BR112015005549A2 - specific human islet amyloid polypeptide antibodies (hiapp) and their uses - Google Patents

Abstract

[0001] The present invention makes use of the MAPP specific immune response of healthy humans to promote the isolation of anti-hIAPP specific human monodonal antibodies. In particular, experiments conducted in accordance with the present invention have been successful in isolating hjAPP and / or proIAPP-specific monoclonal antibodies from a group of healthy humans or groups of obese patients and other groups of patients at increased risk of develop DT2, which at the time of antibody isolation did not exhibit DT2 signals. [0002] The present invention is therefore directed to human antibodies, antigen binding fragments and similar antigen binding molecules that are capable of specifically recognizing IAPP and / or proIAPP. Unless otherwise stated, the sections "specifically recognize IAPP and / or proIAPP", "specific antibody to / for IAPP and / or prolAPP" and "anti-TAPP and / or anti-proIAPP antibody" mean specifically, in general and collectively , antibodies to the native monomeric form of IAPP; antibodies to the precursor proIAPP dè ÍAPP; antibodies that specifically bind to one of two forms, IAPP and proIAPP; antibodies that bind to aggregated, oligomeric, fibrillar and / or non-fibrillar species of IAPP and / or proIAPP. Selective human antibodies for aggregate and / or integral forms are provided herein, for example, oligomeric, fibrillar and non-fibrillar aggregated forms of IAPP and / or proIAPP. [0003] In a particularly preferred embodiment of the present invention, the human antibody or its antigen-binding fragment demonstrates the immunological binding characteristics of an antibody characterized by the variable regions VH and / or VL, as shown in Fig. 1 or Fig . two. [0004] The antigen binding fragment of the antibody can be a single chain Fv fragment, an F (ab ') fragment, an F (ab) fragment and an F (ab') 2 fragment, or any other binding fragment to the antigen. In a specific embodiment, infra, the antibody or fragment thereof is a human antibody of the IgG isotype. Alternatively, the antibody is a rodent-human or rodent chimeric antibody, such as the murine or murine, rat or ratinized antibody, with rodent versions being particularly useful for diagnostic methods and animal studies. [0005] In addition, the present invention relates to compositions comprising the antibody of the present invention or its active fragments and to immunotherapeutic and immunodiagnostic methods that use these compositions for the prevention, diagnosis or treatment of IAPP-related disorders, such as for example, DT2, where an amount. composition is administered to a patient who needs it. [0006] Naturally, the present invention extends to immortalized human memory B lymphocytes and the B cell, respectively, which produces the antibody or its antigen-binding fragment and which has the distinct and unique characteristics, as set out below. [0007] The present invention is also related to polynucleotides that encode at least one variable region of an immunoglobulin chain of the antibody of the invention. Preferably, this variable region comprises at least one complementarity determining region (CDR) of the VH and / or VL of the variable region, as shown in Fig. 1 or Fig. 2. [0008] Accordingly, the present invention also encompasses vectors that comprise such polynucleotides and host cells transformed with them, as well as their use in the production of an antibody and equivalent binding molecules, which are specific for IAPP and / or proIAPP. Means and methods for recombinant antibody production and simulations thereof, as well as methods for screening competing binding molecules, which may or may not be antibodies, are known in the art. However, as described herein, in particular with regard to therapeutic applications in humans, the antibody of the present invention is a human antibody, in the sense that the application of said antibody is substantially free of an immune response directed against that antibody, otherwise observed for chimeric and even humanized antibodies. [0009] In addition, we describe here compositions and methods that can be used to identify IAPP and / or proIAPP in samples and / or in vivo. The described anti-IAPP and / or proIAPP antibodies or their IAPP and / or proIAPP binding fragments can be used for screening, in humans, blood, piasma, serum, saliva, peritoneal fluid, cerebrospinal fluid ("CSF") and urine in search of the presence of IAPP and / or prolAPP in samples, for example, using assays adapted to the surface or based on ELISA. In one embodiment, the present invention relates to a method of diagnosing or monitoring the progression of a disorder related to IAPP and / or proIAPP in an individual, the method comprising the determination of. presence of LAPP and / or proIAPP oligomers, aggregates or fibrils in a sample from the individual who will be diagnosed with at least one antibody of the present invention or an IAPP and / or proIAPP binding molecule that has substantially the same specificities binding of any of them, in which the presence of oligomers, aggregates or fibrils of IAPP and / or proIAPP is indicative of the disorder. [0010] In addition, in one embodiment of the present invention, the described anti-IAPP and / or proiAPP antibodies or their IAPP and / or proLAPP binding fragments, and / or IAPP and / or proIAPP binding molecules comprising at least a CDR of an antibody of the present invention is provided for the preparation of a composition for in vivo detection (also called in vivo imaging) of or targeting a therapeutic and / or diagnostic agent for IAPP and / or projAPP in the human or animal body. The methods and compositions described in this instrument can assist in disorders related to IAPP and characterized, for example, by the occurrence of aggregated oligomeric, fibrillar and non-fibrillar forms of IAPP and / or proIAPP, as in a diagnosis of T2D, for example, and they can be used to monitor the progression of the disease and the therapeutic efficacy of the treatment performed on the individual, for example, in diagnostic methods related to in vivo imaging. Therefore, in one embodiment, the IAPP and / or proIAPP binding molecule of the present invention is provided, wherein the aforementioned in vivo detection (imaging) comprises positron emission tomography (PET), single photon emission tomography '( SPELT), optical imaging by near infrared spectrometry (NIR) or magnetic resonance imaging (MRI). [0011] Therefore, it is a particular objective of the present invention to provide methods for the treatment, diagnosis or prevention of a disease related to IAPP and / or aggregated and / or oligomeric, fibrillar and / or non-fibrillar proIAPP, as for example , Type 2 Diabetes (DT2). The methods comprise administering an effective concentration of a human antibody or an antibody derived to an individual in which the antibody attacks IAPP and / or prolAPP. [0012] In another aspect, the present invention provides a peptide with an IAPP and / or proIAPP epitope recognized specifically by an antibody of the present invention. Such a peptide comprises or is composed of a sequence of amino acids, as indicated below in the detailed description and examples, or a modified sequence thereof, in which one or more amino acids are replaced, deleted and / or added. In addition, the present invention provides a method of diagnosing DT2 or the risk of developing DT2 in an individual, comprising a step of determining the presence of an antibody that binds to said peptide in a biological sample from that individual. [0013] Other embodiments of the present invention are evident in the description and Examples that follow.

Description

SPECIFIC POLYPEPTIDIC HUMAN ISLAND AMYLOID ANTIBODIES (HIAPP) AND THEIR USES Field of the invention

[001] This invention is generally related to new molecules that specifically bind to the human islet amyloid polypeptide (hlAPP), also known as amylin and / or its pro-islet amyloid polypeptide (prolAPP), particularly human antibodies, as well as particularly human antibodies, as well as fragments, derivatives and variants thereof that recognize IAPP and prolAPP proteins, aggregated forms of IAPP, aggregated forms of prolAPP, and / or fibrils of IAPP. In addition, this invention relates to pharmaceutical and diagnostic compositions comprising such binding molecules, antibodies and simulations thereof, valuable both as a diagnostic tool for the identification of IAPP species, prolAPP, aggregated IAPP, aggregated prolAPP and / or plasma IAPP fibrils, and also in passive vaccination strategies in the treatment of disorders related to aggregated IAPP, to aggregated prolAPP and to IAPP fibrils, such as type 2 diabetes mellitus (DT2) and islet rejection after clinical transplantation of pancreatic islets in individuals with type 1 diabetes mellitus (DT1). Fundamentals of the invention

[002] Aggregation, modifications and protein accumulation are pathological aspects of several metabolic diseases, including well-known neurodegenerative diseases, such as Huntington's, Alzheimer's and Parkinson's disease (Taylor et al., Science 296 (2005 ), 199171995). The aggregation of pathological proteins also has a role in metabolic diseases, such as type 2 diabetes mellitus (DT 2) and islet rejection after clinical pancreatic islet transplantation in individuals with type 1 diabetes mellitus (DT1). The abnormal configuration and protein aggregation result in the development of amyloid deposits and appear to be directly related to cell toxicity in these diseases. The islet amyloid polypeptide (IAPP or amylin), a physiological peptide co-secreted with insulin by pancreatic B cells, forms fibrillar aggregates in pancreatic islets (also called islets of Langerhans) in patients with DT2 and has been suggested to play a role in disease development (Westermark et al. (2011), Physiol. Rev. 91 (3): 795-826). In addition, as already mentioned, IAPP aggregates have been found in pancreatic islets after transplantation of isolated islets in patients with type 1 diabetes mellitus (DT1).

[003] Human IAPP (hlAPP) is a peptide hormone consisting of 37 amino acids, with a disulfide bridge between cysteine residues 2 and 7 and an amidated carboxyl terminal. Pancreatic islets are composed of 65 to 80% of B cells, which produce and secrete insulin and IAPP, essential for the regulation of blood glucose levels and cellular metabolism. IAPP is processed from the preprohormone preprolAPP, a precursor of amino acid 89 produced in pancreatic B cells.

[004] PreprolAPP is rapidly cleaved after translation into pro-islet amyloid polypeptide, an amino acid 67 peptide, which undergoes additional proteolysis and post-translational modifications to generate hlAPP, whose expression is regulated together with insulin, as the increase insulin production leads to increased levels of hIAPP, which is released from pancreatic B cells into the bloodstream and is involved in glycemic regulation through gastric emptying and satiety control, in synergy with insulin.

[005] While hlAPP acts as a regulator of cellular metabolism under physiological conditions, it is able to aggregate and form amyloid fibrils (IAPP amyloidosis) associated with B cell failure, the greater number of É cell deaths and the reduced mass of B cells. Several evidences indicate that hlAPP amyloidosis is one of the major triggers of DT2 pathogenesis. First, the deposition of hlAPP fibrils is found in more than 90% of patients with type 2 diabetes (Zraika et al. (2010), Diabetologia, 53 (6): 1046-1056). Second, hlAPP aggregation is toxic to É cells and correlates with the reduction in insulin-producing B cells (Butler et al. (2003), Diabetes 52 (9): 2304-2314; Ritzel et al. (2007) , Diabetes 56 (1): 65-71; Jurgens et al. (2011), Am. J. Pathol. 178 (6): 2632-2640). Third, transgenic murine models that express hlAPP present amyloid deposits from pancreatic islets and spontaneously develop DT2 (Janson et a /. (1996), Proc.

Natl.

Acad, Sci.

USA 93 (14): 7283-7288; Hoppener et a /. (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 a /. (2006), ILAR J. 47 (3): 225-233; Hoppener et al. (2008), Exp.

Diabetes Res. 697035). They recapitulate human disease with É cell dysfunction, deficiency of É cell mass and loss of B cells, comparable to that observed in the tissues of patients with T2D.

The expression hlAPP and the formation of amyloid are directly correlated with B cell apoptosis and the development of diabetes in these models, thus providing evidence for the contribution of human IAPP in the development of the disease.

In addition, treatments that interfere with the aggregation of hlAPP improved the diabetic phenotype and extended the useful life of animals (Aitken et a /. (2009), Diabetes 59 (1): 161-171). Amyloidosis and aggregation of hlAPP are prerequisites for toxicity.

Non-amyloidogenic rodent IAPP (rlAPP), which is unable to form fibrils as a result of substitution of amino acid six, is not toxic to B cells.

In the development of the disease, the pathological aggregation of hlAPP found in human pancreatic islets can cause B-cell dysfunction and death associated with insufficient insulin secretion.

In addition, the compensatory growth of the A cell mass and the secretion of insulin and amylin to maintain normal blood glucose levels may favor the formation of toxic hlAPP oligomers and the deposition of hiAPP fibrils.

While the initial hiAPP oligomers are considered to be the main cytotoxic species, the final hiAPP fibril product may also play a role in the loss of B cells (Meier et a /. (2006), Am.

J.

Physiol.

Endocrinol.

Metab. 291 (6): E1317-1324; Haataja et a /. (2008), Endocr.

Rev. 29 (3): 303-316; Engel et a /. (2008), Proc.

Natl.

Acad.

Sci.

USA 105 (16): 6033-6038). HliAPP fibrils have also been observed in pancreatic islets isolated from donors and associated with loss of B cells after clinical pancreatic islet transplants in individuals with type 1 diabetes (Andersson et al. (2008), Exp.

Diabetes Res. 562985; Udayasankar et a /. (2009), Diabetologia 52 (1): 145-153; Bohman et al. (2012), Amyloid 19 (2): 87-93). The exact mechanism that leads to amyloidosis and hlAPP aggregation in DT2 is unknown.

Insulin resistance in DT2 increases the requirement for insulin secretion together with the cell content of prolAPP and the release of hlAPP, which can cause amyloidosis, as the formation of hiAPP fibrils is concentration-dependent. Another proposed mechanism is the accumulation and aggregation of unprocessed N-terminal prolAPP caused by failure of proteolysis in the configuration of insulin resistance, as partially processed forms of prolAPP are found in amyloid deposits, in particular, the prolAPP1 48 of residue 48 (Marzban et al. (2006), Diabetes 55 (8): 2192-2201). In this context, abnormal processing of prolAPP can act as a sowing for hlAPP amyloidosis and increase the formation of amyloids (Paulsson et al. (2005), Diabetes 54 (7): 211772125; Paulsson et a /. (2006), Diabetologia , 49 (6): 1237-1246; Marzban et al. (2006), Diabetes 55 (8): 2192-2201). ProlAPP is therefore also considered an appropriate therapeutic target.

[006] The clinical features of DT2 are high blood glucose levels and deficiency and / or insulin resistance. Diabetes mellitus is a group of metabolic diseases comprising DT1, DT2 and gestational diabetes. DT2, also called late diabetes, obesity-related diabetes and non-insulin-dependent diabetes mellitus (NIDDM), is the most common form of diabetes, accounting for about 90% of all cases (Gerich et a /. (1998), Endocr. Rev. 19 (4): 491-503). DT2 is characterized by a reduction in the amount of functional insulin-producing B cells. As the pathology progresses, it can lead to long-term complications, such as cardiovascular disease, diabetic retinopathy, which leads to blindness, kidney failure, frequent infections and amputations caused by poor circulation. As a consequence, DT2 is associated with a shorter life expectancy. The disease affects more than 300 million people worldwide, resulting in more than one million deaths each year. Both genetic determinants and environmental factors lead to the development of the disease, with obesity, physical inactivity and aging being considered as the main cause (Kahn et a /. (2006), Nature 444 (7121): 840- 846).

[007] Current treatments for DT2 include lifestyle management (diet and exercise) and pharmacological intervention, such as with the administration of metformin and insulin to lower blood glucose levels or by stimulating the pancreas to release insulin or the increased insulin response. These treatments are based on the symptomatic improvement of diabetes, with the consequence of a lack of durability. In fact, none of the available treatments has proven to neutralize hlAPP aggregation and pancreatic B cell loss. New treatment strategies involving glucagon-like peptide analogs (GLP-1) (Butler et al. (2009), Diabetologia 53 (1): 1-6) and GLP-1 inhibitors by inerting the dipeptidyl-peptidase 4 enzyme ( DDP4) are based on the potent insulinotropic effect of GLP-1 and its effect on increasing the proliferation of RB cells. One important thing is that the increased release of insulin is also coupled with an increased release of amylin. Experimentally, it has been proved that stimulated insulin secretion promotes the development of islet amyloidosis in animal models and that similar effects can be expected in humans (Aston “Mourney et al. (2011), Diabetologia 54 (7): 1756 -1765). These treatments can therefore potentially worsen islet amyloidosis. More recent and promising strategies involve the development of anti-inflammatory drugs or antibodies directed to the IL-10 pathway (Donath et a /. (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): 251572527; Dinarello et al. (2010), Curr. Opin. Endocrinol. Diabetes Obes. 17 (4): 314-321; Bonin 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). It is important to note that recent studies show that hliAPP specifically induces the inflammasome system - IL-16, leading to activation of the innate immune system (Masters et a /. (2010), Nat. Immunol. 11 (10): 897-904; Mandrup - Poulsen et a /. (2010), Nat. Immunol. 11 (10): 881-883), thus supporting a therapeutic strategy aimed at the aggregation of hlAPP.

[008] These findings highlight the potential benefit associated with active or passive immunotherapy approaches targeting hiAPP and / or prolAPP.

[009] Summing up the aforementioned, new therapeutic strategies are urgently needed, and address aggregated proteins of hlAPP, prolAPP and / or fibrils and / or hl! APP oligomers with an effective and safe therapy.

[0010] Passive immunization with human antibodies that are evolutionarily optimized and matured by affinity due to the action of the human immune system would provide a promising new pathway with a high probability of excellent safety and efficacy. Brief description of the drawings - REVIEW

[0011] Fig. 1: Variable region amino acid and nucleotide sequences, ie, heavy chain and kappa / lambda light chain of human IAPP antibodies NI-203.9A2 (A), NI-203.19H8 (B), NI-203.26 C11 (C), NI-203.8E3 (D), NI-203.11B12 (E), NI-

203.205F8 (F), NI-203.9B3 (G), NI-203.19F2 (H), and NI-203.15C7 (Il). Complementary (CDRs) and structural (FR) regions are indicated with the CDRs being accentuated. The heavy chain link region (JH) and the light chain link region (JK) are also indicated. Due to the cloning strategy, the amino acid sequence at the N-terminal end of the heavy chain and the light chain can potentially contain changes induced by primers in FR1, which, however, do not substantially affect the biological activity of the antibody. In order to provide a consensus human antibody, the amino acid and nucleotide sequences of the original clone were aligned with the relevant human germline variable region sequences in the database and adjusted accordingly; see, for example, Vbase (http://vVbase.mrc-cpe.cam.ac.uk/), received by the MRC Center for Protein Engineering (Cambridge, UK). The amino acid sequence of human antibodies is indicated when amino acids at the N-terminus are considered to potentially deviate from the germline consensus sequence due to the polymerase chain reaction (PCR) initiator and thus have been replaced by primer-induced mutation (PIMC). Amino acids modified by PIMC are indicated in bold in the sequences

[0012] Fig. 2: Variable region amino acid and nucleotide sequences, ie, heavy chain and kappa / lambda light chain of NI- human prolAPP antibodies

203.1D10 (A), NI-203.2A11 (B), NI-203.10C4 (C), NI-203.20H9 (D), NI-203.26D2 (E) and NI-203.60H3 (F). Complementary (CDRs) and structural (FR) regions are indicated with the CDRs being accentuated. The heavy chain link region (JH) and the light chain link region (JK) are also indicated. Due to the cloning strategy, the amino acid sequence at the N-terminal end of the heavy chain and the light chain can potentially contain changes induced by primers in FR1, which, however, do not substantially affect the biological activity of the antibody. In order to provide a consensus human antibody, the amino acid and nucleotide sequences of the original clone were aligned with the relevant human germline variable region sequences in the database and adjusted accordingly; see, for example, Vbase (http://vbase.mrc-cpe.cam.ac.uk/), received by the MRC Center for Protein Engineering (Cambridge, UK). The amino acid sequence of human antibodies is indicated when amino acids at the N-terminus are considered to potentially deviate from the germline consensus sequence due to the polymerase chain reaction (PCR) initiator and thus have been replaced by primer-induced mutation (PIMC). PIMC-modified amino acids are indicated in bold in the sequences

[0013] Fig. 3: IAPP binding specificity of human recombinant antibodies assessed by direct ELISA. (A) Electron microscopy image of the IAPP solution (2 mg / ml) used to coat the ELISA plate. The scale bar represents 1 pum. (B) NI-203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3 recombinants showed a specific binding to human IAPP (10 pg / ml). BSA (10 µg / ml) was used as a control to determine non-specific binding. The data are expressed as OD values at 450 nm.

[0014] Fig. 4: EC50 determination of recombinant anti-| APP antibodies of human origin for IAPP and prolAPP. (A) Electron microscopy images of the IAPP and prolAPP solutions (2 mg / ml) used to coat the ELISA plate. The scale bar represents 1 um. (B) Plates were incubated with the indicated concentrations of recombinant antibodies of human origin NI-203.9A2, NI-203.19H8, NI-203.26C11 or NI-203.8E3. The antibodies NI-203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3 bind with high affinity to human IAPP (8, 10 pug / ml) with an EC50 of 9 nM, 22 nM, 6 nM and 4 NM, respectively. NI-203.26C11 also binds to prolAPP (0.10 pg / ml) with an EC50 of 260 nM. The measurements were made in duplicate and the BSA background signal was subtracted. Data are expressed as mean OD values at 450 nm.

[0015] Fig. 5: Anti-antibodies | APPs of human origin are specific for IAPP fibrils. (A) Electron microscopy images of the IAPP (2 mg / ml) and non-fibrillar IAPP (500 pg / ml) solutions used to coat the ELISA plate. While the IAPP solution contains fibrils, IAPP fibrils are missing from the non-fibrillar IAPP solution. The scale bar represents 1 um. (B) The plates were incubated with the indicated concentrations of recombinant antibodies of human origin NIl-203.9A2, NI-203.19H8, NI-

203.26C11 or NI-203.8E3. The antibodies NI-203.9A2, NI-203.19H8, NI-203.26C11 and NI-

203.8E3 bind with high affinity to IAPP fibrils (IAPP solution, 5, 10 water / ml) and with a very low affinity to non-fibrillar IAPP (A, 10 pg / ml), suggesting specificity with respect to fibrils from IAPP. The measurements were made in duplicate and the BSA background signal was subtracted. Data are expressed as mean OD values at 450 nm.

[0016] Fig. 6: IAPP-binding epitopes of human recombinant antibodies evaluated by Pepscan analysis. (A) Pepscan images of recombinant antibodies of human origin NI-203.19H8 and NI-203.26C11 (1 µg / ml). Binding to NI-203.19H8 occurred in peptides 6 and 7 (row A), spanning amino acids 19 to 25 (peptide 6: 16-LVHSSNNFGA-25 SEQ ID NO: 6, peptide 7: 19-SSNNFGAILS-28 ID NO. SEQ: 8, consensus binding sequence: 19-SSNNFGA-25 SEQ ID NO: 4). The connection to NI-

203.26C11 occurred in peptide 1 (row A) encompassing amino acids 1 to 10 (peptide 1: 1-KCNTATCATQ-10 SEQ ID NO: 9) but not in peptide 2 encompassing amino acids 4 through 13 (peptide 2: 47TATCATORLA- 13 SEQ ID NO: 10). The exchange or substitution of alanine in residues 2 to 8 in peptides 33 to 39 and 41 (row C) impaired NI-203.26C11 binding (peptide 33: C2A mutation, peptide 34: N3A mutation, peptide 35 mutation : T4A, peptide 36: ASG mutation, peptide 37: ASP mutation, peptide 38: T6A mutation, peptide 39: C7A mutation, peptide 41 mutation: A8P). Secondary anti-human equine IgG conjugated to horseradish peroxidase (HRP) with Fcy receptors only (1: 20000; Ilary Ab) was used as a control. (B) Binding epitopes identified from different human specific IAPP antibodies within the indicated amino acids of the human IAPP protein sequence. Top panel: amino acid sequence of the integral human IAPP (amino acids 1 to 37). NI: unidentified binding epitope.

[0017] Fig. 7: The antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 specifically recognize the pathological amyloid IAPP in the pancreas of patients diagnosed with type 2 diabetes mellitus (DT2). The antibodies NI-203.9A2, NI-203.19H8 and NI-

203.26C11 demonstrate staining in pancreatic islets with DT2 loaded with IAPP fibrils (amyloid) (A, B), but not in pancreatic islets with DT2 that do not have IAP fibrils (C, D). (A) Tioflavin-S amyloid staining (ThioS, on the left panel) and Congo Red (CR, on the right panel) in pancreatic islets of a patient with DT2. (B) Detection of IAPP fibrils in pancreatic islets with amyloid-positive DT? 2 with antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 (brown-CR) at 50 nM (on the large panel and bottom graph left) and 5 nM (lower right graph). (C) Absence of amyloid in pancreatic islets of a patient with DT2, as shown by the total absence of Tioflavin-S (ThioS, on the left panel) and Congo Red (CR, on the right panel) stains. (D) Absence of staining in pancreatic islets with DT2 negative for amyloid with antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 at 50 nM. Only the equine secondary anti-human antibody (Ilary Ab) was used as a control. Lower graphics: images with high magnification of individual human pancreatic islets. Human pancreatic islets were stained with anti-insulin antibody (blue, originally (i.0.), With strong staining, in this case) and contrast dye was used to visualize cell nuclei (weak blue io, with weak staining, in this case ).

[0018] Fig. 8: The antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 do not recognize the physiological IAPP in the control human pancreas. The antibodies NI-203.9A2, NI-

203.19H8 and NI-203.26C11 (50 nM) show poor staining in human control islets when compared to the control IAPP antibody (1: 100; Ab control). Only the secondary anti-human equine antibody (llary Ab) was used as a control. The human pancreatic islets were stained with anti-insulin antibody (blue, originally (i.0.), With strong staining, in this case) and contrast dye was used to visualize cell nuclei (weak blue io, with weak staining, in this case ).

[0019] Fig. 9: The antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 recognize pathological IAPP fibrils in a feline pancreas with diabetes. Detection of IAPP fibrils in pancreatic islets of feline with DT2 with antibodies NI-203.9A2, NI-

203.19H8 and NI-203.26C11 (50 nM, CR-brown). IAPP fibrils (amyloid) were stained with Congo Red (CR). Only the secondary anti-human equine antibody (llary Ab) was used as a control. Bottom left graphics: images with high magnification of individual pancreatic islets of felines. The pancreatic islets of feline were stained with anti-insulin antibody (blue, originally (i.0.), With strong staining, in this case) and contrast dye was used to visualize cell nuclei (weak blue io, with weak staining, in this case). case).

[0020] Fig. 10: The antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 do not recognize pathological deposits of AB in a human brain with Alzheimer's disease. Absence of staining with antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 (50 nM), in contrast to AB-specific antibody 6E10 (1: 2000; Ab control). Only the secondary anti-human equine antibody (Ilary Ab) was used as a control. Contrast dye was used to visualize cell nuclei (i.0. Weak blue, with weak staining, in this case).

[0021] Fig. 11: Recombinant chimeric human and murine antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 bind with equal affinity to human IAPP. (A) ECso determination of recombinant chimeric human and murine anti-IAPP antibodies to IAPP (8, 10 pg / ml) and BSA (O, 10 ug / ml). The plates were incubated with the indicated concentrations of antibodies. The measurements were made in duplicate. Data are expressed as mean OD values at 450 nm. (B, C) EC50 values of human and murine chimeric antibodies.

[0022] Fig. 12: Recombinant chimeric chimeric antibodies NI-203.9A2, NI-

203.19H8 and NI-203.26C11 recognize pathological IAPP fibrils in the pancreas of patients diagnosed with type 2 diabetes mellitus (DT2). Detection of IAPP fibrils in pancreatic islets of two human patients with DT2 (1 and 2) with chimeric antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 at 50 nM (io brown, with intense dark to in this case). The human pancreatic islets were stained with anti-insulin antibody (ie blue, with strong staining in this case) and contrast dye was used to visualize cell nuclei (ie weak blue, with weak staining in this case).

Description of the invention

[0023] In type 2 diabetes (DT2), genetic determinants and environmental factors lead to the development of insulin resistance, followed by a compensatory growth in the mass of beta cells and by the secretion of insulin and amylin (nIAPP) to maintain normal levels of blood glucose. The resulting high concentrations of amylin favor the formation of toxic oligomers of human islet amyloid polypeptide (hlAPP) and the deposition of fibrils from hlAPP, which is found in more than 90% of patients with type 2 diabetes. The deposition of hlAPP correlates with the reduction of insulin-producing beta cells, and it has also been ventilated to play a role in the loss of B cells in pancreatic islets transplanted to individuals with type 1 diabetes. Various antibodies of human origin from groups of healthy or obese donors with high risk of developing type 2 diabetes, but absence of the disease, were identified and characterized in vitro, cloned and produced recombinantly using Neurimmune's RTMTM technology, as described in detail in international application WO2008 / 081008 and are provided here. Ideal candidates are validated in transgenic mice that have hlAPP and are exposed to a high-fat diet. Therapeutic efficacy is assessed by determining the beta cell mass and amyloid load of hlAPP in the pancreas, as well as plasma levels of hlAPP, and by functional tests of glucose metabolism and insulin secretion.

[0024] Type 2 diabetes is the most common form of diabetes, accounting for about 90% of all cases. The disease affects more than 200 million people worldwide, causing more than one million deaths each year caused directly by diabetes. More than 300,000 patients are affected in Switzerland. The prevalence of diabetes grows dramatically in both developed and developing countries due to demographic growth, aging, urbanization and the increasing prevalence of obesity and inactivity. The global market for type 2 diabetes, currently in the $ 25 billion range, is expected to reach $ 35 billion in 2016, with a compound annual growth rate of 6.4% between 2009 and 2016. Current treatments they comprise diet management and pharmacological intervention, acting in several ways to decrease blood glucose levels or by stimulating the pancreas to release insulin or by increasing insulin sensitivity. None of the available treatments can, however, neutralize the aggregation of hlAPP and the loss of pancreatic beta cells. New treatment strategies for type 2 diabetes include glucagon-like peptide analogs 1 (GLP-1) and dipeptidyl peptidase 4 (DPP 4) inhibitors, the enzyme that inactivates endogenous GLP-1. These strategies are based on the potent insulinotropic effect of GLP-1 and its effect on increasing the proliferation of beta cells. One important thing is that the increased release of insulin is also coupled with an increased release of amylin. Experimentally, it has been proved that stimulated insulin secretion promotes the development of islet amyloidosis in animal models and that similar effects can be expected in humans. In addition to the particular use of IAPP-binding molecules of this invention, another proposed therapeutic approach could therefore be an attractive combination therapy of these molecules and the new treatments indicated above. |. Definitions

[0025] Unless otherwise stated, a term used here has the definition given by the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised in 2000 and reissued in 2003, ISBN 0 19 850673 2.

[0026] It should be noted that the terms "one" or "one" entity refer to one or more of that item; for example, it should be understood that "an antibody" represents one or more antibodies. Thus, the terms "one" (or "one"), "one or more" and "at least one" can be used interchangeably here.

[0027] Unless specifically indicated to the contrary, the term “IAPP” is used interchangeably for specific reference to the native fibrillar, non-fibrillar, nonomeric and oligomeric form of islet amyloid polypeptide (IAPP). The term "IAPP" is also generally used to identify other IAPP conformers, for example, IAPP oligomers and / or aggregates such as IAPP fibrils.

[0028] The term “IAPP” is also used for collective reference to all types and forms of IAPP. The term prolAPP is used interchangeably for specific reference to the native aggregated, monomeric, oligomeric and / or fibrillar form of the islet amyloid polypeptide precursor peptide (prolAPP). Letters added in front of the terms IAPP or prolAPP are used to indicate the organism from which a specific orthologist originates, for example, hlAPP for human IAPP or mIAPP for murine origin.

The amino acid sequence of 37 for human IAPP is KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY (SEQ ID: 1) with a disulfide bridge between cysteine residues 2 and 7 and an amidated C-terminal end.

[0030] IAPP is processed from the prepro-hormone preprolAPP, an amino acid precursor 89 produced in pancreatic B cells. The protein sequence of human preprolAPP is: MGILKLQVFLIVLSVALNHLKATPIESHQVEKRKCNTATCATQRLANFLVHS SNNF GAILSSTNVGSNTYGKRNAVEVLKREPLNYLPL (SEQ ID: 2), and such a sequence can also be found in PID: database, for example: in.

[0031] PreprolAPP is rapidly cleaved after translation and becomes the propyl amyloid polypeptide. The protein sequence of human prolAPP is: TPIE SHQVEKRKCNTATCATOQRLANFLVHS - SNNFGAILS STNV GSNTYGKRNAVEV LKREPLNYLPL (SEQ ID: 3), which undergoes additional proteolysis and post-translational modifications to generate hlAPP.

[0032] The recombinant amino acid sequences of human or "foreign type" IAPP, prolAPP and preprolAPP are represented by the aforementioned sequences according to SEQ ID: 1a3.

