described in the art; see, for example, international applications WO2009005672, WO2010128092, WO2012088157 or European application EP11158212.8.

A therapeutically effective dose or amount refers to an amount of the active ingredient sufficient to ameliorate the symptoms or condition. The therapeutic efficacy and toxicity of such compounds can be determined using standard pharmaceutical procedures in cell cultures or experimental animals, for example, ED ₅₀ (therapeutically effective dose in 50% of the population) and LD ₅₀ (fatal dose in 50% of the population). The ratio of doses that have a therapeutic and toxic effect, is a therapeutic index and can be expressed as a ratio of LD ₅₀ /ED ₅₀ . In a preferred embodiment, the therapeutic agent is present in the composition in an amount sufficient to restore or maintain control of normal blood sugar and/or insulin response in the event of metabolic disorders such as T2DM.

From the foregoing, it is clear that the present invention encompasses any use of an IAPP and/or proIAPP binding molecule containing at least one CDR of an antibody described above, in particular for the diagnosis or treatment of a disease associated with aggregated IAPP and/or proIAPP, as mentioned above, for example, CD2. In a preferred embodiment, the specified binding molecule is an antibody according to the present invention or its immunoglobulin chain. In addition, the present invention relates to anti-idiotypic antibodies of any of the specified antibodies described above. These antibodies or other binding molecules bind to a unique antigenic peptide sequence located on the variable region of the antibody near the antigen binding site; they can be used, for example, to detect antibodies against IAPP and/or proIAPP in a sample obtained from a subject. Thus, in one embodiment, the present invention provides an antibody as described above and hereinafter, or IAPP and/or proIAPP binding molecules having substantially the same specificity as any of said antibodies, polynucleotide, vector, or cell described herein, or a pharmaceutical or diagnostic composition containing any of the above components, for use in prophylactic treatment, therapeutic treatment and/or monitoring the progression or response to treatment of an IAPP and/or proIAPP related disorder, wherein the disorder preferably selected from the group including all types of diabetes, for example, type 1 diabetes mellitus, gestational diabetes, prediabetes (in which blood glycemia does not reach the T2DM threshold or insulin resistance) and latent adult autoimmune diabetes (LADA); any disease that causes damage to the pancreas and thus can lead to diabetes, such as chronic pancreatitis, cystic fibrosis and pancreatic cancer; any disease that increases the risk of T2DM, such as Alzheimer's disease and Huntington's disease or other neurodegenerative diseases that are considered to be associated with diabetes herein; metabolic syndrome in general as a risk factor for diabetes or a condition that may precede diabetes; islet amyloidosis in general as a risk factor for diabetes or a condition that may precede diabetes; obesity in general as a risk factor for diabetes or a condition that may precede diabetes; any cardiovascular disease associated or not associated with obesity and T2DM; all consequences of T2DM that can also increase the risk of developing diabetes, such as heart disease, strokes, diabetic retinopathy, kidney failure, ketoacidosis, and nonketotic hyperosmolar coma. The above group of disorders is referred to as the group of IAPP and/or proIAPP related disorders.

In yet another embodiment, the present invention provides a diagnostic composition comprising any of the IAPP and/or proIAPP binding molecules, antibodies, antigen binding fragments, polynucleotides, vectors, or cells of the invention described above, and optionally a suitable means for detecting such reagents, traditionally used in diagnostic methods based on immunocomponents or nucleic acids. The antibodies of the invention are, for example, suitable for use in immunoassays where they can be used in liquid phase or coupled to a solid phase carrier. Examples of immunoassays in which an antibody of the invention can be used are competitive and non-competitive immunoassays in direct or indirect format. Examples of such immunoassays are radioimmunoassay (RIA), sandwich assay (immunometric assay), flow cytometry and Western blotting. The antigens and antibodies of the invention can be coupled to a variety of carriers and used to isolate cells that specifically bind to them. Examples of well known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified cellulose derivatives, polyacrylamide, agarose, and magnetite. In the present invention, the carrier may be of a soluble or insoluble nature. Those skilled in the art are aware of various labels and labeling methods. Examples of the types of labels that can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds; see also the implementations discussed above.

In a further embodiment, the IAPP or proIAPP binding molecules, in particular the antibodies of the present invention, can also be used in a method for diagnosing a disorder in an individual by obtaining a body fluid sample from the test individual, which may be a blood sample, a plasma sample, a serum sample, a lymph sample or other body fluid sample, such as saliva or urine; and contacting the body fluid sample with an antibody of the present invention under conditions that allow antibody-antigen complexes to form. The level of such complexes is then determined by methods known in the art, with a level significantly elevated compared to the level in the control sample indicating a disease in the individual tested. Similarly, a specific antigen bound by an antibody of the invention can be used. Thus, the present invention relates to an in vitro immunoassay comprising the use of a binding molecule, for example an antibody or antigen-binding fragment thereof, according to the invention.

In this context, the present invention also relates to agents specifically designed for this purpose. For example, an antibody-based matrix, eg, loaded with antibodies or equivalent antigen-binding molecules of the present invention specifically recognizing IAPP and/or proIAPP, can be used. The design of microarrays for immunoassay is given in Kusnezow et al., Mol. Cell Proteomics 5 (2006), 1681-1696. Accordingly, the present invention also relates to microarrays loaded with IAPP and/or proIAPP binding molecules in accordance with the present invention.

In one embodiment, the present invention relates to a method for diagnosing a disease associated with aggregated IAPP and/or proIAPP in a subject, said method comprising determining the presence of IAPP and/or proIAPP and/or aggregated IAPP and/or proIAPP in a sample from the diagnosable subject using at least one antibody of the present invention, its IAPP and/or proIAPP binding fragment, or an IAPP and/or proIAPP binding molecule having substantially the same binding specificity as any of them, wherein the presence of pathologically aggregated IAPP and/ or proIAPP is indicative of a metabolic disorder, eg T2D, and an increase in the level of pathologically aggregated IAPP and/or proIAPP compared to the level of physiological monomeric forms of IAPP and/or proIAPP is indicative of the progression of the metabolic disorder in said subject.

