Antibody-based therapy of transthyretin (TTR) amyloidosis and human-derived antibodies therefor

Abstract

To provide therapeutic strategies for an efficacious and safe therapy of diseases associated with TTR amyloidosis.SOLUTION: The present invention provides a human-derived anti-transthyretin (TTR) antibody or an antigen-binding fragment thereof, which binds to a TTR epitope comprising the amino acid sequence WEPFA (SEQ ID NO: 51), and does not bind to the corresponding E42G mutant.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates generally to antibody-based treatment of transthyretin (TTR) amyloidosis. In particular, the present invention relates to novel molecules that specifically bind to human transthyretin (TTR) and its antigens, in particular to recombinant human-derived antibodies, as well as fragments, derivatives and variants thereof, that recognize misfolded, misassembled or aggregated forms of TTR or fragments thereof and are useful in the treatment of diseases and conditions induced by such pathogenic TTR isotypes.

[0002] In addition, the present invention relates to pharmaceutical and diagnostic compositions comprising such binding molecules, antibodies and mimetics thereof, which are useful both as diagnostic tools for identifying diseases associated with TTR amyloidosis (e.g., familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), senile systemic amyloidosis (SSA), systemic familial amyloidosis, leptomeningeal / central nervous system (CNS) amyloidosis (including Alzheimer's disease), TTR-related ocular amyloidosis, TTR-related renal amyloidosis, TTR-related hyperthyroxinemia, TTR-related ligament amyloidosis (including carpal tunnel syndrome, rotator cuff tear and lumbar spinal stenosis), and preeclampsia, as well as passive vaccination strategies for treating disorders associated with diseases associated with TTR amyloidosis (e.g., the diseases described above).

[0003] Furthermore, the present invention relates to a method for diagnosing a disease or condition induced by a pathogenic TTR isotype (such as misfolded TTR and / or aggregated TTR present in amyloid deposits), wherein the level of the pathogenic TTR isotype is assayed in a sample of body fluid obtained from a subject after administration of an anti-TTR antibody, but when compared with a control sample taken before administration, the presence or change in the level of the pathogenic TTR isotype (e.g., as determined by the presence of an immune complex between TTR and the anti-TTR antibody) is indicative of the disease and / or condition. [Background technology]

[0004] Transthyretin (TTR), formerly called prealbumin, is a 127 amino acid soluble protein (NCBI Reference Sequence: NP_000362.1) involved in the transport of thyroxine and retinol in the body. TTR is secreted into the blood by the liver and into the cerebrospinal fluid by the choroid plexus, and is also expressed in certain tissues such as pancreatic alpha cells or retinal epithelium. TTR synthesis begins during fetal life and continues throughout life. TTR is present in high concentrations in plasma (3.6 μM-7.2 μM) and CSF (0.04 μM-0.4 μM), typically forming a soluble homotetramer of approximately 55 kDa under physiological conditions.

[0005] Under certain conditions, which are not well understood and may include acidic pH, oxidative stress, and local factors, the TTR protein adopts an alternative three-dimensional conformation and becomes toxic.

[0006] The toxicity of misfolded TTR protein has been discovered by investigating a rare autosomal dominant neurodegenerative disorder called familial amyloidotic polyneuropathy (FAP) that affects adults in middle age (Plante-Bordeneuve et al., Lancet Neurol., 10 (2011), 1086-1097). FAP is characterized by progressive sensory, motor and autonomic impairment that leads to death within 10 years after diagnosis. Neuropathy is accompanied by the deposition of amorphous aggregates and amyloid fibrils composed of TTR protein. The Val30Met substitution is the most frequent mutation causing FAP, especially in areas where the disease is endemic (e.g., northern Portugal), but more than 100 different mutations have already been identified in the TTR gene (see Table IV below). The pathophysiological mechanism at work is identical for all of the pathogenic mutations, in that these mutations alter the structural stability of the TTR tetramer, thereby promoting TTR misfolding and leading to the formation of toxic TTR species (Saraiva et al., Curr. Med. Chem., 19 (2012), 2304-2311).

[0007] TTR toxicity has also been observed as a result of the Val122Ile mutation, which is found at high frequency (3%-5%) in African-American and West African populations and is associated with familial amyloidotic cardiomyopathy (FAC), a condition in which excessive accumulation of TTR in the myocardium leads to cardiac weakness and ultimately heart failure (Ruberg et al., Circulation, 126 (2012), 1286-1300).

[0008] Mutations in the TTR protein sequence are not an absolute requirement for TTR toxicity, and wild-type TTR protein is also prone to misfolding and the formation of toxic aggregates. For example, senile systemic amyloidosis (SSA) is characterized by cardiac weakness and accumulation of aggregates of wild-type TTR in the myocardium (Ikeda, Amyloid, 18 Suppl 1 (2011), 155-156; Dungu et al., Heart, 98 (2012), 1546-1554). Deposits of wild-type TTR are also found in numerous cases of ligament and tendon inflammation, including carpal tunnel syndrome, shoulder rotator cuff tears, and lumbar spinal canal stenosis (Sueyoshi et al., Hum. Pathol., 42 (2011), 1259-1264; Gioeva et al., Amyloid, 20 (2013), 1-6). Moreover, TTR amyloidosis has recently been reported in the placenta of mothers suffering from preeclampsia (Kalkunte et al., Am. J. Pathol., 183 (2013), 1425-1436).

[0009] Treatments for diseases involving TTR amyloidosis are limited and mostly invasive, and in these cases, treatment is primarily symptomatic. In the case of FAP, treatments rely on analgesics to manage neuropathic pain, liver transplants to remove the primary source of mutated TTR protein, and treatment with tafamidis. Tafamidis is a small molecule that binds to the TTR tetramer and stabilizes its conformation. Tafamidis acts to prevent dissociation of the TTR tetramer, a rate-limiting step in the misfolding pathway that leads to the formation of toxic TTR species. Although tafamidis has been approved for the treatment of FAP in Europe, it is not approved in the United States, and its therapeutic efficacy is limited to slowing the progression of the disease in the best of cases. Currently, there are no available treatments that target the misfolded TTR protein.

[0010] In view of the above, novel therapeutic strategies are needed for the effective and safe treatment of diseases associated with TTR amyloidosis.

[0011] This technical problem is solved by the embodiments characterized in the claims and further described below and illustrated in the examples and drawings. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Plante-Bordeneuve et al., Lancet Neurol., 10(2011), 1086~1097 [Non-Patent Document 2] Saraiva et al., Curr.Med.Chem., 19(2012), 2304~2311 [Non-Patent Document 3] Ruberg et al., Circulation, 126(2012), 1286~1300 [Non-Patent Document 4] Ikeda, Amyloid, 18 Suppl 1(2011), 155~156 [Non-Patent Document 5] Dungu et al., Heart, 98(2012), 1546~1554 [Non-Patent Document 6] Sueyoshi et al., Hum. Pathol., 42(2011), 1259~1264 [Non-Patent Document 7] Gioeva et al., Amyloid, 20 (2013), 1-6 [Non-Patent Document 8] Kalkunte et al., Am.J. Pathol., 183(2013), 1425~1436 Summary of the Invention

[0013] The present invention provides anti-transthyretin (TTR) antibodies and equivalent TTR binding molecules for use in the prophylactic or therapeutic treatment of diseases and conditions associated with TTR amyloidosis. More specifically, therapeutically useful human-derived recombinant antibodies that recognize misfolded, misassembled or aggregated forms of TTR, as well as fragments and derivatives thereof, are provided.

[0014] Misfolded TTR aggregates are accompanied by markers of cellular stress, oxidative stress, inflammatory responses, and apoptosis years before the onset of symptoms (Macedo et al., Mol. Med., 13 (2007), 584-91). The body's natural ability to recognize and degrade misfolded proteins is protective, and patient-to-patient differences in the ability of patients to eliminate toxic TTR protein undoubtedly contribute to differences in the age of disease onset and rate of disease progression. In support of this hypothesis, patients undergoing liver transplants from FAP donors have been shown to quickly develop antibodies against the pathogenic TTR protein (Ando et al., Transplantation, 73 (2002), 751-755), and FAP patients with high titers of antibodies against the mutated TTR protein have been shown to have a slower disease onset than those without such antibodies (Obayashi et al., Clin. Chim. Acta., 419 (2013), 127-131). In addition, active immunization against pathogenic TTR conformations has been shown to almost completely eliminate TTR deposits in FAP transgenic mice (Terazaki et al., Lab. Invest., 86 (2006), 23-31).

[0015] However, although it may seem attractive to investigate immune-based strategies for therapeutic intervention, so far, anti-TTR antibodies have not been used to treat TTR-related diseases. For example, in International Patent Application Publication WO2010 / 030203, a specific isolated mouse monoclonal antibody against TTR is described and proposed for use in screening for FAP, and in the study and treatment of related diseases. However, mouse monoclonal antibodies are not suitable for human therapy because they induce human anti-mouse antibody (HAMA) responses. Therefore, this international patent application has lapsed, and since no subsequent developments have yet been published, it appears that antibody-based therapeutic efforts have not continued to this day. Rather, anti-TTR antibodies have only been further explored for their diagnostic use for patients with TTR amyloidosis; see, for example, Phay M. et al., Rejuvenation Res., October 28, 2013 [Epub ahead of print].

[0016] In contrast, various experiments carried out according to the present invention have succeeded in isolating human-derived monoclonal TTR-specific antibodies that are specific for misfolded, misassembled, mutated and / or aggregated TTR species and / or fragments thereof, which are matured antibodies in the human body. The human subjects and human patients, which are the source of B cells from which the human-derived monoclonal anti-TTR antibodies and cDNAs encoding their variable domains, respectively, have not shown substantial amounts of misfolded TTR and have been in a condition associated with a pathogenic isotype without symptoms. However, in another embodiment of the present invention, the source of B cells from which the human-derived monoclonal anti-TTR antibodies and cDNAs encoding their variable domains, respectively, may be isolated, is a patient exhibiting symptoms of diseases and / or disorders associated with TTR amyloidosis. Therefore, it is reasonable to expect that the human monoclonal anti-TTR antibodies and derivatives of the present invention will exhibit therapeutically beneficial effects in addition to being non-immunogenic in humans.

[0017] Thus, the present invention relates to recombinant human-derived antibodies, antigen-binding fragments and similar antigen-binding molecules that can specifically recognize TTR. Unless otherwise indicated, by "specifically recognizing TTR", "antibody specific for / for TTR" and "TTR antibody" is meant an antibody that specifically, generally and generally binds to the native monomeric form of TTR; an antibody that specifically binds to any form of TTR, for example, mutated TTR, oligomeric TTR, fibrillar TTR and / or nonfibrillar TTR. Provided herein are human-derived antibodies that are selective for full-length forms and / or fragments and / or misfolded, misassembled and / or aggregated forms of TTR.