[0033] The human anti-lAPP and anti-prolAPP antibodies described herein specifically bind IAPP and / or prolAPP and its epitopes to various conformations of IAPP and / or prolAPP and their epitopes. For example, here are described antibodies that specifically bind pathologically aggregated forms of IAPP and / or prolAPP, such as, for example, non-fibrillar oligomers and / or fibrillar / fibril oligomers and / or aggregates consisting of a mixture of their forms. The term aggregates / aggregates (pathologically) of IAPP and / or prolAPP is used interchangeably for specific reference to the aforementioned forms. The term "aggregates" or "aggregate forms" (pathological [as]), as used herein, describes the products of a

14/139 accumulation or cluster formation due to an erroneous / pathological interaction of IAPP and / or prolAPP with one another. These aggregates, accumulations or forms of agglomerate may be, consist substantially of or consist of both IAPP and / or prolAPP and non-fibrillar oligomers and / or fibrillar oligomers and their fibrils. As used herein, reference to an antibody that "specifically binds", "selectively binds" or "preferably binds" IAPP and / or prolAPP refers to an antibody that does not bind other unrelated proteins. For example, an IAPP and / or prolAPP antibody described herein can bind IAPP and / or prolAPP or one of its epitopes and show no binding above about twice the background for other proteins. An antibody that "specifically binds" or "selectively binds" an IAPP and / or prolAPP conformer refers to an antibody that does not bind all IAPP and / or prolAPP conformations, that is, it does not bind at least one other conformer to IAPP and / or prolAPP. For example, antibodies are described here that can preferentially bind to aggregated forms of IAPP and / or prolAPP, both in vitro and in tissues obtained from patients with overt DT2 or at risk of developing DT2. Since the human IAPP and / or prolAPP antibodies of the present invention were isolated from a group of healthy human individuals or from groups of obese patients and from other groups of patients at greater risk of developing DT2, than at the time of isolation of the antibody showed no signs of DT2, exhibiting a specific immune response to IAPP and / or prolAPP, the IAPP and / or prolAPP antibodies of the present invention can also be called "human autoantibodies", in order to emphasize that these antibodies were actually presented by individuals, and not that they have been isolated, for example, from a phagemid library that has human immunoglobulin, which until now represented a common method of trying to obtain antibodies similar to humans.

[0034] It is understood that the term "peptide" includes the terms "polypeptide" and "protein" (which can sometimes be used interchangeably here) within their meaning. Likewise, fragments of proteins and polypeptides are also contemplated and can be called "peptides" here. However, the term "peptide" preferably means an amino acid polymer comprising at least 5 contiguous amino acids, preferably at least 10 contiguous amino acids, more preferably at least 15 contiguous amino acids, even more preferably at least 20 contiguous amino acids, and especially preferred, at least 25 contiguous amino acids. In addition, the peptide according to the present invention does not usually have more than 100 contiguous amino acids, preferably less than 80 contiguous amino acids and, more preferably, less than 50 contiguous amino acids. Polypeptides

[0035] As used here, the term "polypeptide" is intended to encompass a single "polypeptide", as well as its plural, "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 with two or more amino acids, not a specific size of the product. Thus, “peptides”, “dipeptides”, “tripeptides”, “oligopeptides”, “protein”, “chain of amino acids”, or any other term used to refer to a chain or chains with two or more amino acids are included within the definition of “polypeptide”, and the term “polypeptide” can be used interchangeably with or instead of any of these.

[0036] The term "polypeptide" is also used to refer to post-expression polypeptide modification products, including, without limitation, glycosylation, acetylation, phosphorylation, amidation and derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can originate from a natural biological source or be produced by recombinant technology, but it is not necessarily translated from a sequence called nucleic acid. It can be generated in any way, including by chemical synthesis.

[0037] A polypeptide of the invention can be about 3 or more, 5 or more, or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 in size or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can present themselves in a well-defined three-dimensional structure, although they do not necessarily present themselves in that structure. Polypeptides with a well-defined three-dimensional structure are known as folded, and polypeptides that do not have a well-defined three-dimensional structure, but instead, can adopt a large number of different conformations, are known as splits. As used herein, the term glycoprotein refers to a protein attached to at least one carbohydrate component that is linked to the protein by a side chain containing oxygen or nitrogen from an amino acid residue, for example, a serine or asparagine residue .

[0038] The term "isolated" polypeptide or fragment, variant or its derivative refers to a polypeptide that is not in its natural environment. No particular level of purification is needed. For example, an isolated polypeptide can be removed from its native or natural environment. Polypeptides produced recombinantly and proteins expressed in host cells are considered isolated for the purposes of the invention, as are native or recombinant polypeptides that have been separated, fractionated or purified, either partially or substantially, by any appropriate technique.

[0039] "Recombinant peptides, polypeptides or proteins" are peptides, polypeptides or proteins produced by recombinant DNA techniques, that is, produced from mammalian cells or microbes transformed by an exogenous recombinant DNA expression construct that codes for fusion protein, including the desired peptide Proteins or peptides expressed in most bacterial cultures will normally be free of glycan Proteins or polypeptides expressed in yeast may exhibit a different glycosylation pattern than that expressed in mammalian cells.

[0040] Comprised as polypeptides of the present invention are fragments, derivatives, analogs or variants of the previous polypeptides, and also, any combinations thereof. The terms "fragment", "variant", "derivative" and "analog" comprise peptides and polypeptides that have an amino acid sequence - sufficiently similar to that of the natural peptide. The term "sufficiently similar" means a first amino acid sequence that contains a sufficient or minimal amount of identical or equivalent amino acid residues, relative to a second amino acid sequence, so that the first and second amino acid sequences have a structural domain common and / or a common functional activity. For example, the amino acid sequences that

17/139 comprise a common structural domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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% identical are defined here as sufficiently similar. Preferably, the variants will be sufficiently similar to the amino acid sequence of the preferred peptides of the present invention, and in particular the IAPP and / or prolAPP or fragments, variants, derivatives or analogs of either. In general, these variants exhibit the functional activity of the peptides of the present invention. Among the variants are peptides that differ in the amino acid sequence of the native peptide and wt, respectively, through the elimination (s), addition (s) and / or substitution (s) of one or more amino acids. These variants can occur naturally or be designed artificially.

[0041] In addition, the terms "fragment", "variant", "derivative" and "analog" when referring to the antibodies or polypeptides of the antibodies of the present invention include any polypeptides that exhibit at least some of the binding properties of antigens from the polypeptide, antibody or corresponding native binding molecule. Polypeptide fragments of the present invention comprise proteolytic fragments, as well as fragments of deletion, in addition to fragments of the specific antibody discussed elsewhere in this document. Variants of antibodies and antibody polypeptides of the present invention comprise fragments, as described above, and also, polypeptides with altered amino acid sequences due to amino acid substitutions, deletions or insertions. Variants can occur naturally or not naturally. Non-naturally occurring variants can be produced using mutagenesis techniques known in the art. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of specific binding molecules of IAPP and / or prolAPP, for example, antibodies and antibody polypeptides of the present invention are polypeptides that have been altered in order to have additional characteristics not found in the native polypeptide. Examples include fusion proteins. Variant polypeptides can also be referred to herein as "polypeptide analogs". As used herein, a "derivative" of a binding molecule or fragment thereof, an antibody or antibody polypeptide refers to an object polypeptide that has one or more chemically derived residues by reaction of a functional helper group. Also included as "derivatives" are peptides that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can be replaced with proline; 57 hydroxylysine can be replaced by lysine; 3-methylhistidine can be replaced by histidine; homoserine can be replaced by serine; and ornithine can be replaced by lysine. Determination of similarity and / or identity of the molecules:

[0042] The "similarity" between two peptides is determined by comparing the amino acid sequence of one peptide with the sequence of a second peptide. An amino acid from a peptide is similar to the corresponding amino acid from a second peptide, if it is identical, or a conservative amino acid substitution. Conservative substitutions include those described in Dayhoff, MO, ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, DC (1978), and in Argos, EMBO J. 8 (1989), 779-785 . For example, amino acids belonging to one of the following groups represent conservative substitutions or changes: -Ala, Pro, Gly, GIn, Asn, Ser, Thr; -Cis, Ser, Tir, Thr; -Val, lle, Leu, Met, Ala, Phe; -Lis, Arg, His; -Phe, Tir, Trp, His; and -Asp, Glu.

[0043] The "similarity" between two polynucleotides is determined by comparing the nucleic acid sequence of a polynucleotide with the sequence of a polynucleotide. A nucleic acid of a 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, the respective triplet comprising the nucleic acid codes for the same amino acid or for a substitution conservative amino acid.

[0044] The determination of percentage 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. This algorithm is incorporated into the BLASTn and BLASTp programs by Altschul et a /. (1990) T. Mol. Biol. 215: 403-410 available at NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cge).

[0045] The determination of percentage identity or similarity is carried out with the standard parameters of the BLASTn and BLASTp programs, as recommended on the NCBI website and in the “BLAST Program Selection Guide”, regarding the specific size and composition strings.

[0046] BLAST polynucleotide searches are performed with the BLASTn program.

[0047] For parameters in general, the “Max Target Sequences” box can be set to 100, the "Short searches" box can be checked, the "Threshold expectation" box can be set to 1000 and the "Word size" box can be set to 7, as recommended for short strings (less 20 bases) on the NCBI website. For larger sequences, the “Threshold Expectation” box can be set to 10 and the “Word Size” box can be set to 11. For inlay parameters, “Match / Unmatched Inlays” can be set to 1 , -2 and the box “Hiatus Costs” can be configured as linear. For the Filters and Masks parameters, the “Low complexity regions” box cannot be checked, the “Species specific repetitions” box cannot be checked, the “Mask only for search in the table” box can be checked, the “ DUST Filter Settings "can be checked and the" Mask lowercase letters "box cannot be checked. In general," Search for almost exact short matches "can be used for this purpose, which provides most of the settings indicated above Further information on this subject can be found in the “BLAST Program Selection Guide”, published on the NCBI website.

[0048] Searches for BLAST proteins are carried out with the BLASTp program. For parameters in general, the “Max Target Strings” box can be set to 100, the “Short searches” box can be checked, the “Expectation of

/ 139 threshold ”can be set to 10 and the“ Word Size ”box can be set to“ 3 ”. For incrustation parameters, the “Matrix” box can be configured as “BLOSUMG62”, the “Hiatus Costs” box can be configured as “Existence: 11 Extension: 1”, the “Composition Adjustments” box can be configured as “Adjustment of conditional compositional inlay matrix”. For the Filters and Masks parameters, the “Low complexity regions” box cannot be checked, the “Mask only for searching the table” box cannot be checked, and the “Mask lower case” box cannot be checked.

[0049] Modifications of both programs, for example, regarding the size of the searched strings, are carried out in accordance with the recommendations of the “BLAST Program Selection Guide”, published in HTML and PDF versions on the NCBI website . Polynucleotides:

[0050] The term "polynucleotide" is used to encompass a single nucleic acid, as well as plural nucleic acids, and refers to an isolated nucleic acid construct or molecule, for example, messenger RNA (mRNA) or DNA plasmid ( PDNA). A polynucleotide can comprise an unconventional or conventional phosphodiester bond (for example, an amide bond, such as that found in peptide nucleic acids [PNA]). The term "nucleic acid" refers to any one or more nucleic acid segments, for example, fragments of DNA or RNA, present in a polynucleotide. By "isolated" polynucleotide or nucleic acid is meant a nucleic acid molecule, DNA or RNA, that has been removed from its native environment. For example, a recombinant polynucleotide that encodes an antibody contained in a vector is considered to be isolated for the purposes of the present invention. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified polynucleotides (partially or substantially) in solution. Isolated RNA molecules comprise RNA transcripts in vivo or in vitro of polynucleotides of the present invention. Nucleic acids or polynucleotides isolated in accordance with the present invention further comprise those synthetically produced molecules. In addition, the polynucleotide or a nucleic acid can be or can comprise a regulatory element, such as, for example, a ribosome binding promoter site, or a transcription terminator.

[0051] As used herein, a "coding region" is a portion of nucleic acid made up of codons translated into amino acids. Although a “termination codon” (TAG, TGA or TAA) is not translated into an amino acid, it can be considered part of a coding region, but any flanking sequences, for example, ribosome-binding promoter sites, transcription terminators, introns and the like do not integrate a coding region. Two or more coding regions of the present invention can be present in a single polynucleotide construct, for example, in a single vector, or in separate polynucleotide constructs, for example, in separate (different) vectors. In addition, any vector can contain a single coding region, or can comprise two or more coding regions, for example, a single vector can separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide or nucleic acid of the invention can encode heterologous coding regions, both fused and unfused, in a nucleic acid that encodes a binding molecule, an antibody or its fragment, variant or derivative. The heterologous coding regions comprise, without limitation, specialized elements or motifs, such as, for example, a secretion signal peptide or a heterologous functional domain.

[0052] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid that encodes a polypeptide can normally include a promoter, and / or other operable transcription or translation control elements associated with one or more coding regions. An operable association is when a coding region for a gene product, for example, a polypeptide, is associated with one or more regulatory sequences in order to place the expression of the gene product under the influence or control of the sequence (s) ) regulator (s). Two fragments of DNA (such as a polypeptide coding region and a promoter that is associated with it) are "operable associates" or "operable ligands" if induction of the promoter function results in mRNA transcription, encoding the product of the desired gene, and if the nature of the link between the two DNA fragments does not interfere with the ability of the regulatory expression sequences to direct expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would become operable with a nucleic acid encoding a polypeptide if the promoter were able to transcribe said nucleic acid. The promoter can be a cell-specific promoter that directs substantial DNA transcription only in predetermined cells. Other elements of transcription control, in addition to a promoter, for example, enhancers, operators, repressors, and transcription termination signals can be associated operable with the polynucleotide to direct specific cell transcription. Suitable promoters and other transcription control regions are described herein.

[0053] Several regions of transcription control are known to those who are qualified in the field. These include, without limitation, transcription control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (the immediate early promoter, in conjunction with A-intron), simian virus 40 (the early promoter), and retrovirus (such as Rous's sarcoma virus). Other transcription control regions include those from vertebrate genes, such as actin, heat shock protein, bovine growth hormone and rabbit B-globin, as well as other sequences capable of controlling expression of genes in eukaryotic cells. Other appropriate transcription control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (for example, interferon- or interleukin-inducible promoters).

[0054] Likewise, several elements of translation control are known to those whose skill in the field is common. These include, but are not limited to, ribosome binding sites, termination codons and translation initiation, and elements derived from picornavirus (particularly an internal ribosome entry site, or IRES, also known as an ISCED sequence).

[0055] In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA).

The polynucleotide and nucleic acid coding regions of the present invention can be associated with other coding regions that encode signal and secretion peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretion command sequence, which is cleaved from the mature protein after the growing protein chain has been exported throughout the endoplasmic reticulum rough. Those whose skill in the industry is common are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminal end of the polypeptide, which is cleaved from the complete or "integral" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide, for example, an immunoglobulin light chain or heavy chain signal peptide is used, or a functional derivative of that sequence that has the ability to direct secretion of the polypeptide that is associated with operable with him. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, can be used. For example, the foreign-type leader sequence can be replaced by the leader sequence of human tissue plasminogen activator (TPA) or mouse B-glucuronidase.

A "binding molecule" as used in the context of the present invention refers primarily to antibodies and fragments thereof, but may also refer to molecules other than antibodies that bind to IAPP and / or prolAPP, including, but without being limited to hormones, receptors, ligands, molecules of the main histocompatibility complex (MHC), companions such as heat shock proteins (HSPs), as well as cell adhesion molecules, such as the members of the cadherin superfamilies, integrin, lectin type C and immunoglobulin (lg). Therefore, solely for the sake of clarity and without restricting the scope of the present invention, much of the following embodiments are discussed with respect to antibody and antibody-like molecules that represent the preferred binding molecules for the development of therapeutic agents and diagnostic. Antibodies.

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[0058] The terms "antibody" and "immunoglobulin" are used interchangeably here. An antibody or immunoglobulin is an IAPP and / or prolAPP binding molecule that comprises at least the variable domain of a heavy chain, and normally comprises at least the variable domains of a heavy chain and a light chain. Basic immunoglobulin structures in vertebrate systems are relatively well understood; see, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

[0059] As will be discussed in more detail below, the term "immunoglobulin" comprises several broad classes of polypeptides that can be differentiated biochemically. Those skilled in the art will appreciate the classification of heavy chains as gamma, mu, alpha, delta, or epsilon, (y, 1, a, 8, €) with some subclasses between them (eg: yl-y4). It is the nature of this chain that determines the “class” of the antibody according to IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes), eg: IgGl, I9gG2, 19G3, IgG4, IgAl, etc., are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily apparent to a person skilled in the art, due to the immediate description and, therefore, are within the scope of the present invention. All classes of immunoglobulin are clearly within the scope of the present invention, the discussion that follows will generally be directed to the IgG class of immunoglobulin molecules. As far as IgG is concerned, a standard immunoglobulin molecule comprises two identical light chain polypeptides of approximately 23,000 Daltons molecular weight, and two identical heavy chain polypeptides of between 53,000 to 70,000 molecular weight. The four chains are usually joined by disulfide bonds in a "Y" configuration in which the light chains support the heavy chains that start at the mouth of the "Y" and go through the variable region.

[0060] Light chains are classified as either kappa or lambda (x, 2). Each heavy chain class can be linked to a light chain that can be either kappa or lambda. In general, the light and heavy chains are covalently linked to each other, and the "back" portions of the two heavy chains are linked to each other by means of covalent disulfide bonds or non-covalent bonds when immunoglobulins are generated or by hybridomas , B cells or by genetically engineered host cells. In the heavy chain, the amino acid sequences run from one N-terminal end at the forked ends of the Y configuration towards the C-terminal end at the bottom of each chain.

[0061] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used in a functional way. In this regard, it should be noted that the variable domains of both light chain (V.) and heavy chain (VH) portions determine the specificity and recognition of antigens. On the other hand, the light chain (CL) and heavy chain (CH1, CH2 or CH3) domains confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention, the numbering of constant region domains increases as they move further away from the antigen-binding site or the amino-terminal end of the antibody. The N-terminal portion is a variable region, and at the C-terminal portion there is a constant region; the CH3 and CL domains effectively comprise the carboxy-terminal end of the heavy and light chains, respectively.

[0062] As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes to antigens. That is, the VL and VHn domains, or the subset of the complementarity determining regions (CDRs) of an antibody join to form the variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs on each of the Vr and V chains. Any immunoglobulin fragment or antibody that contains sufficient structure to specifically bind to IAPP and / or prolAPP is interchangeably indicated here as a "binding fragment" or an "immunospecific fragment".

[0063] In naturally occurring antibodies, an antibody comprises six hypervariable regions, sometimes called "complementarity determining regions" or "CDRs" present in each antigen binding domain, which are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain, as the antibody assumes its configuration

26/139 three-dimensional in an aqueous environment. The "CDRs" accompany four "FRs" or "structural" relatively conserved regions, which have a lower intermolecular variability. Structural regions largely adopt a B-type conformation and CDRs form circuits that link the B-type structure, and in some cases, form part of it. Therefore, the structural regions form a support structure that provides the positioning of the CDRs in the correct direction through non-covalent inter-chain interactions. The antigen-binding domain formed by the positioned CDRs defines a complementary surface for the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent attachment of the antibody to its cognate epitope. The amino acids that comprise the CDRs and the structural regions, respectively, can be readily identified for any given variable region of light or heavy chain by someone whose skill in the field is common, as they have been precisely defined; see "Sequences of Proteins of Immunological Interest", Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia & Lesk, J. Mol. Biol., 196 (1987), 901-917, which are incorporated herein by reference in their entirety.

[0064] If there are two or more definitions of a term that is used and / or accepted in the letter, the definition of the term as used here is intended to understand all these meanings, unless explicitly stated otherwise. A specific example is the use of the term “complementarity determining region” (“CDR”) to describe the sites of combination with the non-contiguous antigen found in the variable region of both heavy and light chain polypeptides. That particular region has been described by Kabat et a /., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia & Lesk, J. Mol. Biol., 196 (1987), 901-917, which are hereby incorporated by reference, where definitions comprise the overlap or subsets of amino acid residues when compared to each other. However, the application of any of the two definitions for reference to a CDR of an antibody or variants of it is intended to be within the scope of the term, as defined and used herein. Suitable amino acid residues that comprise CDRs, as defined by each of the aforementioned references are shown below in Table |, for comparison. THE

The exact amount of waste that comprises a particular CDR varies, depending on the size and sequence of the CDR. Those skilled in the art can routinely determine which residues comprise a particular hypervariable region or a CDR of the human IgG subtype of antibody, given the amino acid sequence of the variable region of the antibody.

Table |: CDR definitions 'LE EE e' The numbering of all CDR definitions in Table I is in accordance with the numbering conventions established by Kabat et al. (see below).

[0065] Kabat et al. also defined a numbering system for variable domain strings that applies to any antibody. Someone whose skill in the field is common can unequivocally assign this “Kabat numbering” system to any variable domain sequence, without relying on any experimental data other than the sequence itself. As used here, “Kabat numbering” refers to the system of numbering established by Kabat et a /., US Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise stated, references to the numbering of specific amino acid residue positions in an antibody or antigen-binding fragment, its variant or derivative of the present invention, refer to the Kabat numbering system, which, however, is theoretical and it may not apply equally to all antibodies of the present invention. For example, depending on the position of the first CDR, the following CDRs can be moved in either direction.

[0066] Antigen-binding antibodies or fragments, immunospecific fragments, their variants or derivatives of the present invention comprise, but are not limited to, antibodies - polyclonal, monoclonal, multispecific, human, humanized,

primatized, murinized or chimeric, single chain antibodies, epitope-binding fragments, for example, Fab, Fab 'and F (ab') 2a, Fd, Fvs, single chain Fvs (scFv), single chain antibodies, Fvs with disulfide binding (sdFv), fragments comprising either a V domain or a Vn domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-ld) antibodies (including, for example, anti-ld antibodies to antibodies described here). ScFv molecules are known in the art and are described, for example, in US patent 5,892,019. Antibody or immunoglobulin molecules of the invention can be of any type (for example, IgG, IgE, IgM, 1gD, IgA and IgY), class (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and igA2) or molecule subclass immunoglobulin.

[0067] In one embodiment, the antibody of the present invention is not IgM or one of its derivatives with a pentavalent structure. In particular, in specific applications of the present invention, especially in therapeutic use, IgMs are less useful than IgG and other bivalent antibodies or corresponding binding molecules due to the fact that IgMs, due to their pentavalent structure and lack of affinity maturation, many sometimes demonstrate non-specific cross-reactions and a very low affinity.

[0068] In a particularly preferred embodiment, the antibody of the present invention is not a polyclonal antibody, that is, it consists substantially of a particular species of antibody rather than a mixture obtained from a plasma immunoglobulin sample.

[0069] Antibody fragments, including single chain antibodies, may comprise the variable region (s) alone or in conjunction with all or a portion of the following: articulation region, CH1 domains , CH2 and CH3. Also included in the invention are IAPP and / or prolAPP binding fragments that comprise any combination of variable region (s) with an articulation region, and CH1, CH2 and CH3 domains. Antibodies or their immunospecific fragments of the present invention can be of any animal origin, including birds and mammals. Preferably, the antibodies are human, murine, donkey, cunicular, caprine, guinea pig, camelid, mule, equine or chicken. In another embodiment, the variable region may be chondrictoid in origin (for example, from sharks).

[0070] In one aspect, the antibody of the present invention is a human monoclonal antibody isolated from a human. As an option, the structural region of the human antibody is aligned and adopted in accordance with the relevant human germline variable region sequences in the database; see, for example, Vbase (http://vbase.mrc-cpe.cam.ac.uk/), received by the MRC Center for Protein Engineering (Cambridge, UK). For example, amino acids considered to potentially deviate from the actual germline sequence may occur due to the polymerase chain reaction (PCR) primer sequences incorporated during the cloning process. In comparison to human-like antibodies artificially generated as fragments of single chain antibodies (scFvs) from an antibody library showing phagemids or xenogenic rodents, the human monoclonal antibody of the present invention is characterized by (i) having been obtained using - the human immune response instead of that of animal substitutes, that is, the antibody was generated in response to the natural IAPP or prolAPP in its corresponding conformation in the human body, (ii) having protected the individual or that was at least significant for the presence of IAPP and / or prolAPP, and (iii) as the antibody is of human origin, the risks of cross-reactivity against autoantigens are minimized. Therefore, according to the present invention, the terms "human monoclonal antibody", "human monoclonal autoantibody", "human antibody" and the like are used to denote an IAPP and / or prolAPP binding molecule that is of human origin, or that is, it was isolated from a human cell, such as a B cell or its hybridoma or the cDNA from which it was directly cloned from the mRNA of a human cell, for example, a human memory B cell. A human antibody is still "human", even if amino acid substitutions are made in the antibody, for example, to improve binding characteristics.

[0071] Antibodies derived from libraries of human immunoglobulins or transgenic animals to one or more human immunoglobulins and which do not express endogenous immunoglobulins, as described below and, for example, in U.S. Patent No. 5,939,598 to Kucherlapati et al., Denote antibodies similar to humans are used in order to distinguish them from the truly human antibodies of the present invention.

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[0072] For example, the pairing of heavy and light chains of human-like antibodies, such as synthetic and semi-synthetic antibodies typically isolated from phage display do not necessarily reflect the original pairing as occurred in the original human B cell. Consequently, Fab and scFv fragments obtained from recombinant expression libraries, as commonly used in the prior art, can be considered as artificial with all possible effects associated with immunogenicity and stability.

In contrast, the present invention provides isolated affinity-matured antibodies from selected human individuals, which are characterized by their therapeutic utility and their tolerance in man.

[0074] As used herein, the term "rodentized antibody" or "rodentized immunoglobulin" refers to an antibody comprising one or more CDRs derived from a human antibody of the present invention; and a human structural region that contains insertions and / or substitutions and / or deletions of amino acids that are based on a rodent antibody sequence. When referring to rodents, preferably, the sequences originating in mice and rats are used, in which the antibodies comprising these sequences are cited as "murinized" or "ratinized", respectively. The human immunoglobulin that provides CDRs is called a "mother" or "recipient" and the rodent antibody that provides structural changes is called a "donor". There need not be constant regions, but if there are, they are usually substantially identical to the constant regions of the rodent antibody, that is, at least about 85 to 90%, preferably about 95% or more, identical. Thus, in some embodiments, a murine human heavy or light chain integral immunoglobulin contains a mouse constant region, human CDRs, and a substantially human structure with a number of "murinizing" amino acid substitutions. Typically, a "murinized antibody" is an antibody comprising a murinized variable light chain, and / or a murinized variable heavy chain. For example, a murinized antibody would not encompass a typical chimeric antibody, for example, because the entire variable region of a chimeric antibody is different from that of mice. A modified antibody that has been "murinized" by the "murinization" process binds to the same antigen as the parent antibody that provides CDRs and is usually less immunogenic in mice compared to the parent antibody. The above explanations regarding "murinized" antibodies apply in a similar way to other "rodentized" antibodies, such as "ratinized antibodies", in which the rat sequences are used instead of the murines.

[0075] As used herein, the term "heavy chain moiety" comprises amino acid sequences from an immunoglobulin heavy chain. A polypeptide comprising a portion of the heavy chain comprises at least one of the following: a CH1 domain, a hinge domain (eg, upper, middle and / or lower hinge region), a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, a binding polypeptide for use in the invention can comprise a polypeptide chain that comprises a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain that comprises a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain that comprises a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the invention comprises a polypeptide chain that comprises a CH3 domain. In addition, at least a portion of a CH2 domain (e.g., all or part of a CH2 domain) may be missing from a binding polypeptide for use in the invention. As stated above, someone whose skill in the field is common will understand that these domains (eg, the heavy chain moieties) can be modified to vary in the amino acid sequence from the naturally occurring immunoglobulin molecule.

[0076] In certain antibodies, or antigen binding fragments, variants or derivatives thereof described herein, the heavy chain portions of a polypeptide chain of a multimer are identical to those of a second polypeptide chain of the multimer. Alternatively, the monomers of the invention that contain a heavy chain moiety are not identical. For example, each monomer can comprise a different target binding site, forming, for example, a bispecific antibody or diabody.

[0077] In another embodiment, the antibodies, or antigen-binding fragments, variants or derivatives thereof described in this instrument are composed of a single polypeptide chain, such as scFvs, for example, and must be expressed intracellularly ( intrabodies) for potential therapeutic and in vivo diagnostic applications.

[0078] The heavy chain portions of a binding polypeptide for use in the diagnostic and treatment methods described herein can be derived from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide can comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain portion may comprise a hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, a heavy chain portion may comprise a chimeric joint derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.

[0079] As used herein, the term "light chain moiety" comprises amino acid sequences derived from an immunoglobulin light chain. Preferably, the light moiety portion comprises at least one of a V or CL domain.

[0080] It is thought that the minimum size of an antibody polypeptide or peptide epitope is about four to five amino acids. Polypeptide or peptide epitopes preferably contain at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids. Since a CDR is able to recognize an antigenic polypeptide or peptide in its tertiary form, the amino acids that comprise an epitope do not need to be contiguous, and in some cases, may not even be in the same peptide chain. In the present invention, a polypeptide or peptide epitope recognized by antibodies of the present invention contains 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 between about 15 to about 30 contiguous or non-contiguous amino acids of IAPP or prolAPP.

[0081] By "specifically binding" or "specifically recognizing", used here interchangeably, it is customary to mean that a binding molecule, eg an antibody binds to an epitope through its antigen binding domain, and that the binding leads to some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope, through its antigen-binding domain, more readily than it would bind to a random, unrelated epitope. The term "specificity" is used here to describe the relative affinity by which a given antibody binds to a given epitope. For example, antibody "A" can be considered to have greater specificity for a given epitope than antibody "B", or it can be said that antibody "A" binds to epitope "C" with greater specificity. than that presented for the related epitope “D”.

[0082] Wherever present, the term "immunological binding characteristics", or other binding characteristics of an antibody to an antigen, in all its grammatical forms, refers to specificity, affinity, cross-reactivity, and other binding characteristics of an antibody.

[0083] By "preferential binding", it is understood that the binding molecule, eg, the antibody specifically binds to an epitope more readily than it would bind to a related, similar, homologous or analogous epitope. Thus, an antibody that "preferably binds" to a given epitope would be more likely to bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.

[0084] As a non-limiting example, a binding molecule, eg, an antibody can be considered to bind a first epitope, preferably by binding that first epitope to a dissociation constant (Ko), which is less than Kp of the antibody of the second epitope. In another non-limiting example, an antibody can be considered to bind a first antigen, preferably if it binds the first epitope with an affinity that is at least an order of magnitude lower than the Kp of the antibody of the

34/139 second epitope. In another non-limiting example, an antibody can be considered to bind a first epitope, preferably if it binds the first epitope with an affinity that is at least two orders of magnitude less than the Kp of the antibody of the second epitope.

[0085] In another non-limiting example, a binding molecule, eg an antibody, can be considered to bind a first epitope, preferably if it binds the first epitope with a dissociation rate at (k (off)) that is less than the k (off) of the antibody for the second epitope. In another non-limiting example, an antibody can be considered to bind a first epitope, preferably if it binds the first epitope with an affinity that is at least an order of magnitude less than the k (off) of the antibody to the second epitope. In another non-limiting example, an antibody can be considered to bind a first epitope, preferably if it binds the first epitope with an affinity that is at least two orders of magnitude less than the k (off) of the antibody of the second epitope.

[0086] It can be said that a binding molecule, eg an antibody or antigen-binding fragment, variant or derivative described here, binds IAPP and / or prolAPP or a specific fragment, variant or conformation thereof with a velocity of dissociation (k (off)) less than or equal to 5 x 10? Mon, 10? sec ", 5x 10th sec" or 10th sec 1, More preferably, it can be said that an antibody of the invention binds IAPP and / or prolAPP or a specific fragment, variant or conformation thereof with a dissociation rate (Kk (off) ) less than or equal to 5 x 10th sec, 10th sec, 5x 10th sec 1, or 105 sec? 5x 10th sec, 108 sec, 5x 107 sec or 107 sec ”

[0087] It can be said that a binding molecule, e.g., an antibody or antigen-binding fragment, variant or derivative described here, binds IAPP and / or prolAPP or a fragment, variant or specific conformation thereof with a velocity of dissociation (k (off)) greater than or equal to 10º M sec, 5x 10º M sec, 10º M sec! or 5 x 10º M sec. More preferably, it can be said that an antibody of the invention binds IAPP and / or prolAPP or a fragment, variant or specific conformation thereof with an association speed (k (on)) greater than or equal to 10º M sec ", 5x 10º M 'sec ”, 10º M' sec”, or 5x 10º M 'sec? or 107 M sec?

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[0088] It is said that a binding molecule, eg, an antibody competitively inhibits the binding of a reference antibody to a given epitope if it binds, preferably, to that epitope, until it blocks, in to some degree, binding of the reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art, for example, competitive ELISA assays. It can be said that an antibody competitively inhibits the binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0089] As used herein, the term "affinity" refers to a measure of the strength of binding an individual epitope to the CDR of a binding molecule, eg, an immunoglobulin molecule; see, eg, Harlow et a /., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988) on pages 27 and 28. As used here, the term “avidity” refers to the overall stability of the complex between an immunoglobulin population and an antigen, that is, the strength of the functional combination of an immunoglobulin mixture with antigen; see, for example, Harlow on pages 29 to 34. Avidity is related both to the affinity of individual immunoglobulin molecules in the population with specific epitopes, and to the valences of immunoglobulins and antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repetitive epitopal structure, such as a polymer, would 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 here. General techniques for measuring the affinity of an antibody to an antigen include ELISA, RIA, and surface plasma resonance. The measured affinity of a particular antibody-antigen interaction can vary if it is measured under different conditions, for example, salt concentration, pH. Thus, measurements of affinity and other antigen binding parameters, for example, Ko, ICso, are preferably made with standardized solutions of antibodies and antigens, and a standardized buffer.