The diagnosed subject may have an asymptomatic or preclinical form of the disease. Preferably, the control subject has a disease associated with an aggregated IAPP and/or proIAPP, e.g. to diabetes, including chronic pancreatitis, cystic fibrosis, pancreatic cancer; in diseases that increase the risk of type 2 diabetes, including Alzheimer's disease, Huntington's disease; in cardiovascular diseases associated or not associated with obesity and DM2; and/or T2DM itself, wherein the similarity between the level of pathologically aggregated IAPP and/or proIAPP and the reference standard indicates a diagnosis of the presence or risk of developing a metabolic disease in the subject. Alternatively, or in addition to a second control, a control subject does not suffer from a metabolic disease, wherein the difference between the level of physiological IAPP and/or proIAPP or aggregated IAPP and/or proIAPP monomers and the reference standard is indicative of a diagnosis of the presence or risk of developing a metabolic disease in the subject. In a preferred embodiment, the age of the diagnosed subject and the control subject should match. The analyzed sample can be any body fluid suspected of containing pathologically aggregated IAPP and/or proIAPP, such as blood, plasma, serum, urine, peritoneal fluid, saliva, and cerebrospinal fluid (CSF).

The level of physiological monomers of IAPP and/or proIAPP and/or pathologically aggregated IAPP and/or proIAPP can be determined by any suitable method known in the art, including, for example, analysis of IAPP and/or proIAPP by one or more methods selected from Western blotting, immunoprecipitation , enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS), two-dimensional electrophoresis, mass spectroscopy (MS), matrix-assisted time-of-flight ionization laser desorption (MALDI-TOF), time-of-flight surface-enhanced ionization laser desorption (SELDI-TOF), high performance liquid chromatography (HPLC), rapid protein liquid chromatography (PECP), multidimensional liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS) and laser densitometry. In a preferred embodiment, said in vivo IAPP and/or proIAPP imaging includes positron emission tomography (PET), single photon emission tomography (SPET), near infrared (NIR) optical imaging, or magnetic resonance imaging (MRI).

Antibody-based methods for detecting IAPP and/or proIAPP and diagnosing and monitoring disease progression associated with aggregated IAPP and/or proIAPP, e.g. T2DM, and monitoring treatment of such disease with antibodies and related agents, which can be adapted according to with the present invention are also described in international application WO2003092619, the contents of which are incorporated herein by reference. These methods can be used as described, but using an IAPP and/or proIAPP specific antibody, binding fragment, derivative or variant of the present invention.

Thus, one embodiment includes an antibody of the present invention or an IAPP and/or a proIAPP binding molecule having substantially the same binding specificity as any of them, a polynucleotide, vector, or cell as described above, or a pharmaceutical or diagnostic composition containing any of said components, for use in prophylactic treatment, therapeutic treatment and/or monitoring the progression or response to treatment of an IAPP and/or proIAPP related disorder. Thus, in general, the present invention also relates to a method for diagnosing or monitoring the progression of an IAPP and/or proIAPP-associated disorder (e.g., islet amyloidosis and T2DM, which is usually preceded by islet amyloidosis) in a subject, which method includes determining the presence of oligomers, aggregates or fibrils of IAPP and/or proIAPP in a sample from a diagnosable subject using at least one antibody of the present invention or an IAPP and/or proIAPP binding molecule having substantially the same binding specificity as any of them, where the presence of oligomers, aggregates or fibrils of IAPP and/or proIAPP is indicative of a disorder. In one embodiment, provided is a method for diagnosing or monitoring the progression of islet amyloidosis in a subject, comprising determining the presence of IAPP and/or proIAPP oligomers, aggregates, or fibrils in a sample of the subject to be diagnosed using at least one antibody of the present invention or IAPP- and/ or a proIAPP binding molecule having substantially the same binding specificity as any of them, wherein the presence of IAPP and/or proIAPP oligomers, aggregates, or fibrils is indicative of presymptomatic, prodromal, or clinical type 2 diabetes mellitus (T2DM) and/or deficiency

- 56 042691 beta cells after clinical transplantation of pancreatic islets and an increase in the level of oligomers, aggregates or fibrils of IAPP and / or proIAPP compared with the level of physiological IAPP or compared with a reference sample obtained from the body of a healthy control subject or a control sample from the body of that same subject indicates progression of presymptomatic, prodromal, or established type 2 diabetes mellitus (DM2) and/or islet insufficiency following clinical pancreatic islet transplantation in said subject. One skilled in the art will appreciate that, in one embodiment, this method is also used to diagnose and monitor the progression of any other IAPP and/or proIAPP related disorder as described above.

As indicated above, the antibodies of the present invention, their fragments and molecules with the same binding specificity as the antibodies and their fragments, can be used not only in vitro, but also in vivo, and, in addition to diagnostic, for therapeutic use. Thus, in one embodiment, the present invention also provides an IAPP and/or proIAPP binding molecule comprising at least one CDR of an antibody of the present invention, provided for the manufacture of a composition for in vivo detection or targeting of a therapeutic or diagnostic agent to IAPP, and /or proIAPP in a human or animal body. Potential therapeutic and/or diagnostic agents can be selected from a non-exclusive list of therapeutic agents that can be used in the treatment of metabolic diseases, such as T2DM and potential labels, as described above. With respect to in vivo imaging, in one preferred embodiment of the present invention, said IAPP and/or proIAPP binding molecule is provided comprising at least one CDR of an antibody of the present invention, wherein said in vivo imaging comprises positron emission tomography (PET), single photon emission tomography (SPET), optical imaging in the near infrared spectrum (NIR) or magnetic resonance imaging (MRI). In a further embodiment, the present invention also provides an IAPP and/or proIAPP binding molecule comprising at least one CDR of an antibody of the present invention, or said molecule for making a composition for said in vivo imaging modalities, for use in methods for diagnosing or monitoring the progression of a disorder. associated with IAPP and/or proIAPP in a subject as above.

VII. Peptides with aggregation of specific IAPP epitopes.

In a further aspect, the present invention relates to peptides containing an IAPP and/or proIAPP epitope specifically recognized by an antibody of the present invention. In a preferred embodiment, such a peptide contains or consists of the amino acid sequence specified in SEQ ID NO: 4; SEQ ID NO: 5 or SEQ ID NO: 71 as a unique linear epitope recognized by an antibody or modified sequence thereof in which one or more amino acids are substituted, deleted, or added, the peptide being recognized by any antibody of the present invention, preferably an NI-2O3 antibody .19H8 and, respectively, the NI-203.26d 1 antibody or the NI-2O3.15C7 antibody.