[0018] As mentioned above, preferably the anti-TTR antibodies of the invention are recombinant antibodies, wherein at least one, preferably two or more preferably all three CDRs of the variable heavy and / or variable light chains, and / or substantially the entire variable region are encoded by cDNA derived from mRNA obtained from human memory B cells that produced the anti-TTR antibody. In a preferred embodiment, the anti-TTR antibodies of the invention exhibit at least one of the binding and biological properties as demonstrated for the subject antibodies exemplified in the accompanying Examples and Figures, and preferably at least one of the binding and biological properties as demonstrated for the exemplary antibodies NI-301.59.F1, NI-301.35G11 and NI-301.37F1, in any combination.

[0019] In a particularly preferred embodiment of the invention, the anti-TTR antibody or TTR-binding fragment thereof comprises the variable region V H and / or variable region V L The immunological binding properties of the antibody are characterized by:

[0020] Antigen-binding fragments of antibodies can be single chain Fv fragments, F(ab') fragments, F(ab) fragments and F(ab')2 fragments, or any other antigen-binding fragments. In a specific embodiment, inter alia, the antibody or fragment thereof is a human IgG isotype antibody. Alternatively, the antibody is a human-rodent chimeric antibody or a rodentized antibody, such as a mouse or murine antibody, a rat or ratite antibody, with the rodent forms being particularly useful for diagnostic methods and animal studies.

[0021] Furthermore, the present invention relates to compositions comprising the antibodies or active fragments thereof of the present invention, as well as immunotherapeutic and immunodiagnostic methods using such compositions in the prevention, diagnosis or treatment of disorders associated with TTR amyloidosis, wherein an effective amount of the composition is administered to a patient in need thereof.

[0022] The present invention also relates to a polynucleotide encoding at least the variable region of an immunoglobulin chain of an antibody of the present invention. Preferably, said variable region is a V H and / or V L In a preferred embodiment of the invention, the polynucleotide is a cDNA, preferably derived from mRNA obtained from human memory B cells that produce antibodies reactive with mutant, misfolded, misassembled and / or aggregated TTR species.

[0023] Thus, the present invention also encompasses vectors containing said polynucleotides, and host cells transformed with said vectors, as well as their use for producing antibodies and equivalent binding molecules specific for TTR. In a further aspect of the present invention, the antibodies or binding molecules are capable of binding to misfolded, misassembled, or aggregated TTR species or fragments thereof. Various means and methods for recombinantly producing antibodies and mimetics thereof, as well as various methods for screening for competing binding molecules, which may or may not be antibodies, are known in the art. However, as described herein, particularly with respect to therapeutic applications in humans, the antibodies of the present invention are human antibodies, in the sense that the application of the antibodies is substantially free of the immune response against such antibodies that is otherwise observed for chimeric antibodies, and also for humanized antibodies.

[0024] Further disclosed herein are compositions and methods that can be used to identify TTR (specifically, mutated, misfolded, misassembled, or aggregated TTR species or fragments) in a sample and / or in vivo. The disclosed anti-TTR antibodies and binding fragments thereof can be used to screen human blood, plasma, serum, saliva, peritoneal fluid, cerebrospinal fluid ("CSF"), and urine for the presence of TTR and / or mutated, misfolded, misassembled, or aggregated TTR species or fragments thereof in the sample, for example, by using ELISA-based or surface-compatible assays. In one embodiment, the invention relates to a method for diagnosing or monitoring the progression of a disorder associated with mutated, misfolded, misassembled or aggregated TTR species or fragments in a subject, comprising determining the presence of mutated, misfolded, misassembled or aggregated TTR species or fragments in a sample obtained from the subject to be diagnosed using at least one antibody or TTR binding molecule of the invention and / or a binding molecule for misfolded, misassembled or aggregated TTR species or fragments having substantially the same binding specificity as any one of them, wherein the presence of the misfolded, misassembled or aggregated TTR species or fragments is indicative of the disorder.

[0025] Furthermore, in one embodiment of the present invention, anti-TTR antibodies and TTR binding molecules comprising at least one CDR of the antibody of the present invention are provided for preparing compositions for in vivo detection (also called in vivo imaging) of TTR (particularly mutated, misfolded, misassembled, or aggregated TTR species or fragments) in the human or animal body, or for targeting therapeutic and / or diagnostic agents to TTR (particularly mutated, misfolded, misassembled, or aggregated TTR species or fragments) in the human or animal body. The methods and compositions disclosed herein can be helpful in disorders involving TTR amyloidosis and characterized, for example, by the appearance of various forms of TTR, and can be used to monitor disease progression and therapeutic efficacy of treatment administered to a subject, for example, by diagnostic methods associated with in vivo imaging. Thus, in one embodiment, anti-TTR antibodies and / or TTR binding molecules of the invention are provided, wherein said in vivo detection (imaging) comprises scintigraphy, positron emission tomography (PET), single photon emission computed tomography (SPECT), near infrared (NIR) optical imaging or magnetic resonance imaging (MRI).

[0026] It is therefore a specific object of the present invention to provide a method for treating, diagnosing, or preventing diseases associated with TTR amyloidosis, comprising administering to a subject an effective concentration of a human antibody or antibody derivative, wherein the antibody targets TTR or a fragment thereof, preferably a misfolded, misassembled, or aggregated TTR species or a fragment thereof.

[0027] In a further embodiment, the present invention provides a peptide having an epitope of TTR (preferably misfolded, misassembled or aggregated TTR species or fragments thereof) specifically recognized by the antibody of the present invention. The peptide comprises or consists of an amino acid sequence as shown below in the detailed description and in the examples, or a modified sequence thereof in which one or more amino acids are replaced, deleted and / or added, provided that the peptide is still recognized by the cognate antibody. As mentioned, such peptides can be used as antigens, i.e. immunogens for eliciting an immune response in a subject and stimulating the production of the antibody of the present invention in vivo, and are therefore useful for eliciting an immune response in a subject and stimulating the production of the antibody of the present invention in vivo. Thus, the peptide of the present invention is particularly useful as a vaccine.

[0028] In addition, the present invention provides a method for diagnosing a disease associated with TTR amyloidosis in a subject, the method comprising the step of determining in a biological sample of the subject the presence of an antibody that binds to the peptide.

[0029] In a further aspect, the present invention relates to a method for diagnosing a disease associated with TTR amyloidosis, or for monitoring the treatment of said disease with an anti-TTR antibody, or for determining the diagnostic or therapeutic utility of an anti-TTR antibody, comprising assaying the level of misfolded TTR and / or aggregated TTR in a sample, for example in blood obtained from a subject after administration of an anti-TTR antibody to the subject, wherein the presence or elevated levels of misfolded TTR and / or aggregated TTR in the subject's sample compared to a control (such as a sample obtained from the subject prior to administration of the anti-TTR antibody) is indicative of a disease associated with TTR amyloidosis.

[0030] In one preferred embodiment of the invention, particularly when non-human animals are used to test recombinant human-derived antibodies such as those exemplified in Example 13, but generally when used to test other anti-TTR antibodies intended for use in humans, the levels of misfolded TTR and / or aggregated TTR in a sample are assayed by measuring the complex formed between the anti-TTR antibody and misfolded TTR and / or aggregated TTR, e.g., by immunoprecipitation with an anti-human IgG antibody or an anti-idiotypic antibody.

[0031] With regard to diagnostic aspects, particularly for human subjects and patients, the presence and elevated levels of misfolded TTR and / or aggregated TTR and its complex with anti-TTR antibodies, respectively, indicate the presence of TTR amyloid deposits in the human body, for example in the heart, peripheral nervous system (PNS), eyes, muscles, gastrointestinal tract, kidneys, vascular system and central nervous system (CNS) of a patient or subject. Thus, the method of the present invention allows, on the one hand, the identification and determination of diseases associated with TTR amyloidosis in the subject's body, and, on the other hand, the removal of TTR deposits from the patient's body (which also indicates the therapeutic progress of a given treatment and the efficacy of a drug for treating TTR amyloidosis, such as, for example, an anti-TTR antibody).

[0032] Thus, as demonstrated in Example 13, the anti-TTR antibody of the present invention can bind to misfolded TTR and / or aggregated TTR with sufficient affinity to change the stability of pathological TTR deposits, for example, to capture misfolded TTR and / or aggregated TTR from the deposits and remove them into body fluids, specifically into blood. The designated time interval after administration, i.e., the time frame in which the levels of pathological TTR and the complex with anti-TTR antibody, respectively, are measured, is determined by the practicing physician. Usually, a time interval of less than one week is used. In a preferred embodiment, the level of pathological TTR in a sample from a patient or subject is determined 48 hours or less after administration of an anti-TTR antibody or its antigen-binding fragment to the patient or subject (see also Example 13).

[0033] The present invention also relates to the use of any of the anti-TTR antibodies and TTR binding molecules in the methods described above. However, the use of the anti-TTR antibodies of the invention disclosed herein is preferred because of their advantageous properties, in particular because they are of human origin. In a preferred embodiment, the antibody exhibits substantially the same binding activity and biological activity as any antibody selected from NI-301.59F1, NI-301.35G11, NI-301.37F1, NI-301.2F5, NI-301.28B3, NI-301.119C12, NI-301.5D8, NI-301.9D5, NI-301.104F5, NI-301.21F10, NI-301.9G12, NI-301.12D3, NI-301.37F1-PIMC, NI-301.44E4, NI-301.18C4, NI-301.11A10, NI-301.3C9, NI-301.14D8, NI-301.9X4, and NI-301.14C3. The anti-TTR antibodies can also be modified to facilitate use in the above methods of diagnosis, including labeling of the antibodies as described in detail below.

[0034] Further embodiments of the present invention will be apparent from the following description and examples. [Brief description of the drawings]