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[0090] Binding molecules, for example, antibodies or antigen binding fragments, variants or derivatives thereof of the invention can also be described or specified in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of an antibody, specific for an antigen, to react with a second antigen; a measure of the relationship between two different antigenic substances. Thus, an antibody is cross-reactive if it binds to an epitope that is not the one that induced its formation. The cross-reactive epitope usually contains many of the same complementary structural features as the inducing epitope, and in some cases, may even fit better than the original.

[0091] For example, certain antibodies show a certain degree of cross-reactivity, because they bind related, but not identical, epitopes, such as 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 (as calculated using methods known in the art and described herein) to a reference epitope. An antibody can be said to have little or no cross-reactivity if it does not bind epitopes with 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 (as calculated using methods known in the art and described herein) to a reference epitope. An antibody can be considered “highly specific” for a particular epitope if it does not bind any other analog, ortholog, or homologue to that epitope.

[0092] Binding molecules, e.g., antibodies or antigen binding fragments, variants or derivatives thereof of the invention can also be described or specified in terms of their binding affinity to IAPP and / or prolAPP. Preferred binding affinities include those whose dissociation constant or Kd is less than 5x 102 M, 102 M, 5x 10 ° M, 10 M, 5x 10 ° M, 10 M, 5x 105 M, 10 M, 5x 1086 M, 106 M, 5x 107 M, 107 M, 5x 108 M, 108 M, 5x 10º M, 10 M, 5x 10 M, 10 M, 5x 107 M, 10 M, 5x 102 M, 102 M, 5x 1073 M, 10? M, 5x 107º M, 107º M, 5 x 107 M or 10º M.

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[0093] As previously indicated, the subunit structures and three three-dimensional configurations of the regions contained in the various classes of immunoglobulin are well known. As used herein, the term "VH domain" comprises the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" comprises the first (most amino-terminal) constant region domain of a heavy chain of immunoglobulin. immunoglobulin. The CHI domain is adjacent to the VH domain and is amino-terminal to the hinge region of an immunoglobulin heavy chain molecule.

[0094] As used herein, the term "CH2 domain" comprises the portion of a heavy chain molecule that extends, for example, from approximately residue 244 to residue 360 of an antibody using conventional numbering schemes (residues of 244 360, Kabat numbering system; and residues 231 to 340, EU numbering system; see Kabat EA et al. op. cit.) The CH2 domain is unique in that it is not closely paired with another domain. Instead, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises about 108 residues.

[0095] As used herein, the term "hinge region" comprises the portion of a heavy chain molecule that links the CH1 domain to the CH2 domain. This articulation region comprises about 25 residues and is flexible, thus allowing the two antigen-binding regions of the N-terminal end to move independently. Articulation regions can be subdivided into three distinct domains: domains of upper, middle and lower articulation; see Roux et al., J. Immunol. 161 (1998), 4083-4090.

[0096] As used herein, the term "disulfide bond" includes the covalent bond formed between two sulfur atoms. The cysteine amino acid has a thiol group capable of forming a bridge or disulfide bond with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by means of a disulfide bond and the two heavy chains are linked by means of two disulfidic bonds at the positions corresponding to 239 and 242, using the Kabat numbering (heading 226 or 229, EU numbering system).

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[0097] As used herein, the terms "connected", "fused" or "fusion" are used interchangeably. These terms refer to the joining of two or more elements or components, by any means that includes chemical conjugation or recombinant means. A “phase fusion” refers to the union of two or more ORFs (Open Reading Frames) of polynucleotides to form a longer and more continuous ORF, in a way that the correction of the translational reading frame of the original ORFs is maintained. Thus, a recombinant fusion protein is a single protein that contains two or more segments that correspond to the polypeptides encoded by the original ORFs (whose segments do not usually appear united in nature in this way). Although the reading frame is therefore made continuous across the fused segments, the segments can be physically or spatially separated by, for example, a phase link sequence. For example, polynucleotides that encode the CDRs of an immunoglobulin variable region can be fused, in phase, but separated by a polynucleotide that encodes at least one immunoglobulin structural region or other CDR regions, as long as the "fused" CDRs are co-translated as part of a continuous polypeptide.

[0098] The term "expression", as used herein, refers to a process by which a gene produces a biochemical substance, for example, an RNA or polypeptide. The process comprises any manifestation of the functional presence of the gene within the cell, including, without limitation, the knockdown of genes, as well as both transient and stable expression. It includes, without limitation, the transcription of the gene into messenger RNA (mRNA), carrier RNA (tRNA), clamp-shaped RNA (sShRNA), small interference RNA (siRNA) or any other RNA product, and the translation of mRNA in polypeptide (s). If the desired end product is a biochemical substance, the expression comprises the creation of that biochemical substance and any precursors. The expression of a gene produces a "gene product". As used herein, a gene product can be either a nucleic acid, eg, a messenger RNA produced by the transcription of a gene or a polypeptide that is translated from a transcription. The gene products described herein further comprise nucleic acids with post-transcriptional modifications, for example, polyadenylation, or polypeptides with post-translational modifications, for example

39/139 example, glycosylation, methylation, addition of lipids, association with other subunits of proteins, proteolytic cleavage, and the like.

[0099] As used herein, the term "sample" refers to any biological material obtained from an individual or patient. In one aspect, a sample may comprise blood, peritoneal fluid, CSF, urine or saliva. In other respects, a sample may comprise whole blood, blood plasma, blood serum, B cells enriched with blood samples, and cells in culture (eg, an individual's B cells). A sample can also have a tissue sample or biopsy sample, including neural tissue. In more other respects, a sample may comprise whole cells and / or a cell lysate. Blood samples can be collected by methods known in the art. In one aspect, the pellet can be resuspended by vortexing at 4ºC in 200 µl of buffer (20 mM Tris, pH. 7.5, 0.5% Nonidet, 1 MM EDTA, 1 mM PMSF, 0.1 M NaC1, Inhibitor Protease Sigma IX, and Sigma IX Phosphatase Inhibitors 1 and 2). The suspension can be kept on ice for 20 minutes with an intermittent vortex. After centrifugation at 15,000 x g for 5 minutes at about 4 ° C, aliquots of supernatant can be stored at about -70 ° C. Diseases

[00100] Unless otherwise stated, the terms "disorder" and "disease" are used interchangeably here and include any unwanted physiological changes in an individual, animal, isolated organ, tissue or cell / cell culture.

[00101] In DT2, genetic determinants and environmental factors led to the development of insulin resistance, followed by a compensatory growth in beta cell mass and insulin and amylin secretion (IAPP) to maintain normal blood glucose levels. The resulting high concentrations of amylin favor the formation of toxic hlAPP oligomers and the deposition of hILAPP fibrils, which is found in more than 90% of patients with T2D. The deposition of hiAPP correlates with the reduction of insulin-producing beta cells, and has also been ventilated to play a role in the loss of B cells in transplanted pancreatic islets for individuals with T1D. This application provides several antibodies of human origin from groups of healthy or obese donors at high risk

40/139 to develop DT2, but absence of the disease, which were cloned and produced recombinantly, as described in more detail below.

[00102] However, in one embodiment of the present invention, its antibodies, binding molecules that have substantially the same binding specificities as any of them, the polynucleotides, vectors or cells of the present invention are used in the preparation of a composition of diagnostic or pharmaceutical for prophylactic and therapeutic treatment, monitoring of progress or response to treatment and / or diagnosis of diseases in the group of diseases linked to Diabetes mellitus, comprising type 1 diabetes (DT1), gestational diabetes, pre-diabetes, diabetes latent autoimmune disease in adults (LADA; type 1.5 diabetes) and / or type 2 diabetes (DT2).

[00103] In a preferred embodiment, the antibodies of the present invention, binding molecules that have substantially the same binding specificities as any of them, the polynucleotides, vectors or cells of the present invention are used in the preparation of a composition of diagnostic or pharmaceutical for prophylactic and therapeutic treatment, monitoring of progress or a response to treatment and / or diagnosis of a group of disorders usually characterized by symptoms such as metabolic changes that precede, cause and / or are linked / associated or related to diseases comprised by DT2 that damage the pancreas and can therefore lead to diabetes, comprising chronic pancreatitis, cystic fibrosis, pancreatic cancer; in diseases that increase the risk of T2D, including Alzheimer's disease, Huntington's disease; and / or cardiovascular diseases associated or not with obesity and T2D. In a preferred embodiment, the symptoms that generally characterize the aforementioned diseases comprise sensitivity to disturbed insulin and increased insulin secretion, and / or hiAPP in an individual.

[00104] In one embodiment, the group of disorders associated with the aforementioned specific symptoms comprises gestational diabetes, pre-diabetes (when high blood glucose does not reach the DT2 threshold or insulin resistance); metabolic syndrome in general as a risk factor for the development of diabetes or as a condition that may exist prior to diabetes; islet amyloidosis in general as a risk factor for the development of diabetes or as a condition that may exist prior to diabetes; obesity in general as a risk factor for the development of diabetes or as a condition that may exist prior to diabetes and / or beta cell failure after clinical pancreatic islet transplantation.

[00105] Furthermore, the antibodies of the present invention, binding molecules that have substantially the same binding specificities as any of them, the polynucleotides, vectors or cells of the present invention are used in the preparation of a composition for detecting changes , that is, an increase or decrease in amylin secretion when compared to amylin secretion in a healthy individual with a differential diagnosis of type 1 diabetes, latent autoimmune diabetes in adults (LADA, type 1.5 diabetes) compared to forms of DT2, or in disorders that precede a manifest DT2, such as gestational diabetes or pre-diabetes; in diseases that damage the pancreas and can therefore lead to diabetes such as chronic pancreatitis, cystic fibrosis, pancreatic cancer; in diseases that increase the risk of T2D like Alzheimer's disease, Huntington's disease; and / or cardiovascular diseases associated or not with obesity and T2D.

[00106] Disorders such as obesity and hyperinsulinemia / insulin resistance are very common as a pre-disposition and / or as a symptom of DT2, which can lead to an increase in circulating levels of islet amyloid polypeptide (IAPP) already in the face of the manifest form of DT2. Oligomers, fibrils and amylin plaques (hlAPP) have been found to accumulate not only within the pancreas and kidneys, but also within the heart of patients with obesity and insulin resistance. This accumulation was observed in association with altered cellular homeostasis in felines, which, in turn, may contribute to cardiac dysfunction in these patients.

Therefore, in one embodiment, the antibodies of the present invention, binding molecules that have substantially the same binding specificities as any of them, the polynucleotides, vectors or cells of the present invention are used in the preparation of a composition of diagnostic or pharmaceutical for therapeutic and prophylactic treatment, improvement, monitoring of progress or a response to treatment and / or diagnosis of a group of disorders after DT? 2 or resulting from previous metabolic changes and / or causing DT2 , that is, the metabolic changes that occur in the pre-diabetic phase, in which the group of disorders comprises heart disease, strokes, diabetic retinopathy, renal failure, ketoacidosis and hyperosmolar coma in the ketosis.

[00108] More than 20 neurodegenerative disorders (see, for example, Table 1 on page 511 in M. Ristow, J. Mol. Med 82 (2004), 510-529) are known to be associated with diabetes mellitus, increased insulin resistance and obesity, disturbed insulin sensitivity, and impaired or excessive insulin secretion. Therefore, in one embodiment of the present invention, its antibodies, binding molecules that have substantially the same binding specificities as any of them, the polynucleotides, vectors or cells of the present invention are used in the preparation of a diagnostic or pharmaceutical composition for therapeutic and prophylactic treatment, monitoring progress or a response to the treatment and / or diagnosis of neurodegenerative disorders, including Alzheimer's disease (AD), ataxia telangiectasia (AT), Bardet-Bied! l syndrome (BBS), Friedreich's ataxia (FRDA), Huntington's disease, myotonic dystrophy, narcolepsy, Parkinson's disease, Prader-Willi syndrome and Werner syndrome.

[00109] Reducing glucose tolerance does not induce complications typical of diabetes mellitus, but presents a greater risk for the appearance of diabetes mellitus than in the normal type, which can be a cause of macrovascular diseases.

[00110] "Insulin resistance" is the condition of reduced insulin sensitivity. Insulin is known to have a wide range of effects, for example, in addition to an effect on glucose metabolism, an effect on lipid metabolism or effects on blood vessels and the kidney. After the reduction of insulin sensitivity, not only the metabolic anomaly of glucose can be induced, but also the metabolic anomaly of lipids such as hypertriglycemia, decrease in the level of HDL in the plasma or hypertension due to the anomaly of the effects of insulin in the blood vessels or in the kidney.

[00111] As used herein, the term "diabetic" in a human being generally and presently means a random concentration of glucose in the blood or plasma of 2200 mg / dL (211.1 mmol / L) or a fasting glucose 2126 mg / dL (27.0 mmol / L) or a blood glucose level 2 hours after loading = 200 mg / dL (211.1 mmol / L) during an oral glucose tolerance test. In addition, or as an alternative, the term “diabetic” is used for individuals who have one or more of the clinical symptoms of diabetes, including increased thirst (polydipsia), frequent urination (polyuria), extreme hunger, unexplained weight loss, tiredness and blurred vision, vulnerability to slow healing wounds and frequent infections, including bladder, vagina and gums and / or areas of darkened skin (acanthosis nigricans).

[00112] As used herein, the term “non-diabetic” in a human being generally and currently means a fasting glucose level of <100 mg / dL (5.6 mmol / dL) or a blood glucose level 2 hours afterload <140 mg / dL (<7.8 mmol / dL) during an oral glucose tolerance test.

[00113] As used herein, the term “pre-diabetic” in a human being generally and currently means a fasting glucose level of 100 to 125 mg / dL (5.6 to 6.9 mmol / L) or a 2-hour afterload blood glucose level of 140 to 199 mg / L (7.8 to 11.1 mmol / L) during an oral glucose tolerance test. Unless otherwise indicated, the terms "pre-diabetic", "prodromal" and "pre-symptomatic" are used interchangeably here and describe the pre-clinical phase of DT2.

[00114] In addition or as an alternative, levels of glycosylated hemoglobin (HbAlc) can be used to diagnose diabetes in an individual. At high levels of HbAlc, at the threshold of 6.5% or beyond, the diagnosis of diabetes is made, that is, the term “diabetic” is used in general and currently in a human being, while levels of 5.7% 6.4% point to a high risk of developing both diabetes and cardiovascular diseases, and are indicators of “pre-diabetes”, or of a “pre-diabetic / pre-symptomatic” state in a human being. The term "non-diabetic" in a human being, in general and currently, means HbAlc levels below the 5.7% threshold. Rodents Models of Type II Diabetes Mellitus in Drug Discovery

[00115] Examples of conventionally communicated 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, 503-508, 1995), db / db mice (Hummel KP, et al., Science 153,

44/139 1127-1128, 1966), ob / ob mice (Herberg L. & Kley HK, Horm. Metab. Res. 7, 410-5, 1975), and AKITA mice (Yoshioka M ,, et a /., Diabetes 46, 887-894, 1997).

[00116] Among these animals, the KK-Ay and NSY mice are models with obesity and the db / db and ob / ob mice are models with obesity due to an abnormality in leptin receptors or leptin production. On the other hand, AKITA mice are models for diabetes caused by an abnormality in pancreatic B cells.

[00117] As a non-obese animal model with Type | 1 diabetes, for example, a mouse model is, to date, reported in Japanese Patent Application No. 2004-65181. This mouse has abnormal insulin secretion.

[00118] However, the antibodies of the present invention are preferably tested and characterized in transgenic animals, such as, for example, rodents showing hIAPP as transgenic mice for human amylin in pancreatic B cells (HIP rats) as described in Butler et al ., Diabetes 53 (2004), 150971516 and in Matveyenko & Butler, Diabetes 55 (2006), 2106-2114. Most preferably, murine models with type 2 diabetes with excessive human IAPP are used as described in Matveyenko & Butler (2006), ILAR J. 47 (3): 225-233, and summarized in Table 2, page 228 of the document. mentioned. Due to the excessive amount of hlAPP, these animal models validly exhibit DT2 symptoms, such as, for example, the hyperglycemic state. In general, the term "hyperglycemia" or "hyperglycemic state" refers to significantly higher levels of fasting glucose in two consecutive measurements taken at different time intervals and when compared to that of non-transgenic control subjects. The absolute values measured in hyperglycemic animals may depend on the particular animal model used, for example, in h-IAPP (hemizygous) / ob / + of mice, 2-15-20 mM glucose; in h-IAPP (homozygous / FVB / N of mice 2-11mM glucose.

[00119] Among the methods for the study of diabetes are the management of physiological changes and the analysis of blood or plasma of the diabetic in comparison with healthy and non-diabetic animals. These measures include, but are not limited to growth dynamics, body mass index (BMI), lean mass index (BMI), food and water intake, gender differences, random blood glucose level and fasting, triglycerides (TG), lipoproteins, cholesterol, liver weight and liver lipids, size and kidney function, a glucose tolerance test (TTG), a tolerance test to insulin (TTI), blood insulin concentration, pancreatic islet cell morphology, high fat diets and calorie restrictions.

[00120] As used here, the terms "random" and "no fasting", in general, mean anytime during the day or night, regardless of the time since the last meal.

[00121] As used here, the term “fasting”, in general, means nullity of calorie injection for at least 12 hours.

[00122] It is recognized that these definitions are currently accepted guidelines that, in general, practitioners adopt according to the North American Diabetes Association (ADA) and the German Diabetes Association (GDA). Guidelines may change over time and vary depending on the region or country, and depend on the group or institution (eg ADA, World Health Organization, NIDDK / NIH, CDC, GDA, etc.) that provides the guidelines, known to those skilled in the industry. Doctors can also use clinical experience, the patient's medical history, and / or other information when deciding on a diagnosis and treatment. These definitions can therefore change over time according to advances in science and medicine. Treatment

[00123] As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventive measures whose objective is to prevent or delay (decrease) an disorder or an unwanted physiological change, such as, the development of diabetes. Beneficial or desired clinical results include, but are not limited to, relieving symptoms, decreasing the extent of the disease, stabilizing (ie not worsening) the disease state, delaying or slowing the progression of the disease, improvement or palliation of the disease state, and remission (whether total or partial), whether detectable or undetectable.

46/139 “Treatment” can also mean prolonged survival compared to the expected survival in the case of not receiving treatment. Those in need of treatment include those who already have the disease or disorder, as well as those likely to develop the disease or disorder, or those in which the manifestation of the disease or disorder is to be prevented.

[00124] If there is no indication to the contrary, the terms "drug", "medicine" or "medicine" are used interchangeably here and must include, but are not limited to, all (A) articles, medicines and preparations for use internal or external, and any substance or mixture of substances intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease in both men and other animals; and (B) articles, medicines and preparations (except food) intended to affect the structure or any function of the body of man or other animals; and (C) articles intended for use as a component of any article specified in clauses (A) and (B). The terms "drug", "medicine" or "medicine" must include the complete formula of the preparation intended for use in both man and other animals, containing one or more "agents", "compounds", "substances" or "compositions ( chemicals) ”as and in some other context, including other pharmaceutically inactive excipients such as fillers, disintegrating agents, lubricants, glidants, binders or guaranteeing ease of transportation, disintegration, disintegration, dissolution and biological availability of the“ drug ”,“ medicine ”or “Medicine” to a desired destination within the body of man or other animals, such as on the skin, stomach or intestine. The terms "agent", "compound", "substance" are used interchangeably here and should comprise, in a more particular context, but without being limited to all pharmacologically active agents, that is, agents that induce a biological effect or pharmacological effect or that are investigated or tested in their ability to induce this possible pharmacological effect by the methods of the present invention.

[00125] By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject, particularly a mammal subject, for example, a human patient, for whom diagnosis, prognosis, prevention or therapy. Pharmaceutical administration agents

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[00126] Pharmaceutically acceptable administration agents and routes of administration can be obtained in the corresponding literature known to the qualified members of the field. The pharmaceutical compositions of the present invention can be formulated according to methods well known in the art; see, for example, Remington: The Science and Practice of Pharmacy (2000) by the Philadelphia University of Sciences, ISBN 0-683-306472, Vaccine Protocols. 2 "º 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 & Francis (2006), ISBN: 0 Examples of suitable pharmaceutical administration agents are well known in the art and include phosphate buffered saline solutions, water, emulsions such as oil / water, various types of wetting agents, sterile solutions, etc. Compositions comprising such delivery agents can be formulated by conventional, well known methods.These pharmaceutical compositions can be administered to the subject in an appropriate dose.

[00127] The administration of the appropriate compositions can be carried out in different ways. Examples include administering a composition containing a pharmaceutically acceptable delivery agent by oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods. Aerosol and nasal spray formulations include purified aqueous solutions or other solutions of the active agent with preservative and isotonic agents. These formulations are preferably adjusted based on a pH and an isotonic state compatible with the nasal mucous membranes. Pharmaceutical compositions for oral administration, such as simple domain antibody molecules (eg "nanobodies'Y"), etc., are also provided for in the present invention. These oral formulations can take the form of a tablet, capsule, powder, liquid or semi-solid. A tablet can comprise a solid delivery agent, such as gelatin or an adjuvant. Formulations for rectal or vaginal administration can be presented as a suppository with a suitable delivery agent; see also O'Hagan et al., Nature Reviews, Drug Discovery 2 (9) (2003), 727-735. Other guidelines for formulations that are suitable for various types of administration may

48/139 be found in Remington's work, Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17 "ed. (1985) and corresponding updates. For a brief analysis of methods for drug delivery, see Langer, Science 249 ( 1990), 1527-1533.Ill Antibodies of the present invention

[00128] The present invention relates generally to human anti-APP antibodies and antigen-binding fragments thereof, which preferably demonstrate the immunological binding characteristics and / or biological properties, as described for the antibodies illustrated in the examples . In accordance with the present invention, human monoclonal antibodies specific for IAPP and / or prolAPP have been cloned from a group of healthy human subjects.

[00129] During the experiments carried out in accordance with the present invention, antibodies in conditioned media of cultures with human memory B cells were screened in parallel for binding to aggregated oligomeric, fibrillar and / or non-fibrillar IAPP proteins and / or prolAPP - and bovine serum albumin (BSA). Only cultures of B cells positive for aggregated proteins of IAPP and / or prolAPP, but not for BSA were subjected to antibody cloning.

[00130] Due to this measure, several antibodies could be isolated. Selected antibodies were further analyzed for light chain class and subclass determination. Selected messages of relevant antibodies from memory B cell cultures are then transcribed by RT-PCR, cloned and combined into expression vectors for recombinant production; see the attached examples.

[00131] The recombinant expression of human antibodies in HEK293 or CHO cells and the further characterization of their specificities of binding to the human protein of IAPP and / or prolAPP (Figs. 3 and 4; Example 1), and their distinct binding to pathological forms aggregates of it (Fig. 5; Example 2) confirmed that, for the first time, human antibodies have been cloned and are highly specific for IAPP and / or prolAPP protein and distinctly recognize the pathologically aggregated forms of the IAPP and / or prolAPP protein, such as, IAPP fibrils. A second round of experiments confirmed the above conclusions, as

49/139 shown in Figs. 7 to 9 and in Example 4. In addition, murine chimeric antibodies generated in accordance with the present invention and which comprise CDR domains of the human antibodies of the present invention showed an equal affinity for binding to human IAPP, as the human antibodies illustrated in Figs . 11 and 12 and in the Example

6.

[00132] Therefore, the present invention generally relates to a naturally occurring monoclonal anti-islet human amyloid polypeptide (IAPP) antibody and to binding fragments, derivatives and variants thereof. In one embodiment of the invention, the antibody is capable of binding human IAPP and / or prolAPP.

[00133] In one embodiment, the present invention relates to an anti-| APP and / or anti-prolAPP, or antigen-binding fragment, variant or derivatives thereof, where the antibody specifically binds to the same epitope as IAPP as a reference antibody selected from the group consisting of NI-203.9A2, NI-203.19 H8, NI-

203.26C11, NI-203.8E3, NI-203.11B12, NI-203.205F8, NI-203.9B3, N1I-203.19F2 and NI-

203.15C7. Epitope mapping identified a sequence within the human IAPP, including aa 19-SSNNFGA-25 (SEQ ID: 4) as the only linear epitope recognized by the NI-203.19H8 antibody of this invention, and a sequence within the human IAPP, including aa 2-CNTATCA-8 (SEQ ID No.: 5) as the only linear epitope recognized by the NI-203.26C11 antibody of this invention, and a sequence within the human IAPP, including aa 10-QORLANFLVHS-19 (SEQ ID No.: 71) as the only linear epitope recognized by the NI-203.15C7 antibody of this invention (see Figs. SAe 5Be Example 3). Therefore, in one embodiment, the antibody of the present invention is provided, wherein the antibody specifically binds an IAPP epitope, which comprises the amino acid sequence SSNNFGA (SEQ ID NO: 4), CNTATCA (SEQ ID NO: 5) or QRLANFLVHS (SEQ ID No.: 71). As described in detail in Example 3, the epitopes of the recombinant IAPP antibodies NI-203.9A2, NI-203.8E3 and NI-203.19F2 have not been identified so far, indicating that these antibodies are likely to bind non-linear epitopes .

[00134] Furthermore, in one embodiment, the present invention relates to an anti-| APP and / or anti-prolAPP, or antigen-binding fragment, variant or derivatives thereof, where the antibody specifically binds to the same prolAPP epitope

50/139 as a reference antibody selected from the group consisting of NI-

203.1D10, NI-203.2A11, NI-203.10C4, NI-203.20H9, NI-203.26D2, NI-203.60H3 and NI-

203.26C 11.

[00135] Furthermore, without the intention of being limited by the initial experimental observations, as shown in Example 4 and shown in Fig. 7, human monoclonal antibodies anti- | APP NI-203.9A2, NI-203.19H8, NI- 203.26C11 and NI-203.8E3 of the present invention are preferably characterized by binding specifically to pathological IAPP aggregates (fibrils, in this case) and by not substantially recognizing IAPP in its physiological form in the pancreas. There is an expectation that the same thing will apply to human anti-IAPP NI-203.19F2 and NI-203.15C7 monoclonal antibodies. Therefore, the present invention provides a set of human anti-APP and / or antinprolAPP antibodies with binding specificities, which are therefore particularly useful for therapeutic and diagnostic purposes. Thus, in one embodiment, the present invention provides antibodies, which are capable of specifically binding pathologically aggregated forms of IAPP and / or prolAPP (see Fig. 5 and Figs. 7a 9 and Examples 2 and 4). For more details and a brief overview of the connection specifics of the present invention, see also Figs. 7 and 8 and the description below.

[00136] In one embodiment, the antibody of the present invention exposes the binding properties of exemplary antibodies NI-203.9A2, NI-203.19H8, NI-203.26C11, NI-

203.8E3, NI-203.19F2, and NI-203.15C7, as described in the Examples. In addition, or as an alternative, the anti- | APP and / or anti-prolAPP of the present invention preferably recognizes pathologically aggregated anti-| APP and / or anti-prolAPP as IAPP fibrils, rather than the physiological monomers of IAPP and / or prolAPP, especially when analyzed according to Examples 2 to 4. In such an embodiment, the antibody of the present invention does not substantially recognize the physiological IAPP. The term "does not substantially recognize" when used in this application to describe the binding affinity of a molecule in a group comprising an antibody, a fragment thereof or a binding molecule for an antigen-, conformation- and / or molecule- specific antigen- and / or target molecule targets means that the molecule of the aforementioned group binds that molecule, antigen and / or conformation with a binding affinity that is at least 2 times, 3 times, 4 times, 5 times, 6 times , 7 times, 8 times or 9 times less than the dissociation constant (1 (D) of the molecule in the aforementioned group to bind another molecule, antigen and / or conformation. Preferably, the term “does not substantially recognize”, when used in this application , means that the molecule of the aforementioned group binds that molecule, antigen and / or conformation with a binding affinity that is at least 10 times, 20 times, 50 times, 100 times, 1,000 times or 10,000 times less than the dissociation constant ( 1 (D) city the molecule of the aforementioned group for binding to another molecule, antigen and / or conformation. In addition, or as an alternative, the anti- | APP and / or anti-prolAPP antibody of the present invention binds to the disease-causing aggregate forms of human anti- | APP and / or anti-prolAPP, especially those described in Example 4 In this context, binding specificities may be in the range, as shown for exemplary antibodies NI-203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3 in Fig. 4, respective Fig. 5, ie , which have effective concentrations at half the maximum (EC50) of about 1 pM to 500 nM, preferably an EC50 of about 10 pM to 100 nM, more preferably, an EC50 of about 100 pM to 50 nM for IAPP human, as shown for NI-203.19H8, or an ECB50 of about 100 pM to 10 nM for human IAPP, as shown for NI-

203.9A2, NI-203.26C11 and NI-203.8E3. In this context, the experimental results provided in Example 4 and illustrated in Fig. 4, respective Fig. 5, also indicate that, in addition or as an alternative, some of the anti- | APPs of the present invention do not substantially recognize prolAPP as shown for exemplary antibodies NI-203.9A2, NI-203.19H8 and NI-203.8E3. In such an embodiment, the antibody of the present invention does not substantially recognize prolAPP.

[00137] In addition, or as an alternative, the anti-lAPP antibody of the present invention binds specifically, in addition to maturing IAPP to the precursor form of IAPP, that is, prolAPP too, especially as described in Example 1. In this context , the binding specificities can be in the range, as shown for the exemplary antibody NI-203.26C11 of Fig. 3, respective Fig. 4, that is, which have the effective concentrations at half the maximum (EC50) of about 1 pM at 500 nM, preferably an EC50 from about 10 pM to 400 nM, more preferably an EC50 from about 100 pM to 400 nM, or an EC50 from about 100 pM to 300 nM, more preferably an EC50 from about 1 nM to 300nM for aggregated pro-IlAPP, as shown for NI-203.26C11.

[00138] In addition, or as an alternative, the anti-| lAPP antibody of the present invention binds specifically to mature IAPP and does not bind or substantially bind to the precursor form of IAPP, that is, prolAPP, especially as described in Example 1. In this context, binding specificities may be in the range, as shown for exemplary antibodies NI-203.9A2, NI-203.19H8 and NI-

203.8E3 in Fig. 4, respective Fig. 5 and indicated above.

[00139] In one embodiment, the antibody of the present invention exhibits the binding properties of exemplary antibodies NI-203.1D10, NI-203.2A11, NI-203.10C4, NI-

203.20H9, NI-203.26D2 and NI-203.60H3, preferably linking prolAPP over prolAPP. However, prolAPP antibodies were obtained by screening for antibodies that specifically bind to the N-terminal flanking region of prolAPP, which is not present in IAPP, as there is some evidence of the role of unprocessed prolAPP with N- terminal, instead of the integral prolAPP peptide in amyloid formation and cell death. Thus, in one embodiment, the anti-prolAPP antibody of the present invention, such as the exemplary antibodies NI-203.1D10, NI-203.2A11, NI-203.10C4, NI-203.20H9, NI-203.26D2 and NI- 203.60H3 do not bind substantially or do not bind IAPP.

[00140] Some purified antibodies bind to a wide variety of biomolecules, for example, proteins. As someone who is specialized in the field can attest, the specific term is used here to indicate that biomolecules other than IAPP and / or prolAPP proteins or fragments thereof do not bind significantly to the antigen binding molecule, for example, one of the antibodies of the present invention. Preferably, the level of binding to a biomolecule other than IAPP and / or prolAPP results in a binding affinity that is at most only 20% or less, 10% or less, only 5% or less, only 2% or less or only 1% or less (ie at least 5, 10, 20, 50 or 100 times less) of affinity for IAPP and / or prolAPP, respectively; see, for example, Example 1 and Fig. 3. In addition, the anti- | APP and / or anti-prolAPP antibodies of the present invention specifically bind to aggregates of IAPP and / or prolAPP, as validated by experiments that demonstrate that the antibodies of the present invention do not recognize pathological AB amyloid in a human brain with Alzheimer's disease and have merely minimal cross-reactive binding qualities to several candidate proteins with abnormalities of configuration / aggregation, as shown with exemplary NI antibodies -

203.9A2, NI-203.19H8 and NI-203.26C11 in paraffin-embedded brain sections of a patient diagnosed with Alzheimer's disease and by direct ELISA experiments; see Example 5 and Fig. 10. Therefore, in one embodiment, the antibody of the present invention is provided, and it does not substantially recognize the amyloid B peptide (AB 1-42).