In one embodiment of the present invention, such a peptide can be used to diagnose or monitor a disease associated with aggregated IAPP and/or proIAPP in a subject, for example, T2DM, comprising the step of determining the presence of an antibody that binds to the peptide in a biological sample from said subject, and used to diagnosing such a disease in said subject by measuring the level of antibodies recognizing the above-described peptide of the present invention and comparing the measurement results with levels in healthy subjects of comparable age and sex. Thus, in one embodiment, the present invention relates to a method for diagnosing islet amyloidosis indicative of presymptomatic or clinical type 2 diabetes mellitus (DM2) and/or beta cell deficiency following clinical transplantation of pancreatic islets in a subject, comprising the step of determining the presence of an antibody, binding said peptide as described above in a biological sample from said subject. According to this method, an elevated level of a measurable antibody specific for said peptide of the present invention is indicative of a diagnosis of presymptomatic or clinical type 2 diabetes mellitus (T2DM) and/or beta cell deficiency following clinical transplantation of pancreatic islets in said subject or for diagnosis in said subject. the subject of any other disease from the group of disorders associated with IAPP and/or proIAPP, as described above. The peptide of the present invention can be formulated into a matrix, kit and composition, respectively, as described above. In this context, the present invention also relates to a kit that can be used in the diagnosis or monitoring of the progression of islet amyloidosis, said kit comprising at least one antibody of the present invention or an IAPP and/or proIAPP binding molecule having substantially the same specificity , as well as any of them, a polynucleotide, a vector, a cell and/or a peptide, as respectively indicated above, optionally together with reagents and/or instructions for use.

These and other implementation options are described and included in this description and examples of this

- 57 042691 inventions. Additional literature relating to any materials, methods, applications and compounds used in accordance with the present invention can be obtained from and public libraries and databases, using, for example, electronic devices. For example, you can use the public database Medline, which is hosted by the National Center for Biotechnology Information and/or the National Library of Medicine of the National Institutes of Health. Additional databases and web addresses, such as the European Bioinformatics Institute (EBI), part of the European Molecular Biology Laboratory (EMBL), are known to those skilled in the art and can also be obtained using Internet search engines. For a review of patent information in the field of biotechnology and a review of relevant sources of patent information suitable for retrospective search and understanding of the current state of the art, see Berks, TIBTECH 12 (1994), 352-364.

The above information contains a general description of the present invention. Unless otherwise indicated, the term used herein is defined as provided in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0198506732. In the text of this specification several documents have been cited. Full bibliographic citations can be found at the end of the description, just before the claims. The contents of all cited references (including literature, issued patents, published patent applications cited throughout this application and in manufacturer's specifications, instructions, etc.) are expressly incorporated herein by reference; however, it is implied that none of the cited documents is actually a precursor of the present invention.

A more complete understanding of the present invention may be obtained by reading the following specific examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention.

Examples

Example 1 Validation of the target and binding specificity of human anti-IAPP antibodies.

To check whether IAPP is a target recognized by isolated antibodies, a direct ELISA was performed. For representative recombinant human antibodies NI-203.9A2, NI203.19H8, NI-2O3.26C11 and NI-203.8E3, 96-well microplates (Costar, Corning, USA) were coated with either human IAPP or BSA solution (Sigma-Aldrich, Bux, Switzerland) diluted to 10 μg/ml in carbonate solid phase ELISA immobilization buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO 3 , pH 9.42) and tested for antibody binding efficiency. Importantly, the human IAPP solution used for the ELISA contained IAPP fibrils as shown by electron microscopy; see fig. 3A. Representative antibodies NI-203.9A2, NI-2O3.19H8, NI-203.26C11 and NI-203.8E3 specifically bound to human IAPP fibrils according to ELISA. Binding to BSA was not observed; see fig. 3b. Similar characteristics appear to apply to the NI-203.19F2 and NI-203.15C7 antibodies.

To determine the half-maximal effective concentration (EC50) of typical antibodies NI203.9A2, NI-2O3.19H8, NI-203.26d1 and NI-203.8E3, additional experiments were performed by direct ELISA with various concentrations of antibodies. 96-well microplates (Costar, Corning, USA) were coated with a solution of human IAPP or human proIAPP diluted to a concentration of 10 μg/ml in carbonate buffer for immobilization in solid phase ELISA (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9, 42) and tested the binding efficiency of the antibody. While the human IAPP solution used for the ELISA contained IAPP fibrils, human proIAPP formed large aggregates in the solution, as shown by electron microscopy; see fig. 4A. Binding was determined with donkey anti-human IgG antibodies (Jackson ImmunoResearch, Newmarket, UK) conjugated to HRP followed by measurement of HRP activity in a standard colorimetric assay.

EC50 values were estimated by non-linear regression using GraphPad Prism software (San Diego, USA). Human recombinant antibodies NI203.9A2, NI-2O3.19H8, NI-203.26C11, and NI-203.8E3 bound human IAPP fibrils with high affinity and EC50s of 9, 22, 6, and 4 nM, respectively. The NI-203.26d1 antibody also bound to aggregated human proIAPPs with an EC50 in the nanomolar range (260 nM); see fig. 4B.

Example 2 Antibody specificity for human IAPP fibrils, but not for non-fibrillar human IAPP, indicated preferential binding to conformational epitopes.

To determine the binding capacity of typical antibodies NI-2O3.9A2, NI-2O3.19H8, NI203.26C11 and NI-203.8E3 to conformational epitopes, experiments were performed based on direct ELISA using IAPP solutions and non-fibrillar human IAPP diluted to a concentration of 10 μg/ml in carbonate solid phase ELISA immobilization buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.42) and tested the binding capacity of the antibody. Whereas the human IAPP solution used for ELISA contained IAPP fibrils, the non-fibrillar human IAPP solution did not contain IAPP fibrils and showed only small

- 58 042691 amorphous aggregates, as shown by electron microscopy; see fig. 5A. Binding was determined using donkey anti-human IgG antibodies (Jackson ImmunoResearch, Newmarket, UK) conjugated to HRP followed by measurement of HRP activity in a standard colorimetric assay.

Recombinant antibodies NI-203.9A2, NI-2O3.19H8, NI-203.26C11 and NI-203.8E3 showed high binding affinity for IAPP fibrils when coated with IAPP solution (FIG. 5B) as observed previously (FIG. 4). For non-fibrillar IAPP, insufficient affinity was observed compared to IAPP fibrils (FIG. 5B), demonstrating preferential binding of NI-2O3.9A2, NI-2O3.19H8, NI-203.26d1, and NI-203.8E3 antibodies to IAPP fibrils. Preliminary results showed the presence of effects similar to those described above for antibodies NI-203.19F2 and NI-2O3.15C7.

These data strongly indicate that NI-203.9A2, NI-2O3.19H8, NI-203.26C11, NI203.8E3, NI-203.19F2, and NI-2O3.15C7 antibodies bind epitopes that are predominantly located on the surface and are accessible during formation. IAPP fibrils, in contrast to the linear epitopes present in the physiological conformation of the human IAPP protein. Pathological IAPP fibrils are observed in the pancreatic islets of patients with type 2 diabetes mellitus. Since antibodies NI-203.9A2, NI-2O3.19H8, NI-203.26C11, NI-203.8E3, NI-203.19F2, and NI-2O3.15C7 showed preferential binding to therapeutically relevant pathological human IAPP fibrils, these human-derived antibodies have a therapeutic potential for DM2.