[0035] [Figure 1] Amino acid sequences of the variable regions of the human antibodies NI-301.59F1, NI-301.35G11, NI-301.37F1, NI-301.2F5, NI-301.28B3, NI-301.119C12, NI-301.5D8, NI-301.9D5, NI-301.104F5, NI-301.21F10, NI-301.9G12, NI-301.12D3, NI-301.37F1-PIMC, NI-301.44E4, NI-301.18C4, NI-301.11A10, NI-301.3C9, NI-301.14D8, NI-301.9X4 and NI-301.14C3. The framework regions (FR) and complementarity determining regions (CDRs) are indicated and the CDRs are underlined. The Kabat numbering scheme was used (see http: / / www.bioinf.org.uk / abs / ). [Diagram 2] Binding to aggregated wild-type and mutant TTR by direct ELISA. A, B, C: ELISA plates were coated with aggregated human wild-type TTR (●), aggregated recombinant V30M-TTR (▼), and bovine serum albumin (BSA) (■) at 10 μg / ml and incubated with the following human monoclonal antibodies at concentrations ranging from 4 pM to 400 nM: A) NI-301.59F1, B) NI-301.35G11, and C) NI-301.37F1. EC50 values ​​were estimated by least-squares fitting of the data points. NI-301.59F1: EC50 for aggregated wt-TTR = 3.0 nM, EC50 for aggregated V30M-TTR = 15.5 nM NI-301.35G11: EC50 for aggregated wt-TTR = 3.9 nM, EC50 for aggregated V30M-TTR = 5.0 nM NI-301.37F1: EC50 for aggregated wt-TTR = 0.35 nM, EC50 for aggregated V30M-TTR = 0.15 nM [Diagram 3]Specificity for aggregated TTR on dot blots. Human wild-type TTR protein in native conformation (1) or aggregated conformation (2), and aggregated recombinant V30M-TTR protein (3) were deposited on nitrocellulose membranes and incubated with the following antibodies: A) a commercial rabbit polyclonal antibody against TTR (Dako-A0002; 150 ng / ml), B) NI-301.59F1, C) NI-301.35G11, and D) NI-301.37F1 (B, C, and D: human monoclonal antibodies, 50 nM). [Figure 4] Specificity for aggregated TTR on Western blot. Human wild-type TTR protein (300 ng) in native conformation (1) or aggregated conformation (2) and wild-type mouse liver extracts (10 μg total protein) (3) were loaded onto SDS-PAGE gels and processed for Western blot with the following antibodies: A) a commercial rabbit polyclonal antibody against TTR (Dako-A0002; 150 ng / ml), B) NI-301.59F1, C) NI-301.35G11, and D) NI-301.37F1 (B, C, and D: human monoclonal antibodies, 50 nM). To prevent dissociation of high molecular weight aggregates, samples of aggregated TTR were cross-linked with glutaraldehyde (1%, 5 min) before loading onto the gel. [Diagram 5] Absence of binding to TTR in human plasma on Western blot. Plasma samples (0.5 μl) from controls (n=5), asymptomatic mutation carriers (n=5) and FAP patients (n=4) were loaded onto SDS-PAGE gels and processed for Western blot with the following antibodies: A) a commercial rabbit polyclonal antibody against TTR (Dako-A0002; 150 ng / ml), B) secondary antibody only (anti-human IgG-HRP, diluted 1 / 10000), C) NI-301.35G11, and D) NI-301.37F1 (C and D: human monoclonal antibodies, 50 nM). [Figure 6]Absence of binding to TTR in human plasma on dot blots. Pure wild-type and mutant TTR proteins in native and aggregated conformations, as well as plasma samples from controls, asymptomatic mutation carriers, and FAP patients, were placed on nitrocellulose membranes and incubated with the following antibodies: A) a commercial rabbit polyclonal antibody against TTR (Dako-A0002; 150 ng / ml), B) secondary antibody only (anti-mouse IgG2a-HRP, 1 / 10000 dilution), and C) mouse chimeric antibody NI-301.mur35G11 (10 nM). Samples 1-6: 150 ng of 1) aggregated wt-TTR, 2) native wt-TTR, 3) BSA, 4) native V30M-TTR, 5) native L55P-TTR, and 6) native Y78F-TTR. Samples 7–18: 2 μl of collected plasma, 7–10) controls (n=4), 11–14) asymptomatic mutation carriers (n=4), and 15–18) FAP patients (n=4). [Figure 7] Specific binding to aggregated TTR in solution. Human wild-type and recombinant TTR proteins in native and aggregated conformations, as well as human plasma samples at three different dilutions, were used for TTR immunoprecipitation (IP) using the following antibodies: A) a commercial rabbit polyclonal antibody against TTR (Dako-A0002), B) NI-301.35G11, and C) NI-301.37F1. Immunoprecipitated proteins were subjected to SDS-PAGE and detected by Western blot (WB) with Dako-A0002 antibody (150 ng / ml). Lanes 1-2: WB loading controls: 300 ng of 1) human wt-TTR, 2) recombinant wt-TTR. Lanes 3-6: IP against pure TTR protein: 3) human native wt-TTR, 4) human aggregated wt-TTR, 5) recombinant native wt-TTR, and 6) recombinant aggregated wt-TTR. Lanes 7-10: IP against human plasma diluted in PBS, 7) 10x, 8) 100x, 9) 1000x, and 10) PBS only. [Figure 8]Specific binding to TTR in FAP mouse tissues. Transgenic mice expressing the human V30M-TTR allele in a TTR knockout (KO) background recapitulate the histopathological features of FAP, including amorphous and amyloid TTR deposits in various tissues. Liver and intestinal tissue sections collected from A) FAP and B) TTR-KO mice were processed for immunohistochemistry using the following antibodies: 1) a commercially available rabbit polyclonal antibody against TTR (Dako-A0002; dilution of 1 / 1000), 2) NI-301.35G11, and 3) NI-301.37F1 (2 and 3: human monoclonal antibodies, 50 nM). [Figure 9] Specific binding to misfolded, but not native, TTR deposits in human tissues. Various antibodies were characterized for their ability to bind to TTR in sections from FAP patient skin biopsies and healthy control pancreases: accumulations of misfolded TTR that are characteristic for FAP are present in patient skin biopsies, whereas pancreatic alpha cells show endogenous expression of TTR. Sections were processed for immunohistochemistry using the following antibodies: 1A) a commercially available rabbit polyclonal antibody against TTR (Dako-A0002; dilution 1 / 1000), 1B) an HRP-conjugated anti-rabbit IgG antibody (dilution 1 / 125), 2A) mouse chimeric antibody NI-301.mur35G11 (50 nM), 2B) an HRP-conjugated anti-mouse IgG2a antibody (dilution 1 / 125), 3A) NI-301.37F1 (50 nM), and 3B) an HRP-conjugated anti-human IgG (dilution 1 / 125). [Figure 10]TTR binding epitopes assessed by pep scan analysis. Antibody binding epitopes for TTR were determined using peptide scan methodology. In addition to peptides covering the complete human wild-type TTR sequence (spots 1-29), selected TTR mutations were also represented on the membrane (spots 30-44). Peptide scan membranes were incubated with the following antibodies at 50 nM: A) NI-301.59F1, B) NI-301.35G11, and C) NI-301.37F1. As summarized in Table D): NI-301.59F1 binds EEEFVEGIY (TTR61-69); NI-301.35G11 binds GELHGLTTEEE (TTR53-63); the L55P mutation prevents antibody binding; NI-301.37F1 binds WEPFA (TTR41-45); the E42G mutation prevents antibody binding. To determine the sequence requirements of the described epitopes, antibody binding epitopes for TTR were further identified using an alanine scanning method. The entire sequence of the human wild-type TTR protein was displayed on a membrane as a set of 151 consecutive peptides, 15 amino acids in length, starting at any amino acid of the TTR protein. For each peptide, the amino acid at position 10 was replaced by alanine or, when the first amino acid was alanine, by glycine or proline. Peptide scanning membranes were incubated with the following antibodies at 20 nM: E) NI-301.59F1, F) NI-301.35G11, and G) NI-301.37F1. As summarized in Table H): NI-301.59F1 binds EEFXEGIY (TTR62-69); NI-301.35G11 binds ELXGLTXE (TTR54-61); NI-301.37F1 binds WEPFA (TTR41-45), where X represents an amino acid; substitution of E42 with alanine did not prevent binding, but substitution with guanine prevented antibody binding as reported in C. [Figure 11]Antibody binding kinetics to TTR protein in solution assessed by surface plasmon resonance. The binding kinetics of antibody NI-301.37F1 to TTR protein was measured by surface plasmon resonance (SPR). Antibody NI-301.37F1 was captured on the sensor by anti-human IgG antibody, and TTR protein solutions were flowed over the sensor surface at concentrations ranging from 3.2 nM to 316 nM. A simple 1:1 binding model was used to fit the data and derive the respective association constant (ka) and dissociation constant (kd) as well as affinity (KD). Binding properties were determined for A) human wild-type TTR protein in native conformation, B) denatured human wild-type TTR protein (misfolded conformation), and C) recombinant mutant TTR-L55P protein. Native wild-type TTR: ka = not determined, kd = not determined, KD > 316 nM Denatured wild-type TTR: ka = 2.1 104 M-1 s-1, kd = 2.6 10-5 s-1, KD = 1.2 nM Recombinant TTR-L55P: ka = 3.3 104 M-1 s-1, kd = 4.6 10-5 s-1, KD = 1.4 nM [Figure 12] Chronic treatment with anti-TTR antibodies reduces pathological TTR deposition in a FAP mouse model. FAP mice (Tg(6.0hMet30)×muTTR-KO) were given weekly doses of mouse chimeric NI-301.37F1 antibody or isotype control antibody at 3 mg / kg (ip) for 12 weeks. At the end of the treatment period, tissues were collected and the extent of TTR deposition was quantified by immunofluorescence. A) Effect of treatment in 7-month-old mice (n=14-15 mice / group); B) Effect of treatment in 17-month-old mice (n=10 mice / group). Group comparison by two-tailed unpaired t-test. [Figure 13]Antibody binding to pathological TTR deposits in vivo. Target engagement was characterized in adult FAP mice (July) 48 hours after administration of a single dose of antibody NI-301.37F1 at 30 mg / kg (ip) or PBS. Pathological TTR deposits and localization of injected antibodies were detected simultaneously by immunofluorescence. A, D) Pathological TTR deposits in the kidneys of mice injected with (A) NI-301.37F1 or (D) PBS. B, E) Detection of human antibodies in mice injected with (B) NI-301.37F1 or (E) PBS. C, F) Overlaid images showing TTR and NI-301.37F1, (C) colocalization and (F) absence of nonspecific staining. [Figure 14] Detection of misfolded TTR in vivo without tissue. Adult FAP mice were given a single dose of NI-301.37F1 antibody or isotype control antibody at 3 mg / kg (ip). Blood samples were collected before antibody injection (t=0) and 48 hours after antibody injection (t=48h). Plasma samples were processed by immunoprecipitation with anti-human IgG antibody and analyzed by Western blot using A) conformation-independent anti-TTR polyclonal antibody (Dako A0002, 150 ng / ml) and B) NI-301.37F1 (20 nM) for detection. In parallel, plasma samples from uninjected FAP mice were incubated with antibody NI-301.37F1 in vitro before processing. [Figure 15] Antibody specificity assessed against aggregating proteins by ELISA. Antibody specificity for TTR protein was assessed by measuring binding to selected aggregating proteins by direct ELISA. Antibody binding was assessed at 4 nM and 20 nM and signal intensity was expressed as fold change over background levels measured for each assay in the absence of anti-TTR antibody. A-B) NI-301.37F1 binding assayed at 20 nM (A) and 4 nM (B) C-D) NI-301.44E4 binding assayed at 20 nM (C) and 4 nM (D). Detailed Description of the Invention

[0036] The present invention generally relates to immunotherapeutic and non-invasive methods for detecting diseases and conditions associated with the presence of pathogenic isotypes of transthyretin (TTR), often mutant and / or misfolded isotypes. More specifically, the present invention relates to recombinant human-derived monoclonal antibodies and antigen-binding fragments thereof, generated based on sequence information obtained from a selected population of human donors, and capable of binding to such TTR isotypes and their antigens. The recombinant human-derived monoclonal antibodies of the present invention are advantageously characterized by specific binding to misfolded, misassembled, mutated and / or aggregated TTR species and / or fragments thereof, allowing targeting of pathological altered TTR species for treatment and / or diagnosis. Being of human origin, the resulting recombinant antibodies of the present invention can be reasonably expected to be effective and safe as therapeutic agents and highly specific as diagnostic reagents for detecting pathological TTR without giving false positives.