[00141] In one embodiment, the anti- | APP and / or anti-prolAPP antibody of the present invention preferably binds to aggregated forms of IAPP and / or prolAPP, fibrils of IAPP and / or prolAPP and / or oligomers; see experimental results by direct ELISA in Example 2 and in pancreases of patients diagnosed with T2D and in pancreases of diabetic cats by means of the immunohistochemical staining described in Example 4 and illustrated in Figs. 7 and 9, respectively. In one embodiment, the antibody of the present invention preferably binds to aggregated forms of IAPP and / or prolAPP, in which the aggregates comprise, are essentially composed of or are composed of fibrillar forms and / or fibrillar oligomers of IAPP. In another embodiment, the antibody of the present invention preferentially binds to aggregated forms of IAPP and / or prolAPP, in which the aggregates comprise, are essentially composed of or are composed of non-fibrillar forms and / or non-fibrillar oligomers of IAPP. In yet another embodiment, the antibody of the present invention preferably binds to aggregated forms of IAPP and / or prolAPP, in which the aggregates comprise, are essentially composed of or are composed of, or are fibrillar or non-fibrillar forms of IAPP and / or prolAPP and / or fibrillar and non-fibrillar oligomers of IAPP and / or prolAPP. In yet another embodiment, the anti-lAPP and / or anti-prolAPP antibody of the present invention preferably binds both to the native form of IAPP and / or prolAPP and to the pathologically aggregated forms of IAPP and / or prolAPP; see experimental results as exemplified in Examples 2 and 3 by direct ELISA.

[00142] As mentioned before, aggregates comprising IAPP and / or prolAPP can also be found associated with amyloid deposits in islets

54/139 pancreatic patients of T2D patients. Therefore, in one embodiment, the antibody of the present invention can be useful in the treatment of DT 2.

[00143] The present invention is also attracted to an antibody, or antigen-binding fragment, variant or derivatives thereof, where the antibody comprises an antigen-binding domain identical to that of an antibody selected from the group consisting of NI-203.9A2 , NI-203.19H8, NI-203.26C11, NI-203.8E3, N1I-203.11B12, NI-

203.205F8, NI-203.9B3, NI-203.19F2, NI-203.15C7, NI-203.1D10, NI-203.2A11, NI-

203.10C4, NI-203.20H9, NI-203.26D2 and NI-203.60H3.

[00144] The present invention further exemplifies several of these binding molecules, eg, antibodies and binding fragments thereof, which can be characterized by comprising, in their variable region, eg: binding domain in at least one determining region of complementarity (CDR) of the VH and / or Vi variable region comprising any of the amino acid sequences described in Fig. 1 or Fig.

2. The corresponding nucleotide sequences encoding the variable regions identified above are shown in Table | 1, respective Table III below. Exemplary sets of CDRs from the amino acid sequences above the Vr and / or V. regions are represented in Fig. 1 and Fig. 2. However, as discussed below, a person skilled in the field is well aware of the fact that, in addition to In addition or as an alternative, CDRs can be used, which differ in their amino acid sequence from those shown in Fig. 1, respectively in Fig. 2 by one, two, three or even more amino acids in the case of CDR2 and CDR3. Therefore, in one embodiment, the antibody of the present invention or an IAPP and / or prolAPP binding fragment thereof is provided, comprising, in its variable region, at least one complementarity determining region (CDR), as shown in Fig. 1 and / or one or more CDRs thereof, comprising one or more amino acid substitutions.

[00145] In one embodiment, the antibody of the present invention is any of the antibodies that comprise an amino acid sequence of the VH and / or V. region, as illustrated in Fig. 1 or a Vnx and / or Vi region thereof, which comprises one or more amino acid substitutions. Preferably, the antibody of the present invention is characterized by preserving the cognate pairing of the heavy and light chain as it was present in the human cell.

55/139

[00146] As an alternative, the antibody of the present invention is an antibody or antigen-binding fragment, derived or variant thereof, which competes to bind IAPP and / or prolAPP with at least one of the antibodies having the Vx region and / or V. as shown in Fig. 1.

[00147] The experimental results provided in Example 4 suggest that some of the anti-| APP and / or anti-prolAPP antibodies of the present invention preferentially bind disease-causing aggregate forms of human anti-| APP and / or anti-prolAPP about the physiological forms of proteins. In such an embodiment, the antibody of the present invention preferably recognizes aggregates of IAPP and / or prolAPP over physiological IAPP and / or prolAPP. In addition, in one embodiment, the antibody of the present invention preferably recognizes aggregates of IAPP that comprise fibrils and / or IAPP oligomers over the physiological IAPP. In another embodiment, the antibody of the present invention preferably recognizes aggregates of IAPP which comprise oligomers of non-fibrillary IAPP and / or non-fibrillary IAPP over the physiological IAPP.

[00148] As previously indicated, some of the antibodies of the present invention have proved capable of binding both IAPP and its precursor form, prolAPP. In addition, some of the antibodies of the present invention have been isolated from human patients due to their ability to bind prolAPP. Therefore, in one embodiment, the antibody of the present invention is capable of binding prolAPP.

Therefore, as an alternative or in addition to the above, in one embodiment, the antibody of the present invention or an IAPP and / or prolAPP binding fragment thereof is provided, comprising in its variable region at least one complementarity determining region (CDR), as illustrated in Fig. 2, and / or one or more CDRs thereof, comprising one or more amino acid substitutions.

[00150] In one embodiment, the antibody of the present invention is any of the antibodies that comprise an amino acid sequence of the VH and / or V. region, as illustrated in Fig. 2 or a Vx and / or V. region thereof, comprising one or more amino acid substitutions. Preferably, the antibody of the present invention

56/139 is characterized by the preservation of the cognate pairing of the heavy and light chain as it was present in human cell 8.

[00151] Alternatively, the antibody of the present invention is an antibody or antigen-binding fragment, derived or variant thereof, which competes to bind IAPP and / or prolAPP with at least one of the antibodies that have the VH region and / or V. as shown in Fig. 2.

[00152] The antibody of the present invention can be human, especially for therapeutic applications. Alternatively, the antibody of the present invention is a rodent, rodent or chimeric rodent-human, preferably murine, murinized or chimeric murine-human or rat, ratinized or chimeric rat-human antibody, which are particularly useful for diagnostic methods and animal studies. In one embodiment, the antibody of the present invention is a rodent-human or chimeric rodent antibody.

[00153] Furthermore, in one embodiment, the chimeric antibody of the present invention exhibits the binding properties of exemplary murine chimeric antibodies NI-

203.9A2, NI-203.19H8 and NI-203.26C11 as described in the Examples. In addition, the chimeric antibodies of mice of the present invention bind with high affinity to human IAPP fibrils, as described in Example 6. Preferably, the binding affinity of the chimeric antibodies is similar to that of their human counterparts. In this context, binding specificities may be in the range, as shown for the exemplary murine chimeric antibodies NI-203.9A2, NI-

203.19H8 and NI-203.26C11 with an EC50 of 18.6 nM, 23.9 nM and 11.5 AM, respectively as described in Example 6 and shown in Fig. 11 and Table C thereof. There was no link to the BSA.

[00154] In one embodiment, the antibody of the present invention is supplied by cultures of single or oligoclonal B cells that are cultured and the culture supernatant, which contains antibodies produced by said B cells, is screened for verification. presence and affinity of anti- | APP and / or prolAPP of the same. This screening process comprises a screening in search of binding to native monomeric, fibrillar or non-fibrillar aggregates such as hlAPP oligomers from

57/139 of a synthetic hlAPP peptide integral to the amino acid sequence represented by SEQ ID No.: 1; and / or a separate and independent screening for binding to a synthetic peptide derived from human prolAPP (N-terminal fragment) of the amino acid sequence TPIESHQVEKRKCNTATCATOR represented by SEQ ID No.: 7.

[00155] In addition or as an alternative, the screening process for the presence and affinity of anti- | APP and / or prolAPP antibodies may comprise the steps of a sensitive tissue amyloid plaque (TAPIR) immunoreactivity assay as described in international application W02004 / 095031, whose content disclosure is hereby incorporated by reference, made here by analogy for amyloid deposits in pancreatic islets. In addition or as an alternative, screening in pancreatic sections for binding to anti- | APP and / or prolAPP as described in analogy in international application W02008 / 081008 for sections of the brain and spinal cord can be performed.

[00156] As mentioned above, due to its generation through a human immune response, the human monoclonal antibody of the present invention will recognize epitopes that are of particular pathological relevance and that may not be accessible or less immunogenic in the case of immunization processes for the generation of, for example, mouse monoclonal antibodies and in vitro screening of phage display libraries, respectively. Thus, it is prudent to stipulate that the epitope of the human anti-APP and / or prolAPP antibody of the present invention is unique and that there is no other antibody capable of binding to the epitope recognized by the human monoclonal antibody of the present invention; see also Fig. 5A and showing the only epitope of the NI-203.19H8 antibody, respective NI-203.26C11 antibody of the present invention. Another indication of the uniqueness of the antibodies of the present invention is the fact that, as indicated in Example 3, the NI-203.9A2 and NI-203.8E3 antibodies of the present invention bind assumptible conformational epitopes of IAPP aggregates, which, as indicated above, they are of particular pathological relevance and may also be impossible to obtain by the usual antibody generation processes such as immunization or in vitro library screenings.

58/139

[00157] Therefore, in one embodiment, the present invention also generally extends to anti-| APP and IAPP binding molecules that compete with the human monoclonal antibody of the present invention for specific binding to IAPP. The present invention is more specifically directed to an antibody, or antigen-binding fragment, variant or derivative thereof, where the antibody specifically binds to the same epitope of IAPP as a reference antibody selected from the group consisting of NI-203.9A2, NI-203.19H8, NI-203.26C11, NI-

203.8E3, NI-203.19F2 and NI-203.15C7.

[00158] In addition, in one embodiment, the present invention also generally extends to anti-| APP and / or anti-prolAPP and IAPP and / or anti-prolAPP binding molecules that compete with the human monoclonal antibody of the present invention for specific binding to prolAPP. The present invention is therefore more specifically also directed to an antibody, or antigen-binding fragment, variant or derivatives thereof, where the antibody specifically binds to the same prolAPP epitope as a reference antibody selected from the group consisting of NI -203.1D10, NI-203.2A11, NI-203.10C4, N1I-203.20H9, N1I-203.26D2, NI-

203.60H3 and NI-203.26C 11.

[00159] Furthermore, or as an alternative, the present invention also generally extends to bispecific anti-| APP and / or anti-prolAPP and IAPP antibodies and / or to anti-prolAPP binding molecules that compete with the human monoclonal antibody of the present invention for specific binding to both IAPP and prolAPP. The present invention is therefore more specifically also directed to an antibody, or antigen-binding fragment, variant or derivatives thereof, where the antibody specifically binds to the same epitope of IAPP and prolAPP as the exemplary antibody NI-203.26C11. In view of the above, therefore, in one embodiment, the present invention also relates to an antibody or antigen-binding molecule that competes with an antibody of the present invention for specific binding to IAPP and / or prolAPP.

[00160] Antibody competition is determined by an assay in which the immunoglobulin under test inhibits the specific binding of a reference antibody to a common antigen, such as, for example, IAPP and / or prolAPP. Various types of

59/139 competitive binding are known, for example: solid phase radioimmunoassay (RIA), direct or indirect, solid phase enzyme immunoassay (EIA), direct or indirect, sandwich competition assay; see Stahli et al., Methods in Enzymology 9 (1983), 242-253; Direct phase solid avidin-biotin EIA; see Kirkland et al., J. Immunol. 137 (1986), 3614-3619 & Cheung et a / l., Virology 176 (1990), 546-552; marked, direct phase solid assay; direct solid phase marked sandwich test; see Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988); Direct, solid phase labeled RIA, using 1125 marking; see Morel et al., Molec. Immunol. 25 (1988), 7-15 and Moldenhauer et a /., Scand. J. Immunol. 32 (1990), 77-82. Typically, this assay involves the use of purified IAPP and / or prolAPP or aggregates, such as oligomeres and / or fibrils thereof, attached to a solid surface or cells carrying any of these, an unmarked test immunoglobulin and a labeled reference immunoglobulin, that is, the human monoclonal antibody of the present invention. Competitive inhibition is measured by determining the amount of markers bound to the solid surface or cells in the presence of the test immunoglobulin. The test immunoglobulin is normally present in excess. Preferably, the competitive binding assay is carried out under conditions such as those described for the ELISA assay in the attached Examples. Antibodies identified by competitive assay (competing antibodies) comprise antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an adjacent epitope close enough to the epitope bound by the reference antibody for stereochemical impairment to occur. Typically, when a competing antibody is present in excess, it will inhibit the specific binding of a reference antibody to a common antigen by at least 50% or 75%. Thus, the present invention is even more attracted to an antibody, or antigen-binding fragment, variant or derivative thereof, where the antibody competitively inhibits a reference antibody selected from the group consisting of NI-203.9A2, NI-203.19 H8, NI-203.26C11, NI-

203.8E3, NI-203.19F2 and NI-203.15C7 to connect to IAPP.

[00161] Furthermore, the present invention is even more attracted to an antibody, or fragment of binding to the antigen, variant or derivatives thereof, where the antibody competitively inhibits a reference antibody selected from the compound group

60/139 by NI-203.1D10, NI-203.2A11, NI-203.10C4, NI-203.20H9, NI-203.26D2, NI-203.60H3 and NI-203.26C11 to bind prolAPP.

[00162] Even more than that, or as an alternative, the present invention is even more attracted to a bispecific antibody, or antigen-binding fragment, variant or derivatives thereof, where the antibody competitively inhibits a reference antibody, such as for example, the exemplary NI-203.26C11 antibody to bind to either IAPP or prolAPP.

[00163] In another embodiment, the present invention provides an isolated polypeptide that comprises, composed essentially of, or composed of an immunoglobulin heavy chain (VH) variable region, wherein at least one of the Vx-CDRs of the variable chain region heavy or at least two of the heavy chain variable region VH-CDRs are at least 80%, 85%, 90% or 95% identical to the reference heavy chain VA-CDR1, VA-CDR2 or VH-CDR3 amino acid sequences from the antibodies described here. Alternatively, Vx VH-CDR1, Vn-CDR2 and Vx-CDR3 regions are at least 80%, 85%, 90% or 95% identical to the VHA-CDR1, V - CDR2 and VH-CDR3 amino acid sequences reference heavy chain from the antibodies described here. Thus, according to that embodiment, a heavy chain variable region of the invention has the VH-CDR1, VH-CDR2 and VH-CDR3 polypeptide sequences related to the groups shown in Fig. 1 or Fig. 2, respectively. While Figs. 1 and 2 show VH-CDRs defined by the Kabat system, other definitions of CDRs, for example, V4-CDRs defined by the Chothia system, are also included in the present invention, and can be easily identified by a person with common skill in the field , using the data presented in Fig. 1 and Fig. 2.

[00164] In another embodiment, the present invention provides an isolated polypeptide comprising, essentially composed of, or composed of an immunoglobulin heavy chain (VH) variable region, where the V-CDR1, V4-CDR2 and Vn- CDR3 show the polypeptide sequences that are identical to the Vn-CDR1, Vr-CDR2 and V4-CDR3 groups shown in Fig. 1 or Fig. 2, respectively.

[00165] In another embodiment, the present invention provides an isolated polypeptide comprising, essentially composed of, or composed of an immunoglobulin heavy chain (Vx) variable region in which the VH-CDR1, V4-CDR2 and Vn-CDR3 regions show the polypeptide sequences that are identical to the Vn-CDR1, V4-CDR2 and VH-CDR3 groups shown in Fig. 1 or Fig. 2, respectively, except for one, two, three, four, five or six amino acid substitutions on any individual VH-CDR. In certain embodiments, amino acid substitutions are conservative.

[00166] In another embodiment, the present invention provides an isolated polypeptide comprising, composed essentially of, or composed of an immunoglobulin light chain (VL) variable region, wherein at least one of the VL-CDRs of the variable chain region light or at least two of the light chain variable region VL-CDRs are at least 80%, 85%, 90% or 95% identical to the light chain VL-CDR1, V.-CDR2 or VL-CDR3 amino acid sequences reference from the antibodies described here. Alternatively, the V1-CDR1, V-CDR2 and VL-CDR3 regions of V. are at least 80%, 85%, 90% or 95% identical to the amino acid sequences VL-CDR1, VL7CDR? 2 and V.-CDR3 of the reference light chain from the antibodies described here. Therefore, according to that embodiment, a light chain variable region of the invention has the VL-CDR1, VL-CDR2 and V.- CDR3 polypeptide sequences relative to the polypeptides shown in Fig. 1 or Fig. 2, respectively. While Figs. 1 and 2 show the V.-CDRs defined by the Kabat system, other definitions of CDR, for example, VL-CDRs defined by the Chothia system, are also included in the present invention.

[00167] In another embodiment, the present invention provides an isolated polypeptide comprising, composed essentially of, or composed of an immunoglobulin (V.) light chain variable region, in which the V.-CDR1, V.-CDR2 regions and VL-CDR3 have polypeptide sequences that are identical to the VL-CDR1, V.- CDR? 2 and V.-CDR3 groups shown in Fig. 1 or Fig. 2, respectively.

[00168] In another embodiment, the present invention provides an isolated polypeptide comprising, composed essentially of, or composed of an immunoglobulin heavy chain (VL) variable region in which the VL-CDR1, V.-CDR2 and V. -CDR3 have polypeptide sequences that are identical to the VL-CDR1, V.- CDR? 2 and V.-CDR3 groups shown in Fig. 1 or Fig. 2, respectively, except for one, two, three, four, five or six amino acid substitutions on any individual VL-CDR. In certain embodiments, amino acid substitutions are conservative.

[00169] An immunoglobulin or its encoding cDNA can be further modified. Thus, in a later embodiment, the method of the present invention comprises any of the step (s) of producing a chimeric antibody, a murinized antibody, a single chain antibody, a Fab fragment, an antibody bispecific, a fusion antibody, a labeled antibody or analog of any of these. Corresponding methods are known to those skilled in the art and are described, for example, in Harlow & Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor (1988). When the derivatives of these antibodies are obtained by the phage display technique, surface plasma resonance, as used in the BlAcore system, can be used to increase the efficiency of phage antibodies that bind to the same epitope as that of any of the antibodies described herein (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). The production of chimeric antibodies is described, for example, in international application WO89 / 09622. Methods of producing humanized antibodies are described, for example, in European patent application EP-A1 O 239 400 and international application WO90 / 07861. Other sources of antibodies to be used in accordance with the present invention are called xenogenic antibodies. The general principle of producing xenogenic antibodies, such as antibodies similar to those of humans in mice, is described, for example, in international applications WO91 / 10741, WO94 / 02602, WO96 / 34096 and WO 96/33735. As discussed above, the antibody of the invention can exist in a variety of forms in addition to complete antibodies; including, for example, Fv, Fab and F (ab) 2, as well as in single strands; see, for example, international application WO88 / 09344. In one embodiment, therefore, the antibody of the present invention is provided, which is selected from the group consisting of a single chain Fv fragment (scFv), an F (ab ') fragment, an F (ab) fragment, and a fragment F (ab ') 2

[00170] The antibodies of the present invention or their corresponding immunoglobulin chain (s) can be further modified using conventional techniques known in the art, for example, using elimination (s), insertion (s), substitution (s) ), addition (s) and / or recombination (s) and / or any other modification (s) of amino acids known in the art, either alone or in combination. Methods for introducing these 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 the antibody of the invention include chemical and / or enzymatic derivatizations into one or more constituent amino acids, including side chain modifications, skeleton modifications and NedCcC terminal modifications, including acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrates or portions of lipids, cofactors, and the like. Likewise, the present invention comprises the production of chimeric proteins that comprise the described antibody or some fragment thereof at the end of the amino terminus fused to the heterologous molecule, such as, for example, an immunostimulatory linker at the end of the carboxyl terminus; see, for example, international application WO000 / 30680 for the corresponding technical details.

[00171] Furthermore, the present invention comprises peptides, including those containing a binding molecule, as described above, for example, containing the CDR3 region of the variable region of any of the mentioned antibodies, in particular, the heavy chain CDR3 , since it has often been observed that heavy chain CDR3 (HCDR3) is the region with the highest degree of variability and a predominant participation in the antigen-antibody interaction. Such peptides can be easily synthesized or produced by recombinant means to produce a useful linker according to the invention. These methods are well known to those with common skill in the field. Peptides can be synthesized, for example, using automated peptide synthesizers that are commercially available. Peptides can also be produced by recombinant techniques by incorporating the DNA that expresses the peptide into a vector

64/139 of expression, and transforming cells with the expression vector to produce the peptide.

[00172] Therefore, the present invention relates to any binding molecule, for example, an antibody or binding fragment thereof, which is directed to the human anti-| lAPP and / or anti-prolAPP antibodies of the present invention. and displays the mentioned properties, that is, that it specifically recognizes IAPP and / or prolAPP. Such antibodies and binding molecules can be tested for their binding specificity and affinity by ELISA and immunohistochemistry as described herein, see, e.g., the Examples. These characteristics of antibodies and binding molecules can also be tested by Western Blot. Preliminary results of subsequent experiments carried out in accordance with the present invention revealed that the human anti-lAPP and / or anti-prolAPP antibody of the present invention, in particular the antibodies NI-203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3 were able to differentially bind to IAPP fibrils in an ELISA test. In addition, antibodies 203.9A2, NI-203.19H8 and NI-203.26C11 of the present invention have been found to bind preferentially to pathologies in humans, such as large amyloid deposits in pancreatic islets corresponding to IAPP fibrils pathological, as viewed by ThioS and Congo Red staining (see Fig. 7A). The same properties are expected to apply to antibodies NI-203.19F2 and NI-203.15C7.

[00173] Human antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 showed prominent staining in pancreatic islets in amyloid-positive sections, but showed no staining in the pancreatic islets of a patient with DT2 with deposit deficiency. amyloid and a control patient not diagnosed with DT2 (see Example 4 and Fig. 7). The antibodies of the present invention also tested positive in the pancreas of diabetic felines, showing islet amyloid deposits; see Fig. 9. This binding specificity for pathological forms of IAPP and / or prolAPP in human and animal tissue emphasizes, in addition to the biochemical experiments shown here (see Examples 2 and Fig. 5), the ability to use the antibodies of the present invention in the treatment and diagnosis of diseases associated with the occurrence of IAPP and / or prolAPP aggregated in the pancreas.

65/139

[00174] As an alternative for obtaining immunoglobulins directly from the culture of B cells or B memory cells, the cells can be used as a source of rearranged sites of the heavy and light chains for later expression and / or genetic manipulation. Rearranged antibody genes can be reverse transcribed from appropriate RNAms to produce cDNA. If desired, the heavy chain constant region can be exchanged for a different isotype or completely eliminated. The variable regions can be linked to encode single chain Fv regions. Several Fv regions can be linked to confer binding capacity to more than one target or heavy and light chain chimeric combinations can be employed. Once the genetic material is available, creating analogues, as described above, that retain their ability to bind the intended target, is simple. Methods of cloning variable regions of antibodies and generating 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.

[00175] After the appropriate genetic material has been obtained and, if desired, modified to encode an analogue, the coding sequences, including those that encode at least the variable regions of the heavy and light chain, can be inserted into the expression contained in vectors that can be transfected into standard recombinant host cells. A variety of these host cells can be used; for efficient processing, however, mammalian cells are preferred. Typical useful mammalian cell lines for this purpose include, but are not limited to, CHO cells, HEK 293 cells, or NOS cells.

[00176] The production of the antibody or analogue is then carried out by culturing the modified recombinant host under culture conditions suitable for the growth of the host cells and the expression of the coding sequences. Then, the antibodies are recovered when isolated from the culture. The expression systems are preferably designed to include signal peptides so that the resulting antibodies are secreted into the medium; however, intracellular production is also possible.

66/139

[00177] In accordance with the above, the present invention also relates to a polynucleotide encoding the antibody or equivalent binding molecule of the present invention, in the case of the antibody, preferably at least one variable region of a chain. immunoglobulin of the antibody described above. Typically, that variable region encoded by the polynucleotide comprises at least one complementarity determining region (CDR) of the Vr and / or V. of the variable region of said antibody.

[00178] The person skilled in the art will readily appreciate that the antibody variable domain that has the variable domain described above can be used for the construction of other polypeptides or antibodies of desired biological function and specificity. Thus, the present invention also encompasses polypeptides and antibodies that comprise at least one CDR of the variable domain described above, and which advantageously have substantially the same or similar binding properties as the antibody described in the attached examples. The person skilled in the art knows that binding affinity can be enhanced by making amino acid substitutions within CDRs or within hypervariable circuits (Chothia & Lesk, J. Mol. Biol. 196 (1987), 901-917) that overlap partially to CDRs as defined 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 the mentioned CDRs comprise one or more, but preferably not more than two amino acid substitutions. Preferably, the antibody of the invention comprises, in one or both of its immunoglobulin chains, two or all three CDRs of the variable regions, as determined in Fig. 1, or respectively, in Fig. 2.

[00179] Binding molecules, for example, antibodies, or antigen binding fragments, variants or derivatives thereof, as are known to those whose skills in the field are merely common, may comprise a constant region that mediates one or more functions effector. For example, binding of the complement component C1 to a constant region of the antibody can activate the complement system. Complement activation is important in opsonization and lysis of pathogen cells. Complement activation also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity.

67/139 In addition, antibodies bind to receptors in various cells via the Fc region, with an Fc receptor binding site in the Fc region of the antibody that binds to an Fc receptor (FCcR) in a cell. There are a number of Fc receptors that are specific to different classes of antibodies, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). The binding of the antibody to Fc receptors on cell surfaces triggers a series of important and diverse biological responses, including immersion and destruction of antibody-coated particles, removal of immune complexes, lysis of antibody-coated target cells by killer cells (called cytotoxicity) cell-mediated, antibody-dependent, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.

Accordingly, certain embodiments of the present invention comprise an antibody, or antigen-binding fragment, variant or derivative thereof, in which at least a fraction of one or more of the domains of the constant region has been deleted or altered in some other way. Thus, in order to provide desired biochemical characteristics such as reduction of effector functions, the ability to dimerize non-covalently, an increase in the ability to locate, in the aggregation and deposition site of IAPP and / or prolAPP, a lower serum half-life, or a longer serum half-life compared to an integral antibody, unchanged from approximately the same immunogenicity. For example, certain antibodies for use in the diagnostic and treatment methods described herein are domain-excluded antibodies, which comprise a polypeptide chain similar to an immunoglobulin heavy chain, but which do not have at least a portion of one or more chain domains heavy. For example, in certain antibodies, an entire domain of the modified antibody constant region will be excluded, for example, all or part of the CH2 domain will be deleted. In other embodiments, certain antibodies for use in the diagnostic and treatment methods described herein have a constant region, for example, an IgG heavy chain constant region, which is altered to eliminate glycosylation, mentioned elsewhere in this document as aglycosylated antibodies or "agli". These "agli" antibodies can be prepared enzymatically, as well as by engineering the consensus glycosylation site (s) in the constant region. Although they are not limited, in theory,

68/139 it is believed that “agli” antibodies may have an enhanced profile of safety and stability in vivo. Methods of producing aglycosylated antibodies that have the desired effector function are found, for example, in international application WO02005 / 018572, which is incorporated by reference in its entirety.

[00181] In certain antibodies, or antigen-binding fragments, variants or derivatives thereof described herein, the Fc portion may be mutated to reduce effector function, using techniques known in the art. For example, the elimination or inactivation (by point mutations or by other means) of a constant region domain can reduce the binding to the Fc receptor of the circulating modified antibody, thereby increasing the localization of IAPP and / or prolAPP. In other cases, it may be that the modifications of the constant region, consistent with the present invention, moderate the complement binding, thereby reducing serum half-life and the non-specific association of a conjugated cytotoxin. Further other modifications of the constant region can be used to modify disulfide bonds or oligosaccharide fractions that allow for improved localization due to increased antigen specificity or antibody flexibility. The resulting physiological profile and bioavailability, as well as other biochemical effects of the modifications, such as the location and biodistribution of IAPP and / or prolAPP, as well as their serum half-life, can be easily measured and quantified through the use of well-known immunological techniques, without undue experimentation.

[00182] In certain antibodies, or antigen-binding fragments, variants or derivatives thereof described herein, the Fc portion may be mutated or replaced by alternative protein sequences to increase cellular antibody absorption by, for example, improving antibody receptor-mediated endocytosis via Fcy, LRP, or Thyl receptors, or by Superantibody Technology ', which is seen as allowing antibodies to be transported to living cells without being harmed (Expert Opin. Biol. Ther. (2005 ), 237-241). For example, the generation of fusion proteins from the antibody binding region and cognate protein ligands from cell surface receptors or bispecific or multispecific antibodies with specific sequence binding to IAPP

69/139 and / or prolAPP, as well as a receptor on the cell surface can be manipulated, using techniques known in the art.

[00183] In certain antibodies, or antigen-binding fragments, variants or derivatives thereof described herein, the Fc portion may be mutated or replaced by alternative protein sequences or the antibody may be chemically modified to increase its penetration into the blood-brain barrier.

[00184] The modified forms of antibodies, or antigen-binding fragments, variants or derivatives thereof of the invention can be made from integral precursors or parent antibodies, using techniques known in the art. Exemplary techniques are discussed here in greater detail. Antigen-binding antibodies or fragments, variants or derivatives of the invention can be made or manufactured using techniques that are known in the art. In certain embodiments, antibody molecules or fragments are "produced recombinantly", that is, they are produced using recombinant DNA technology. Exemplary techniques for making antibody molecules or fragments are discussed in greater detail elsewhere in this document.

[00185] Antigen-binding antibodies or fragments, variants or derivatives thereof of the invention also comprise derivatives that are modified, for example, by covalent attachment of any type of molecule to the antibody, so that covalent attachment does not prevent the antibody from specifically link to your cognate epitope. For example, but not by way of limitation, antibody derivatives comprise antibodies that have been modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, ligand binding cell or other protein, etc. Any of the various chemical modifications can be accomplished 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.

[00186] In particularly preferred embodiments, antibodies or antigen-binding fragments, variants or derivatives of the invention do not elicit a response

70/139 harmful immune in the animal to be treated, for example, in a human. In certain embodiments, binding molecules, for example, antibodies or fragments binding to their antigen of the present invention come from a patient, for example, a human patient, and are subsequently used in the same species as their origin, for example , human, relieving or minimizing the occurrence of harmful immune responses.

[00187] Deimmunization can also be used to decrease the immunogenicity of an antibody. As used herein, the term "de-immunization" includes the alteration of an antibody to modify T cell epitopes; see, for example, international applications WO98 / 52976 and WO00 / 34317. For example, the Vx and V. sequences of the initial antibody are analyzed and a human T cell epitope “maps” based on each V region, showing the location of epitopes relative to the complementarity determining regions (CDR) and other key residues within of the sequence. Individual T cell epitopes originating from the T cell epitope map are analyzed in order to identify alternative amino acid substitutions with a low risk of altering the activity of the last antibody. A series of alternative Vr and V sequences is designed comprising combinations of amino acid substitutions, and these sequences are subsequently incorporated into a variety of binding polypeptides, for example, specific IAPP and / or prolAPP antibodies or immunospecific fragments of the same for use in the diagnostic and treatment methods described here, which are then tested to verify their function. Typically, between 12 and 24 variant antibodies are generated and tested. Full genes of the heavy and light chains that comprise the human C and modified V regions are then cloned into expression vectors and the subsequent plasmids, introduced into cell lines to produce the entire antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the ideal variant is identified.

[00188] Monoclonal antibodies can be prepared using a wide range of techniques known in the art, including the use of hybridoma, recombinant and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques,

including those known in the art and taught, 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), such references incorporated 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 that is derived from a single clone, including any eukaryotic, prokaryotic, or phagemid clone, and not the method by which it is produced. Thus, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. In certain embodiments, antibodies of the present invention originate from human B cells, which have been immortalized by transformation with Epstein-Barr virus, as described herein.

[00189] In the well-known hybridoma process (Kohler et a /., Nature 256 (1975), 495) relatively short-lived, or deadly lymphocytes, originating from a mammal, for example, B cells from a human individual, as described herein, they are fused to an immortal tumor cell line (for example, a myeloma cell line), thus producing hybrid cells or "hybridomas", which are immortal and capable of producing the B cell genetically encoded antibody. The resulting hybrids are secreted into individual genetic strains through selection, dilution and regrowth with each of the individual strains comprising specific genes for the formation of a single antibody. They produce antibodies, which are homogeneous against a desired antigen and, in reference to their pure genetic ancestry, are called "monoclonal".