Example 3 Evaluation of the binding epitope of antibodies NI-203.9A2, NI-2O3.19H8, NI-2O3.26C11, NI-203.8E3, NI-203.19F2 and NI-203.15C7.

To determine the binding epitope of representative antibodies NI-203.9A2, NI-2O3.19H8, NI-203.26d1, NI-203.8E3, NI-203.19F2, and NI-2O3.15C7, pepscan and alanine scan analysis were performed using overlapping peptides by mapping the complete amino acid sequence of human IAPP and replacing the first 22 amino acids of proIAPP with alanine. Antibody binding capacity was tested on indicated peptides spotted on a nitrocellulose membrane (JPT Peptide Technologies, Berlin, Germany) with donkey HRP-conjugated anti-human secondary IgG antibody (Jackson ImmunoResearch, Newmarket, UK) followed by activity detection. HRP (Fig. 6A).

Recombinant antibodies NI-2O3.19H8, NI-203.26d1 and NI-2O3.15C7 (1 μg/ml) showed binding to the sequence 19-SSNNFGA-25 (SEQ ID NO: 4), 2-CNTATCA-8 (SEQ ID NO : 5) and 10-QRLANFLVHS-19 (SEQ ID NO: 71) human IAPP (FIGS. 6A and 6B) corresponding to the sequences of the putative binding epitope of these antibodies. Epitopes of recombinant antibodies NI203.9A2, NI-203.8E3 and NI-203.19F2 (1 and 10 µg/ml) were not detected.

Example 4 Binding of NI-203.9A2, NI-2O3.19H8 and NI-203.26C11 antibodies to pathological IAPP fibrils in the pancreas of patients diagnosed with type 2 diabetes mellitus, but not control patients.

Paraffin-embedded pancreatic sections of two patients diagnosed with type 2 diabetes mellitus (T2DM) were selected for the amyloid loading of pancreatic islets observed with ThioS and Congo red staining and then used to evaluate the binding characteristics of a representative antibody NI-203.9A2, NI-2O3. 19H8 and NI-203.26d1. Paraffin-embedded pancreatic sections of a patient without a diagnosis of type 2 diabetes mellitus were used as controls. After pretreatment with formic acid, sections were incubated with human antibodies NI-203.9A2, NI203.19H8 and NI-203.26d1 (5 and 50 nM) or mouse monoclonal antibody against IAPP (1:100; Abcam, Cambridge, UK), followed by incubation with biotinylated donkey anti-human secondary (1:500; Jackson ImmunoResearch, Newmarket, UK) or biotinylated goat anti-mouse secondary (1:500; Jackson ImmunoResearch, Newmarket, UK). The antibody signal was amplified using the Vectastain ABC-AP kit (Vector Laboratories, USA) and detected using the substrate, diaminobenzidine (Thermo Fisher Scientific, USA). After avidin/biotin blocking (Avidin/Biotin blocking kit, Vector Laboratories, USA), pancreatic islet β-cells were visualized with guinea pig polyclonal antibodies against insulin (1:5; Dako, USA) in combination with biotinylated donkey anti-guinea pig secondary antibody (1:500; Jackson ImmunoResearch Laboratories, USA); the antibody signal was amplified with the Vectastain ABC-AP kit (Vector Laboratories, USA) and detected using an alkaline phosphatase substrate (Vector Laboratories, USA).

The first T2DM patient demonstrated large amyloid deposits in pancreatic islets consistent with abnormal IAPP fibrils visualized by ThioS and Congo red staining (Fig. 7A). Human antibodies NI-203.9A2, NI-2O3.19H8, and NI-203.26d1 showed preferential staining of pancreatic islets on these amyloid-positive sections (Fig. 7B). Antibody staining was observed at 5 nM and increased at 50 nM; staining with a single secondary antibody was not observed, indicating specific binding of human anti-IAPP antibodies. These data were confirmed by the second

- 59,042,691 T2DM patients who demonstrated amyloid deposits in pancreatic islets (data not shown). In contrast, human antibodies NI-2O3.9A2, NI-2O3.19H8, and NI-2O3.26C11 did not show pancreatic islet staining in the third patient without amyloid deposits (Fig. 7C and D) and in the control patient without a diagnosis of T2DM (Fig. . 8). A commercially available mouse anti-IAPP monoclonal antibody stained physiological IAPP in pancreatic islets of a non-diabetic control patient; see fig. 8. Antibodies according to the present invention also allowed to obtain positive results in the pancreas of cats with diabetes and deposits of islet amyloid; see fig. 9. Similar binding properties appear to apply to NI-203.19F2 and NI-203.15C7 antibodies.

These data demonstrate that the NI-203.9A2, NI-2O3.19H8, NI-2O3.26C11, NI203.19F2, and NI-2O3.15C7 antibodies specifically recognize abnormal IAPP fibrils and, in accordance with the biochemical binding properties of these antibodies, which exhibited a high fibril binding specificity of IAPP in vitro (FIG. 5).

Example 5 Antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 do not cross-react with abnormal amyloid Ae in the brain of a patient diagnosed with Alzheimer's disease.

Paraffin-embedded brain sections from a patient diagnosed with Alzheimer's disease were used to assess cross-reactivity of representative antibodies NI-203.9A2, NI-2O3.19H8 and NI-2O3.26C11. After pretreatment with formic acid, sections were incubated with human antibodies NI203.9A2, NI-2O3.19H8 and NI-2O3.26C11 (50 nM) or mouse monoclonal antibody 6E10 against β-amyloid (1:2000; Covance, Alschwil, Switzerland), followed by by incubation with biotinylated donkey anti-human secondary (1:500; Jackson ImmunoResearch, Newmarket, UK) or biotinylated goat anti-mouse secondary (1:500; Jackson ImmunoResearch, Newmarket, UK). The antibody signal was amplified using the Vectastain ABC-AP kit (Vector Laboratories, USA) and detected using the substrate, diaminobenzidine (Thermo Fisher Scientific, USA).

Antibodies NI-203.9A2, NI-2O3.19H8, and NI-203.26C11 did not recognize abnormal amyloid Ae in human Alzheimer's brain, in contrast to the specific antibody 6E10 against β-amyloid (Fig. 10). Apparently, similar properties apply to antibodies NI-203.19F2 and NI203.15C7.

These data show that NI-203.9A2, NI-2O3.19H8, and NI-203.26C11 did not cross-react with pathological Ae amyloid. Accordingly, direct ELISA also demonstrated minimal cross-linking of NI-203.9A2, NI-2O3.19H8, and NI-2O3.26C11 antibodies with several misfolded/aggregate candidate proteins, including the best-known amyloid-forming proteins. including but not limited to alpha-synuclein, superoxide dismutase 1 (SOD1), Tau and TAP binding protein 43 (TDP-43).