[0037] In addition, the antibodies of the present invention, as well as derivatives thereof, can be used for combination therapy of patients after organ transplantation who are nevertheless at risk of developing TTR amyloidosis, for example due to its deposition, for example due to inherited mutations in TTR or lack of production of TTR in the liver. Thus, as a specific advantageous embodiment, the present invention relates to the human monoclonal antibodies and any derivatives thereof described herein for use either alone in the treatment of patients, or in the treatment of patients receiving, for example, immunosuppressive drugs after organ transplantation or other drugs utilized for symptoms associated with TTR amyloidosis, with the proviso that in this case, any of the antibodies and various derivatives of the present invention are designed for administration simultaneously with immunosuppressive drugs and / or drugs suppressing further side effects, or for administration consecutively before or after administration of immunosuppressive drugs and / or drugs suppressing further side effects. In this regard, the anti-TTR antibodies and TTR binding fragments of the present invention are preferably substantially non-immunogenic in humans. In one embodiment of the invention, a pharmaceutical composition comprising both a human monoclonal antibody of the invention, or any derivative thereof, and one or more immunosuppressant drugs is provided and / or utilized for symptoms associated with TTR amyloidosis.

[0038] I. Definition Unless otherwise stated, terms as used herein are given the definitions as provided in the Oxford Dictionary of Biochemistry and Molecular Biology (Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0 19 850673 2).

[0039] It should be noted that an entity accompanied by the term "a" or "an" refers to one or more of such entities; for example, "an antibody" is understood to refer to one or more antibodies. As such, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0040] Unless otherwise specifically indicated, the term "TTR" is used interchangeably to specifically indicate various forms of transthyretin (TTR). The term "TTR" is also used to generally identify other conformers of TTR (e.g., oligomers and / or misfolded, misassembled and / or aggregated forms of TTR). The term "TTR" is also used collectively to indicate all types and forms of TTR (e.g., mutated TTR, etc.). Additional letters in front of the TTR term are used to indicate the organism from which the specific ortholog originates, e.g., hTTR is used for human TTR or mTTR is used for mouse origin. In addition, unless otherwise indicated, the numbering system for TTR amino acid sequence used herein refers to the mature TTR protein, i.e., the TTR protein as secreted by cells after cleavage of the signal peptide. This numbering system is used to define TTR mutations found in patients (e.g., TTR-V30M or TTR-L55P, etc.), but differs from that used for the transthyretin precursor protein sequence (NCBI Reference Sequence: NP_000362.1). In this regard, positions and substituted amino acids in mutant TTR are sometimes designated in a different, but equivalent manner; see, e.g., "TTR-V30M" and "V30M-TTR".

[0041] The anti-TTR antibodies disclosed herein specifically bind to TTR and its epitopes, and also specifically bind to various conformations of TTR and its epitopes. For example, antibodies are disclosed herein that specifically bind to pathologically altered TTR species or fragments thereof, such as oligomers / fibrils and / or mutated, misfolded, misassembled and / or aggregated forms or fragments thereof of TTR. The terms (pathologically) mutated, misfolded, misassembled, aggregated / aggregates of TTR are used interchangeably to specifically refer to the above forms. The terms (pathologically) "aggregated forms" or "aggregates" as used herein describe the products of accumulation or clustering due to the mutual incorrect / pathological interaction of TTR. These aggregates, deposits or clusters may be, or may consist essentially of, both TTR and / or TTR fragments, and their nonfibrillar and / or fibrillar oligomers and fibrils. As used herein, references to an antibody that "specifically binds," "selectively binds," or "preferentially binds" to TTR refer to an antibody that does not bind to other unrelated proteins. In one example, the TTR antibodies disclosed herein can bind to TTR or an epitope thereof, and do not exhibit more than about two-fold background binding to other proteins. In a preferred embodiment, the antibodies of the present invention do not substantially recognize unrelated amyloidogenic proteins selected from the group consisting of alpha-synuclein (α-syn), tau, transactivation response DNA binding protein 43 (TDP-43), serum amyloid A (SAA), and huntingtin protein (HTT) (see, e.g., FIG. 15). An antibody that "specifically binds" or "selectively binds" to a given TTR conformer refers to an antibody that does not bind to all conformations of TTR, i.e., an antibody that does not bind to at least one other TTR conformer.

[0042] For example, disclosed herein are antibodies capable of preferentially binding to misfolded, misassembled and / or aggregated forms of TTR both in vitro and in tissues obtained from patients with or at risk for developing disease associated with TTR amyloidosis. Because the sequences of the TTR antibodies of the invention are obtained from human subjects, the antibodies of the invention may also be referred to as "human autoantibodies" or "human-derived antibodies," which is intended to emphasize that such antibodies are in fact expressed primarily by a human subject and are not synthetic constructs, such as those generated by phage libraries expressing human immunoglobulins (which has heretofore represented one common method for attempting to provide human-like antibodies).

[0043] The term "peptide" is understood to include within its meaning the terms "polypeptide" and "protein" (sometimes used interchangeably herein). Similarly, fragments of proteins and polypeptides are also included, and may be referred to herein as "peptides". Nevertheless, the term "peptide" preferably refers to an amino acid polymer comprising at least 5 consecutive amino acids, preferably at least 10 consecutive amino acids, more preferably at least 15 consecutive amino acids, even more preferably at least 20 consecutive amino acids, and particularly preferably at least 25 consecutive amino acids. In addition, a peptide according to the invention typically comprises at most 100 consecutive amino acids, preferably less than 80 consecutive amino acids, more preferably less than 50 consecutive amino acids, and even more preferably less than 15 consecutive amino acids of a TTR polypeptide.

[0044] Polypeptides: As used herein, the term "polypeptide" is intended to encompass a single "polypeptide" as well as multiple "polypeptides" and refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, "peptide," "dipeptide," "tripeptide," "oligopeptide," "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms.

[0045] The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, and modifications with known protecting / blocking groups, derivatization by proteolytic cleavage, or non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced by recombinant technology, but are not necessarily translated from a designated nucleic acid sequence. Polypeptides may be made in any manner, including by chemical synthesis.

[0046] The polypeptides of the invention may be about 3 or more amino acids in size, 5 or more amino acids in size, 10 or more amino acids in size, 20 or more amino acids in size, 25 or more amino acids in size, 50 or more amino acids in size, 75 or more amino acids in size, 100 or more amino acids in size, 200 or more amino acids in size, 500 or more amino acids in size, 1,000 or more amino acids in size, or 2,000 or more amino acids in size. Although a polypeptide may have a defined three-dimensional structure, a polypeptide does not necessarily have such a structure. A polypeptide that has a defined three-dimensional structure is referred to as folded. A polypeptide that does not have a defined three-dimensional structure, but rather can adopt a large number of different conformations, is referred to as unfolded. As used herein, the term glycoprotein refers to a protein linked to at least one carbohydrate moiety that is attached to the protein through an oxygen- or nitrogen-containing side chain of an amino acid residue (e.g., a serine or asparagine residue).

[0047] By "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not present in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered to be isolated for various purposes of the present invention, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0048] "Recombinant peptide, polypeptide or protein" refers to a peptide, polypeptide or protein produced by recombinant DNA technology, i.e., produced from a cell (microbial or mammalian cell) transformed with an exogenous recombinant DNA expression construct encoding a fusion protein containing the desired peptide. Proteins or peptides expressed in most bacterial cultures will typically be free of glycans. Proteins or peptides expressed in yeast may have a different glycosylation pattern than those expressed in mammalian cells.

[0049] The polypeptides of the present invention also include fragments, derivatives, analogs or variants of said polypeptides, and any combination thereof. The terms "fragment", "variant", "derivative" and "analog" include peptides and polypeptides having an amino acid sequence sufficiently similar to that of a natural peptide. The term "sufficiently similar" refers to a first amino acid sequence that contains a sufficient or a minimum number of identical or equivalent amino acid residues to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and / or a common functional activity. For example, amino acid sequences that are 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, including amino acid sequences that share a common structural domain, are defined herein as being sufficiently similar. Preferably, the variants will be sufficiently similar to the amino acid sequences of the preferred peptides of the invention, particularly to the amino acid sequences of TTR or fragments, variants, derivatives or analogs of either thereof. Such variants generally retain the functional activity of the peptides of the invention. Variants include peptides that differ in amino acid sequence from the native and wt peptides, respectively, by one or more amino acid deletions, additions and / or substitutions. These may be naturally occurring variants as well as artificially designed variants.

[0050] Furthermore, the terms "fragment," "variant," "derivative," and "analog," when referring to an antibody or antibody polypeptide of the invention, include any polypeptide that retains at least a portion of the antigen-binding properties of the corresponding native binding molecule, antibody, or polypeptide. Fragments of the polypeptides of the invention include proteolytic fragments, as well as deletion fragments, in addition to the specific antibody fragments discussed elsewhere herein. Variants of the antibodies and antibody polypeptides of the invention also include fragments as described above, and polypeptides with altered amino acid sequences resulting from amino acid substitutions, deletions, or insertions. Variants may be naturally occurring or non-naturally occurring. Non-naturally occurring variants may be generated using mutagenesis techniques known in the art. Variant polypeptides may include conservative or non-conservative amino acid substitutions, amino acid deletions, or amino acid additions. Derivatives of TTR-specific binding molecules, such as derivatives of the antibodies and antibody polypeptides of the invention, are polypeptides that have been altered to exhibit additional characteristics not found for the native polypeptide. Examples include fusion proteins. Variant polypeptides may also be referred to herein as "polypeptide analogs." As used herein, the "derivative" of a binding molecule or its fragment, an antibody or an antibody polypeptide refers to the polypeptide having one or more residues that are chemically derivatized by reaction of a functional side group.The "derivative" also includes such peptides that contain one or more naturally occurring amino acid derivatives of the 20 standard amino acids.For example, 4-hydroxyproline may be used instead of proline; 5-hydroxylysine may be used instead of lysine; 3-methylhistidine may be used instead of histidine; homoserine may be used instead of serine; and ornithine may be used instead of lysine.

[0051] Determining Molecular Similarity and / or Identity: "Similarity" between two peptides is determined by comparing the amino acid sequence of one peptide to the sequence of a second peptide. An amino acid of one peptide is similar to a corresponding amino acid of a second peptide if the amino acids of the first peptide are identical or have conservative amino acid substitutions. Conservative substitutions include those described in The Atlas of Protein Sequence and Structure 5, edited by Dayhoff, MO, National Biomedical Research Foundation, Washington, DC (1978), and those described by Argos, EMBO J. 8 (1989), 779-785. For example, amino acids belonging to one of the following groups represent conservative changes or substitutions: - Ala, Pro, Gly, Gln, Asn, Ser, Thr; - Cys, Ser, Tyr, Thr; - Val, Ile, Leu, Met, Ala, Phe; - Lys, Arg, His; - Phe, Tyr, Trp, His; and - Asp, Glu.