[00190] Hybridoma cells thus prepared are seeded and grown in an appropriate culture medium which preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. Those skilled in the art will appreciate that reagents, cell lines and the means for the formation, selection and growth of hybridomas are commercially available based on a number of sources and standardized protocols are well established. In general, the culture medium in which the hybridoma cells grow is assayed for the production of monoclonal antibodies against the desired antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells is determined by in vitro assays, such as, for example, immunoprecipitation, radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA), as described herein. After identifying the hybridoma cells that produce antibodies with the desired specificity, affinity and / or activity, the clones can be subcloned, limiting dilution procedures and cultured by standard methods; see, for example, Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986). It will be even more appreciated that the monoclonal antibodies secreted by the subclones can be separated from the culture medium, ascites fluid or serum by means of conventional purification procedures, for example, protein-A, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

[00191] In another embodiment, lymphocytes can be selected by micromanipulation, and the variable genes, isolated. For example, peripheral blood mononuclear cells can be isolated from an immunized or naturally immune mammal, for example, a human, and cultured for about 7 days in vitro. Cultures can be screened for specific IgGs that meet the selection criteria. Positive cavity cells can be isolated. Individual Ig-producing B cells can be isolated by FRCS or by their identification in a complement-mediated assay with hemolytic plaques. Ig-producing B cells can be micromanipulated into a tube, and the Vr and V genes can be amplified using, for example, RT-PCR. The Vh and V genes can be cloned into an antibody expression vector and transfected into cells (for example, eukaryotic or prokaryotic cells) for expression.

[00192] As an alternative, antibody-producing cell lines can be selected and cultured using techniques well known to those skilled in the art. These techniques are described in a series of laboratory manuals and major publications. In this regard, techniques suitable for use in the 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), which is incorporated herein by reference in its entirety, including supplements.

[00193] Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, Fab and F (ab ') fragments can be produced recombinantly or by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab') fragments 2 ). F (ab ') 2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain. These fragments are sufficient for use, for example, in immunodiagnostic procedures involving the coupling of immunospecific portions of immunoglobulins to detect reagents such as radioisotopes.

[00194] Human antibodies, such as those described herein, are particularly desirable for therapeutic use in human patients. The human antibodies of the present invention are isolated, for example, from healthy human beings who, due to overweight or obesity, may be suspected of being at risk of developing a metabolic disorder, for example, DT2, or a patient with the disorder, but with an unusually stable course of the disease or an unusually mild form of the disease. However, the healthy human being suspected of being at risk of developing a metabolic disorder, for example, DT2 from whom antibodies, as described here, can be isolated, can also be selected based on the presence of other known risks to increase the chances of a person developing a metabolic disorder, for example, T2D. These risks can be deduced from an examination of the person, researching the risk factors associated with the development of a metabolic disorder, for example, T2D, such as the age of 45 years or more; overweight or obesity; close family members with diabetes; family history being African-American, Alaskan Native, Indian-American, Asian-American, Hispanic / Latino, Pacific Islander American, Asian or Arab; history of gestational diabetes; having given birth to at least one baby weighing more than 4.5 kg; blood pressure of 140/90 or higher; cholesterol levels above normal, with, for example, high density lipoprotein (HDL) level below 40 mg / dL (equivalent to less than 1 mmol / L), or triglyceride level above 200-499 mg / dl (equivalent to more than 2.3-5.6 mmol / L);

74/139 sedentary lifestyle; diagnosis of polycystic ovary syndrome (PCOS); diagnosis of prediabetes on previous examination - a CIC level (also called HbAlc or glycosylated hemoglobin) of 5.7 to 6.4 percent, anomaly of fasting glucose (AGJ), or anomaly of glucose tolerance (ATG ); diagnosis of other clinical conditions associated with insulin resistance, such as acanthosis nigricans, for example; history of cardiovascular disease.

[00195] In case a person's obesity or overweight is used as an indicator of their propensity to develop a metabolic disease, for example DT2, although it is prudent to expect that healthy and asymptomatic obese individuals, respectively, will more often have developed protective anti-IAPP and / or anti-prolAPP antibodies than individuals who are diagnosed with lower risk, for example, because they are not classified as obese, but as overweight people, or even as people of normal weight, individuals belonging to the two latest classifications can be used, as well as source, too, to obtain a human antibody of the present invention.

[00196] An individual can be classified as having normal weight, overweight or obesity based on their height and weight measurements, and calculating the individual's Body Mass Index by the following calculation: BMI = weight [kg] / (height [m]) 2. Based on the result, individuals are classified as having normal weight (BMI 18.5 to 24.9 kg / m2), overweight (25.0 to 29.9 kg / m2) or obesity (> 30 kg / m2 ) based on current World Health Organization (2000) criteria "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, an individual's waist circumference (WC) can be measured and used based on gender specific standards to define WC as normal (<94 cm [<34.6 inches] in men and < 80 cm [31.5 inches] in women), moderately increased (between 94 and 102 cm [34.6 and 40 inches] in men and 80 to 88 cm [31.5 to 35 inches] in women) or large ( > 102 cm [> 40 inches] in men and> 88 cm [> 35 inches] in women) as described in the InterAct Consortium, Langenberg et a., PLoS Med. 2012 Jun; 9 (6): e1001230, where a healthy individual with a high waist circumference (large WC) will more often have developed protective anti-lAPP and / or anti-prolAPP antibodies comparable to the classification of obese, the highest risk of developing T2D, and can be used preferably for the isolation of these antibodies, but people with moderately increased or normal CC can also be used for and anti-lAPP and / or anti-prolAPP antibodies.

[00197] In one embodiment, an antibody of the invention comprises at least one heavy or light chain CDR of an antibody molecule. In another embodiment, an antibody of the invention comprises at least two CDRs of one or more antibody molecules. In another embodiment, an antibody of the invention comprises at least three CDRs of one or more antibody molecules. In another embodiment, an antibody of the invention comprises at least four CDRs of one or more antibody molecules. In another embodiment, an antibody of the invention comprises at least five CDRs of one or more antibody molecules. In another embodiment, an antibody of the invention comprises at least six CDRs of one or more antibody molecules. Exemplary antibody molecules that comprise at least one CDR that can be included in the individual's antibodies are described herein.

[00198] Antibodies of the present invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably by recombinant expression techniques, as described herein.

[00199] In one embodiment, an antibody or antigen-binding fragment, variant or derivative of the invention comprises a constant region of synthesis in which one or more domains are partially or totally eliminated ("antibodies excluded from the domain"). In certain embodiments, compatible modified antibodies will comprise constructs excluded from the domain or variants in which the entire CH? 2 domain has been removed (ACH2 constructs). For other embodiments, a short-linked peptide can be replaced by the excluded domain to provide flexibility and freedom of movement for the variable region. Those skilled in the art will appreciate that these constructions are particularly preferred due to the regulatory properties of the CH2 domain in the catabolic rate of the antibody. Constructions excluded from the domain can be derived using a vector that encodes a human IgG1 constant domain, see, for example, international applications WO0O02 / 060955 and

76/139 WOO02 / 096948A? 2. This vector is designed to exclude the CH2 domain and provide a synthetic vector that expresses an IgGi constant region excluded from the domain.

[00200] In certain embodiments, antibodies or antigen-binding fragments, variants or derivatives thereof of the present invention are minibodies. Minibodies can be made using methods described in the art, see, for example, US patent 5,837,821 or international application WO94 / 09817.

[00201] In one embodiment, an antibody or antigen-binding fragment, variant or derivative of the invention comprises an immunoglobulin heavy chain that has elimination or replacement of some or even a single amino acid, as long as it allows association between the subunits monomeric. For example, mutating a single amino acid in selected areas of the CH2 domain may be sufficient to substantially reduce binding to Fc and thus increase the localization of IAPP and / or prolAPP. Similarly, it may be desirable to simply exclude that part of one or more domains of the constant region that controls the effector function (eg, complement link) to be modulated. These partial exclusions of the constant regions can improve the selected characteristics of the antibody (serum half-life), while leaving other desirable functions associated with the domain of the individual's constant region intact. In addition, as mentioned above, the regions contained in the described antibodies can be synthetic by mutating or replacing one or more amino acids that enhance the profile of the resulting construct. In this regard, it may be possible to interrupt the activity provided by a preserved binding site (eg, binding to Fc) while substantially maintaining the configuration and immunogenic profile of the modified antibody. However, other embodiments include adding one or more amino acids to the constant region to enhance desirable characteristics, such as an effector function, for example, or to provide other cytotoxin or carbohydrate fixations. In these embodiments, it may be desirable to insert or replicate specific sequences from selected domains from constant regions.

[00202] The present invention also provides antibodies that comprise, are essentially composed of, or are composed of variants (including derivatives) of antibody molecules (eg, the Vx and / or V regions) described herein, whose antibodies or

77/139 fragments of them bind immunospecifically to IAPP and / or prolAPP. Standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding an antibody, including, but not limited to, site-directed mutagenesis and PCR (Polymerase Chain Reaction) mutagenesis that result in amino acid substitutions. Preferably, 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 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 in relation to the reference Vr region, V4- CDR1, V- CDR2, VH "CDR3, region V., VI-CDR1, VL-CDR2, or VL-CDR3. A" conservative amino acid substitution "is one in which the amino acid residue is replaced by an amino acid residue with a side chain of similar charge. Families of amino acid residues with side chains of similar charges have been defined in the industry. These families include amino acids with basic side chains (eg lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg: alanine , valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), side chains with beta branches (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly throughout all or part of the coding sequence, for example, by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity ( eg the ability to link IAPP and / or prolAPP).

[00203] For example, it is possible to introduce mutations only in structural regions or only in the CDR regions of an antibody molecule. Introduced mutations can be silent or neutral missense mutations, for example, that have little or no effect on the antibody's ability to bind antigens. In fact, some of these mutations do not alter the amino acid sequence in any way. These types of mutations can be useful to optimize the use of codons, or to improve the production of antibodies to a hybridoma. Coding regions optimized for codons that encode antibodies of the present invention are described elsewhere in this document. Alternatively, non-neutral missense mutations can alter an antibody's ability to bind antigens. The location of most silent and neutral Missense mutations is prone to appear in structural regions, while the location of most non-neutral Missense mutations is likely to appear in the CDR, although it is not an absolute requirement. Someone who is skilled in the art would be able to design and test mutant molecules with desired properties, such as, without changing the activity of binding to the antigen or changing the activity of binding (eg, improvements in the activity of binding to the antigen or changing the antibody specificity). After mutagenesis, the encoded protein can be expressed routinely and the functional and / or biological activity of the encoded protein (eg, ability to immunospecifically bind at least one epitope of IAPP and / or prolAPP) can be determined using techniques described herein or by routinely modify the techniques known in the art.

Il. Antibody Coding Polynucleotides

[00204] A polynucleotide that encodes an antibody, or antigen-binding fragment, variant or derivative thereof may be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide encoding an antibody, or antigen-binding fragment, variant or derivative thereof, may be composed of single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, Single-stranded and double-stranded RNA, and RNA which is a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of regions single-stranded and double-stranded. In addition, a polynucleotide encoding an antibody, or antigen-binding fragment, variant or derivative thereof, may be composed of triple-stranded regions, comprising 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 structures of DNA or RNA modified to confer stability or for other reasons. Among the “modified” bases, there are, for example, triturated bases and unusual bases, such as, for example, inosine. Several modifications can be made to DNA and RNA; thus, the term "polynucleotide" encompasses chemically, enzymatically or metabolically modified forms.

[00205] An isolated polynucleotide encoding an unnatural variant of an immunoglobulin-derived polypeptide (e.g., a portion of the light chain or a portion of the immunoglobulin heavy chain) can be created by introducing one or more exclusions, additions or nucleotide substitutions of the immunoglobulin nucleotide sequence, such that one or more amino acid deletions, additions or substitutions are introduced into the encoded protein. Mutations can be introduced using standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis (Polymerase Chain Reaction). Preferably, conservative amino acid substitutions are made on one or more non-essential amino acid residues.

[00206] As is well known, RNA can be isolated from the original B cells, from the hybridoma cells or from other cells transformed by standard techniques, for example, extraction of guanidine isothiocyanate and precipitation, followed by centrifugation or chromatography. When desirable, mRNA can be isolated from total RNA using standard techniques, such as chromatography on oligo (dT) cellulose. Suitable techniques are well known in the art. In one embodiment, DNAcs encoding the antibody heavy and light chains can be made, either simultaneously or separately, using reverse transcriptase and DNA polymerase according to well-known methods. PCR can be initiated by primers from the consensus constant region or by more specific primers, based on the published amino acid sequences and heavy and light chain DNA. As discussed above, PCR can also be used to isolate DNA clones that encode antibody heavy and light chains. In this case, libraries can be screened by larger homologous probes or consensus initiators, such as probes from the human constant region.

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[00207] DNA, typically DNA plasmids, can be isolated from cells using techniques known in the art, strictly mapped and sequenced according to very popular standard techniques shown in detail, eg in previous references regarding Recombinant DNA. Naturally, DNA can be synthetic according to the present invention at any time during the process of isolation or further analysis.

[00208] In this context, the present invention also relates to a polynucleotide that encodes at least the binding domain or variable region of an immunoglobulin chain of the antibody of the present invention. In one embodiment, the present invention provides an isolated polynucleotide that comprises, composed essentially of, or composed of a nucleic acid encoding an immunoglobulin heavy chain (Vs) variable region, wherein at least one of the CDRs of the variable chain region heavy or at least two of the heavy chain variable region V4-CDRs are at least 80%, 85%, 90% or 95% identical to the reference heavy chain Vn-CDR1, VH-CDR2 or VH-CDR3 amino acid sequences antibodies described here. Alternatively, the Va-CDR1, Va-CDR2 or VH-CDR3 regions of Vx are at least 80%, 85%, 90% or 95% identical to the VA-CDR1, Vn-CDR2 and V4-CDR3 amino acid sequences reference weight of the antibodies described here. Thus, according to this embodiment, a heavy chain variable region of the invention has the Vx-CDR1, Va-CDR2 or V4H-CDR3 polypeptide sequences relative to the polypeptide sequences shown in Fig. 1, or, respectively, the sequences of VH-CDR1, V4-CDR2 or V4-CDR3 polypeptides relative to the polypeptide sequences shown in Fig. 2.

[00209] In another embodiment, the present invention provides an isolated polynucleotide comprising, composed essentially of, or composed of a nucleic acid encoding an immunoglobulin light chain (VL) variable region, in which at least one of the VL-CDRs of the light chain variable region or at least two of the V-CDRs of the light chain variable region are at least 80%, 85%, 90% or 95% identical to the amino acid sequences VI-CDR1, V-CDR2 or VL- CDR3 of the reference light chain of the antibodies described herein. Alternatively, the VL-CDR1, V.- CDR2 or V.-CDR3 regions of VL are at least 80%, 85%, 90% or 95% identical to the amino acid sequences VL-CDR1, VI-CDR2 and VL-CDR3 of the reference light chain of the antibodies described here. Thus, according to this embodiment, a light chain variable region of the invention has the VL-CDR1, VL-CDR2 or V.-CDR3 polypeptide sequences relative to the polypeptide sequences shown in Fig. 1, or, respectively, the VL-CDR1, VI-CDR2 or VL-CDR3 polypeptide sequences relative to the polypeptide sequences shown in Fig. 2.

[00210] In another embodiment, the present invention provides an isolated polynucleotide that comprises, composed essentially of, or composed of a nucleic acid encoding an immunoglobulin (VH) heavy chain variable region, wherein the VH-CDR1, VH regions -CDR2 and V4H-CDR3 have polypeptide sequences that are identical to the groups VH-CDR1, VH-CDR2 and Vx-CDR3 shown in Fig. 1 or that, respectively, are identical to the groups VH-CDR1, VH-CDR2 and VH-CDR3 , as shown in Fig. 2.

[00211] As those in the field know, the "sequence identity" between two polypeptides or two polynucleotides is determined by comparing the amino acid or nucleic acid sequence of a polypeptide or polynucleotide with the sequence of a second polypeptide or polynucleotide. When discussed here, whether any specific polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide, it can be determined using methods and computer programs / software popular in the field, such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). BESTFIT uses Smith & Waterman's local homology algorithm, Advances in Applied Mathematics 2 (1981), 482-489, to find the best homology segment between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence in accordance with the present invention, the parameters are naturally adjusted in such a way that the percent identity is calculated based on the completeness of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.

[00212] In a preferred embodiment of the present invention, the polynucleotide comprises, is essentially composed of, or is composed of a nucleic acid that has a sequence of polynucleotides from the Vr or V region of an anti-IAPP and / or anti- prolAPP, as illustrated in Table || or in Table III. In this regard, the person skilled in the art will readily appreciate that polynucleotides that encode at least the variable domain of the heavy and / or light chain can encode the variable domain of both immunoglobulin chains or just one. In one embodiment, therefore, the polynucleotide comprises, is essentially composed of, or is composed of a nucleic acid that has a Vx and V region polynucleotide sequence of an anti-| l | APP and / or anti-prolAPP antibody as illustrated in Table || or the sequence of the VH and V.L region of an anti-| APP and / or anti-prolAPP antibody as shown in Table III. Table II: Nucleotide sequences of the Vr and V. region of IAPP antibodies. Nucleotide sequences of the heavy chains Antibody 1 1. variable (V4) and light variable (VLNK)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGG GGGGTCCCTGAGGCTCTCCTGTGCAGCCTCTGGATTCACGTTTA GCACCTTTGCCATGAGCTGGGTCCGCCAGECTCCAGGGAAGEGG

CTGGAGTGGGTCTCAACTATTAGTGGTAGTGGTGATAATACATA NI-203.9A2-Vy CTATGCAGACTCCCTGAAGGGCCGGTTCACCATCTCCAGAGACA

ATTCCAAGAACACACTATATCTGCAAGTGAACAGCCTGAGACCC GAGGACACGGCCGTTTATTACTGTGCGAAAAGTCCCTCGTCACT

TCTGGCCACCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCA CCGTCTCCTCGE SEQ ID NO: 11

GAAATTGTGTTGACACAGTCTCCTTCCACCCTGTCTGCATCTGT AGGAGACAGAGTCACCATCACTTGCCGGGCCAGTGAGAGTATTA ATAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGGCCCT

AAGCTCCTGATCTATAAGGCGTCTAGTTTACAAAGTGGGGTCCC NI-203.9A2 -Vx ATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCA

CCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGC

CAACAGCACAATAGTTATTGGACGTTCGGCCAAGGGACCAAGGT GGAAATCAAA SEQ ID NO: 13

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGEG GACGTCCCTGAGACTCTCCTGTGCAGCGTCTGGGTTCACCTTCA

GCAGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGC NI-203.19H8-Vn CTGGAGTGGGTGGCAATTATATGGTATGATGGAAGTAAGGAATA

TTATGCAGACTCCCTGAAGGGCCGAGTCACCATCTCCAGAGACA ATTCCGAGAACACTCTCTATCTGCAACTGCACACCCTGAGAGTC GAGGACACGGCTGTGTATTTCTGTGCGAGGACAATCGCATCGGC

CACCGTGGACCACGGTATGGACGTCTGGGGCCAAGGCACCCTGG TCACCGTCTCCTCG SEQ ID NO: 15

GATGTTGTGATGACTCAGTCTCCTTCGTCCGTGTCTGCATCTGT AGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCACGATATTA GCACCTGGTTAGCCTGGTATCAGCAGAGACCAGGGAAAGCCCCT

AACCTCCTGATCTTTGGAGCATCGAGGTTGCAAAGTGGGGTCTC NI-203.19H8-Vx ACCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCA

CCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGT

CAACAGACTAACAATTTCCCTCCCACCTTCGGCCAAGGGACACG ACTGGAGATTAAA SEQ ID NO: 17

CAGGTGCAGCTGCAGGAGTCGGGCCCAGGATTGGTGAAGCCTTC TCAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCA GCAGTGGTAATTACTACTGGACCTGGATCCGGCAGCCCGCCEGG

AAGGGACTGGAGTGGATTGGGCATATCTATTCCAGTGGGACCAC NI-203.26C | | -Vy CAATTACAACCCCTCCCTCGAGAGTCGAGTCACCATTTCAGTAG

ACACGTCCAAGAACCAGTTCTCCCTGAGCCTGAACTCTGTGACC GCCGCAGACACGGCCGTTTATTACTGTGCGAGACCACTGGCTAC

AGTTCCGGATGCTTTTAATATCTGGGGCCAAGGGACAATGGTCA CCGTCTCTTCG SEQ ID NO: 19

GAAATTGTGATGACTCAGTCTCCAGACTCCCTGGCTGTGTCTCT GGGCGAGAGGGCCACCATCAAGTGCAAGTCCAGCCAGAGTGTTT TATACAGCAATAAGAACTTCTTAGCTTGGTACCAGCAGAAACCA

GGACAGCCTCCTAAATTACTCATTTACTGGGCATCTACTCGGGA NI-203.26C | | -Vx ATCCGGGGTCCCTGACCGATTCAGTGGCAGCGEGTCTGGGACAGS

ATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCA

GTTTATTACTGTCAGCAGTATTATAGTAATCCTAACACTTTTGG CCAGGGGACCAAGGTGGAGATCAAA SEQ ID NO: 21

CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAMACCTGG GTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCA GTAGTCACACTATCAGCTGGGTGCGACAGGCCCCTGGGCAAGGEGS

CTTGAGTGGATGGGAGGGATCATCCCCATCTTTGGTACAGCAAA NI-203.8E3-Vu CTACGCACAGAAGTTTCAGGACAGAGTCACGGTTACCGCGGACA

AATCCACGAATACAGCCTACATGGAGTTGAGTAGCCTCAGACCT GAGGACACGGCCGTGTATTACTGTGCGAAGGGGGAACTGGAACC

ACGAATCCTCTACTACTACGGTATGGACGTCTGGGGCCGAGEGA CCACGGTCACCGTCTCCTCG SEQ ID NO: 23

GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCT TGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAMAGCCTCG TATACAGTGATGGAAACACCTACTTGAATTGGTTTCACCAGAGG

CCAGGCCAATCTCCAAGGCGGCTAATTTATAAGGTTTCTAATCG NI-203.8E3-Vx TGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCA

CTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTT

GGGGTTTATTACTGCATGCAAGGTTCAAATTGGCCAGGGACGTT CGGCCAAGGGACCAAGGTGGAAMATCAMA - SEQ ID NO: 25

CAGGTGCAGCTGGTGCAATCTGGGGCTCGAGGTGAAGAAGCCTEG

GGCCTCAATGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCA NI-203. | 1B | 2-Vu CCAACTACTATTTACACTGGGTGCGACAGGCCCCTGGACAAGGA

CTTGAGTGGATGGGAATAATCAACCCTAGTGCTGGTAGCACAAG CTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACA

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CGTCCACGAGCACAGTCTACATGGAACTGAGCAGCCTGAAATCT GAAGACACGGCCGTCTATTACTGTGCGAGAGATTCCGCTGGGAT

ACAGATATGGTTCAGGGATGCTTTTGATATCTGGGGCCAAGEGA CAATGGTCACCGTCTCTTCG SEQ ID NO: 27

CAGCCTGTGCTGACTCAGCCACCCTCTGCCTCTGCTTCCCTGGG ATCCTCGGTCAAGCTCACCTGCACTCTGAACAGTGGGCACAGTA GCTACACCATCGCATGGCATCAGCAGCAGCCAGGGAAGGCCCCT

CGGTACTTGATGAAGGTTGAACATAATGGAAACTACAACAAGGG NI-203. 116 | 2-V. GAGCGGACTTCCTGATCGCTTCTCAGGCTCCAGCTCTGGGEGCTG

ACCGCTACCTCGCCATCTCCAACCTCCAGTCTGAGGATGAGGCT

GATTATTACTGTGAGACCTGGGACACTAGCACTAGGGTCTTCGG CGGAGGGACCAAGCTGACCGTCCTA SEQ ID NO: 29

CAGGTGCAGCTGCAGGAGTCGGGCCCCGGACTGGTGAAGCCTTC GGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGACTCCGTCA GCAGTGGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGEG AAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCAC

CAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAG NI-203 .205F8-VH ACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACC

GCTGCGGACACGGCCGTATATTCCTGTGCGAGAGTCCCCTATGG TTACGGATATAGGGGCTACGATGGGGCTTGGTACTTTGACTACT

GGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 31

GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCC AGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTA GCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCC

AGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCC NI-203.205F8-Vx AGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCA

CCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGT

CAGCAGCGTAGCAACCGGTTCACTTTCGGCCCTGGGACCAAAGT GGATATCAAA SEQ ID NO: 33

GAGGTGCAGCTGGTGGAGTCTGGGEGAGECETEGETCCAGCCTGEG GGGGTCCCTGAGACTCTCCTGTGCAGCGTCAGGATTCACCTTCA GTAGCTATGGCATGCACTGGGTCCGCCAGECTCCAGECAAGEGG

CTGGAGTGGGTGGCAGTTATCTGGTATGATGGAACTAAGAAGTA NI-203.983-Vu CTATGCAGACTCCGTGAAGGGCCGATTCACCACCTCCAGAGACA

ATTCCAAGAATACGCTGTCTCTGCAAATGAACAGCCTGAGAGCC GAGGACTCGGCTGTGTATTACTGTGCGAGAGGCTTTAGCAGCAG

CTGGGAGTTTGGGTTCTGGGECCAGGGAACCCTEGGTCACCGTCT CCTCG SEQ ID NO: 35

CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCEEETCTCCTEG ACAGTCAGTCACCATCTCCTGCACTGGAACCAGCGGTTACATTT ATGGTTATAACTATGTCTCCTGGTACCAACAGCACCCCGGCAAA

GCCCCCAAAGTCATGATTTATGAGGTCACTAAGCGGCCCTCAGG NI-203.983-V. GGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGEGCCT

CCCTGACCGTCTCTGGGCTCCAGGCTGAGGATGAGGCTGTTTAT

TACTGCGCCTCATATGCAGGCAGCAACAATGTAGTATTCGGCGG AGGGACCAAGCTGACCGTCCTA SEQ ID NO: 37

GAGGTGCAGCTGGTGCAGTCTGGGECTEAGETCGAGGAAGCCTGEG GTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCAACTTCT TGAGCTATTCCATCAGTTGGGTGCGACAGGCCCCTGGACAAGGEG CTTGAGTGGATGGGAGGGATCATCCCGATCTTTGGTACACCAAA

CTACGCACAGAAGTTCCAAGGAAGAGTCACAATTACGGCGGACA NI-203,19F2-Vr AATCGACGAGGACAGCCTACATGGAGCTGAGCAGCCTGAGATTT

GATGACACGGCCGTCTATTATTGTGCGGATGCGACAAGACCGEGG TACAGCAGCCTCTGGTTTCTATTACTACGGTATGGACGTCTGGG

GCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 63

GAAATTGTGATGACACAGTCTCCAGACACCCTGTCTGTGTCTCC AGGTGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTA ACAACAACTTAGCCTGGTTCCAGCAGAAACCTGGCCAGGCTCCC

AGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATTCC NI-203.19F2 -Vk AGCCAGATTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCA

CCATCAGCAGCCTACAGTCTGAAGATTTTGCAGTTTATTTCTGT

CAGCAGAGTCACAATTGGCCCACTTTCGGCCCTGGGACCAAAGT GGATATCAAA SEQ ID NO: 65

GAGGTGCAGCTGGTGGAGACTGGGECGAGECETEGTCCAGCCTEG GATGTCCCTGAAMACTCTCCTGTGCAGCCTCTGGATTCACCTTCA GTACCTATACTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGEGG

CTGGAGTGGGTGTCATTTATATCATATGATGGAAGGGATAAATA NI-203.15C7-Vn CTACGCAGATTCCGTGAAGGGCCGATTCACCATCTCCAGAGACA

ATTCCAAGAACATGTTGTATCTGCAAATGAACAGCCTGAGAGAT GAGGACATGGCTGTGTATTACTGTGCGACTCTGCAAGTATGGCA

ACTCTACGATTACTACGGAATGGACGTCTGGGGCCAAGGGACCA CGGTCACCETCTCCTCG SEQ ID NO: 67

CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCEGCCCCAGE ACAGAAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTG GGAATAATTATGTATCTTGGTATCAGCAACTCCCAGGAACAGCC

CCCAAACTCCTCATTTATAACAGTGATAAGCGACCCTCAGGGAT NI-203.15C7-V. TCCTGACCGATTCTCTGCCTCCAAGTCTGGCACGTCAGCCACCC

TGGGCATCACCGGGCTCCAGACTGGGEACGAGGCCGATTATTAC

TGCGCAACATGGGATACCAGACTGAGTGCTGGGGTATTCGGCGG AGGGACCAAGCTGACCGTCCTT SEQ ID NO: 69 Table III: Nucleotide sequences of the Vx and V. region of prolAPP antibodies. [anteoro The RES raise variable Antibody (Vy) and mild variable (VLNK)

GAGGTGCAGCTGGTGCAGTCTGGCGCAGAAGTGAAGAAGCCCGGE GGAGTCTCTCAGAATCTCCTGTAAGGCTTCTGGATACAGCTTCA

CCAACTCTTGGATCGCCTGGETGCECCAGATGCCCGGGAAAGEC NI-203. 1D10-V "CTGGACTACGTGGGTATCATCTATCCTGGTGACTCTGATACCAA

GTATGGCCCGTCCTTCCAAGGCCACGTCACTATCTCAGCCGACA ACTTCGCCAACACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCC TCCGACACCGCCATCTATTATTGTGCGAGACGGGCAGCAGCGEGC

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TATTAACTGGTTCGACTCCTGGEGGCCAGEGAACCCTGGETCACCG TCTCCTCG SEQ ID NO: 39

GACATCCAGTTGACCCAGTCTCCACTCTCCCTGTCCGTCACCCC TGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGCCAGAGCCTCC TGCATCCTAATGGAAACGACTATTTGGATTGGTACGTGCAGAAG

CCAGGGCAGTCTCCACAGATCGTGATCTACATGGGTTCTAATCG NI-203. 1 D | The -Vx GGCCECCEGEGTCCCTEGACAGGTTCAGTGGCAGTGGATCAGGCA

CAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTT

GGGACTTATTACTGCCTGCAAGCTCTACGCGGGTACACTTTTGG CCAGGGGACCAAGGTGGAAATCAAA SEQ ID NO: 41

CAGGTGCAGCTGGTGGAGTCTGGGEGAGECETEGTCCAGCCTEG GGGGTCCCTGAGACTCTCCTGTGTAGCGTCTGGATTCACCTTCA GCAGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGEGG

CTGGAGTGGGTGGCATTTGTACGGTATGATGGAAGTAATAAGTA NI-203.2A || -Vu CTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACA

ATTCCAAGAACTCGCTGTCTCTTCAMATGAACAGTCTGAGAACT GAAGACACGGCTGTATATTACTGCGCGAAAGAACAGGAGGACCA

CAAGGAAGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCG TCTCCTCG SEQ ID NO: 43

GAAATTGTGATGACACAGTCTCCAGCCACCCTGTCTGTGTCTCC AGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGAGTTA CCACCATAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG

CTCCTCATCTATGGTGCATCCAGCAGGGCCACTGATATTCCCGC NI-203.2A | | -Vx CAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCA

TCAGCAGTCTGCAGTCTGAAGACTTTGCAGTTTATTACTGTCAG

CAGTATAACCAGTGGCCCCTCACTTTCGGCGGAGGGACCAAGCT GGAGATCAAA SEQ ID NO: 45

GAGGTGCAGCTGGTGGAGTCTGEGGECTEGAAGTGAGGAAGCCTGEG GGCCTCAGTGAGGGTCTCCTGCCAGACATCTGGATACAGCGTCA CCGACTACTATCTACACTGGGTGCGACAGGCCCCTGGACAGEGC

CTTGAGTGGATGGGAGTGATGAACCCGAGCAATGGAAACGTGGG NI-203.10C4-Vn CTACCCACAGAAGTTTCAGGGCCGAGTCACCATGACCGCAGACA

CGTCCACGGGCACAGTGTACATGGTGTTGACCGGCCTTACEGCT GGGGACACGGCCGTCTACTACTGTGCCAGAGGCGEGTCCACGECC

GGGTCAGGAAGTAAGGAGTCCCCACGTCCTTGACCTCTGGGGECC AGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 47

GATGTTGTGATGACTCAGTCTCCCCTCTCTCTGTCCGTCACCCC TGGACAGCCGGCCTCCATCTCCTGCAGGTCTGATGAGAGCCTCC TGCATAGTGATGGAAGGACCTATTTGTATTGGTATCTACAGAAG

CCCGECCAGCCTCCTCAGCTCCTGATCTATGAAGTTTCCAACCG NI-203. 10C4-Vx GTTCTCGGGAGTGCCAAATAGGTTCAGTGGCAGCEGETCAGEGA

CAGATTTCACACTGAAAATCAGCCGGGTGGAGGCTGAGGATGTT GGCGTTTATTACTGCATGCAGGGTGTACACTTTCCTCAGACGTT

CGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 49

CAGGTGCAGCTGGTGCAATCTGGGTCTGAGTTGAAGAAGCCTGG

GGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACATCTTCA NI-203.20H9-Vn GTAAACATGGTATCAACTGGGTGCGACAGGCCCCTGGACAAGEC

CTTGAGTGGATAGGATGGATCAACACCAATACGGGGAACCCAAC

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ATATGCCCAGGACTTCACAGGACGATTTGTCTTCTCCTTGGACA CCTCTGTCAGCACGGCATATCTGGAGATCAGCAGCCTAAAGGCT GAGGACACTGCCGTGTATTACTGTGCGAGAGAATCAGAGCCGAT

TTTTGGAGTTATCTATTACATGGACGTCTGGGGCAAAGGGACCA CGGTCACCGTCTCCTCG SEQ ID NO: 51

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGT AGGAGACAGCGTCACCATCACTTGCCGGGCACACCAGAGCATAA GCACTAATTTAAATTGGTATCAGAAGAAACCAGGACAAGCCCCT

ACGGTCTTGATCTATGCTGCGTCCAGTTTGCAAGGTGGEGETCCC NI-203.20H9-Vx ATCAAGGTTCAGGGGCCGGGGATCTGGGACATATTTCACTCTCA

CCATCAGCGGTCTTCAACCTGAAGATTTTGCAACTTATTACTGT

CAACACAATTACAATGATTTGTGGACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA SEQ ID NO: 53

CAGGTGCAGCTGGTGGAGTCTGEGEGAGECETECTCCAGCCGEGGE GGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGGTTCACGTTCAG AACCTGTGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGEGEC

TGGAATGGGTGGCATTTGTTCGGTCTGATGGAACTACTAGATAT NI-203 .26D2-Vn TACGCAGACTCCCTGATGGGCCGCTTCACCATCTCCAGAGACAA

TTCCAAGAACTCGCTGTATCTTCAAATGAACAGTCTGAGACCTG AGGACACGGCTCTTTATTACTGTGCGAGGGAAAAGGAGGATCAC

AGGGAAGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGT CTCCTCG SEQ ID NO: 55

GAAATTGTGATGACACAGTCTCCAGCCACCCTGTCTGTGTCTCC AGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGCGTGTTA GCACTGTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG

CTCCTCATCTATGATGCATCCACCAGGGCCACTGATATCCCCGC NI-203.26D2-Vx CAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCA

TCAGCACTCTGCAATCTGAAGACTCTGCAGTTTATTACTGTCAG

CAGTATAATAGGTGGCCCCTCACTTTCGGCGGAGGGACCAAGGT GGAGATCAAA SEQ ID NO: 57

GAGGTGCAGCTGGTGGAGTCTGGGEGAGGATTGGCACGCCCTGEG AGGCTCCCTGAGACTCTCCTGTGCAGTCGCTGGATTCACTTTCA GTGGTTATGAAATGAATTGGGTCCGCCAGECACCAGGGAAGEGG

CTGGAGTGGATTTCATATATTAGCGGTCCTGGGGATGTGATATA NI-203 .60H3-Vu CTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACA

ACGCCAAGAACTCACTGTTTCTACAGATGAACAGCCTGAGAGCC GAGGACACGGCTGTTTATTATTGTACGAGAGTCCCCCCTGACAT

CAGCTATGGATTTGATTACTGGGGCCAGEGCACCCTGGTCACCG TCTCCTCG SEQ ID NO: 59

GACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGT ACGAGACAGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTA GCACCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCT

AACCTCCTGATCCATGATACAGACATTTTGCAAAGTGGGGTCCC NI-203.60H3-Vx ATCAAGGTTCAGTGGCACTGGATCTGGGACAGATTTCACTCTCA

CCATCAGCGGTCTGCAACCTGAAGATTTTGCAACTTACTACTGT

CAACAGAGTTACAGTACCCCTCCTACTTTTGGCCAGGGGACCAA GCTGGAGATCAAA SEQ ID NO: 61

88/139

[00213] The present invention also includes fragments of the polynucleotides of the invention, as described elsewhere. In addition, polynucleotides encoding fusion polynucleotides, Fab fragments and other derivatives, as described herein, are also contemplated by the invention.