Example 6 Quality control of mouse chimeric antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11.

For validation of typical mouse chimeric antibodies NI-203.9A2, NI-2O3.19H8 and NI-203.26C11, direct ELISA was performed as described above. Chimeric antibodies were compared with corresponding human antibodies. For typical recombinant chimeric antibodies NI-203.9A2, NI203.19H8, NI-2O3.26C11 and NI-203.8E3 and their corresponding human antibodies, 96-well microplates (Costar, Corning, USA) were coated with a solution of human IAPP or BSA (Sigma-Aldrich , Bux, Switzerland) diluted to 10 μg/ml in carbonate solid phase ELISA immobilization buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.42) and tested the binding efficiency of chimeric and human antibodies. Binding was determined with donkey anti-human IgG antibodies (Jackson ImmunoResearch, Newmarket, UK) conjugated to HRP followed by measurement of HRP activity in a standard colorimetric assay. Importantly, the human IAPP solution used for the ELISA contained IAPP fibrils as shown by electron microscopy; see fig. 3A. EC ₅₀ values were evaluated by non-linear regression using GraphPad Prism software (San Diego, USA).

Chimeric mouse antibodies NI-203.9A2, NI-2O3.19H8, and NI-203.26d1 bind to human IAPP fibrils with high affinity and EC ₅₀ of 18.6, 23.9, and 11.5 nM, respectively. Binding to BSA was not observed. The binding affinity of the chimeric antibodies was similar to that of their corresponding human antibodies, with _EC50 for human antibodies NI-203.9A2, NI-2O3.19H8, and NI-203.26C11 being 9.4, 22.9, and 6.8 nM, respectively; see fig. eleven.

Representative mouse chimeric antibodies NI-203.9A2, NI-2O3.19H8, and NI-203.26C11 were then tested on paraffin-embedded pancreas sections of two selected patients diagnosed with type 2 diabetes mellitus (DM2) and islet amyloid deposits. After pretreatment with formic acid, sections were incubated with chimeric antibodies NI-203.9A2, NI-2O3.19H8, and NI-203.26C11 (50 nM) followed by incubation with a biotinylated donkey anti-anab secondary antibody.

- 60 042691 human titel (1:500; Jackson ImmunoResearch, Newmarket, UK). The antibody signal was amplified using the Vectastain ABC-AP kit (Vector Laboratories, USA) and detected using the substrate diaminobenzidine (Thermo Fisher Scientific, USA). After avidin/biotin blocking (Avidin/Biotin blocking kit, Vector Laboratories, USA), pancreatic islet β-cells were visualized with guinea pig polyclonal antibodies against insulin (1:5; Dako, USA) in combination with biotinylated donkey anti-guinea pig secondary antibody (1:500; Jackson ImmunoResearch, Newmarket, UK); the antibody signal was amplified with the Vectastain ABC-AP kit (Vector Laboratories, USA) and detected using an alkaline phosphatase substrate (Vector Laboratories, USA).

Chimeric antibodies NI-203.9A2, NI-2O3.19H8, and NI-203.26C11 showed preferential staining of pancreatic islets on amyloid-positive sections of two patients with T2DM (Fig. 12).

These data demonstrate that the NI-203.9A2, NI-2O3.19H8, and NI-203.26d1 chimeric antibodies specifically recognized pathological IAPP fibrils with comparable efficacy to their respective human antibodies (Fig. 12).

Example 7 Validation of the therapeutic effect of anti-IAPP and/or proIAPP antibodies in animal models of T2DM.

Lead candidate antibodies were tested in two transgenic mouse models and a rat model expressing hIAPP:

1) h-IAPP (hemizygous)/C57BL/6/DBA mice fed a high fat diet (Hull et al. (2003) Diabetes 52: 372-379);

2) h-IAPP (hemizygous/A/ ^vy A mice fed standard diet (Butler et al. (2003) Diabetes 52: 2304-2314);

3) h-IAPP (homizygous)/CD rats fed standard diet (Butler et al. (2004) Diabetes 53: 1509-1516).

Therapeutic efficacy is assessed by measuring beta cell mass and pancreatic hIAPP-amyloid load, as well as plasma hIAPP levels, and functional assays of glucose metabolism and insulin secretion.

1) Physiological characteristics.

To see if the use of antibodies according to the present application has a prophylactic or therapeutic effect, the following physiological characteristics (i)-(vii) of type II diabetes models in animals were investigated.

(i) Blood glucose.

The blood glucose level in animal models of T2DM was analyzed and compared with the level in animals not receiving treatment, and in animals of healthy (non-model T2DM) lines.

The term "healthy mouse" herein is not limited to a specific definition, and may be any mouse as long as there are no abnormalities in blood glucose, urine sugar, insulin secretion, and the like. a laboratory mouse used as a recurrent parent when creating a congenic mouse (eg, C3H/He mice, BALB/c mice, and C57BL/6 mice).

The term "healthy rat" herein is not limited to a specific definition, and may be any rat as long as there are no abnormalities in blood glucose, urine sugar, insulin secretion, and the like.

Since diabetes in mouse and rat models is preferably induced by transgenic expression of hIAPP, it is preferable to use as controls the same lines that were originally used to create transgenic animals.

An expression with elevated blood glucose compared to a healthy mouse/rat means that the fasting blood glucose (blood glucose concentration) is higher than that of a healthy fasting mouse/rat. A blood glucose level of diabetic animals of 130 mg/dL or higher, more preferably 140 mg/dL or higher, more preferably 200 mg/dL, and even more preferably 300 mg/dL or higher was considered high blood glucose. In addition, the term fasted herein means the state about 12 hours after the start of the rat/mouse fasting.

In the present invention, the blood glucose level can be measured by a conventional method known to those skilled in the art, for example, using a commercially available measuring device (for example, Medisafe Reader; Terumo Co., Ltd.) in accordance with the method described later in this example.

A typical way to measure blood glucose levels.

The blood glucose level (blood glucose concentration) of the animal subject was measured with a commercially available meter (eg, Medisafe Reader; Terumo Co., Ltd). The measurement principle of this instrument can be explained as follows. The measurement was based on colorimetric analysis. A measuring chip was prepared, glucose oxide was placed on the chip

- 61 042691 zu and peroxidase as catalysts and 4-aminoantipyrine and N-ethyl-N-(2-hydroxy-3sulfopropyl)-t-toluidine as chromogenic agents. A blood sample absorbed by capillary events was placed on this chip, and blood glucose was subjected to oxidation by glucose oxidase. The chromogenic agents on the chip were then oxidized with hydrogen peroxide formed up to that point and with peroxidase, resulting in a red-violet color. The amount of glucose in the blood was calculated by measuring the degree of this staining.