[0052] "Similarity" between two polynucleotides is determined by comparing the nucleic acid sequence of one polynucleotide to the sequence of a given polynucleotide. A nucleic acid of one polynucleotide is similar to the corresponding nucleic acid of a second polynucleotide if the nucleic acids are identical or, if the nucleic acid is part of a coding sequence, if each triplet comprising the nucleic acid encodes the same amino acid or a conservative amino acid substitution.

[0053] The determination of percent identity or similarity between two sequences is preferably accomplished using the mathematical algorithm of Karlin and Altschul (1993) (Proc. Natl. Acad. Sci USA 90:5873-5877). Such an algorithm is incorporated into the BLASTn and BLASTp programs of Altschul et al. (1990) (J. Mol. Biol. 215:403-410), available at NCBI (http: / / www.ncbi.nlm.nih.gov / blast / Blast.cge).

[0054] The determination of percent identity or percent similarity is performed using the standard parameters of the BLASTn program for BLAST nucleotide searches and the standard parameters of the BLASTp program for BLAST protein searches, as recommended on the NCBI web page and as described in the "BLAST Program Selection Guide" for sequences of a particular length and composition.

[0055] BLAST polynucleotide searches are performed with the BLASTn program. For general parameters, the "Max Target Sequences" box may be set to 100, the "Short queries" box may be checked, the "Expect threshold" box may be set to 1000, and the "Word Size" box may be set to 7, as recommended on the NCBI webpage for short sequences (<20 bases). For longer sequences, the "Expect threshold" box may be set to 10, and the "Word Size" box may be set to 11. For scoring parameters, the "Match / mismatch Scores" may be set to 1, -2, and the "Gap Costs" box may be set to linear. For filter and masking parameters, the "Low complexity regions" box may not be checked, the "Species-specific repeats" box may not be checked, the "Mask for lookup table only" box may be checked, the "DUST Filter Settings" may be checked, and the "Mask lower case letters" box may not be checked. Generally, "Search for short nearly exact matches" may be used in this regard, which provides most of the settings shown above. Further information on this may be found in the "BLAST Program Selection Guide" published on the NCBI web page.

[0056] BLAST protein searches are performed using the BLASTp program. For general parameters, the "Max Target Sequences" box may be set to 100, the "Short queries" box may be checked, the "Expect threshold" box may be set to 10, and the "Word Size" box may be set to "3". For scoring parameters, the "Matrix" box may be set to "BLOSUM62", the "Gap Costs" box may be set to "Existence:11 Extension:1", and the "Compositional adjustments" box may be set to "Conditional compositional score matrix adjustment". For filter and masking parameters, the "Low complexity regions" box may not be checked, the "Mask for lookup table only" box may not be checked, and the "Mask lower case letters" box may not be checked.

[0057] Modifications of both programs, for example with regard to the length of the sequences searched, are made according to the recommendations in the "BLAST Program Selection Guide" published in HTML and PDF versions on the NCBI web page.

[0058] Polynucleotides: The term "polynucleotide" is intended to encompass a single nucleic acid as well as multiple nucleic acids, and refers to an isolated nucleic acid molecule or nucleic acid construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide may contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)). The term "nucleic acid" refers to any one or more nucleic acid segments (e.g., DNA or RNA fragments) present in a polynucleotide. By "isolated" nucleic acid or polynucleotide is intended a nucleic acid molecule (DNA or RNA) that has been removed from its native environment. For example, a recombinant polynucleotide encoding an antibody, contained in a vector, is considered to be isolated for various purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polypeptides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules that are synthetically produced. In addition, a polynucleotide or nucleic acid can be or can include a regulatory element, such as a promoter, ribosome binding site, or transcription terminator.

[0059] As used herein, a "coding region" is a portion of a nucleic acid consisting of codons that are translated into amino acids. Although "stop codons" (TAG, TGA, or TAA) may be considered to be part of the coding region even though they are not translated into amino acids, any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. Two or more coding regions of the invention can be present in one polynucleotide construct, such as in one vector, or in separate polynucleotide constructs, such as in separate (different) vectors. Furthermore, any vector may contain only one coding region or may include two or more coding regions, such as one vector separately encoding an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, vectors, polynucleotides, or nucleic acids of the invention may encode heterologous coding regions, fused or unfused to a nucleic acid encoding a binding molecule, an antibody, or a fragment, variant, or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

[0060] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid encoding a polypeptide may usually include a promoter and / or other transcription or translation control elements operably linked to one or more coding regions. Operable linkage is when a coding region for a gene product (e.g., a polypeptide) is linked to one or more regulatory sequences in such a manner that expression of the gene product is under the influence or control of the regulatory sequences. Two DNA fragments (e.g., a polypeptide coding region and a promoter linked thereto) are "operably linked" or "operably linked" if induction of promoter function results in transcription of an mRNA encoding a desired gene product, and if the nature of the link between these two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct expression of the gene product or the ability of the DNA template to be transcribed. Thus, a promoter region will be operably linked to a nucleic acid encoding a polypeptide if the promoter is able to effect transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in a given cell. In addition to a promoter, other transcription control elements, such as enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.

[0061] A variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as promoter and enhancer segments from cytomegalovirus (immediate early promoter, in combination with intron A), promoter and enhancer segments from Simian Virus 40 (early promoter), and promoter and enhancer segments from retroviruses (e.g., Rous sarcoma virus). Other transcription control regions include transcription control regions from vertebrate genes (e.g., actin, heat shock protein, bovine growth hormone, rabbit β-globin, etc.) and other sequences that can control gene expression in eukaryotic cells. Further suitable transcription control regions include tissue-specific promoters and enhancers, and lymphokine-inducible promoters (e.g., promoters that can be induced by interferon or interleukin).

[0062] Similarly, various translation control elements are known to those skilled in the art, including, but not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses, in particular internal ribosome entry sites, or IRES, also referred to as CITE sequences.

[0063] In other embodiments, a polynucleotide of the invention is RNA, for example in the form of messenger RNA (mRNA).

[0064] The polynucleotide and nucleic acid coding regions of the present invention may be linked to additional coding regions that encode secretory or signal peptides, thereby directing the secretion of the polypeptides encoded by the polynucleotides of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once translocation of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those skilled in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the complete or "full-length" polypeptide to yield a secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide is used, such as the signal peptide of an immunoglobulin heavy chain or an immunoglobulin light chain, or a functional derivative of that sequence that retains the ability to direct the secretion of a polypeptide to which it is operably linked. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader sequence may be replaced by the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0065] "Binding molecule", as used in the context of the present invention, refers primarily to antibodies and fragments thereof, but may also refer to other non-antibody molecules that bind to TTR, including, but not limited to, hormones, receptors, ligands, major histocompatibility complex (MHC) molecules, chaperones (e.g., heat shock proteins (HSPs) and the like), and cell-cell adhesion molecules (e.g., members of the cadherin superfamily, integrin superfamily, C-type lectin superfamily and immunoglobulin (Ig) superfamily and the like). Thus, for clarity only, and without limiting the scope of the invention, most of the embodiments below are discussed with respect to antibodies and antibody-like molecules, which represent preferred binding molecules for developing therapeutic and diagnostic agents.

[0066] antibody: The terms "antibody" and "immunoglobulin" are used interchangeably herein. An antibody or immunoglobulin is a binding molecule that contains at least the variable domain of a heavy chain, and usually contains at least the variable domains of a heavy chain and a light chain. Basic immunoglobulin structure in vertebrate systems is relatively well understood (see, e.g., Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed., 1988)).

[0067] As discussed in more detail below, the term "immunoglobulin" includes various broad classes of polypeptides that can be biochemically distinguished. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), with several subclasses therein (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of an antibody as IgG, IgM, IgA, IgG or IgE, respectively. Subclasses (isotypes) of immunoglobulins, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible to the skilled artisan in view of the present disclosure and are therefore within the scope of the present invention. While all immunoglobulin classes are clearly within the scope of the present invention, the following discussion will generally relate to the IgG class of immunoglobulin molecules. For IgG, a standard immunoglobulin molecule contains two identical light polypeptide chains with a molecular weight of approximately 23,000 daltons and two identical heavy polypeptide chains with a molecular weight of 53,000-70,000. The four chains are typically joined by disulfide bonds in a "Y" configuration, with the light chains supporting the heavy chains as arms starting at the mouth of the "Y" and continuing to the end of the variable region.

[0068] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can associate with either a kappa or lambda light chain. Generally, when immunoglobulins are produced by either hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequences run from the N-terminus at the forked ends of the Y-shaped configuration to the C-terminus at the bottom of each chain.

[0069] Both the light and heavy chains are divided into regions that are structurally and functionally homologous. The terms "constant" and "variable" are used functionally. In this context, the variable domains (V L and V H It will be appreciated that the constant domains of the light chain (CL) and the constant domains of the heavy chain (CH1, CH2 or CH3) determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the constant domains of the heavy chain (CH1, CH2 or CH3) confer various important biological properties, such as secretion, transplacental mobility, Fc receptor binding and complement binding. By convention, the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino terminus of the antibody. The N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CL domains actually comprise the carboxy termini of the heavy and light chains, respectively.

[0070] As described above, the variable region enables an antibody to selectively recognize an epitope on an antigen and specifically bind to this epitope. L Domains and V H Domains, or subsets of the complementarity determining regions (CDRs) of an antibody, combine to form the variable region that defines the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding sites at the end of each arm of the Y. More specifically, the antigen-binding site is a V H Chain and V LIt is defined by three CDRs in each of the chains. Any antibody or immunoglobulin fragment that contains sufficient structure to specifically bind to TTR is referred to herein interchangeably as a "binding fragment" or an "immunospecific fragment."

[0071] In naturally occurring antibodies, antibodies contain six hypervariable regions (sometimes called "complementarity determining regions" or "CDRs" present in each antigen-binding domain) that are short, non-contiguous sequences of amino acids that are specifically arranged to form an antigen-binding domain in which the antibody assumes its three-dimensional configuration in an aqueous environment. The "CDRs" are flanked by four relatively conserved "framework" regions or "FRs" that show less inter-molecular variability. These framework regions largely adopt a β-sheet conformation, and the CDRs form loops that connect, and sometimes form part of, this β-sheet structure. Thus, the framework regions act to form a scaffold that provides for the positioning of the CDRs in the correct orientation by non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope in the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids which constitute the CDRs and framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of skill in the art because they have been precisely defined; see "Sequences of Proteins of Immunological Interest," Kabat, E. et al., eds., USDepartment of Health and Human Services (1983), and Chothia and Lesk, J. Mol. Biol. 196 (1987), 901-917.

[0072] In the event that there are more than one definition of a term used and / or accepted in the art, the definition of the term as used herein is intended to encompass all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the discontinuous antigen-binding site found in the variable regions of both heavy and light chain polypeptides. This particular region is described by Kabat et al., USDept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983), and by Chothia and Lesk, J. Mol. Biol. 196 (1987), 901-917, which are incorporated herein by reference, where the definitions encompass overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of either definition to refer to the CDRs of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 for comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. One skilled in the art can routinely determine which residues comprise a particular hypervariable region or CDR of a human IgG subtype of antibody given the antibody variable region amino acid sequence. [Table 1] 1 The numbering of all CDR definitions in Table 1 follows the numbering convention set forth by Kabat et al. (see below).