[00214] Polynucleotides can be produced or manufactured by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide that encodes the antibody can be assembled from chemically synthesized oligonucleotides, eg as described in Kutmeier et al., BioTechniques 17 (1994) 242, which briefly involves the synthesis of overlapping oligonucleotides that contain portions of the sequence encoding the antibody, the hybridization and ligation of those oligonucleotides, and then, the amplification of the PCR-linked oligonucleotides.

[00215] Alternatively, a polynucleotide encoding an antibody, or antigen-binding fragment, variant or derivative thereof, can be generated from 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, a nucleic acid encoding the antibody can be chemically synthesized or obtained from a suitable source (e.g. : a library of cDNA antibodies, or a cDNA library generated from, or nucleic acid, preferably polyA + RNA, isolated from any tissue or cells that express the specific antibody of IAPP and / or prolAPP, such as cells of hybridoma selected to express an antibody) by PCR amplification, using synthetic primers hybridizable to the 3 'and 5' ends of the sequence or by cloning, using an oligonucleotide probe specific to the particular genetic sequence to be identified, eg, a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR can then be cloned as replicable cloning vectors using any method very popular in the art.

[00216] After the nucleotide sequence and the corresponding amino acid sequence of the antibody, or antigen-binding fragment, variant, or derivative thereof, its nucleotide sequence can be manipulated by

89/139 using popular methods in the field for the manipulation of nucleotide sequences, eg: recombinant DNA techniques, site-directed mutagenesis, POR (Polymerase Chain Reaction), etc. (see, for example, the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1990) & Ausubel et a., eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998), which are both incorporated herein by reference in their entirety), to generate antibodies that have a different amino acid sequence, for example, to create insertions, exclusions and / or amino acid substitutions.

IV. Expression of Antibody Polypeptides

[00217] After manipulating the isolated genetic material to provide antibodies or antigen-binding fragments, variants or derivatives thereof, the polynucleotides encoding the antibodies are typically inserted into an expression vector for introduction into host cells that can be used to produce the desired amount of antibodies. Recombinant expression of an antibody, or its fragment, derivative or analog, e.g., an antibody heavy or light chain that binds to a target molecule is described herein. Once a polynucleotide encoding an antibody molecule or an antibody heavy or light chain, or portion thereof (preferably containing the heavy or light chain variable domain) of the invention has been obtained (a), the vector for the production of the antibody molecule it can be produced by means of recombinant DNA technology, using techniques very popular in the field. Therefore, methods for preparing a protein by expressing a polynucleotide that contains an antibody that encodes a nucleotide sequence are described herein. Methods that are well known to those skilled in the art can be used to construct expression vectors that contain antibody coding sequences and appropriate translation and transcription control signals. These methods include, for example, in vitro recombinant DNA techniques, synthesis techniques, and in vivo genetic recombination. The invention, therefore, provides replicable vectors that comprise a nucleotide sequence that encodes an antibody molecule of the invention, either a heavy or light chain, or a heavy chain variable domain.

90/139 or light, operationally linked to a promoter. These vectors can include the 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. 5,122,464) and the variable domain of antibody can be cloned into this vector for expression of the entire heavy or light chain.

[00218] The term "vector" or "expression vector" is used herein to refer to vectors used in accordance with the present invention as a vehicle for introducing and expressing a desired gene in a host cell. As is known to those skilled in the art, these vectors can be easily selected from the group consisting of plasmids, phagemids, viruses and retroviruses. In general, vectors compatible with the immediate invention will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and / or replicate in eukaryotic or prokaryotic cells. For the purposes of the present invention, various expression vector systems can be used. For example, a vector class uses DNA elements that come from animal viruses, such as bovine papillomatosis virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV ) or the SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. In addition, cells that have integrated DNA into their chromosomes can be selected by introducing one or more markers, which allows selection of transfected host cells. The marker may 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 either directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal mRNA synthesis. These elements can include signal sequences, junction signals, as well as transcription promoters, enhancers and termination signals,

[00219] In particularly preferred embodiments, the cloned genes of the variable region are inserted into an expression vector, along with the genes of the heavy and light chain constant region (preferably human) as discussed above. In one embodiment, this is accomplished using a proprietary expression vector from Biogen IDEC, Inc., entitled NEOSPLA, and disclosed in US patent No. 6,159,730. This vector contains the cytomegalovirus promoter / enhancer, the main promoter of mouse beta globin, the SV40 origin of replication, the bovine growth hormone polyadenylation sequence, exon 1 and exon 2 of the neomycin phosphotransferase, the neomycin gene. dihydrofolate reductase and the leader sequence. It was noticed that this vector results in expression at a very high level of antibodies through the incorporation of genes from the constant and variable regions, transfection in CHO cells, followed by selection in a medium containing G418 and amplification of methotrexate. Of course, any expression vector that is capable of inducing expression in eukaryotic cells can be used in the present invention. Examples of suitable vectors include, but are not limited to, plasmids pcDNA3, pHCMV / Zeo, pCR3.1, pEF1i / His, piND / GS, pRc / HCMV2, pSV40 / Zeo ?, pTRACER-HCMV, puUB6 / V5-His , pyvAX1, and pZzeoSV2 (available from Invitrogen, San Diego, CA), and the plasmid pCl (available from Promega, Madison, WI). In general, screening for large numbers of transformed cells for those expressing adequately high levels if light and heavy immunoglobulin chains represent routine experimentation, which can be performed, for example, by robotic systems. Vector systems are also taught in US Patent No.

5,736,137 and 5,665,570, each of which is incorporated herein by reference in its entirety. This system provides for high levels of expression, eg:> 30 pg / cell / day. Other exemplary vector systems are described, eg, in US patent No. 6,413,777.

[00220] In other preferred embodiments, the antibodies, or antigen-binding fragments, variants or derivatives of the invention can be expressed using polycistronic constructs, such as those described in US patent application publication 2003-0157641 A1 and here incorporated in their entirety. In these expression systems, several genetic products of interest, for example, light and heavy chains of antibodies can be produced from a simple polycistronic construction. Such systems advantageously use an internal ribosome entry site (IRES) that provides relatively high levels of antibodies. IRES compatible strings are described in US patent No. 6,193,980, which is also incorporated herein. Those skilled in the art will appreciate that these expression systems can be used to effectively produce the full range of antibodies described in the present application. Therefore, in one embodiment, the present invention provides a vector comprising the polynucleotide that encodes at least the binding domain or variable region of an antibody immunoglobulin chain, optionally in combination with a polynucleotide that encodes the variable region of the other chain of immunoglobulin from that binding molecule.

[00221] More generally, after the vector or DNA sequence encoding a monomeric subunit of the antibody has been prepared (a), the expression vector can be introduced into an appropriate host cell. The introduction of the plasmid into the host cell can be carried out by several very popular techniques for those skilled in the art, which include, but are not limited to transfection, including lipotransfection using, for example, FugeneO & O or lipofectamine, protoplast fusion, precipitation with calcium phosphate, cell fusion with encapsulated DNA, micro-injection and infection with intact virus. Typically, the introduction of the plasmid into the host occurs through a standard method of calcium phosphate coprecipitation. The host cells that harbor the expression construct are cultured under conditions suitable for the production of light and heavy chains, and tested for protein synthesis in light and / or heavy chains. Exemplary assay techniques include enzyme-linked immunoabsorbent assay (ELISA), radioimmunoassay (RIA) or fluorescence-activated cell separation analysis (FRCS), immunohistochemistry and the like.

[00222] The expression vector is transferred to a host cell using conventional techniques and the transfected cells are then cultured using conventional techniques to produce an antibody for use in the methods described herein. Therefore, the invention includes host cells that comprise a polynucleotide that encodes an antibody of the invention, or a heavy or light chain thereof, or at least the binding domain or an immunoglobulin variable region thereof, which are preferably linked operational to a heterologous promoter. In addition or as an alternative, the invention also includes host cells that comprise a vector, as defined above, comprising a polynucleotide that encodes at least the binding domain or the variable region of

93/139 an antibody immunoglobulin chain, optionally in combination with a polynucleotide that encodes the variable region of the other immunoglobulin chain of said binding molecule. In preferred embodiments for the expression of double chain antibodies, a single vector or vectors encoding both the heavy and light chains can be coexpressed in the host cell for the expression of the entire immunoglobulin molecule, as detailed below.

[00223] The host cell can be cotransfected with two expression vectors of the invention, the first vector to encode a polypeptide from the heavy chain and the second vector to encode a polypeptide from the light chain. The two vectors can contain identical selectable markers that allow equal expression of the polypeptides of the heavy and light chains. Alternatively, a simple vector can be used, which encodes the polypeptides of both heavy and light chains. In these situations, the light chain is positioned advantageously in front of the heavy chain to avoid an excess of the toxic-free heavy chain; see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad. Sci. EUA 77 (1980), 2197. The coding sequences of the heavy and light chains can comprise cDNA or genomic DNA.

[00224] As used herein, "host cells" refer to cells that harbor vectors constructed using recombinant DNA techniques and that encode at least one heterologous gene. In descriptions of recombinant host antibody isolation processes, the terms "cell" and "cell culture" are used interchangeably to indicate the source of the antibody, unless clearly specified otherwise. In other words, the recovery of the polypeptide from the "cells" can be either from rapidly centrifuged whole cells, or from the cell culture containing the medium and cells in suspension.

[00225] Various host vector expression systems can be used to express antibody molecules for use in the methods described herein. These host expression systems represent vehicles through which the coding sequences of interest can be produced and subsequently purified, but they also represent cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, express

94/139 an antibody molecule of the invention in situ. They include, but are not limited to, microorganisms such as bacteria (eg E. coli, B. subtilis) transformed with expression vectors with cosmid DNA, plasmid DNA and recombinant bacteriophage DNA containing antibody coding sequences ; yeast (eg, Saccharomyces, Pichia) transformed with recombinant yeast expression vectors, containing antibody coding sequences; cellular systems of insects infected with recombinant virus expression vectors (eg, baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus 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 mammalian cell systems (eg, COS, CHO, NSO, BLK, 293, 3T3 cells) that harbor recombinant expression constructs that contain promoters from the mammalian cell genome (eg, metallothionein promoter) or mammals (eg, the adenovirus late promoter; the 7.5K promoter of the vaccinia virus). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of the integral recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as the Chinese Hamster Ovary (CHO), together with a vector, such as, for example, the main promoter element of early and intermediate genes from the human cytomegalovirus is an efficient expression system for antibodies; see, e.g., Foecking et a /., Gene 45 (1986), 101; Cockett et al., Bio / Technology 8 (1990), 2.

[00226] The host cell line used for protein expression is often of mammalian origin; those skilled in the art are credited with the ability to preferentially determine the particular host cell lines, which are most suitable for the desired gene product to be expressed there. Exemplary host cell lines include, but are not limited to CHO (Chinese Hamster Ovary), DG44 and DUXB 11 (Chinese Hamster Ovary lines, minus DHFR), HELA (human cervical carcinoma), CVI (kidney line) monkey), COS (a derivative of CVI with SV40 T antigen), VERY, BHK (baby kidney

95/139 hamster), MDCK, WI38, R1610 (Chinese hamster fibroblasts) BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney line), SP2 / O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-1cC1IBPT (bovine endothelial cells), RAJI (human lymphocytes) and 293 (human kidney). CHO and 293 cells are particularly preferred. Host cell lines are typically available with commercial services, with the American Tissue Culture Collection (or the North American Fabric Culture Collection) or with the literature already published.

[00227] In addition, a host cell strain can be chosen that modulates the expression of the inserted sequences or modifies and processes the gene product in the specific form desired. These modifications (eg, glycosylation) and processing (eg, cleavage) of protein products can be important for protein function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells that have the cellular machinery for proper processing of the primary transcript, glycosylation and phosphorylation of the gene product can be used.

[00228] For high-yield, long-term production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the antibody molecule can be manipulated. Instead of using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate elements of expression control (eg promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.). ), and a selectable marker. After introducing the foreign DNA, the manipulated cells can be grown for 1 to 2 days in an enriched medium, and then changed to a selective medium. The selectable marker on the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which, in turn, can be cloned and expanded in lines

96/139 cell phones. This method can be used advantageously to manipulate cell lines that stably express the antibody molecule.

[00229] A range of selection systems can be used, including, but not limited to, the herpes simplex virus thymidine kinase (Wigler et al /., Cell 11 (1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48 (1992), 202), and adenine phosphoribosyltransferase (Lowy et a /., Cell 22 (1980), 817), the genes can be used in tk, hgprt cells or aprt, respectively. In fact, antimetabolite resistance can be used as the basis for selecting the following genes: dhfr, which confers resistance to methotrexate (Wigler et a /., 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), 4887505; Wu & Wu, Biotherapy 3 (1991), 87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32 (1993), 573-596; Mulligan, Science 260 (1993), 926-932; and Morgan & 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. The methods commonly known in the field of recombinant DNA technology that can be used 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.

[00230] The expression levels of an antibody molecule can be increased by means of vector amplification. For access to an analysis, 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). When a marker in the antibody expressing the vector system is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. As the amplified region is associated with the antibody gene, antibody production will also increase; see Crouse et al., Mol. Cell.

97/139 Biol. 3 (1983), 257. In vitro production allows scaling up to obtain large quantities of the desired polypeptides. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include homogeneous suspension culture, eg in a fluidized bed reactor or in a continuous agitation reactor or immobilized or trapped cell culture, eg .: in hollow fibers, microcapsules, in agarose microspheres or ceramic cartridges. If necessary and / or desired, polypeptide solutions can be purified using the usual chromatography methods, for example, gel filtration, ion exchange chromatography, DEAE cellulose chromatography or (immuno-) affinity chromatography, eg: after the preferential biosynthesis of a synthetic polypeptide from the articulation region or before or after the HIC chromatography step described here.

[00231] Genes encoding antibodies, or antigen-binding fragments, variants or their derivatives of the invention can also be expressed in non-mammalian cells, such as bacteria or insects or yeast or vegetables. Bacteria that easily occupy nucleic acids include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococceus; Streptococcus and Haemophilus influenzae. It will be even more appreciated if, when expressed in bacteria, heterologous polypeptides normally become part of inclusion bodies. The heterologous polypeptides must be isolated, purified and then assembled into functional molecules. Where tetravalent forms of antibodies are desired, the subunits will then self-organize into tetravalent antibodies; see, eg, international application WO002 / 096948.

[00232] In bacterial systems, a series of expression vectors can be selected advantageously, depending on the intended use for the antibody molecule being expressed. For example, when a large amount of this protein needs to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors that direct the expression of high levels of fusion protein products that are readily purified may be desirable. These vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2 (1983), 1791), in which the antibody coding sequence can be individually linked within the grid vector with the region of

98/139 encoding lacZ so that a fusion protein is produced; plN vectors (Inouye & Inouye, Nucleic Acids Res. 13 (1985), 3101-3109; Van Heeke & Schuster, J. Biol. Chem. 24 (1989), 5503-5509); and the like. PGEX vectors can also be used to express foreign polypeptides such as glutathione S-transferase (GST) fusion proteins. In general, these fusion proteins are soluble and can be easily purified from cells lysed by adsorption and binding to a matrix of glutathione-agarose beads, followed by elution in the presence of free glutathione. The pGEX vectors are designed to include factor Xa or thrombin protease cleavage sites, so that the cloned target gene product can be released from the GST portion.

[00233] In addition to prokaryotes, eukaryotic microbes can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms, although some other strains are commonly available, eg Pichia pastoris. For expression in Saccharomyces, the plasmid YRp7, for example, (Stinchcomb et a /., Nature 282 (1979), 39; Kingsman et al., Gene 7 (1979), 141; Tschemper et al., Gene 10 (1980) , 157) is commonly used. This plasmid already contains the TRP 1 gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, Genetics 85 (1977), 12 ). The presence of the trilus lesion as a characteristic of the yeast host cell genome, then, provides an effective environment for the detection of transformation through growth in the absence of tryptophan.

[00234] In an insect system, the nuclear polyhedrosis virus Autographa californica (ACNPV) is typically used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into non-essential regions (for example, in the polyhedrin gene) of the virus and placed under the control of an ACNPV promoter (for example, the polyhedrin promoter).

[00235] Once an antibody molecule of the invention has been expressed recombinantly, the integral antibodies, their dimers, individual light and heavy chains, or other forms of immunoglobulin of the present invention can be purified

99/139 according to standard industry procedures, including, for example, by chromatography (eg ion exchange, affinity, particularly by affinity of the specific antigen after Protein A, and column sizing chromatography), centrifugation, differential solubility , eg, precipitation with ammonium sulfate, or by any other standard technique for protein purification; see, e.g., Scopes, "Protein Purification", Springer Verlag, N.Y. (1982). Alternatively, a preferred method for increasing the antibody affinity of the invention is described in US patent publication 2002-0123057 A1. In one embodiment, therefore, the present invention also provides a method for preparing an anti-APAP or anti-prolAPP antibody or the immunoglobulin chain (s) thereof, with this method comprising: (a) culturing the host cell, as defined above, which cell comprising a polynucleotide or vector as defined above; and (b) isolating that antibody or immunoglobulin chain (s) from the culture.

[00236] Furthermore, in one embodiment, the present invention also relates to an antibody or immunoglobulin chain (s) thereof, encoded by a polynucleotide, as defined above or obtained by said method for the preparation of an anti- | APP or anti-prolAPP or immunoglobulin chain (s) thereof. V. Fusion Proteins and Conjugates

[00237] In certain embodiments, the antibody polypeptide comprises an amino acid sequence or one or more fractions that are not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, a single chain Fv antibody fragment of the invention can comprise a flexible linker sequence, or can be modified to add a functional group (eg, PEG, a drug, a toxin, or a label as a fluorescent heavy metal, radioactive, enzymatic, nuclear magnetic, and the like).

[00238] An antibody polypeptide of the invention can comprise, be composed essentially of, or be composed of a fusion protein. Fusion proteins are chimeric molecules that comprise, for example, an IAPP-binding domain

Immunoglobulin 100/139 and / or prolAPP with at least one target binding site, and at least one heterologous moiety, i.e., a moiety with which it does not naturally bind in nature. The amino acid sequences can normally exist in separate proteins that are assembled in the fusion polypeptide or can normally exist in the same protein, but are placed in a new arrangement in the fusion polypeptide. Fusion proteins can be created, for example, by chemical synthesis, or by creating and translating a polynucleotide, in which the regions of the peptide are encoded in the desired relationship.

[00239] The term "heterologous", as applied to a polynucleotide or polypeptide, means that that polynucleotide or polypeptide is derived from an entity distinct from the rest of the entity to which it is being compared. For example, as used herein, a "heterologous polypeptide" that will be fused to an antibody or antigen-binding fragment, variant or analog thereof, originates from a polypeptide without immunoglobulin of the same species, or a polypeptide with immunoglobulin or without immunoglobulin from different species.

[00240] As discussed in greater detail elsewhere in this document, antibodies, or antigen-binding fragments, variants or derivatives of the invention can be further recombinantly fused to a heterologous polypeptide at the N- or C-terminus or conjugated by chemical reaction (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies can be fused recombinantly or conjugated to molecules useful as markers in detection assays and effector molecules such as heterologous polypeptides, drugs, radioisotopes, or toxins; see, eg international orders VWOS92 / 08495; WO091 / 14438; WOB89 / 12624; US patent no.

5,314,995; and European patent application EP 0 396 387.

[00241] Antigen-binding antibodies or fragments, variants or derivatives thereof of the invention may be composed of amino acids linked to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres, and may contain amino acids other than amino acids encoded with 20 genes. Antibodies can be modified by natural processes, such as post-translational processing, or by chemical modification techniques that are very popular in the industry. These changes are described in detail in basic texts and in more detailed monographs, as well as in a large research literature. Modifications can occur anywhere in the antibody, including the backbone of the peptide, the secondary chains of amino acids and the ends of the amino or carboxyl termini, or in groups such as carbohydrates. It will be appreciated if the same type of modification is present to the same degree or to varying degrees in different locations of a given antibody. Even a given antibody can contain many types of modifications. Antibodies can be branched, for example, as a result of ubiquitination, and can be cyclical, with or without branching. Cyclic, branched and cyclic branched antibodies can result from natural post-translational processes or can be prepared by synthetic methods. Modifications include acetylation, acylation, ADP ribosylation, amidation, covalent bonding of flavin, covalent bonding of a heme fraction, covalent bonding of a nucleotide or nucleotide derivative, covalent bonding of a lipid or lipid derivative, covalent phosphatidylinositol bonding, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking formation, cysteine formation, pyroglutamate formation, formulation, gamma carboxylation, glycosylation , the formation of GPI anchors, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, RNA-mediated addition transferring amino acids to proteins such as arginylation, and ubiquitination; see, e.g., Proteins - Structure And Molecular Properties, T. E. Creighton, W. H. Freeman & 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).

[00242] The present invention provides for fusion proteins that comprise an antibody, or antigen-binding fragment, variant or derivative thereof, and a heterologous polypeptide. In one embodiment, a fusion protein of the invention comprises, is essentially composed of, or is composed of a polypeptide that has the amino acid sequence of any one or more of the Vx regions of an antibody of the invention or the amino acid sequence of any one or more of the V. regions of an antibody of the invention, or fragments or variants thereof, and a heterologous polypeptide sequence. In another embodiment, a fusion protein for use in the diagnostic and treatment methods described herein comprises, is essentially composed of, or is composed of a polypeptide that presents the amino acid sequence of any one, two or three of the V4-CDRs of an antibody , or fragments, variants or derivatives thereof, or the amino acid sequence of any one, two or three of the V1-CDRs of an antibody, or fragments, variants or derivatives thereof, and a heterologous polypeptide sequence. In one embodiment, the fusion protein comprises a polypeptide that has the amino acid sequence of a VH-CDR3 of an antibody of the present invention, or fragment, derivative or variant thereof, and a heterologous polypeptide sequence, whose fusion protein is specifically links to IAPP and / or prolAPP. In yet another embodiment, a fusion protein comprises a polypeptide that has the amino acid sequence of at least one Vr region of an antibody of the invention and the amino acid sequence of at least one V region of an antibody of the invention or fragments, derivatives thereof or variants thereof, and a heterologous polypeptide sequence. Preferably, the Vx and VL regions of the fusion protein correspond to a single source antibody (or Fab or scFv fragment), which specifically binds IAPP and / or prolAPP. In yet another embodiment, a fusion protein for use in the diagnostic and treatment methods described herein comprises a polypeptide that has an amino acid sequence of any one, two, three or more of an antibody's V4 - CDRs and the amino acid sequence of any one, two, three or more of an antibody's V-CDRs, or fragments or variants thereof, and a heterologous polypeptide sequence. Preferably, two, three, four, five, six or more of the Vi-CDR (s) or V.-CDR (s) correspond to the single source antibody (or Fab or scFv fragment) of the invention . Nucleic acid molecules that encode such fusion proteins are also understood by the invention.

[00243] Exemplary fusion proteins reported in the literature include T cell receptor fusions (Gascoigne et a / l., Proc. Natl. Acad. Sci. USA 84 (1987), 2936-2940; CD4 (Capon et a /., Nature 337 (1989), 525-531; Traunecker et al., Nature 339 (1989), 68-70; Zettmeissl et a /., DNA Cell Biol. USA 9 (1990), 347-353; and Byrn et al.,

103/139 Nature 344 (1990), 667-670); L-selectin (receptor homing) (Watson et a., J. Cell. Biol. 110 (1990), 2221-2229; and Watson et a., Nature 349 (1991), 164-167); CD44 (Aruífo et al., Cell 61 (1990), 1303-1313); CD28 and B7 (Linsley et al., J. Exp. Med. 173 (1991), 721-730); CTLA- (Lisley et al., J. Exp. Med. 174 (1991), 561-569); CD22 (Stamenkovic et al., Cell 66 (1991), 1133-1144); TNF receptor (Ashkenazi et a / l., 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 a receptor (Ridgway & Gorman, J. Cell. Biol. 115 (1991), Abstract No. 1448).

[00244] As discussed elsewhere in this document, antibodies, or antigen-binding fragments, variants or derivatives of the invention can be fused to heterologous polypeptides to increase the in vivo half-life of the polypeptides or for use in immunoassays using methods popular in the field . For example, in one embodiment, PEG can be conjugated to the antibodies of the invention to increase its half-life in vivo; see, e.g., Leong et al., Cytokine 16 (2001), 1067119; Adv. In Drug Deliv. Rev. 54 (2002), 531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

[00245] Furthermore, antibodies, or antigen-binding fragments, variants or derivatives of the invention can be fused to marker sequences, such as a peptide, for example, to facilitate their purification or detection. In preferred embodiments, the amino acid marker sequence is a hexa-histidine (HIS) peptide, as well as the identification provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86 (1989), 821-824, for example, hexanhistidine provides convenient purification of the fusion protein. Other peptide identifications useful for purification include, but are not limited to, the “HA” identification, which corresponds to an epitope derived from the flu hemagglutinin protein (Wilson et a /., Cell 37 (1984), 767), GST, C -MYC and the “flag” marker; see, eg, Bill Brizzard, BioTechniques 44 (2008) 693-695 for an analysis of epitope tagging techniques, and Table 1 on page 694 of the same, listing the most common epitope tags usable in the present invention, the subject of which is expressly incorporated by reference in this instrument.

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[00246] Fusion proteins can be prepared using methods that are well known in the art; see, for example, US patents No. 5,116,964 and No. 5,225,538. The exact location at which the fusion is performed can be selected empirically to optimize the binding or secretion characteristics of the fusion protein. The DNA encoding the fusion protein is then transfected into a host cell for expression, which is performed as described above.

[00247] Antibodies of the present invention can be used in unconjugated form or can be conjugated to at least one of a series of molecules, eg to improve the therapeutic properties of the molecule, to facilitate detection of the target, or to imaging or patient therapy. Antigen-binding antibodies or fragments, variants or derivatives of the invention can be labeled or conjugated, either before or after purification, when carried out. In particular, antibodies or antigen-binding fragments, variants or derivatives thereof of the invention can be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, lipids, viruses, biological response modifiers, pharmaceutical agents, or PEG.

[00248] Conjugates that are immunotoxins, including conventional antibodies, have already been widely described in the art. Toxins can be coupled to antibodies using conventional coupling techniques, or immunotoxins that contain toxin portions of the protein can be produced as fusion proteins. The antibodies of the present invention can be used correspondingly to obtain these immunotoxins. Illustrative of these immunotoxins are those described by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and by Fanger, Immunol. Today 12 (1991), 51-

54.

[00249] Those who are qualified in the field will appreciate the fact that the conjugates can also be assembled using a series of techniques, depending on the selected agent to be conjugated. For example, biotin conjugates are prepared, eg by reacting an IAPP and / or prolAPP binding polypeptide with an activated biotin ester, such as the biotin ester N-hydroxysuccinimide. Likewise, conjugates with a fluorescent marker can be prepared in the presence of a coupling agent, for example, those listed here, or by reaction with an isothiocyanate, preferably fluorescein isothiocyanate. The conjugates of

105/139 antibodies or antigen-binding fragments, variants or derivatives of the invention are prepared in an analogous manner.

The present invention further encompasses antibodies or antigen-binding fragments, variants or derivatives of the invention conjugated to a therapeutic or diagnostic agent. Antibodies can be used in a diagnostic manner, for example, to demonstrate the presence of a metabolic disease, eg DT2, to indicate the risk of contracting a metabolic disease, to monitor the development or progression of a metabolic disease, or that is, a disease that presents the occurrence of IAPP and / or aggregated prolAPP, or related to them, as part of a clinical testing procedure for, eg, determining the effectiveness of a particular treatment and / or prevention regime. In such an embodiment, the present invention relates to an antibody, which is detectably labeled. In addition, in one embodiment, the present invention relates to an antibody that is linked to a drug. Detection can be facilitated by coupling the antibody or antigen-binding fragment, variant or derivative of it to a detectable substance. The detectable substances or the marker can, in general, be an enzyme; a heavy metal, preferably gold; a dye, preferably fluorescent or luminescent; or a radioactive marker. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals that use various positron emission tomographs, non-radioactive paramagnetic metal ions; see, e.g., US Patent No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostic agents in accordance with the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, B-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbeliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin; and examples of suitable radioactive materials include 1251, 1311, In or Tc. Therefore, in a

106/139 embodiment, the present invention provides a detectably labeled antibody, wherein the detectable marker is selected from the group consisting of an enzyme, a radioisotope, a fluorophore and a heavy metal.

[00251] An antibody or antigen-binding fragment, variant or derivative of the same can also be detectably labeled, being coupled to a chemiluminescent compound. The presence of the chemiluminescent-labeled antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, thermal acridinium ester, imidazole, acridinium salt and oxalate ester.

[00252] One of the ways in which an antibody or antigen-binding fragment, variant or derivative of it can be detectably labeled is by binding it to an enzyme, and using the product bound in an enzyme immunoassay (EIA ) (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 enzyme, which is bound to the antibody, will react with an appropriate substrate, preferably a chromogenic substrate, in order to produce a chemical portion that can be detected, for example, by visual, spectrophotometric or fluorimetric means. Enzymes that can be used to detectably detect the antibody include, but are not limited to, malate dehydrogenase, micrococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase , triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glycoamylase and acetylcholinesterase. In addition, detection can be achieved by colorimetric methods that employ a chromogenic substrate for the enzyme. Detection can also be achieved by visually comparing the extent of a substrate's enzymatic reaction compared to similarly prepared standards.