In this case, 4 μl of whole blood was obtained from an animal subject as a blood sample and measured over a measurement time of 18 seconds.

(ii) glycosylated hemoglobin concentration (HbA1c).

Animal models of type II diabetes were examined for elevated blood glycosylated hemoglobin concentrations and compared with untreated animal models of type 2 diabetes and healthy, non-diabetic animal models. Concentrations of 2.5% or more, 2.6% or more, 2.8% or more, and 3.0% or more were considered elevated.

The term glycosylated hemoglobin concentration as used herein refers to the proportion of hemoglobin molecules with glucose attached to them in erythrocytes. The concentration of glycosylated hemoglobin can be used as an indicator of the appropriateness of therapeutic monitoring of patients with diabetes; it is known that it correlates better with blood sugar levels that occurred 1-2 months earlier than at present.

The concentration of glycosylated hemoglobin can be measured in a conventional manner known to those skilled in the art, for example, using a commercially available meter (eg, DCA 2000 system; Bayer Medical Ltd.) according to the method described later in this example. More specifically, for example, when using the aforementioned DCA 2000 system as a measuring instrument, the amount of total hemoglobin was measured by the thiocyanmethemoglobin method, and the amount of glycosylated hemoglobin was measured by the inhibition of the latex coagulation reaction.

(iii) Urine sugar.

In the present example, urine sugar was analyzed from control animals and animal models of T2D.

The term positive urine sugar as used herein means that the urine glucose concentration excreted by the animals was 100 mg/dL or higher. Urinary glucose concentration can be measured in a conventional manner, for example, as described later in this example, using a commercially available kit (eg, Pretest; Wako Pure Chemical Industries, Ltd.). In particular, for example, when using the Pretest kit as a measurement kit, a urine sample of the animal was first placed on the Pretest paper for analysis, and after 30 seconds the result was determined by the color chart provided in this kit for evaluation on a five-point scale from - to +4. Urinary glucose concentration as measured by the result was 100-250 mg/dl for +1, 250-500 mg/dl for +2, 500-2000 mg/dl for +3, and 2000 mg/dl or higher for +4 . A result of +1 or higher was considered a positive urine sugar test result.

A typical way to measure sugar levels in urine.

Urinary glucose (urine sugar) of the animal subject was measured by methods known to those skilled in the art, for example, using a commercially available kit (Pretest; Wako Pure Chemical Industries, Ltd.). First, a urine sample of the animal was placed as a spot on the analysis paper from the above-mentioned Pretest kit, and after 30 seconds the result was determined according to the presented color chart for evaluation on a five-point scale from - to +4. Urinary glucose concentrations estimated from the results were 100-250 mg/dl for +1, 250-500 mg/dl for +2, 500-2000 mg/dl for +3, and 2000 mg/dl or higher for +4 . A result of +1 or higher was considered a positive urine sugar test result.

(iv) Blood insulin concentration.

In this example, we tested whether the insulin concentration in the blood of animal models of type II diabetes is comparable, equivalent or increased compared to the level in animals not treated, and control non-diabetic animals (animals of healthy lines).

The expression that the concentration of insulin in the blood is equivalent to or increased compared to an animal of a healthy line means that the concentration of fasting blood insulin is equivalent to or increased compared to the concentration in an animal of a healthy line on an empty stomach, for example, is 90 pg / ml or higher, or 110 pg / ml or higher.

In the present invention, the blood insulin concentration can be measured by a conventional method, for example, using a Lewis U-type insulin assay kit (Shibayagi Co.) according to the method described later in this example. More specifically, for example, an anti-insulin monoclonal antibody (mouse) was immobilized on a plate, insulin present in the sample was bound to it, after which a biotin-labeled anti-insulin monoclonal antibody was reacted with it, recognizing another insulin site, then a peroxidase-avidin conjugate was added to bind with biotin, and finally a chromogenic substrate was added to measure insulin by color development.

- 62 042691

An example of a method for measuring the concentration of insulin in the blood.

The blood insulin concentration of the mouse subject was measured by a method known to one skilled in the art, for example, using a commercially available kit (Lewis U-type insulin assay kit, Shibayagi Co.). The insulin concentration was measured as follows. The anti-insulin monoclonal antibody was immobilized on the plate, the insulin present in the sample was bound to it, after which the biotin-labeled anti-insulin monoclonal antibody reacted with it, recognizing another site of insulin, then the peroxidaseavidin conjugate was added to bind to biotin, and finally the chromogenic substrate for measuring insulin by staining development. The measurement range was usually from 39 to 2500 pg/ml for a healthy animal (mice).

(v) Glucose tolerance.

Treated and untreated model animals with T2DM were tested for abnormal glucose tolerance.

Normal or abnormal glucose tolerance in an animal can be confirmed using a glucose tolerance test. The glucose tolerance test can be performed according to a conventional procedure known to those skilled in the art, for example, by intraperitoneally administering fasting glucose (at least 12 hours later) to an animal at 2 mg per gram of body weight and measuring the animal's blood glucose level at a specific time after a glucose tolerance test (eg, every 15 minutes for 240 minutes). If the result shows that the blood glucose level does not show a downward trend over time compared to a healthy mouse, the glucose tolerance was considered abnormal. If the result in treated animals shows a decreasing trend over time compared to untreated animals, treatment with the antibodies of the present invention is considered effective.

An example of a method for assessing glucose tolerance.

Glucose tolerance in an animal subject can be assessed using a glucose tolerance test.

The glucose tolerance test was performed by first administering glucose intraperitoneally to an animal fasted for 12 hours at a rate of 2 mg per gram of body weight, and then measuring the blood (peripheral blood) glucose level of the animal every 15 minutes for 240 minutes. The blood glucose level was measured using the above method. If the result showed that the blood glucose level did not show a downward trend over time compared to a healthy animal, the glucose tolerance was considered abnormal. A faster decline over time in treated animals compared to untreated T2DM animal models was considered an indication of the effectiveness of the treatment according to the present invention.

(vi) Insulin sensitivity.

Treatment and untreated animal models of T2DM were tested for abnormal insulin sensitivity.