[0073] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One skilled in the art can uniquely assign this system of "Kabat numbering" to any variable domain sequence without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., US Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in the antibody of the present invention or its antigen-binding fragment, variant, or derivative are in accordance with the Kabat numbering system; however, the Kabat numbering system is theoretical and may not be equally applicable to every antibody of the present invention. For example, depending on the position of the first CDR, subsequent CDRs may be shifted in either direction.

[0074] Antibodies or antigen-binding or immunospecific fragments, variants or derivatives thereof of the invention include polyclonal, monoclonal, multispecific, human, humanized, primatized, murine or chimeric antibodies, single chain antibodies, epitope-binding fragments (e.g., Fab, Fab' and F(ab')2), Fd, Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), VFv, ... L Domain or V HThe scFV molecules include, but are not limited to, fragments comprising either the IgG1 or IgG2 domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies, including, for example, anti-Id antibodies to the antibodies disclosed herein. scFV molecules are known in the art and are described, for example, in U.S. Pat. No. 5,892,019. The immunoglobulin or antibody molecules of the present invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule.

[0075] In one embodiment, the antibody of the present invention is not IgM or its derivatives having a pentavalent structure. In particular, in the specific application of the present invention, especially in therapeutic use, IgM is less useful than IgG and other bivalent antibodies or corresponding binding molecules. This is because IgM often shows non-specific cross-reactivity and very low affinity due to its pentavalent structure and lack of affinity maturation.

[0076] In a particularly preferred embodiment, the antibodies of the invention are not polyclonal, i.e., they are not mixtures obtained from plasma immunoglobulin samples, but rather consist essentially of one particular antibody species.

[0077] Antibody fragments, including single chain antibodies, may comprise the variable region(s) alone or in combination with all or a portion of the following: hinge region, CH1 domain, CH2 domain, and CH3 domain. The invention also includes TTR-binding fragments comprising any combination of the variable region(s) with the hinge region, CH1 domain, CH2 domain, and CH3 domain. The antibodies or immunospecific fragments thereof of the invention may be from any animal origin, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be chondricthoid in origin (e.g., from sharks).

[0078] In one embodiment, the antibody of the present invention is a human monoclonal antibody isolated from humans.Optionally, the framework region of human antibody is aligned according to and matched with the corresponding human germline variable region sequence in database; see, for example, Vbase (http: / / vbase.mrc-cpe.cam.ac.uk / ) provided by MRC Centre for Protein Engineering (Cambridge, UK).For example, it is considered that the amino acid that is considered to potentially deviate from the true germline sequence may be due to the PCR primer sequence that is incorporated during the cloning process. In comparison with artificially produced human-like antibodies, such as single-chain antibody fragments (scFV) derived from phage-displayed antibody libraries or xenogeneic mice, the human monoclonal antibodies of the present invention are characterized by (i) being obtained using a human immune response, rather than that of a surrogate animal, i.e., the antibodies are produced in response to the native TTR in its relevant conformation in the human body, (ii) protecting the individual or at least significant for the presence of TTR, and (iii) the risk of cross-reactivity to autoantigens is minimized since the antibodies are of human origin. Thus, according to the present invention, the terms "human monoclonal antibody", "human monoclonal autoantibody" and "human antibody" etc. are used to indicate TTR-binding molecules that are of human origin, i.e., isolated from human cells (e.g., B cells or hybridomas thereof) or whose cDNAs have been directly cloned from the mRNA of human cells (e.g., human memory B cells). A human antibody remains "human" even if amino acid substitutions are made in the antibody, e.g., to improve binding properties.

[0079] In one embodiment, the humanized antibodies of the invention contain heterologous regions compared to naturally occurring antibodies, such as amino acid substitutions in framework regions, constant regions exogenously fused to variable regions, and different amino acids at the C-terminus or N-terminus.

[0080] Antibodies derived from human immunoglobulin libraries or from animals that are genetically modified for one or more human immunoglobulins and do not express endogenous immunoglobulins (as described below and, for example, in U.S. Pat. No. 5,939,598 (Kucherlapati et al.)) are referred to as human-like antibodies to distinguish them from the truly human antibodies of the present invention.

[0081] 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, does not necessarily reflect the original pairing as it occurred in the original human B cell. Thus, Fab and scFv fragments obtained from recombinant expression libraries as commonly used in the prior art are considered artificial with respect to all possible related effects on immunogenicity and stability.

[0082] In contrast, the present invention provides isolated affinity matured antibodies obtained from selected human subjects that are characterized by their therapeutic utility and their tolerance in humans.

[0083] As used herein, the term "rodentified antibody" or "rodentified immunoglobulin" refers to an antibody comprising one or more CDRs derived from a human antibody of the invention and a human framework region containing amino acid substitutions and / or deletions and / or insertions based on rodent antibody sequences. When referring to rodents, preferably sequences originating from mouse and rat are used, in which case antibodies comprising such sequences are designated as "mouse-modified" or "rat-modified", respectively. The human immunoglobulin providing the CDRs is referred to as the "parent" or "acceptor", and the rodent antibody providing the framework changes is referred to as the "donor". Constant regions need not be present, but if they are present, they are usually substantially identical to the constant regions of the rodent antibody, i.e., at least about 85%-90% identical, preferably about 95% or more identical. Thus, in some embodiments, a full-length murine-ized human heavy or light chain immunoglobulin contains a murine constant region, human CDRs, and a substantially human framework with some "murine-izing" amino acid substitutions. Typically, a "murine-ized antibody" is an antibody that comprises a murine-ized variable light chain and / or a murine-ized variable heavy chain. For example, a murine-ized antibody would not encompass a typical chimeric antibody, for example, because the entire variable region of the chimeric antibody is non-murine. A modified antibody that has been "murine-ized" by the process of "murine-ization" binds to the same antigen as the parent antibody that provides the CDRs, and is usually less immunogenic in mice compared to the parent antibody. The above explanation regarding "murine-ized" antibodies is equally applicable to other "rodent-ized" antibodies, such as "rat-ized antibodies" in which rat sequences are used instead of mouse.

[0084] As used herein, the term "heavy chain portion" includes amino acid sequences derived from immunoglobulin heavy chains. A polypeptide comprising a heavy chain portion includes at least one of a CH1 domain, a hinge (e.g., 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 present invention may include a polypeptide chain comprising 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 comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the present invention includes a polypeptide chain comprising a CH3 domain. Furthermore, a binding polypeptide for use in the present invention may lack at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). As noted above, it will be understood by those skilled in the art that these domains (eg, the heavy chain portions) may be altered to differ in amino acid sequence from naturally occurring immunoglobulin molecules.

[0085] In certain antibodies or antigen-binding fragments, variants or derivatives thereof disclosed herein, the heavy chain portion of one polypeptide chain of the multimer is identical to the heavy chain portion of the second polypeptide chain of the multimer. Alternatively, the monomers containing the heavy chain portion of the present invention are not identical. For example, each monomer may contain a different target binding site, thereby forming, for example, a bispecific antibody or diabody.

[0086] In another embodiment, the antibodies, or antigen-binding fragments, variants, or derivatives thereof disclosed herein are composed of a single polypeptide chain (e.g., scFv) and will be expressed intracellularly (intrabodies) for potential in vivo therapeutic and diagnostic applications.

[0087] The heavy chain portion of a binding polypeptide for use in the diagnostic and treatment methods disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain portion of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the 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, the heavy chain portion may comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

[0088] As used herein, the term "light chain portion" includes an amino acid sequence derived from an immunoglobulin light chain. Preferably, the light chain portion is V L The protein comprises at least one of a CL domain or a CL domain.

[0089] The minimum size of a peptide or polypeptide epitope for an antibody is believed to be about 4 to 5 amino acids. A peptide or polypeptide epitope preferably contains at least 7, more preferably at least 9, and most preferably at least about 15 to about 30 amino acids. Since the CDR can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids constituting the epitope do not have to be contiguous, and in some cases may not even be present on the same peptide chain. In the present invention, a peptide or polypeptide epitope recognized by an antibody 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 about 15 to about 30 contiguous or non-contiguous amino acids of TTR.

[0090] By "specifically bind" or "specifically recognize", which are used interchangeably herein, it is generally meant that a binding molecule, e.g., an antibody, binds to an epitope through its antigen-binding domain, and that this binding involves 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 the epitope through its antigen-binding domain more readily than the antibody would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have a greater affinity for a given epitope than antibody "B", or antibody "A" may be said to bind epitope "C" with greater specificity than it has for related epitope "D".

[0091] When present, the term "immunological binding properties" or other binding properties of an antibody with an antigen, in all of its grammatical forms, refers to the specificity, affinity, cross-reactivity and other binding properties of the antibody.

[0092] By "preferentially binds," it is meant that a binding molecule, e.g., an antibody, specifically binds to an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope. Thus, an antibody that "preferentially binds" to a given epitope will more likely bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.

[0093] As a non-limiting example, a binding molecule, e.g., an antibody, can be used to bind a second epitope, such that the antibody has a K D A dissociation constant (K D ) may be considered to preferentially bind to the first epitope. In another non-limiting example, an antibody may be considered to preferentially bind to the first epitope if the antibody binds to the first epitope via a K D In another non-limiting example, an antibody may be considered to preferentially bind a first antigen if it binds to the first epitope with an affinity that is at least one order of magnitude less than the K of the antibody for a second epitope. D A peptide may be considered to preferentially bind a first epitope if it binds the first epitope with an affinity that is at least two orders of magnitude less than

[0094] As another non-limiting example, a binding molecule, e.g., an antibody, may be considered to preferentially bind a first epitope if the antibody binds to the first epitope with an off-rate (k(off)) that is less than the antibody's k(off) for the second epitope. In another non-limiting example, an antibody may be considered to preferentially bind a first epitope if the antibody binds to the first epitope with an affinity that is at least one order of magnitude less than the antibody's k(off) for the second epitope. In another non-limiting example, an antibody may be considered to preferentially bind a first epitope if the antibody binds to the first epitope with an affinity that is at least two orders of magnitude less than the antibody's k(off) for the second epitope.

[0095] A binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative disclosed herein, may be used to bind 5×10 -2 sec -1 Below, 10 -2 sec -1 Below, 5 x 10 -3 sec -1 Less than or equal to 10 -3 sec -1 An antibody of the invention may be said to bind TTR or a fragment, variant, or specific conformation thereof with an off rate (k(off)) that is less than or equal to 5×10 -4 sec -1 Below, 10 -4 sec -1 Below, 5 x 10 -5 sec -1 Less than or equal to 10 -5 sec -1 Below, 5 x 10 -6 sec -1 Below, 10 -6 sec -1 Below, 5 x 10 -7 sec -1 Less than or equal to 10 -7 sec -1 They may be said to bind TTR, or a fragment, variant, or specific conformation thereof, with an off rate (k(off)) that is less than or equal to

[0096] The binding molecules, e.g., antibodies or antigen-binding fragments, variants, or derivatives disclosed herein, may be 3 M -1 sec -1 That's it, 5 x 10 3 M -1 sec -1 That's it, 10 4 M -1 sec -1 or more than 5×10 4 M -1 sec -1 Antibodies of the invention may be said to bind TTR, or a fragment, variant, or specific conformation thereof, with an on-rate (k(on)) that is greater than or equal to 10 5 M -1 sec -1 That's it, 5 x 10 5 M -1 sec -1 That's it, 10 6 M -1 sec -1 or more than 5x10 6 M -1 sec -1 Greater than or equal to 10 7 M -1 sec -1 An antibody may be said to bind TTR or a fragment, variant, or specific conformation thereof with an on-rate (k(on)) equal to or greater than 1.