107/139

[00253] Detection can also be achieved by using any of a number of other immunoassays. For example, by radiolabeling the antibody or antigen-binding fragment, variant or derivative of it, it is possible to detect the antibody using a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, (March, 1986)), which is hereby incorporated by reference). The radioactive isotope can be detected by means that include, but are not limited to, a gamma ray counter, a scintillation counter, or autoradiography.

[00254] An antibody or antigen-binding fragment, variant or derivative thereof, can also be detectably labeled using fluorescence-emitting metals, such as 152Eu, or others in the lanthanide series. These metals can be linked to the antibody using metal chelating groups such as diethylenetriamine pentaacetic acid (DTPA) or ethylenediamine tetraacetic acid (EDTA).

[00255] Techniques for conjugating several portions to an antibody or antigen-binding fragment, variant or derivative thereof, are well known, see, eg, Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy" , in Monoclonal Antibodies And Cancer Therapy, Reisfeld et a /. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et a. (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 a. (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 a. (Eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-To xin Conjugates ”, Immunol. Rev. 62 (1982), 119-158.

[00256] As mentioned in certain embodiments, a unit that enhances the stability or effectiveness of a binding molecule, e.g., a binding polypeptide, e.g., an antibody or immunospecific fragment thereof can be conjugated.

108/139 For example, in one embodiment, PEG can be conjugated to the binding molecules of the invention to increase its half-life in vivo. Leong et a / l., Cytokine 16 (2001), 106; Adv. In Drug Deliv. Rev. 54 (2002), 531; or Weir et al., Biochem. Soc. Transactions (2002), 512. Vl. Compositions and Methods of Use

[00257] The present invention relates to compositions that comprise the aforementioned IAPP and / or prolAPP binding molecule, e.g., the antibody or its binding fragment to the antigen of the present invention or derivative or variant thereof, or the polynucleotide , vector or cell of the invention, as defined hereinbefore. In one embodiment, the composition of the present invention is a pharmaceutical composition and further comprises a pharmaceutically acceptable delivery agent. In addition, the pharmaceutical composition of the present invention can comprise other agents, for example, interleukins or interferons, depending on the intended use of the pharmaceutical composition. For use in the treatment of a metabolic disease that presents or related to IAPP and / or prolAPP, eg DT2, the other agent can be selected from the group consisting of small organic molecules, anti-lAPP antibodies and / or anti-prolAPP, and their combinations. Therefore, in a particularly preferred embodiment, the present invention relates to the use of the IAPP and / or prolAPP binding molecule, e.g., antibody or its antigen binding fragment of the present invention or a binding molecule that has substantially the same binding specificities as any of these, the polynucleotide, the vector or the cell of 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 for a response to a metabolic disease treatment in an individual or to determine an individual's risk of developing a metabolic disease.

[00258] Therefore, in one embodiment, the present invention relates to a method of treating a metabolic disorder characterized by abnormality in the accumulation and / or deposition of IAPP and / or prolAPP in islets of Langerhans, the method of which comprises administration, for an individual who needs, an amount

109/139 therapeutically effective of any of the aforementioned binding molecules of IAPP and / or prolAPP, as well as antibodies, polynucleotides, vectors or cells of the present invention. The terms “metabolic disorder” include, but are not limited to, the group of disorders generally characterized by symptoms such as metabolic changes that precede, cause and / or connected / associated with or linked to DT2, comprising diseases that cause damage to the pancreas and are therefore capable of leading to diabetes, comprising chronic pancreatitis, cystic fibrosis, pancreatic cancer; in diseases that increase the risk of T2D, including Alzheimer's disease, Huntington's disease; in cardiovascular diseases linked or not to obesity and DT2; and / or DT2 itself.

[00259] A particular advantage of the therapeutic approach of the present invention resides in the fact that the antibodies of the present invention originate in the B cells or the B memory cells of healthy humans and without signs of a disease that presents the occurrence of IAPP and / or related prolAPP, or related to them, such as DT2 and, therefore, are, with some probability, capable of preventing a clinically manifest disease related to IAPP and / or aggregated prolAPP, or of decreasing the risk of occurrence of the clinically manifest disease , or to delay the spread or progression of the clinically manifest disease. Typically, the antibodies of the present invention have also successfully passed somatic maturation, that is, optimization regarding selectivity and efficacy in binding high affinity to the target molecule of IAPP and / or prolAPP through the somatic variation of the variable regions of the antibody .

[00260] The knowledge that these cells in vivo, eg in a human being, were not activated by means of physiological proteins or related or different cellular structures, in the sense of an allergic or autoimmune reaction is also of great medical importance, since it means a considerably greater chance of living successfully through the phases of clinical trials. In a way, efficiency, acceptance and tolerance have already been demonstrated before the pre-clinical and clinical development of the prophylactic or therapeutic antibody in at least one human being. Therefore, the human anti-lAPP and / or anti-prolAPP antibodies of the present invention can be expected, both their specific efficiency for the target structure as a therapeutic agent and their reduced likelihood of side effects significantly increase their likelihood. successful clinic.

[00261] The present invention also provides a pharmaceutical and diagnostic product, respectively, in the form of a package or kit, comprising one or more containers filled with one or more of the above described ingredients, eg anti-lAPP and / or anti antibody -prolAPP, binding fragment, derivative or variant thereof, polynucleotide, vector or cell of the present invention. Associated with this (these) container (s), there may be a notice in the form prescribed by some government agency that regulates the manufacture, use or sale of pharmaceutical or biological products, and that this notice reflects the approval by the agency , from the manufacture, use or commercialization for administration to human beings. In addition, or as an alternative, the kit comprises reagents and / or instructions for use in appropriate diagnostic tests. The composition, eg, the kit of the present invention is, of course, particularly suitable for risk assessment, diagnosis, prevention and treatment of a disorder that is accompanied by the presence of aggregated IAPP and / or prolAPP, and in particular, applicable to treatment of disorders usually characterized by symptoms “such as metabolic changes that precede, cause and / or connected / associated with or linked to DT2, comprising diseases that cause damage to the pancreas and capable, therefore, of leading to diabetes, comprising chronic pancreatitis, cystic fibrosis, cancer pancreatic; in diseases that increase the risk of T2D, including Alzheimer's disease, Huntington's disease; in cardiovascular diseases linked or not to obesity and DT2; and / or DT2 itself, for example.

[00262] The pharmaceutical compositions of the present invention can be formulated according to methods very popular in the art; see, eg, Remington: The Science and Practice of Pharmacy (2000) by the Philadelphia University of Sciences, ISBN 0-683-

306472. Examples of suitable pharmaceutical delivery agents are well known in the art and comprise phosphate buffered saline solutions, water, emulsions, such as oil / water, various types of wetting agents, sterile solutions, etc. Compositions comprising such delivery agents can be formulated by well-known conventional methods. Such pharmaceutical compositions can be administered to the individual through an appropriate dose. The administration of the appropriate compositions can be carried out in several ways, eg: intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, topical or intradermal or spinal or cerebral penetration. Aerosol formulations, such as nasal spray formulations, include purified aqueous or other solutions of the active agent with preservative agents and isotonic agents. These formulations are preferably adjusted to a pH and an isotonic state compatible with the nasal mucosa membranes. Formulations for rectal or vaginal administration can be presented in the form of a suppository with a suitable delivery agent.

[00263] The dosage regimen will be determined by the doctor treating the patient and by clinical factors. As the medical community is well aware, dosages for any patient depend on many factors, including patient size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs that are being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 µg (or nucleic acid for expression or for inhibiting expression in that range); however, doses lower or higher than this exemplary interval are foreseen, especially considering the factors mentioned above. In general, the dosage can vary, eg from about 0.0001 to 100 mg / kg, and more generally from 0.01 to 5 mg / kg (eg 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 2 mg / kg, etc.), of the host's body weight. For example, dosages can be 1 mg / kg body weight or 10 mg / kg body weight or within the range of 1 to 10 mg / kg, preferably at least 1 mg / kg. Intermediate doses in the above ranges are also intended to be within the scope of the invention. Individuals can receive these doses daily, on alternate days, weekly or according to any other schedule determined by empirical analysis. An exemplary treatment involves the administration of several dosages over a prolonged period, for example, of at least six months. Other exemplary treatment regimens involve administration once every two weeks or once a month or once every 3 to 6 months. Exemplary dosages include 1710 mg / kg or 15 mg / kg on consecutive days, 30 mg / kg on alternate days or 60 mg / kg per 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 fits within the indicated ranges. Progress can be monitored through periodic evaluation. Preparations for parenteral administration include sterile aqueous or 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 administration agents include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate or fixed oils. Intravenous vehicles include fluid, nutrient or electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. In addition, the pharmaceutical composition of the invention can comprise other agents such as dopamine or other psychopharmacological drugs, depending on the intended use of the pharmaceutical composition.

[00264] Furthermore, in a preferred embodiment of the present invention, the pharmaceutical composition can be formulated as a vaccine, for example, if the pharmaceutical composition of the invention comprises an anti-| APP and / or anti-prolAPP antibody or binding fragment , derivative or variant thereof, for passive immunization. As mentioned in the background section, several lines of evidence were shown, indicating that aggregated species of IAPP and / or prolAPP are an important trigger for the pathogenesis DT2 (Zraika et a /. (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 treatments that interfered with the aggregation of hlAPP improved the diabetic phenotype and increased the life expectancy of the animals (Aitken et a /. (2009), Diabetes 59 (1): 161- 171 ). Thus, it is prudent to expect that passive immunization with human anti-IAPP and / or anti-prolAPP antibodies and equivalent IAPP and / or prolAPP binding molecules of the present invention will help to achieve various adverse effects of the concepts of active immunization therapy, as already discussed in the background section. Therefore, the present anti-| lAPP and / or antinprolAPP antibodies and their equivalents of the present invention will be particularly useful as a vaccine for the prevention or amelioration of diseases that have the presence of aggregated IAPP and / or prolAPP, or that are caused by these. , such as, for example, metabolic changes that precede, cause and / or are linked / associated with or related to diseases comprised by DT2 that damage the pancreas and can therefore lead to diabetes, comprising chronic pancreatitis, cystic fibrosis, pancreatic cancer; in diseases that increase the risk of T2D, including Alzheimer's disease, Huntington's disease; in cardiovascular diseases associated or not with obesity and T2D; and / or DT2 itself, for example.

[00265] In one embodiment, it may be beneficial to use bispecific or multispecific recombinant constructs of the antibody of the present invention. For a reference, see Fischer & Léger, Pathobiology 74 (2007), 3-14. This bispecific molecule can be designed to attack IAPP with one connecting arm and another entity known in the pathogenesis of diabetes with the second arm, eg: prol | APP (in addition to the antibodies of the present invention, which are bispecific against IAPP and prolAPP, as indicated above for the exemplary antibody NI-203.26C11). Or a bispecific molecule in these molds may be designed to bind other entities known in the pathogenesis of diabetes to the second connecting arm, such as IL-16 and IL-6, or blocking the sodium-glucose type 2 (SGLT2) or CD33 cotransporter, whose intervention it is thought to reverse diabetes in NOD mice (non-obese diabetics) by inducing adaptive regulatory T cells (Ablamunits et al., Diabetes 61 (2012), 1457154; Belghith et a /., Nat Med 9 (2003), 1202 -1208).

[00266] In one embodiment, it may be beneficial to use recombinant Fab (Fabr) and single chain fragments (scFvs) of the antibody of the present invention, which can more readily infiltrate a cell membrane. For example, Robert et al., Protein Eng. Des. Sel. (2008) Oct 16; S1741-0134, published on the internet in advance, describes the use of the chimeric recombinant Fab (Fabr) and single chain fragments (scFvs) of the monoclonal antibody WO-2 that recognizes an epitope in the N-terminal region of A (3. worked fragments were able to (i) prevent amyloid fibrillation, (ii) disintegrate preformed AB1-42 fibrils and (iii) inhibit neurotoxicity mediated by the AB1-442 oligomer in vitro as efficiently as the entire IgG molecule The perceived advantages of using the small formats of the worked Fab and scFv antibodies that do not have an effective function, include the more efficient passage through the blood-brain barrier and the minimization of the risk of triggering inflammatory side reactions, in addition to scFv and antibodies single-domain proteins retain the specificity of integral antibody binding, can be expressed as simple genes and, intracellularly, in mammalian cells as intrabodies, with the potent alteration of folding, interactions, modifications or subcellular location of their targets; see, for analysis, eg Miller & Messer, Molecular Therapy 12 (2005), 394-401.

[00267] In a different approach, Muller et al /., Expert Opin. Biol. The R. (2005), 237-241, describes a technological platform, called “Super Antibody Technology ', which supposedly allows antibodies to be transported into living cells without harming them. These antibodies that penetrate cells open new therapeutic and diagnostic opportunities. The term TransMabs' was coined for these antibodies.

[00268] In another embodiment, co-administration or sequential administration of other antibodies useful for the treatment of a disease related to the occurrence of IAPP and / or aggregated prolAPP may be desirable. In one embodiment, the additional antibody is comprised in the pharmaceutical composition of the present invention. Examples of antibodies that can be used to treat an individual include, but are not limited to, antibodies that attack CD33, SGLT2, IL-6 and IL -1.

[00269] In another embodiment, co-administration or sequential administration of other agents useful for the treatment of a disease related to IAPP and / or aggregated prolAPP, and / or diabetes may be desirable. In one embodiment, the additional agent is comprised in the pharmaceutical composition of the present invention. Examples of agents that can be used to treat an individual include, but are not limited to: Insulin and insulin analogs; modulators of the insulin signaling pathway, such as protein tyrosine phosphatase inhibitors (PTPases), non-small molecule mimetic compounds and glutamine inhibitors: fructose-6-phosphate amidotransferase (GFAT), DPP-IV inhibitors, agents that influence unregulated hepatic glucose production, such as glucose-6-phosphatase inhibitors

(G6Pase), fructosen-1,6-biphosphatase inhibitors (F-1,6-BPase), glycogen phosphorylase (GP) inhibitors, glucagon receptor antagonists, phosphoenolpyruvate carboxykinase (PEPCK) inhibitors, kinase inhibitors pyruvate dehydrogenase (PDHK), insulin sensitivity enhancers, insulin secretion enhancers, alpha-glucosidase inhibitors, gastric emptying inhibitors; Glucagon-like Peptide 1 receptor agonists (GLP-1); Sulphonylurea agents; Biguanide agents like Metformin; alpha-glucosidase inhibitors; Peroxisome proliferator activated receptor (PPAR) agonists; Medglitinide agents; dipeptidyl peptidase IV (DPP-4) inhibitors; PDE1, PDES5, PDE9, PDE10 or PDE11 inhibitors (PDE = Phosphodiesterase); amylin agonists (eg, pramlintide and other amylin analogues); Cinnamon; Glucagon receptor antagonists; Glycogen-Phosphorylase inhibitors; Fructose-1,6-Bisphosphate inhibitors; Cannabinoid receptor (CBI) antagonists; anti-obesity drugs, such as appetite suppressants, substances that increase satiety and drugs that promote increased energy expenditure; anti-inflammatory agents or any combination thereof. Examples of agents that can be used for the treatment or prevention of islet rejection following clinical pancreatic islet transplantation include, but are not limited to, agents in the group comprising sirolimus (rapamycin), calcineurin inhibitors (e.g. tacrolimus), cyclosporine, mycophenolate mofetil, FTY 720, cyclosporine, corticosteroids and anti-IL2 receptor monoclonal antibodies (eg daclizumab), Glucagon-like Peptide 1 receptor agonists (GLP-1) (see, eg Noguchi et a / l., Acta Med. Okayama, 60 (2006), and the international application WO02012088157). Therefore, in one embodiment, a composition is provided further, comprising an additional agent useful for the treatment of type 2 diabetes mellitus (DT2) and / or the treatment or prevention of islet rejection after clinical pancreatic islet transplantation. Examples of other agents that can be used concurrently with a pharmaceutical composition of the present invention are described in the material; see, eg, international orders VWO2009005672, WO02010128092, WO02012088157 or European order EP11158212.8.

[00270] A therapeutically effective dose or amount refers to that amount of the active ingredient sufficient to alleviate the symptoms or condition. The therapeutic efficacy and toxicity of these compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, eg ED50 (the therapeutically effective dose in 50% of the population) and LD50 (the lethal dose for 50% of the population). The dosage relationship between the toxic effect and the therapeutic effect is the therapeutic index, and this can be expressed as the LD50 / ED50 ratio. Preferably, the therapeutic agent of the composition is present in an amount sufficient to restore or preserve control of the normal blood glucose content and / or the response to insulin in the case of metabolic disorders such as DT2.

[00271] Based on the foregoing, it is evident that the present invention encompasses any use of an IAPP and / or prolAPP binding molecule, comprising at least one CDR of the antibody described above, in particular to diagnose and / or treat a related disease to IAPP and / or aggregated prolAPP, as mentioned above, for example, DT2. Preferably, that binding molecule is an antibody of the present invention or an immunoglobulin chain thereof. In addition, the present invention relates to anti-idiotypic antibodies to any of the antibodies mentioned hereinabove described. These are antibodies or other binding molecules that bind to the unique sequence of antigenic peptides, located in the variable region of an antibody, close to the antigen binding site and are useful, eg, for the detection of anti- | APP antibodies and / or anti-prolAPP in a sample obtained from an individual. In such an embodiment, the present invention provides an antibody as defined above and below, or an IAPP and / or prolAPP binding molecule that has substantially the same binding specificities as either polynucleotide, vector or cell , as defined herein, or a pharmaceutical or diagnostic composition, comprising any of these for use in prophylactic and therapeutic treatment and / or in monitoring the progression or response to treatment of a disorder related to IAPP and / or prolAPP, preferably where the disorder is selected based on the group comprising all types of diabetes, such as type 1 diabetes, gestational diabetes, pre-diabetes (when high blood glucose does not reach the DT2 threshold or insulin resistance ) and latent autoimmune diabetes in adults (LADA); any disease that damages the pancreas and can therefore lead to diabetes, for example, chronic pancreatitis, cystic fibrosis and pancreatic cancer; any disease that increases the risk of T2D, such as Alzheimer's disease and Huntington's disease or

117/139 other neurodegenerative diseases that have been defined here as associated with diabetes; metabolic syndrome, in general, as a risk factor for the development of diabetes or as a condition that may exist before diabetes; islet amyloidosis, in general, as a risk factor for the development of diabetes or as a condition that may exist before diabetes; obesity in general, as a risk factor for the development of diabetes or as a condition that may exist before diabetes; any cardiovascular disease linked or not to obesity and T2D; all the consequences of T2D that can also increase the risk of developing diabetes, such as heart disease, stroke, diabetic retinopathy, kidney failure, ketoacidosis and hyperglycemic hyperosmolar coma. The above group of disorders will be called the group of disorders related to IAPP and / or prolAPP.

[00272] In another embodiment, the present invention relates to a diagnostic composition comprising any of the aforementioned binding molecules, antibodies, antigen binding fragments, polynucleotides, vectors or cells of IAPP and / or prolAPP of the invention and, optionally, appropriate means for detection, such as, for example, reagents conventionally used in diagnostic methods based on nucleic acids or immunology. The antibodies of the invention are, for example, suitable for use in immunoassays, where they can be used in liquid phase or attached to a solid phase delivery agent. Examples of immunoassays that can use the antibody of the invention are competitive and non-competitive immunoassays, either of direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay), flow cytometry and the Western Blot assay. The antigens and antibodies of the invention can be linked to a number of different delivery agents and used to isolate cells specifically linked to them. Examples of well-known delivery agents include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agarose and magnetite. The nature of the delivery agent can be either soluble or insoluble for the purposes of the invention. There are many different markers and marking methods known to those whose skills in the field are merely common. 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 embodiments discussed here above.

[00273] By means of another embodiment, the binding molecules of IAPP and / or prolAPP, in particular the antibodies of the present invention can also be used in a method to diagnose a disorder in an individual by obtaining a sample of body fluid of the subject being examined which may be a blood, plasma, serum, lymph sample or any other sample of a body fluid, such as saliva or urine, and placing that body fluid sample in contact with an antibody of the present invention under conditions that allow the formation of antibody-antigen complexes. The level of these complexes is then determined by methods known to everyone in the industry, a level significantly higher than that formed in a control sample, indicating the disease in the subject examined. Likewise, the specific antigen bound by the antibodies of the invention can also be used. Therefore, the present invention relates to an in vitro immunoassay that comprises the binding molecule, e.g., its antibody or antigen binding fragment of the invention.

[00274] In this context, the present invention also relates to means designed specifically for this purpose. For example, an antibody-based matrix can be used, which is, for example, filled with antibodies or equivalent antigen-binding molecules of the present invention that specifically recognizes IAPP and / or prolAPP. The design of micromatrix immunoassays is summarized in Kusnezow et al., Mol. Cell Proteomics 5 (2006), 1681-1696. Thus, the present invention also relates to microarrays filled with IAPP and / or prolAPP binding molecules identified in accordance with the present invention.

[00275] In one embodiment, the present invention relates to a method of diagnosing a disease related to IAPP and / or prolAPP aggregated in an individual, to the method comprising determining the presence of IAPP and / or prolAPP and / or IAPP and / or prolAPP aggregated in a sample from the subject to be diagnosed with at least one antibody of the present invention, an IAPP and / or prolAPP-binding fragment of the same, or an IAPP and / or prolAPP-binding molecule that substantially presents the same binding specificities as any of these, where the presence of a pathologically aggregated IAPP and / or prolAPP is indicative of a metabolic disorder, such as DT2 and an increase in the level of pathologically aggregated IAPP and / or prolAPP compared to the level of the monomeric forms of IAPP and / or physiological prolAPP is indicative of the progression of a metabolic disorder in that individual.

[00276] The individual to be diagnosed may be asymptomatic or preclinical for the disease. Preferably, the control individual should have a disease related to IAPP and / or aggregated prolAPP, such as, for example, metabolic changes that precede, cause and / or connected / associated or linked to DT2, comprising diseases that cause damage to the pancreas and capable therefore leading to diabetes, comprising chronic pancreatitis, cystic fibrosis, pancreatic cancer; in diseases that increase the risk of T2D, including Alzheimer's disease, Huntington's disease; in cardiovascular diseases linked or not to obesity and DT2; and / or DT2 itself, in which a similarity between the pathologically aggregated IAPP level and / or prolAPP and the reference standard indicates that the individual to be diagnosed has a metabolic disease or is at risk of developing one. Alternatively, or in addition, as a second control, the control subject has no metabolic disease, in which the difference between the level of physiological monomers of IAPP and / or prolAPP and / or IAPP and / or aggregated prolAPP and the reference standard indicates that the individual to be diagnosed has a metabolic disease or is at risk of developing one. Preferably, the individual to be diagnosed and the control individual (s) are matched for age. The sample to be analyzed can be any body fluid suspected of containing pathologically aggregated IAPP and / or prolAPP, for example, blood, blood plasma, blood serum, urine, peritoneal fluid, saliva or cerebrospinal fluid (CSF).

[00277] The level of physiological monomers of IAPP and / or prolAPP and / or of a pathologically aggregated IAPP and / or prolAPP can be assessed by any suitable method known in the art comprising, for example, analysis of IAPP and / or prolAPP by one or more techniques chosen from Western Blot, immunoprecipitation, enzyme linked immunosorbent assays (ELISA), radioimmunoassay (RIA), fluorescence activated cell separation analysis (FRCS), two-dimensional gel electrophoresis, spectroscopy of

120/139 mass (MS), ionization / desorption laser assisted by matrix-time of flight (MALDI-TOF), ionization by surface-time enhanced laser desorption (SELDI-TOF), high performance liquid chromatography ( HPLC), rapid protein liquid chromatography (FPLC), multidimensional liquid chromatography (LC), followed by tandem mass spectrometry (MS / MS), and laser densitometry. Preferably, the aforementioned in vivo imaging of IAPP and / or prol! APP comprises positron emission tomography (PET), single photon emission tomography (SPELT), optical imaging by near infrared spectrometry (NIR) or magnetic resonance (MRI).

[00278] Antibody-based methods for detecting IAPP and / or prolAPP and for diagnosing or monitoring the progression of a disease related to IAPP and / or aggregated prolAPP, such as DT2, for example, and monitoring the treatment of that disease using antibodies and other related means that can be adapted in accordance with the present invention are also described in international application WO02003092619, the content of which is hereby incorporated by reference. These methods can be applied as described, but with an antibody, binding fragment, derivative or specific variant of IAPP and / or prolAPP of the present invention.

[00279] In such an embodiment, an antibody of the present invention or an IAPP and / or prol! APP binding molecule that has substantially the same binding specificities as any of these, the polynucleotide, the vector or the cell, as defined above, or a pharmaceutical or diagnostic composition, comprising any of these, is provided for use in prophylactic and therapeutic treatments and / or in monitoring the progression or response to treatment of an IAPP and / or prolAPP-related disorder. In general, therefore, the present invention also relates to a method of diagnosing or monitoring the progression of a disorder related to IAPP and / or prolAPP (such as islet amyloidosis and DT2, which is usually preceded by islet amyloidosis ) in an individual, with the method comprising determining the presence of oligomers, aggregates or fibrils of IAPP and / or prolAPP in a sample from the individual to be diagnosed with at least one antibody of the present invention or an IAPP-binding molecule and / or prolAPP that has substantially the same binding specificities as any of these, where the presence of oligomers, aggregates or fibrils of IAPP and / or prolAPP is indicative of the disorder. In one embodiment, the aforementioned method of diagnosing or monitoring the progression of islet amyloidosis in an individual is provided, with the method comprising determining the presence of oligomers, aggregates or fibrils of IAPP and / or prolAPP in a sample from the individual to be diagnosed with at least one antibody of the present invention or an IAPP and / or prolAPP binding molecule that has substantially the same binding specificities as any of those, wherein the presence of IAPP and / or prolAPP oligomers, aggregates or fibrils is indicative of pre-symptomatic, prodromal or clinical type 2 (DT2) diabetes mellitus and / or beta cell failure after clinical pancreatic islet transplantation and after the increase in the level of IAPP and / or prolAPP oligomers, aggregates or fibrils compared to the physiological IAPP level or compared to a reference sample from a healthy control individual or a sample of c control from the same individual, be it indicative of progression of pre-symptomatic, prodromal or established type 2 (DT2) diabetes mellitus and / or insufficiency of islets after the clinical transplantation of pancreatic islets to that individual. Anyone skilled in the field's techniques would find it interesting that, in one embodiment, this method would also be used to diagnose or monitor the progression of any other disorder in the group of disorders related to IAPP and / or prolAPP, as defined above.

[00280] As indicated above, the antibodies of the present invention, their fragments and molecules with the same binding specificity as the antibodies and fragments thereof can be used not only in vitro, but also in vivo, in which, in addition to diagnostic applications , therapeutic applications can also be sought. In such an embodiment, the present invention also relates to an IAPP and / or prolAPP-binding molecule that comprises at least one CDR of an antibody of the present invention for the preparation of a composition for in vivo detection of, or for the purpose of, a therapeutic and / or diagnostic agent for IAPP and / or prolAPP in the human or animal body. Potential therapeutic and / or diagnostic agents can be chosen from the non-exhaustive list of therapeutic agents useful in the treatment of metabolic diseases such as DT2 and potential markers, as indicated above. With reference to in vivo imaging, in a preferred embodiment, the present invention provides that IAPP and / or prolAPP binding molecule that comprises at least one CDR of an antibody of the present invention, wherein that in vivo imaging comprises emission tomography positron (PET), single photon emission tomography (SPELT), optical imaging by near infrared spectrometry (NIR) or magnetic resonance imaging (MRI). In another embodiment, the present invention also relates to that IAPP and / or prolAPP binding molecule, comprising at least one CDR of an antibody of the present invention, or that molecule for the preparation of a composition for in vivo imaging methods. specified above, for use in the method of diagnosing or monitoring the progression of an IAPP and / or prolAPP-related disorder in an individual, as defined above. VILE. Peptides with specific aggregation specific IAPP epitopes

[00281] In another aspect, the present invention relates to peptides that have an IAPP and / or prolAPP epitope specifically recognized by any antibody of the present invention. Preferably, that peptide comprises or is composed of an amino acid sequence, as indicated in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 71, as the only linear epitope recognized by the antibody or a sequence modified from it in which one or more amino acids are replaced, deleted and / or added, in which the peptide is recognized by any antibody of the present invention, preferably by the NI-203.19H8 antibody, respectively by the NI-203.26C11 antibody or by the NI antibody -203.15C7.

[00282] In one embodiment of the present invention, such a peptide can be used to diagnose or monitor a disease related to IAPP and / or prolAPP aggregated in an individual, such as DT2, for example, comprising a step of determining the presence of an antibody that binds to a peptide in a biological sample of that individual, and being used for the diagnosis of a disease in that individual by measuring the levels of antibodies that recognize the peptide described above of the present invention, and comparing the measurements with the levels that are found in healthy individuals of comparable age and sex. Therefore, in one embodiment, the present invention relates to a method of diagnosing islet amyloidosis, indicative of pre-symptomatic or clinical type 2 diabetes mellitus (DT2) and / or beta cell failure after clinical islet transplantation pancreatic infections in an individual, comprising a step of determining the presence of an antibody that binds to a peptide, as defined above in a biological sample of that individual. According to this method, a high level of specific antibodies measured for that peptide of the present invention is indicative for the diagnosis, in that individual, of pre-symptomatic or clinical type 2 diabetes mellitus (DT2) and / or of insufficiency of beta cells after the clinical transplantation of pancreatic islets or for the diagnosis, in that individual, of any other disease in the group of disorders related to IAPP and / or prolAPP, as defined above. The peptide of the present invention can be formulated into a matrix, kit and composition, respectively, as described hereinabove. In this context, the present invention also relates to a kit useful for the diagnosis or monitoring of islet amyloidosis progression, with that kit comprising at least one antibody of the present invention or an IAPP and / or prolAPP binding molecule that has substantially the same binding specificities as any of these, the polynucleotide, the vector or the cell, and / or the peptide, as respectively defined hereinbefore, and optionally with reagents and / or instructions for use.

[00283] These and other embodiments are described and encompassed in the description and examples of the present invention. Other academic materials relating to any of the materials, methods, uses and compounds to be employed in accordance with the present invention can be consulted in public databases and libraries, using, for example, electronic devices. For example, the public database “Medline” can be used, it is organized by the National Biotechnology Information Center and / or the National Medicine Library of the National Health Institute. Other databases and electronic addresses, such as those of the European Bioinformatics Institute (EBI), which is part of the European Molecular Biology Laboratory (EMBL), are known to those who are qualified in the field and can also be obtained with the help of internet search engines. An overview of biotechnology patent information and a survey of sources

124/139 relevant information on patents useful for retrospective research and for the science of how things are at the moment is given in Berks, TIBTECH 12 (1994), 352-364.

[00284] The foregoing description generically describes the present invention. Unless otherwise specified, a term that has been used here receives the definition provided in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised in 2000 and reissued in 2003, ISBN O 19 850673 2. Several documents are cited throughout the text of this specification. Complete bibliographic citations can be found at the end of the specification immediately prior to the claims. The content of all references cited (including bibliographic references, issued patents, published patent applications, as cited throughout this application, and manufacturer's specifications, instructions, etc.) are hereby expressly incorporated by reference; however, there is no admission that any document cited is, in fact, previously conceived with respect to the present invention.

[00285] A more complete understanding can be obtained by reference to the specific examples that follow, which are provided here for the sole purpose of illustration, and not with the intention of limiting the scope of the invention. Examples of embodiments of the invention Example 1: Validation of binding and target specificity of human IAPP antibodies

[00286] To validate IAPP as a recognized target for isolated antibodies, direct ELISA assays were performed. For the exemplary recombinant human antibodies NI-203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3, platelets with 96 wells (Costar, Corning, USA) were coated with a solution of human IAPP or with BSA (Sigma -Aldrich, Buchs, Switzerland) diluted to a concentration of 10 µg / ml in carbonate coating buffer for ELISA (15 mM Na2COs, 35 mM NaHCO ;, pH 9.42) and the binding efficiency of the antibody was tested. The important thing is that the human IAPP solution used for the ELISA matrix contained IAPP fibrils, as shown by electron microscopy; see Fig. 3A. Exemplary NI-antibodies

203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3 specifically bind to human IAPP fibrils by ELISA. There is no link to BSA; see Fig. 3B. The same characteristics seem to apply to the antibodies NI-203.19F2 and NI-203.15C7.

[00287] To determine the effective concentration at half the maximum (EC50) of the exemplary antibodies NI-203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3, other experiments with direct ELISA with different concentrations of antibodies. Platelets with 96 wells (Costar, Corning, USA) were coated with solutions of human IAPP and human prolAPP diluted to a concentration of 10 µg / ml in carbonate coating buffer for ELISA (15 MM Na2CO; 3, 35 mM NaHCO ;, pH

9.42) and the binding efficiency of the antibody was tested. While the human IAPP solution used for the ELISA assay contains IAPP fibrils, human prolAPP formed large aggregates in solution, as revealed by electron microscopy; see Fig. 4A. Binding was determined using an anti-human IgGy equine antibody (Jackson ImmunoResearch, Newmarket, UK) conjugated to HRP, followed by measuring HRP activity in a standard colorimetric assay.