Normal or abnormal insulin sensitivity in an animal can be confirmed with an insulin sensitivity test. An insulin sensitivity test can be performed according to a conventional procedure known to one skilled in the art, for example, by intraperitoneally administering 0.5 to 0.85 units of fasting insulin to an animal at a rate of 0.5-0.85 units per kg of body weight and measuring the blood glucose level of the animal at a specific time. after an insulin sensitivity test (eg, every 15 minutes for 240 minutes). If the result shows that the blood glucose level does not show a tendency to decrease over time compared to a healthy mouse, insulin sensitivity was considered abnormal. If the result in treated animals shows a decreasing trend over time compared to untreated animals, treatment with the antibodies of the present invention is considered effective.

An example of a method for assessing insulin sensitivity.

Insulin sensitivity in an animal subject can be assessed using an insulin sensitivity test.

The study of insulin sensitivity was carried out by first administering insulin intraperitoneally to an animal fasting for 12 hours, at the rate of 0.5-0.85 units per kg of body weight, and then measuring the level of glucose in the blood (peripheral blood) of the animal every 15 minutes for 240 minutes . The blood glucose level was measured by the above method. If the result showed that the blood glucose level did not show a tendency to decrease over time compared to a healthy animal, insulin sensitivity was considered abnormal. A faster decline over time in treated animals compared to untreated T2DM animal models was considered an indication of the effectiveness of the treatment according to the present invention.

(vii) Other.

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Claims (3)