[0097] A binding molecule, for example, an antibody, is said to competitively inhibit the binding of a reference antibody to a given epitope if it preferentially binds to that epitope to such an extent that it blocks the binding of the reference antibody to that epitope to some extent. Competitive inhibition may be determined by any method known in the art, for example, by competitive ELISA assay. An antibody may be said to competitively inhibit 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%.

[0098] As used herein, the term "affinity" refers to a measure of the strength of binding of an individual epitope with the CDR of a binding molecule (e.g., an immunoglobulin molecule) (see, e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd Edition (1988), pp. 27-28). As used herein, the term "avidity" refers to the overall stability of the complex between a population of immunoglobulins and an antigen, i.e., the functional binding strength of an immunoglobulin mixture with an antigen (see, e.g., Harlow, pp. 29-34). Avidity is related to both the affinity of individual immunoglobulin molecules in a population with a specific epitope, and also to the binding valency of the immunoglobulin and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure (e.g., a polymer, etc.) would be one of high avidity. The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method (see, e.g., Berzofsky et al., "Antibody-Antigen Interactions," Fundamental Immunology, Paul, WE ed., Raven Press New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company, New York, NY (1992)) and the methods described herein. Common techniques for measuring the affinity of an antibody for an antigen include ELISA, RIA and surface plasmon resonance. The measured affinity of a particular antibody-antigen interaction may differ if measured under different conditions (e.g., salt concentration, pH). Thus, affinity and other antigen binding parameters (e.g., K D ,I C 50 Measurements of ) are preferably performed using standardized solutions of antibody and antigen and standardized buffers.

[0099] Binding molecules, such as the antibodies of the present invention or their antigen-binding fragments, variants or derivatives, may also be described or designated in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of an antibody (which is specific for one antigen) to react with a second antigen, i.e., the degree of relatedness between two different antigenic substances. Thus, an antibody is cross-reactive if it binds to an epitope different from the epitope that induced its formation. Cross-reactive epitopes generally contain many of the same complementary structural features as the inducing epitope, and in some cases may actually fit better than the original epitope.

[0100] For example, certain antibodies have some degree of cross-reactivity in that they bind to related, but non-identical epitopes, e.g., epitopes that have 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 the reference epitope. An antibody may be said to have little or no cross-reactivity if it does not bind to epitopes that have 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 the reference epitope. An antibody may be considered to be "highly specific" for a particular epitope if it does not bind to any other analogs, orthologs, or homologs of that epitope.

[0101] Binding molecules, such as antibodies of the invention or antigen-binding fragments, variants or derivatives thereof, may also be described or specified in terms of their binding affinity to TTR and / or mutated, misfolded, misassembled, and / or aggregated TTR species and / or fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd of 5×10 -2 Less than M, 10 -2 Less than M, 5×10 -3 Less than M, 10 -3 Less than M, 5×10 -4 Less than M, 10 -4 Less than M, 5×10 -5 Less than M, 10 -5 Less than M, 5×10 -6 Less than M, 10 -6 Less than M, 5×10 -7 Less than M, 10 -7 Less than M, 5×10 -8 Less than M, 10 -8 Less than M, 5×10 -9 Less than M, 10 -9 Less than M, 5×10 -10 Less than M, 10 -10 Less than M, 5×10 -11 Less than M, 10 -11 Less than M, 5×10 -12 Less than M, 10 -12 Less than M, 5×10 -13 Less than M, 10 -13 Less than M, 5×10 -14 Less than M, 10 -14 Less than M, 5×10 -15 Less than M or 10 -15 Binding affinities that are less than M are included.

[0102] In one embodiment, the antibodies of the invention have a given Kd for various TTR isotypes as illustrated for the exemplary antibodies in Table V below, i.e., a Kd of greater than 300 nM for wild-type native TTR, and / or a Kd of 15 nM or less, preferably 5 nM or less, and most preferably 2 nM or less for denatured TTR, and / or a Kd of 35 nM or less, preferably 20 nM or less for native TTR-V30M, and / or a Kd of 150 nM or less, preferably 5 nM or less, and most preferably 2 nM or less for native TTR-L55P.

[0103] As noted above, the subunit structures and three-dimensional configurations of the constant regions of the various immunoglobulin classes are widely known. H The term "CH1 domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain. H It is adjacent to the domain and is amino-terminal to the hinge region of an immunoglobulin heavy chain molecule.

[0104] As used herein, the term "CH2 domain" includes, for example, a portion of a heavy chain molecule extending from about residue 244 to residue 360 ​​of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; 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. Rather, two N-linked branched carbohydrate chains are placed between the two CH2 domains of an intact native IgG molecule. It has also been widely documented that the CH3 domain extends from the CH2 domain to the C-terminus of an IgG molecule and comprises approximately 108 residues.

[0105] As used herein, the term "hinge region" includes the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle and lower hinge domains (see Roux et al., J. Immunol. 161 (1998), 4083-4090).

[0106] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or crosslink with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by disulfide bonds, and the two heavy chains are linked by two sulfide bonds at positions corresponding to 239 and 242 (226 or 229, EU numbering system) using the Kabat numbering system.

[0107] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably. These terms refer to the joining together of two or more elements or components by any means, including chemical conjugation or recombinant means. An "in-frame fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous, longer ORF in a manner that maintains the correct translational reading frame of these original ORFs. 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 (such segments are not normally so joined in nature). Thus, although the reading frame is made continuous throughout the fused segments, these segments may be physically or spatially separated, for example, by in-frame linker sequences. For example, polynucleotides encoding the CDRs of immunoglobulin variable regions may be fused in-frame, but may be separated by polynucleotides encoding at least one immunoglobulin framework region or additional CDR regions, so long as the "fused" CDRs are co-translated as part of a continuous polypeptide.

[0108] The term "expression" as used herein refers to the process by which a gene results in a biochemical (e.g., RNA or polypeptide). This process includes any manifestation of the functional presence of a gene in a cell, including, but not limited to, gene knockdown and both transient and stable expression. This process includes, but is not limited to, transcription of a gene into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and translation of mRNA into a polypeptide. If the final desired product is a biochemical, expression includes the production of such biochemical and any precursors. Expression of a gene results in a "gene product". As used herein, a gene product can be either a nucleic acid, e.g., messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. The gene products described herein further include nucleic acids with post-transcriptional modifications (e.g., polyadenylation) or polypeptides with post-translational modifications (e.g., methylation, glycosylation, addition of lipids, association with other protein subunits, proteolytic cleavage, and the like).

[0109] As used herein, the term "sample" refers to any biological material obtained from a subject or patient. In one embodiment, the sample can include blood, peritoneal fluid, CSF, saliva, or urine. In another embodiment, the sample can include whole blood, plasma, serum, B cells enriched from blood samples, and cultured cells (e.g., B cells from a subject). Samples can also include biopsy samples or tissue samples, including neural tissue. In yet other embodiments, the sample can include whole cells and / or cell lysates. Blood samples can be collected by methods known in the art. In one embodiment, the pellet can be resuspended by vortexing in 200 μl of buffer (20 mM Tris (pH.7.5), 0.5% Nonidet, 1 mM EDTA, 1 mM PMSF, 0.1 M NaCl, IX Sigma protease inhibitor, and IX Sigma phosphatase inhibitors 1 and 2) at 4°C. The suspension can be kept on ice for 20 minutes with intermittent vortexing. After centrifugation at 15,000×g for 5 minutes at about 4° C., aliquots of the supernatant can be stored at about −70° C.

[0110] disease: Unless otherwise noted, the terms "disorder" and "disease" are used interchangeably herein and include any unwanted physiological alteration in a subject, animal, isolated organ, tissue or cell / cell culture.

[0111] Transthyretin (TTR) amyloidosis is a pathophysiological mechanism operative in many different diseases characterized by abnormal deposition of TTR protein in various tissues as a result of structural (i.e., conformational) changes in the TTR protein. Misfolded and misassembled TTR protein is toxic, often resulting from mutations in the TTR gene. The toxicity of misfolded TTR causes localized tissue damage, which, when accumulated over time, can cause organ dysfunction and even organ failure. There are many types of tissues and organs susceptible to TTR amyloidosis (e.g., peripheral and autonomic nervous systems, heart, pia mater, eye, tendons, ligaments, or kidneys). The wide range of tissues that can be affected by TTR amyloidosis is the reason for the variety of symptoms exhibited by patients with TTR amyloidosis. Indeed, patients with TTR amyloidosis are clinically classified as suffering from various diseases, depending on the tissue or organ most affected by TTR amyloidosis and the corresponding symptoms.

[0112] Based on this, TTR amyloidosis is classified into neuropathic form, in which the peripheral and autonomic nervous systems are mainly affected, and patients usually show pain, paresthesia, muscle weakness and autonomic dysfunction.There is also a cardiac form of TTR amyloidosis, in which the heart is mainly affected, and patients usually show orthostatic hypotension or hypertension, arrhythmia and cardiac hypertrophy.These two forms are not mutually exclusive, and there are many patients with the combination of these two.When TTR amyloidosis affects other tissues, it can cause vitreous opacity, dry eye or glaucoma, proteinuria, hyperthyroxinemia, carpal tunnel syndrome or preeclampsia.

[0113] Thus, in one embodiment of the invention, the antibodies of the invention, binding molecules having substantially the same binding specificity of any one of said antibodies, polynucleotides, vectors, cells and / or peptides of the invention are used to prepare pharmaceutical or diagnostic compositions for the prophylactic and / or therapeutic treatment of TTR amyloidosis diseases, to monitor disease progression and / or treatment response, and to diagnose diseases associated with TTR amyloidosis, including familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), senile systemic amyloidosis (SSA), leptomeningeal / central nervous system (CNS) amyloidosis (including Alzheimer's disease), ocular amyloidosis, renal amyloidosis, hyperthyroxinemia, carpal tunnel syndrome, rotator cuff tears and lumbar spinal stenosis, and pre-eclampsia.

[0114] treatment: As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, where the purpose is to prevent or inhibit (alleviate) an undesired physiological change or disorder, such as the onset of diabetes. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, attenuation of the extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or mitigation of disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with a condition or disorder as well as those prone to have the condition or disorder, or those in whom the onset of the condition or disorder must be prevented.