[00288] EC50 values were estimated by non-linear regression, using the GraphPad Prism software (San Diego, USA). Recombinant antibodies of human origin NI-203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3 bind with great affinity to fibrils of human IAPP with EC50 of 9 nM, 22 nM, 6 nM and 4 AM, respectively. The NI-203.26C11 antibody also binds to prolAPP. human aggregated with ECB50 in the nanomolar range (260 nM); see Fig. 4B. Example 2: Specificity of antibodies to human IAPP fibrils and not to non-fibrillar human IAPP, therefore, preferentially binding to conformational epitopes

[00289] To determine the binding capacity of exemplary NI-antibodies

203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3 to conformational epitopes, direct ELISA experiments were performed with solutions of human IAPP and non-fibrillar IAPP diluted to a concentration of 10 ug / m! in carbonate coating buffer for ELISA (15 mM Na2zCO; 3.35 mM NaHCO ;, pH 9.42) and the binding capacity of the antibody was tested. While the human IAPP solution used for the

126/139 ELISA contains IAPP fibrils, the non-fibrillar human IAPP solution was deficient in IAPP fibrils and only small amorphous aggregates, as revealed by electron microscopy; see Fig. SA. Binding was determined using an equine anti-human IgGy antibody (Jackson ImmunoResearch, Newmarket, UK) conjugated to HRP, followed by measuring HRP activity in a standard colorimetric assay.

[00290] NI-203.9A2, NI-203.19H8, NI-203.26C11 and NI- recombinant antibodies

203.8E3 showed a high affinity bond to IAPP fibers after coating with the IAPP solution (Fig. 5B), as previously noted (Fig. 4). A certain loss of affinity was observed in non-fibrillar IAPP when compared to IAPP fibrils (Fig. 5B), thus demonstrating a preferential binding of the antibodies NI-203.9A2, NI-203.19H8, NI-203.26C11 and NI-203.8E3 to IAPP fibrils. Preliminary results show the same effects as described above for NI-antibodies.

203.19F2 and NI-203.15C7.

[00291] These conclusions strongly point to the antibodies NI-203.9A2, NI-

203.19H8, NI-203.26C11, NI-203.8E3, NI-203.19F2 and NI-203.15C7 that connect epitopes that are predominantly exposed and accessible by the formation of IAPP fibrils, in contrast to linear epitopes that are present in the physiological conformation of the human IAPP protein. Pathological IAPP fibrils are seen in pancreatic islets in patients with T2D. Since NI-

203.9A2, NI-203.19H8, NI-203.26C11, NI-203.8E3, NI-203.19F2 and NI-203.15C7 have a prominent connection with pathological fibrils of IAPP. therapeutically relevant human antibodies, these antibodies of human origin have a therapeutic potential for DT2. Example 3: Evaluation of the binding epitope of antibodies NI-203.9A2, NI-

203.19H8, NI-203.26C11, NI-203.8E3, NI-203.19F2 and NI-203.15C7

[00292] To determine the binding epitope of exemplary NI-antibodies

203.9A2, NI-203.19H8, NI-203.26C11, NI-203.8E3, NI-203.19F2 and NI-203.15C7, an alanine and Pepscan scan analysis was performed with overlapping peptides mapping the entire amino acid sequence of human IAPP , and with the replacement of

127/139 alanine in the first 22 amino acids of prolAPP. The binding capacity of the antibody was tested on these peptides that were stained on a nitrocellulose membrane (JPT Peptide Technologies, Berlin, Germany) and using an HRP-conjugated equine anti-human IgGy secondary antibody (Jackson ImmunoResearch, Newmarket, UK ) followed by HRP activity detection (Fig. 6A).

[00293] The recombinant antibodies NI-203.19H8, NI-203.26C11 and NI-203.15C7 (1 pg / ml) showed binding to the sequence 19-SSNNFGA-25 (SEQ ID: 4), 2- CNTATCA-8 (No. SEQ ID: 5) and 10-Q0RLANFLVHS-19 (SEQ ID No.: 71) in human IAPP (Figs. 6A and 6B), thus corresponding to the putative binding epitope sequences of these antibodies. The epitope of recombinant antibodies NI-203.9A2, NI-203.8E3 and NI-203.19F2 (1 and 10 pug / ml) were not identified. Example 4: Binding of NI-203.9A2, NI-203.19H8 and NI-203.26C11 antibodies to pathological IAPP fibrils in the pancreas of patients diagnosed with type 2 diabetes mellitus, but not in control patients

[00294] Paraffin-embedded pancreas sections of two patients diagnosed with type 2 diabetes mellitus (DT2) were selected based on the amyloid load of the pancreatic islets observed based on the staining technique with ThioS and Congo Red, and later used for the characterization the binding of exemplary antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11. Sections of pancreas soaked in paraffin from a patient not diagnosed with type 2 diabetes mellitus were used as controls. After pretreatment with formic acid, sections were incubated with human antibodies N1I-203.9A2, NI-203.19H8 and NI-

203.26C11 (5 and 50 nM) or with the anti- | Murine APP (1: 100; Abcam, Cambridge, United Kingdom), followed by incubation with the equine anti-human biotinylated secondary antibody (1: 500; Jackson ImmunoResearch, Newmarket, United Kingdom) or the anti-murine goat biotinylated secondary antibody (1 : 500; Jackson ImmunoResearch, Newmarket, United Kingdom). The antibody signal was amplified with the Vectastain ABC-AP kit (Vector Laboratories, USA) and detected with diaminobenzidine substrate (Thermo Fisher Scientific, USA). After avidin / biotin block (Avidin / Biotin block kit, Vector Laboratories, USA), pancreatic islet B cells were visualized using a polyclonal guinea pig anti-insulin antibody (1: 5; Dako, USA) coupled to a biotinylated secondary equine anti-goat antibody (1: 500; Jackson ImmunoResearch Laboratories, USA) and the antibody signal was amplified with the Vectastain ABC-AP kit (Vector Laboratories, USA) and detected with the alkaline phosphatase substrate (Vector Laboratories , USA).

[00295] The first patient with DT2 presented vast amyloid deposits in pancreatic islets that corresponded to pathological fibrils of IAPP, as visualized based on the technique of staining with ThioS and Congo Red (Fig. 7A). Human antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 showed prominent pancreatic islet staining in these amyloid-positive sections (Fig. 7B). Antibody staining was observed at 5 nM and enlarged to 50 nM, with no staining seen with only the secondary antibody, suggesting specific binding of human IAPP antibodies. These results were confirmed in a second DT2 patient who had amyloid deposits on pancreatic islets (data not shown). In contrast, the human antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 did not show any coloration in the pancreatic islets of a third patient with DT2 with amyloid deposit deficiency (Figs. 7C and D) and a patient of control not diagnosed with DT2 (Fig. 8). The commercially available murine anti-| APP monoclonal antibody colored the physiological IAPP of the pancreatic islets of the non-diabetic control patient; see Fig. 8. The antibodies of the present invention also gave positive results in feline diabetic pancreas that showed amyloid deposits in the islets; see Fig. 9. The same binding properties appear to apply to NI-203.19F2 and N1I-203.15C7 antibodies.

[00296] These data demonstrate that the antibodies NI-203.9A2, NI-203.19H8, NI-

203.26C11, NI-203.19F2 and NI-203.15C7 specifically recognize pathological IAPP fibrils and conform to the biochemical binding properties of these antibodies, which have a strong specificity of binding to IAPP fibrils in vitro (Fig. 5).

Example 5: NI-203.9A2, NI-203.19H8 and NI-203.26C11 antibodies do not cross-react with pathological AB amyloid in the brain of patients diagnosed with Alzheimer's disease

[00297] Paraffin-embedded brain sections of a patient diagnosed with Alzheimer's disease were used to assess the cross-reactivity of the exemplary antibodies NI-203.9A2, NI-203.19H8 and N1I-203.26C11. After pretreatment with formic acid, sections were incubated with human antibodies NI-203.9A2, NI-

203.19H8 and NI-203.26C11 (50 nM) or with the murine monoclonal anti-B 6E10 antibody (1: 2000; Covance, Allschwill, Switzerland), followed by incubation with the biotinylated secondary anti-human equine antibody (1: 500; Jackson ImmunoResearch, Newmarket, United Kingdom) or by the anti-murine goat biotinylated secondary antibody (1: 500; Jackson ImmunoResearch, Newmarket, United Kingdom). The antibody signal was amplified with the Vectastain ABC-AP kit (Vector Laboratories, USA) and detected with diaminobenzidine substrate (Thermo Fisher Scientific, USA).

[00298] The antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 did not recognize the pathological AB amyloid in the human brain with Alzheimer's disease, in contrast to the specific anti-B amyloid antibody 6E10 (Fig. 10). The same seems to apply to NI-203.19F2 and NI-203.15C7 antibodies.

[00299] These data demonstrate that the antibodies NI-203.9A2, NI-203.19H8 and NI-

203.26C11 do not cross-react with pathological AB amyloid. Thus, a minimally reactive cross-linking of the antibodies NI-203.9A2, NI-203.19H8 and NI-

203.26C11 to several candidate proteins with a propensity to aggregation / unfolding by direct ELISA has been demonstrated, including the most prominent amyloid-forming proteins, including, but not limited to alpha-synuclein, superoxide dismutase 1 (SOD1), and Taue TAR 43 binding protein (TDP-43). Example 6: Quality control of chimeric murine antibodies NI-203.9A2 NI-203.19H8 and NI-203.26C11

[00300] To validate exemplary chimeric murine antibodies NI-203.9A2, NI-

203.19H8 and NI-203.26C11, tests were performed with direct ELISA, as

130/139 described above. Chimeric antibodies were compared to the corresponding human antibodies. For exemplary NI-recombinant chimeric antibodies

203.9A2, NI-203.19H8 and NI-203.26C11, and their corresponding human antibodies, 96-well platelets (Costar, Corning, USA) were coated with a human IAPP solution or with BSA (Sigma-Aldrich, Buchs, Switzerland) diluted to pg / ml concentration in carbonate coating buffer for ELISA (15 mM Na2CO; z, 35 mM NaHCO ;, pH 9.42) and the binding efficiency of chimeric and human antibodies was tested. Binding was determined using an anti-human IgGy equine antibody (Jackson ImmunoResearch, Newmarket, UK) conjugated to HRP, followed by measuring HRP activity in a standard colorimetric assay. The important thing is that the human IAPP solution used for the ELISA matrix contained IAPP fibrils, as shown by electron microscopy; see Fig. 3A. ECso values were estimated using non-linear regression, using the GraphPad Prism software (San Diego, USA).

[00301] Murine chimeric antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 bind with high affinity to human IAPP fibrils with an ECrso of 18.6 nM, 23.9 nM and 11.5 nM , respectively. There was no link to the BSA. The binding affinity of chimeric antibodies was similar to that of their human counterparts, with an ECso of 9.4 nM, 22.9 Nm and 6.8 Nm for human antibodies NI-203.9A2, NI-

203.19H8 and NI-203.26C11, respectively; see Fig. 11.

[00302] The exemplary murine chimeric antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 were subsequently validated in paraffin-embedded pancreas sections of two selected patients diagnosed with type 2 diabetes mellitus (DT2), and presenting deposits amyloid in the islets. After pretreatment with formic acid, the sections were incubated with chimeric antibodies NI-203.9A2, NI-203.19H8 and NI-203.26C11 (50 nM), followed by incubation with the biotinylated secondary anti-human equine antibody ( 1: 500; Jackson ImmunoResearch, Newmarket, United Kingdom). The antibody signal was amplified with the Vectastain ABC-AP kit (Vector Laboratories, USA) and detected with diaminobenzidine substrate (Thermo Fisher Scientific, USA). After avidin / biotin block (Avidin / Biotin block kit, Vector Laboratories, USA), pancreatic islet B cells were visualized using a polyclonal guinea pig anti-insulin antibody (1: 5; Dako, USA) coupled to a biotinylated secondary equine anti-goat antibody (1: 500; Jackson ImmunoResearch, New Market, UK) and the antibody signal was amplified with The Vectastain ABC-AP kit (Vector Laboratories, USA) and detected with the alkaline phosphatase substrate (Vector Laboratories, USA).

[00303] The chimeric antibodies NI-203.9A2, NI-203.19H8 and Nl-203.26C11 showed a prominent staining in the pancreatic islets in amyloid-positive sections of two patients with DT2 (Fig. 12).

[00304] These data demonstrate that chimeric antibodies NI-203.9A2, NI-

203.19H8 and NI-203.26C11 specifically recognize pathological fibrils of IAPP with efficiency comparable to that of their human counterparts (Fig. 12). Example 7: In vivo validation of the therapeutic effect of IAPP and / or prolAPP antibodies in animal models with DT2

[00305] The main candidate antibodies are validated in two transgenic rodent models and in a murine model that expresses hlAPP: 1) h- | lAPP (hemizygous / C57BL / 6 / DBA mice exposed to a high calorie diet (Hull et al . (2003), Diabetes 52: 372-379); 2) h-lAPP (hemizygous) / AY // To mice exposed to a standard diet (Butler et a /. (2003), Diabetes 52: 2304-2314); 3) h- |) lAPP (homozygous) / CD rats exposed to a standard diet (Butler et a /. (2004), Diabetes 53: 1509-1516). Therapeutic efficacy is assessed by determining the beta cell mass and amyloid load of hlAPP in the pancreas, as well as plasma levels of hlAPP, and by functional tests of glucose metabolism and insulin secretion. 1) Physiological characteristics

[00306] The physiological characteristics described below (i) to (vii) of type 1 diabetes in animal models are tested to verify whether the application of the antibodies of the present patent application has any preventive and / or therapeutic effect. (i) Glucose in Sanque

[00307] The blood glucose level of animal models with DT2 is tested and compared to that of untreated animals and animals of normal strain (not of the model with DT2).

[00308] The aforementioned "normal strain mouse" is not particularly limited and can be any mouse, as long as it does not show any abnormality in blood glucose, sugar in urea, insulin secretion and the like. Preferred examples of the “normal strain mouse” include a KOR mouse, an NC mouse and a laboratory mouse that is used as a recurrent parent in the generation of congenic mice (eg, C3H / He mouse, BALB / ce mouse) mouse C57BL / 6).

[00309] The aforementioned "normal strain rat" is not particularly limited and can be any rat, as long as it does not show any abnormality in blood glucose, sugar in urea, insulin secretion and the like.

[00310] As diabetes in mouse and rat models is preferably induced by the transgenic expression of hlAPP, preferably the same strains that were originally used for the generation of transgenic animals are used as control.

[00311] The expression “with a higher blood glucose level compared to a normal mouse / rat strain” means that the fasting blood glucose level (blood glucose concentration) is higher than that of a mouse / fasting normal strain rat. Blood glucose levels in diabetic animals of 130 mg / d1i or more, more preferably 140 mg / d1 or more, more preferably 200 mg / d1 and even more preferably 300 mg / d1 or more are considered to be higher levels of blood glucose. In addition, the term "fasting", as used herein, means a condition of about 12 hours after the start of a mouse / rat fast.

[00312] 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 commercial measuring device (eg, Medisafe reader; Terumo Co. , Ltd.) according to the method described in the following example.

Exemplary method of measuring the Blood Glucose Level

[00313] The blood glucose level (blood glucose concentration) of a given animal is measured using a commercial measuring device (Reader Medisafe, Terumo Co., Ltd.). The measuring principle of the device will be explained below. The measure is based on colorimetric analysis. A measurement chip is prepared, and on the chip, glucose oxidase and peroxidase are placed as catalysts and 4-aminoantipyrin and N-ethyl-N (2-hydroxy-3-sulfopropyl) -n-toluide as chromogenic agents. When a blood sample absorbed through the capillary phenomenon is placed on that chip and then the glucose in the blood is oxidized by glucose oxidase, the chromogenic agents on the chip are oxidized by the hydrogen peroxide generated at that moment and by the peroxidase, which produces a purple-red color. The amount of glucose in the blood is calculated by measuring the degree of that color tone.

[00314] Here, 4 ul of all blood is obtained from a given animal as a blood sample and measured using a measurement time of 18 seconds. (ii) Glycosylated Hemoglobin (HbAlc) concentration:

[00315] Animal models with Type II diabetes are tested to see if there was an increase in the concentration of glycosylated hemoglobin in the blood and compared to animal models with DT2, however, untreated, and to normal animal models, without DT2. Concentrations of 2.5% or greater, 2.6% or greater, still 2.8% or greater, and still 3.0% or greater are classified as increased.

[00316] The "concentration of glycosylated hemoglobin", as used here, means the proportion of hemoglobin molecules with glucose attached to them in a red blood cell. The concentration of glycosylated hemoglobin can be used as an index to judge the adequacy of the therapeutic control to patients with diabetes, and it has been proven that it correlates better with the blood sugar level 1 to 2 months earlier than with the of the present moment.

[00317] The "concentration of glycosylated hemoglobin" can be measured by a conventional method known to those skilled in the art, for example, through

134/139 using a commercial measuring device (eg DCA 2000 System; Bayer Medical Ltd.) according to the method described in the following example. More specifically, for example, when the aforementioned DCA 2000 System is used as a measuring device, the amount of total hemoglobin is measured by the method of thiocyanatomethemoglobin and the amount of glycosylated hemoglobin is measured by means of the latex coagulation inhibition reaction. (iii) Sugar in the Urine:

[00318] The urine sugar of control animals and animal models with DT2 is tested here.

[00319] The term "positive test for sugar in the urine", as used here, means that the glucose concentration in the urine excreted by the animals is 100 mg / d1 or higher. The concentration of glucose in the urine can be measured by a conventional method, for example, by the method described in the following example, using a commercial kit (eg Pretest; Wako Pure Chemical Industries, Ltd.), Specifically, for example, when Pretest is used as a measurement kit, a urine sample from the animal is first placed on a Pretest test strip, and after 30 seconds, judged according to the color table specified in that Kkit for classification between five intervals ranging from - to +4. The estimated urine glucose concentrations resulting from the trial are 100 to 250 mg / d1i for +1, 250 to 500 mg / dl for +2, 500 to 2000 mg / dl for +3, and 2000 mg / d1 or more to +4. Results judged to be +1 or higher are assessed as a “positive urine sugar test”. Exemplary urine sugar measurement method

[00320] The urine glucose (sugar in the urine) of a given animal is measured by methods known to those skilled in the art, eg using a commercial kit (Pretest; Wako Pure Chemical Industries, Ltd.). First, a urine sample from the animal is placed on a test strip of the aforementioned Pretest, and after 30 seconds, judged according to the specified color table for classification between five intervals ranging from - to +4. The estimated urine glucose concentrations resulting from the trial are 100 to 250 mg / dl for

+1, from 250 to 500 mg / dl for +2, from 500 to 2000 mg / dl for +3, and 2000 mg / d1 or more for +4. Results judged to be +1 or higher are assessed as a “positive urine sugar test”. (iv) Insulin Concentration in Sanque:

[00321] It is tested whether the Blood Insulin Concentration levels of animal models with Type diabetes || are equivalent or superior to those of non-diabetic control animals and untreated control animals (animals of the normal strain).

[00322] The expression that the blood insulin concentration is "equivalent or higher than that of a normal strain animal" means that the fasting blood insulin concentration is equivalent to or higher than that of a fasting normal strain animal, eg 90 pg / ml or more, or 110 pg / ml or more.

[00323] In the present invention, the "Blood Insulin Concentration" can be measured by a conventional method, for example, using a Levis type U insulin assay kit (Shibayagi Co.) in accordance with the method described in the following example. More specifically, for example, an anti-insulin (murine) monoclonal antibody is immobilized on a plate, and an insulin sample is attached to it, and then a biotin-labeled anti-insulin monoclonal antibody that recognizes another insulin sample is reacted with it, and a peroxidase-avidin conjugate is added to these to bind to biotin, and, finally, a chromogenic substance is added to determine insulin by color development. Exemplary method of measuring Insulin Concentration in Sanque

[00324] The concentration of insulin in the blood of a given mouse is measured using a method known to those skilled in the art, eg, incorporated into a commercial kit (Levis type U insulin test kit; Shibayagi Co. ). Insulin concentration is measured as follows: an anti-insulin monoclonal antibody is immobilized on a plate, and an insulin sample is attached to it, and then a biotin-labeled anti-insulin monoclonal antibody, which recognizes another portion of insulin, it is reacted with it, and a

136/139 peroxidase-avidin conjugate is also added to these to bind to biotin, and, finally, a chromogenic substance is added to determine insulin by color development. The measurement range is usually 39 to 2,500 pg / ml for a normal animal (mouse). (v) Glucose Tolerance

[00325] Animal models with treated and untreated DT? 2 and normal animals are tested for abnormal glucose tolerance.

[00326] The normality or abnormality of an animal's glucose tolerance can be confirmed by means of a glucose tolerance test, which can be performed according to a conventional procedure known to those skilled in the field, eg by administering glucose intraperitoneally to a fasting animal (at least 12 hours) at 2 mg per gram of body weight and subsequently measuring the blood glucose level of the animal at certain times during the glucose tolerance test (eg Every 15 minutes over an interval of 240 minutes). When the result shows that the blood glucose level has no tendency to decrease over time compared to that of a normal rodent, glucose tolerance is assessed as abnormal. When the result in treated animals shows a tendency to decrease over time compared to that of untreated animals, treatment with the antibodies of the present invention is classified as effective. Exemplary Glucose Tolerance Assessment Method

[00327] The glucose tolerance of certain animals is assessed by the glucose tolerance test.

[00328] The glucose tolerance test is performed, first, by administering glucose intraperitoneally to an animal fasting for 12 hours at 2 mg per gram of body weight and subsequently measuring the blood glucose level (peripheral) of the animal every 15 minutes for an interval of 240 minutes. The blood glucose level is measured by the above method. When the result shows that the blood glucose level has no tendency to decrease with the

137/139 over time compared to that of a normal animal, glucose tolerance is assessed as abnormal. A faster reduction over time in animal models with treated DT? 2, compared to untreated, is evaluated as a sign of treatment effectiveness with the present invention. (vi) Insulin sensitivity

[00329] Animal models with treated DT2 and untreated, in addition to normal animals, are tested for abnormal insulin sensitivity.

[00330] The normality or abnormality of an animal's insulin sensitivity can be confirmed by means of an insulin sensitivity test, which can be performed according to a conventional procedure known to those skilled in the art, eg by administering insulin intraperitoneally to a fasting animal of between 0.5 and 0.85 U per kg of body weight and subsequent measurement of the blood glucose level of the animal at certain times during the insulin sensitivity test (eg Every 15 minutes over an interval of 240 minutes). When the result shows that the blood glucose level has no tendency to decrease over time compared to that of a normal rodent, insulin sensitivity is assessed as abnormal. When the result in treated animals shows a tendency to decrease over time compared to that of untreated animals, treatment with the antibodies of the present invention is classified as effective. Exemplary Insulin Sensitivity Assessment Method

[00331] Insulin sensitivity of certain animals is assessed by the insulin sensitivity test.

[00332] The insulin sensitivity test is carried out, first, by administering insulin intraperitoneally to an animal fasting for 12 hours of between 0.5 and 0.85 U per kg of body weight and later measurement of the blood glucose level (peripheral) of the animal every 15 minutes over an interval of 240 minutes. The blood glucose level is measured by the above method. When the result shows that the blood glucose level has no tendency to

138/139 decrease over time compared to that of a normal animal, insulin sensitivity is assessed as abnormal. A faster reduction over time in treated DT2 animal models, compared to untreated, is evaluated as a sign of treatment effectiveness with the present invention. (vii) Others Assessment of polydipsia and polyuria

[00333] Animal models with Type | 1 diabetes are tested for trends in increasing water intake and increased urinary frequency after the onset of diabetes and during treatment with the antibodies of the present invention. The trend of increasing water intake can be confirmed, for example, by meticulous monitoring of the proportion of decreased water volume in a water bottle placed in a breeding cage compared to that of a normal strain animal and untreated animals. In addition, the tendency to increase urinary frequency can be confirmed, for example, by observing the wetting point of a floor sheet in the breeding cage, if compared to those previously indicated. Judgment of Presence or Absence of Tendency to Obesity in Mice

[00334] The body weight of treated and untreated DT2 animal models, in addition to that of normal animals, is measured and compared. The weight reduction of treated animals, compared to that of untreated animals and / or the comparable weight of treated and normal animals, is evaluated as a sign of the effectiveness of the treatment with the present invention. 2) Histopathological examination

[00335] Pancreatic tissue from animals with Type II diabetes, from control models of animals with untreated DT2 and from normal animals is fixed, embedded in paraffin, stained and analyzed according to the methods of Example 4 for amyloid deposits of amylin in cells 8 of the pancreatic islets (islets of Langerhans). Likewise, B cell apoptosis and survival is assessed by means of

139/139 immunostaining with the use of appropriate markers (eg staining via cleaved caspase-3 and by the TUNEL technique for apoptosis and insulin staining for the B cell area). A reduction in amyloid deposits and / or B cell apoptosis and / or an increase in B cell survival in pancreatic islets in treated animals compared to untreated animals or at comparable levels in treated and normal animals is assessed as a sign of effectiveness of the preventive and / or therapeutic methods of the present inventions.

3) Animal housing

[00336] Animal models with Type Il diabetes are kept protected against specific pathogens and in accordance with what they described previously (Hull et al. (2003), Diabetes 52: 372-379; Butler et a /. (2003), Diabetes 52: 2304-2314; Butler et al. (2004), Diabetes 53: 1509-1516); At 6 weeks of age, mice with h- IAPP (hemizygous / C57BL / 6 / DBA receive a calorie-rich diet in order to promote the formation of islet amyloid (Hull et a /. (2003), Diabetes 52: 372 -379).

Claims (37)

CLAIMS 1 Human monoclonal antibody characterized by being polypeptide and amyloid anti-islets (IAPP). 2. Antibody according to claim 1, characterized by being able to unite human prolAPP and / or IAPP. Antibody according to claim 1 or 2, characterized in that it does not substantially recognize the amyloid-B peptide (AB1-2). Antibody according to any one of claims 1 to 3, characterized in that it does not substantially recognize the physiological IAPP. Antibody according to any one of claims 1 to 4, characterized in that it does not substantially recognize prolAPP. Antibody according to any one of claims 1 to 5, characterized in that it specifically joins an IAPP epitope comprising the amino acid sequence SSNNFGA (SEQ ID NO: 4), CNTATCA (SEQ ID NO: 5) , or QRLANFLVHS (SEQ ID NO: 71). Antibody according to any one of claims 1 to 6 or its prolAPP and / or IAPP binding fragment, characterized in that it comprises, in its variable region, at least one complementarity determining region (CDR), as shown in Fig. 1 and / or one or more of its CDRs, comprising one or more amino acid substitutions. Antibody or binding fragment of claim 7, characterized in that it comprises an amino acid sequence of the VL and / or VH region, as shown in Fig. 1 or its VL and / or VH region, comprising one or more amino acid substitutions. Antibody according to any one of claims 1 to 8, characterized in that it preferably recognizes aggregates of IAPP, comprising fibrils and / or oligomers of IAPP over the physiological IAPP 10. —An antibody, according to any one of claims 1 to 4, characterized in that it is capable of joining prol | APP 11. An antibody according to claim 10 or its prolAPP binding fragment, characterized by comprising, in its variable region, at least one CDR, as represented in Fig. 2 and / or one or more of its CDRs, comprising a or more amino acid substitutions. 12. The binding antibody or fragment according to claim 11, characterized in that it comprises an amino acid sequence of the VL and / or VH region, as shown in Fig. 2 or its VL and / or VH region, comprising one or more amino acid substitutions. 13. An antibody according to any one of claims 1 to 12, characterized in that it is a rodent-human or rodent chimeric antibody 14. An antibody or antigen-binding molecule characterized by competing with an antibody of any one of claims 1 to 13 for specific binding to IAPP and / or prolAPP 15. An antibody according to any one of claims 1 to 14, characterized in that it 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. 16. - Polynucleotide, characterized in that it encodes at least the binding domain or the variable region of an immunoglobulin chain of the antibody of any one of claims 1 to 15 17. Vector, characterized in that it comprises the polynucleotide of claim 16, optionally linked to a polynucleotide of claim 16 that encodes the variable region of the other immunoglobulin chain of such binding molecule 18. “Host cell, characterized by comprising a polynucleotide of claim 16 or a vector of claim 17 19. Method of preparing the anti-| lAPP or anti-prolAPP antibody or its immunoglobin chain (s), characterized by comprising (a) the cell culture of Claim 18; and (b) isolating such an antibody or its immunoglobulin chain (s) from the culture Antibody or its immunoglobulin chain (s) encoded by a polynucleotide according to claim 16, characterized in that it is capable of being obtained by the method of claim 19 21. —An antibody according to any one of claims 1 to 15 or 20, characterized in that it is detectably marked 22. Antibody according to claim 21, characterized by having a detectable marker selected from the group consisting of an enzyme, a radioisotope, a fluorophore and a heavy metal 23. An antibody according to any one of claims 1 to 15 or 20 to 22, characterized in that it is linked to a drug. 24. "Composition characterized by comprising the antibody of any one of claims 1 to 15 or 20 to 23, the polynucleotide of claim 16, the vector of claim 17 or the cell of claim 18. 25. "Composition according to claim 24, characterized in that it is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier 26. "Composition according to claim 25, characterized by being a vaccine. 27. Composition according to either of Claims 25 or 26, characterized in that it further comprises an additional agent useful for the treatment of type 2 diabetes mellitus (DT2) and / or for the treatment or prevention of islet rejection following a clinical pancreatic islet transplantation 28. "Composition according to claim 24, characterized in that it is a diagnostic composition, and optionally comprises reagents that are conventionally used in nucleic acid or immunological diagnostic methods. 29. —An antibody according to any one of claims 1 to 15 or 20 to 23 or an IAPP and / or prolAPP-binding molecule, characterized by having substantially the same binding specificities as any of these, the polynucleotide of Claim 16, the Claim 17 vector or the Claim 18 cell or a pharmaceutical or diagnostic composition comprising any of these for use in prophylactic treatment, therapeutic treatment and / or monitoring progression or a response to the treatment of a disorder related to IAPP and / or prolAPP 30. Process of diagnosing or monitoring the progression of islet amyloidosis in an individual, characterized by determining the presence of oligomers, aggregates or fibrils of IAPP and / or prolAPP in a sample taken from the individual who will be diagnosed with at least one antibody of any one of claims 1 to or from 20 to 23 or a molecule binding to IAPP and / or prolAPP, having substantially the same binding specificities as any of those, wherein the presence of oligomers, aggregates or fibrils of IAPP and / or prolAPP is indicative of pre-symptomatic, prodromal or clinical type 2 (DT2) diabetes mellitus and / or beta cell failure following a clinical transplantation of pancreatic islets and an increase in the level of IAPP oligomers, aggregates or fibrils and / or prolAPP compared to the physiological IAPP level or compared to a reference sample from a healthy individual representing the control group or a sample d and control of the same individual is indicative for progression of pre-symptomatic, prodromal or established type 2 (DT2) diabetes mellitus and / or islet failure after clinical pancreatic islet transplantation in that individual. 31. The IAPP and / or prolAPP binding molecule, characterized in that it comprises at least one CDR of an antibody of any one of claims 1 to 15 or of a 23 for the preparation of a composition for in vivo detection or to achieve a therapeutic and / or diagnostic agent for IAPP and / or prolAPP in the human or animal body. 32. - The molecule according to claim 31, wherein the aforementioned in vivo detection is characterized by comprising positron emission tomography (PET), single photon emission tomography (SPELT), optical imaging by near infrared spectrometry ( NIR) or magnetic resonance imaging (MRI). 33. A molecule according to either of Claims 31 or 32, characterized in that it is used in the process of Claim 30. 34. "Peptide characterized by having a prolAPP epitope recognized specifically by an antibody of any of Claims 1 to 15 or 20 to 23. 35. A peptide according to claim 34, characterized in that it comprises an amino acid sequence, as defined in claim 6 or a modified sequence thereof, in which one or more amino acids are substituted, deleted and / or added, in which the peptide is recognized by the antibody of any one of claims 6 to 15. 36. Process to diagnose islet amyloidosis indicative of pre-symptomatic or clinical type 2 diabetes mellitus and / or beta cell failure following a clinical pancreatic islet transplant in an individual, characterized by comprising a step of determining the presence of an antibody that binds to a peptide of claims 34 or 35 in a biological sample from such an individual 37. Kit for diagnosing or monitoring the progression of islet amyloidosis, comprising at least one antibody of any of claims 1 to 15 or 20 to 23 or a molecule binding to IAPP and / or prolAPP characterized by having substantially the same binding specificities of any of these, the polynucleotide of claim 16, the vector of claim 17 or the cell of claim 18 and / or the peptide of claims 34 or 35, optionally with reagents and / or instructions for use.