Assessment of polydipsia and polyuria. Animal models of type II diabetes were examined for a tendency to increase water intake and urination after the first onset of diabetes and during treatment with the antibodies of the present invention. A trend towards increased water drinking can be confirmed, for example, by carefully monitoring the rate of water volume decline in a water bottle in a cage for young animals and comparing it with a similar rate for an animal of a healthy line and for animals not treated. In addition, the tendency to increase urination can be confirmed, for example, by observing the degree of wetting of the floor covering in the cage for young animals and comparing it, as indicated earlier. Conclusions on the presence or absence of a trend towards obesity in mice. Body weights were measured and compared in treated and untreated model animals with T2D, as well as in healthy animals. Reducing the body weight of treated animals compared to untreated animals and/or comparable weight of treated animals and healthy animals was considered an indication of the effectiveness of the treatment according to the present invention. 2) Histopathological examination. Pancreatic tissue from type II diabetic animals, untreated T2DM controls, and healthy animals were fixed, paraffin-embedded, stained, and analyzed according to the methods of Example 4 for amyloid deposits of amylin in pancreatic islet (islets of Langerhans) β-cells. . Similarly, β-cell apoptosis and β-cell survival were assessed by immunostaining using appropriate markers (eg, TUNEL and cleaved caspase-3 staining to detect apoptosis and insulin staining to assess β-cell area). Reduction in amyloid deposits or β-cell apoptosis or increase in β-cell survival in pancreatic islets in treated animals compared to untreated animals, or a comparable level in treated animals and healthy animals, was assessed as as an indication of the effectiveness of the prophylactic or therapeutic methods according to the present invention. 3) Keeping animals. Type II diabetic model animals were maintained under SPF conditions as previously described (Hull et al. (2003), Diabetes 52: 372-379; Butler et al. (2003), Diabetes 52: 2304-2314; Butler et al (2004), Diabetes 53: 1509-1516); At 6 weeks of age, h-IAPP (hemizygous)/C57BL/6/DBA mice fed a high fat diet demonstrated stimulation of islet amyloid production (Hull et al. (2003), Diabetes 52: 372-379). CLAIM 1. Human monoclonal antibody against islet amyloid polypeptide (IAPP) or IAPP and/or proIAPP-binding fragment, characterized in that said antibody: (i) recognizes human IAPP and/or proIAPP aggregates containing fibrils and/or oligomers of IAPP and/or proIAPP preferentially over physiological IAPP and/or proIAPP, (ii) does not recognize pathological amyloid-β peptide deposits (A1-42 ), and (iii) binds to a peptide containing an IAPP epitope containing the amino acid sequence KCNTATCATQ (SEQ ID NO: 9); wherein the antibody or IAPP-binding fragment thereof contains in its variable region the following six complementarity determining regions (CDRs) of the V _H and VL regions: V _H -CDR1: positions 26-37 of SEQ ID NO: 20, V _H -CDR2: positions 52-67 of SEQ ID NO: 20, V H-CDR3: positions 100-110 of SEQ ID NO: 20, V L-CDR1: positions 24-38 of SEQ ID NO: 22, V L-CDR2: positions 54-60 of SEQ ID NO: 22, V L-CDR3: positions 93-101 of SEQ ID NO: 22, wherein one or more CDRs may contain one or two conservative amino acid substitutions. 2. A human monoclonal antibody against islet amyloid polypeptide (IAPP) or an IAPP-binding fragment thereof, characterized in that said antibody: (i) recognizes human IAPP aggregates containing IAPP fibrils and/or oligomers preferentially over physiological IAPP, (ii) does not recognize abnormal deposits of the amyloid-β peptide (A1-42), and (iii) binds to the peptide containing the epitope IAPP containing the amino acid sequence KCNTATCATQ (SEQ ID NO: 9); wherein the antibody or IAPP-binding fragment thereof contains in its variable region the following six complementarity determining regions (CDRs) of the V _H and VL regions: VH-CDR1: positions 26-35 of SEQ ID NO: 28, VH-CDR2: positions 50-66 of SEQ ID NO: 28, - 64 042691 VH-CDR3: positions 99-113 of SEQ ID NO: 28, VL-CDR1: positions 23-34 of SEQ ID NO: 30, VL-CDR2: positions 50-56 of SEQ ID NO: 30, VL-CDR3: positions 93-101 of SEQ ID NO: 30, wherein one or more CDRs may contain one or two conservative amino acid substitutions. 3. An antibody according to claim 1 that specifically binds an IAPP epitope that contains the amino acid sequence CNTATCA (SEQ ID NO: 5). 4. Antibody or IAPP/proIAPP-binding fragment according to claim 1, containing the amino acid sequence of the following V _H and V _L region: NI-203.26C11-VH (VH heavy chain variable region sequence) FR1-------------------------------------------------- ----------------CDR1---------------------FR2---- -------CDR2-----QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGNYYWTWIRQPAGKGLEWIGHIYSSGTTNY NPSLES FR3------------------------------------------------- -------------CDR3------JH--------RVTISVDTSKNQFSLSLNSVTAADTAVYYCARPLATVPDAFNIWGQGTMVTVSS NI-203.26C11-VK (VK Light Chain Variable Region Sequence) FR1---------------------------------- -------------CDR1--------------------FR2--------- ---CDR2-----FR3 EIVMTQSPDSLAVSLGERATIKCKSSQSVLYSNKNFLAWYQQKPGQPPKLLIYWASTRESG VPDRFS -------------------------------------- ------------CDR3----------------------JK-----GSGSGTDFTLTISSLQAEDVAVYYCQQYYSNPNTFGQGTKVEIK. 5. Antibody or IAPP-binding fragment according to claim 2, containing the amino acid sequence of the following V _H and V _L region: NI-203.11B12-VH (VH heavy chain variable region sequence) FR1---------------------------------- -------------------CDR1------------------FR2----------- CDR2------------QVQLVQSGAEVKKPGASMKVSCKASGYTFTNYYLHWVRQAPGQGLEWMGIINPSAGSTSY AQKFQG FR3--------------------------------- ----------------------CDR3--------------------- -------JH--------RVTMTRDTSTSTVYMELSSLKSEDTAVYYCARDSAGIQIWFRDAFDIWGQGTMVTVSS NI-203.11B12-VL (VL light chain variable region sequence) FR1--- ----------------------------CDR1-------------------FR2- -----------CDR2-----------FR3QPVLTQPPSASASLGSSVKLTCTLNSGHSSYTIAWHQQQPGKAPRYLMKVEHNGNYNKGS GLPDRFS ------------------------ ----------------------CDR3-------------JK----- --GSSSGADRYLAISNLQSEDEADYYCETWDTSTRVFGGGTKLTVL 6. An antibody according to any one of claims 1 to 5, which is a chimeric human rodent antibody or a rodentized antibody. 7. Antibody or IAPP/proIAPP binding fragment according to any one of claims 1 to 6, wherein the IAPP/proIAPP binding fragment is selected from the group consisting of single chain Fv fragment (scFv), F(ab') fragment, F(ab)- fragment and F(ab') ₂ -fragment. 8. A polynucleotide encoding an antibody or IAPP/proIAPP binding fragment thereof according to any one of claims 1, 3, 4, 6 and 7. 9. A polynucleotide encoding an antibody or IAPP/proIAPP binding fragment thereof according to any one of claims 2 and 5-7. 10. An antibody expression vector according to any one of claims 1, 3, 4, 6 and 7, containing a polynucleotide according to claim 8. 11. An antibody expression vector according to any one of claims 2 and 5-7, containing a polynucleotide according to claim 9. 12. A host cell for expressing an antibody according to any one of claims 1, 3, 4, 6 and 7, containing a polynucleotide according to claim 8 or a vector according to claim 10. 13. A host cell for expressing an antibody according to any one of claims 2 or 5-7, containing a polynucleotide according to claim 9 or a vector according to claim 11. 14. A method for producing an antibody or its IAPP/proIAPP-binding fragment according to any one of claims 17, including: (a) culturing the cell according to claim 12 or 13; and (b) isolating said antibody or immunoglobulin chain(s) thereof from the culture. 15. An antibody or IAPP/proIAPP binding fragment thereof, encoded by a polynucleotide according to claim 8 or 9 or obtained by the method of claim 14. 16. Antibody according to any one of claims 1-7 or 15 labeled with a detectable label. 17. An antibody according to claim 16, characterized in that the detectable label is selected from the group consisting - 65 042691 from an enzyme, a radioisotope, a fluorophore and a heavy metal. 18. An antibody according to any one of claims 1-7 or 15-17 attached to a drug substance. 19. A pharmaceutical composition for the treatment or prevention of a metabolic disease, comprising an antibody according to any one of claims 1 to 7 or 15, a polynucleotide according to claim 8 or 9, a vector according to claim 10 or 11, or a cell according to claim 12 or 13 and is pharmaceutically acceptable carrier. 20. Pharmaceutical composition according to claim 19, which is a vaccine. 21. A pharmaceutical composition according to claim 19 or 20, further comprising an agent that can be used for the treatment of type 2 diabetes mellitus (DM2) and/or in the treatment or prevention of islet rejection after clinical pancreatic islet transplantation. 22. Diagnostic composition for identifying oligomers, aggregates or fibrils of IAPP/proIAPP, IAPP and/or proIAPP, containing an antibody according to any one of claims 1-7 or 15-18, polynucleotide(s) according to claim 8 or 9, vector(s ) according to claim 10 or 11 or a cage according to claim 12 or 13 and reagents conventionally used in diagnostic methods based on immunological components or nucleic acids. 23. The use of an antibody according to any one of claims 1-7 or 15-18 in the prophylactic or therapeutic treatment of an IAPP and/or proIAPP related disorder. 24. The use of an antibody according to any one of claims 1-7 or 15-18 in monitoring the progression or response to treatment of an IAPP and/or proIAPP related disorder. 25. The use of a vector according to claim 10 or 11 in prophylactic treatment, therapeutic treatment and/or monitoring the progression or response to treatment of an IAPP and/or proIAPP related disorder. 26. A method for diagnosing or monitoring the progression of islet amyloidosis in a subject, comprising determining the presence of IAPP and/or proIAPP oligomers, aggregates, or fibrils in a sample of the diagnostic subject, using an antibody according to any one of claims 1-7 or 15-18 by bringing said sample to contact with the specified antibody under conditions that allow the formation of antibody-antigen complexes, and by determining the level of such complexes, and the presence of oligomers, aggregates or fibrils of IAPP and/or proIAPP indicates presymptomatic, prodromal or clinical type 2 diabetes mellitus (DM2) and/ or beta-cell deficiency after clinical pancreatic islet transplantation, and an increase in IAPP and/or proIAPP oligomers, aggregates or fibrils compared to physiological IAPP or compared to a reference sample obtained from a healthy control subject or a control sample from an organism of that in the same subject, indicates progression of presymptomatic, prodromal, or established type 2 diabetes mellitus (DM2) and/or islet insufficiency following clinical pancreatic islet transplantation in said subject. 27. The use of an antibody according to any one of claims 1-7 or 15-18 for preparing a composition for in vivo detection or for targeting a therapeutic or diagnostic agent to IAPP and/or proIAPP in a human or animal body. 28. Use according to claim 27, characterized in that said in vivo detection includes positron emission tomography (PET), single photon emission tomography (SPET), near infrared (NIR) optical imaging, or magnetic resonance imaging (MRI). 29. A method for diagnosing islet amyloidosis indicative of presymptomatic or clinical type 2 diabetes mellitus and/or beta cell deficiency after clinical transplantation of pancreatic islets in a subject, comprising the step of measuring the level of an antibody that binds the KCNTATCATQ peptide (SEQ ID NO: 9), in biological sample from the specified subject and comparing the measurement result with the level present in healthy subjects of comparable age and sex. 30. A kit for use in diagnosing or monitoring the progression of islet amyloidosis, said kit comprising an antibody according to any one of claims 1-7 or 15-18, polynucleotide(s) according to claim 8 or 9, vector(s) according to claim 10 or 11 and/or a cage according to claim 12 or 13. -