[0115] Unless otherwise noted, the terms "drug," "medicine," or "pharmaceutical product" are used interchangeably herein and are intended to include, but are not limited to, all of the following: (A) articles, medicaments, and preparations for internal or external use, and any substance or mixture of substances intended to be used for the diagnosis, cure, mitigation, treatment, or prevention of disease in either humans or other animals; and (B) articles, medicaments, and preparations (other than food) intended to affect the structure or any function of the body of humans or other animals; and (C) articles intended for use as components of any of the articles specified in clauses (A) and (B). The terms "drug", "medicine" or "pharmaceutical product" are intended to include the complete formulation of a preparation intended for use in either humans or other animals, containing one or more "agents", "compounds", "substances" or "(chemical) compositions" as fillers, disintegrants, lubricants, glidants, binders, and in some other contexts also other medicamentously inactive excipients as fillers, disintegrants, lubricants, glidants, binders, or ensuring easy transport, disintegration, dissociation, dissolution and bioavailability of the "drug", "medicine" or "pharmaceutical product" at the intended target location (e.g., skin, stomach or intestine) in the human or other animal body. The terms "agent", "compound" or "substance" are used interchangeably herein, and in a more specific context are intended to include, but are not limited to, all pharmacologically active agents, i.e. agents that induce a desired biological or pharmacological effect or that are studied or tested for their ability to induce such possible pharmacological effect by the methods of the present invention.

[0116] By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject for which diagnosis, prognosis, prevention, or treatment is desired, particularly a mammalian subject, e.g., a human patient.

[0117] Pharmaceutical carriers: Pharmaceutically acceptable carriers and administration routes can be selected from the corresponding literature known to those skilled in the art. The pharmaceutical composition of the present invention can be formulated according to the method generally known in the art (see, for example, Remington: The Science and Practice of Pharmacy (2000) (University of Sciences in Philadelphia, ISBN 0-683-306472); Vaccine Protocols, 2nd Edition (Robinson et al., Humana Press, Totowa, New Jersey, USA, 2003); Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, 2nd Edition (Taylor and Francis (2006), ISBN: 0-8493-1630-8)). Examples of suitable pharmaceutical carriers are generally known in the art, and include phosphate buffered saline solution, water, emulsions (such as oil / water emulsions), various types of wetting agents, sterile solutions, etc. The composition containing such carriers can be formulated according to the conventional method generally known. These pharmaceutical compositions can be administered to subjects in suitable doses. Administration of suitable compositions can be accomplished by various modes. Examples include administering compositions containing pharma- ceutically acceptable carriers via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods. Aerosol formulations, such as nasal spray formulations, include purified aqueous or other solutions of active agents with preservatives and isotonicity agents. Such formulations are preferably adjusted to a pH and isotonicity state compatible with the nasal mucosa. Pharmaceutical compositions for oral administration, such as single domain antibody molecules (e.g., "Nanobodies™"), are also contemplated in the present invention. Such oral formulations may be in the form of tablets, capsules, powders, liquids, or semisolids. Tablets may include solid carriers, such as gelatin or adjuvants.Formulations for rectal or vaginal administration may be presented as suppositories with a suitable carrier (see also O'Hagan et al., Nature Reviews, Drug Discovery 2(9)(2003), 727-735). Further guidance on suitable formulations for various types of administration can be found in Remington's Pharmaceutical Sciences (Mace Publishing Company, Philadelphia, PA, 17th Edition (1985) and corresponding updates). For a brief review of methods for drug delivery, see Langer, Science 249(1990), 1527-1533.

[0118] II. Antibodies of the Invention The present invention generally relates to human-derived anti-TTR antibodies and antigen-binding fragments thereof, which preferably exhibit immunological binding characteristics and / or biological properties as outlined for the antibodies exemplified in the Examples. According to the present invention, human monoclonal antibodies specific for TTR have been cloned from a pool of healthy human subjects. However, in another embodiment of the present invention, human anti-TTR monoclonal antibodies may also be cloned from patients exhibiting symptoms of diseases and / or disorders associated with TTR amyloidosis.

[0119] In the course of experiments performed according to the present invention, various antibodies present in the conditioned medium of cultured human memory B cells were evaluated for their binding ability to TTR and to more than 10 other proteins, including bovine serum albumin (BSA). Only B cell supernatants capable of binding to TTR protein in this screen, but not to any of the other proteins, were selected for further analysis, including determination of antibody class and light chain subclass. The selected B cells were then processed for antibody cloning.

[0120] Briefly, this consists in extracting messenger RNA from selected B cells, reverse transcribing by RT-PCR, amplifying the antibody coding region by PCR, cloning into a plasmid vector and sequencing. The selected human antibodies were then produced by recombinant expression and purification in HEK293 or CHO cells, and subsequently characterized for their ability to bind to human TTR protein. By a combination of various tests, for example recombinant expression of antibodies in HEK293 or CHO cells, and subsequent characterization of their binding specificity to human TTR protein, and their differential binding to its pathologically misfolded, misassembled and / or aggregated forms, it was confirmed that human antibodies have now been cloned for the first time that are highly specific for TTR and differentially recognize and selectively bind to pathologically aggregated forms of TTR protein (such as TTR fibrils). Optionally, mouse chimeric antibodies were also produced based on the variable domains of the human antibodies of the present invention. These murine chimeric antibodies demonstrate binding affinity, specificity and selectivity for human TTR comparable to the human antibodies, as shown in Figures 6 and 9 and in Examples 4 and 8.

[0121] Thus, the present invention generally relates to recombinant, human-derived, monoclonal anti-TTR antibodies, and binding fragments, derivatives and variants thereof. In one embodiment of the invention, the antibodies are capable of binding to human TTR.

[0122] In one embodiment, the invention relates to an anti-TTR antibody, or an antigen-binding fragment, variant, or derivative thereof, wherein the antibody specifically binds to the same epitope of TTR as a reference antibody selected from the group consisting of NI-301.59F1, NI-301.35G11, NI-301.37F1, and NI-301.12D3. Epitope mapping identified a sequence in human TTR comprising amino acids 61-EEEFVEGIY-69 (SEQ ID NO: 49) as the unique linear epitope recognized by the antibody NI-301.59F1 of the present invention, a sequence in human TTR comprising amino acids 53-GELHGLTTEEE-63 (SEQ ID NO: 50) as the unique linear epitope recognized by the antibody NI-301.35G11 of the present invention, and a sequence in human TTR comprising amino acids 41-WEPFA-45 (SEQ ID NO: 51) as the unique linear epitope recognized by the antibody NI-301.37F1 (see FIG. 10 and Example 9). Thus, in one embodiment, an antibody of the present invention is provided that specifically binds to a TTR epitope comprising the amino acid sequence EEEFVEGIY (SEQ ID NO: 49), GELHGLTTEEE (SEQ ID NO: 50) or WEPFA (SEQ ID NO: 51).

[0123] In this regard, as described in Example 9, the binding epitopes of the exemplary antibodies NI-301.59F1, NI301.35G11 and NI-301.37F1 are 29 consecutive peptides having a length of 15 amino acids and an overlap of 11 amino acids (i.e., TTRaa 1-15 The first peptide, TTRaa 5-19 (the second peptide being, etc.), where antibodies NI-301.59F1 and NI301.35G11 recognize two overlapping peptides ((15 and 16) and (13 and 14)), respectively, and antibody NI-301.37F1 recognizes three overlapping peptides (9, 10 and 11) (see Example 9 and Figure 10).

[0124] Thus, with respect to the amino acid sequence and corresponding peptide mapping of the mature TTR polypeptide, this indicates that the antibody NI-301.59F1, which binds to the epitope EEEFVEGIY (SEQ ID NO:49), has the amino acid sequence GLTT EEEFVEGIY KV (SEQ ID NO: 85) and the amino acid sequence EEEFVEGIY KVEIDT This means that it is capable of recognizing a peptide having the sequence of SEQ ID NO:86.

[0125] Similarly, the anti-TTR antibody NI-301.35G11, which binds to the epitope GELHGLTTEEE (SEQ ID NO:50), has the amino acid sequence TSES A peptide having the amino acid sequence GELHGLTTEEE (SEQ ID NO: 87) FVEG It is capable of recognizing a peptide having the sequence shown in SEQ ID NO:88.

[0126] Similarly, the anti-TTR antibody NI-301.37F1, which binds to the epitope WEPFA (SEQ ID NO:51), has the amino acid sequence FRKAADDT WEPFA SG A peptide having the amino acid sequence ADDT WEPFA SGKTSE (SEQ ID NO: 90) and a peptide having the amino acid sequence WEPFA SGKTSESGEL It is capable of recognizing a peptide having the sequence shown in SEQ ID NO:91.

[0127] Thus, the subject antibodies of the invention exemplified in the Examples are distinct from antibodies that recognize any of these mentioned epitopes in the context of only additional amino acids at the N-terminus and / or C-terminus. Thus, in a preferred embodiment of the invention, specific binding of anti-TTR antibodies to TTR epitopes comprising the amino acid sequence EEEFVEGIY (SEQ ID NO: 49), the amino acid sequence GELHGLTTEEE (SEQ ID NO: 50) or the amino acid sequence WEPFA (SEQ ID NO: 51) is demonstrated by contiguous peptides having a length of 15 amino acids and an overlap of 11 amino acids according to Example 9 and Figures 10A-D.

[0128] In this regard, in an extended epitope mapping carried out according to the invention and described in Example 9, using a panel of 151 consecutive peptides with a length of 15 amino acids and an overlap of 14 amino acids (wherein for each peptide the amino acid at position 10 was replaced by alanine for amino acids other than alanine, whereas alanine was replaced by glycine or proline), antibody NI-301.59F1 was found to bind to the epitope EEFXEGIY (TTRaa 62-69 ) and antibody NI-301.35G11 binds to ELXGLTXE (TTRaa 54-61 ), whereas no additional sequence requirements were determined for the epitope of antibody NI-301.37F1. Thus, in another embodiment, determining whether a given antibody binds to the same epitope as the NI-301.59F1, NI301.35G11 and NI-301.37F1 antibodies is performed according to Example 9 and Figures 10E-10H.

[0129] It will be appreciated that epitope mapping and determining whether a given antibody binds to the same epitope as the subject antibody used in Example 9 and shown in FIG. 10 can also be applied to any other anti-TTR antibodies of the invention described in the Examples having the variable regions shown in FIGS. 1A-1T.

[0130] Thus, the invention generally relates to any anti-TTR antibodies and antibody-like molecules that bind to the same epitope as the antibodies having at least the CDRs and / or variable heavy and light chain regions as exemplified in the Examples and shown in any one of Figures 1A-1T.

[0131] In further embodiments, the antibody specifically binds to the amino acid sequence GELHGLTTEEE (SEQ ID NO: 50) but not to the amino acid sequence GELHGPTTEEE corresponding to the mutant epitope of TTR-L55P, or the antibody specifically binds to the amino acid sequence WEPFA (SEQ ID NO: 51) but not to the amino acid sequence WGPFA corresponding to the mutant epitope of TTR-E42G.

[0132] Furthermore, without intending to be bound by earlier experimental findings as described in Examples 3-8 and shown in Figures 2, 3, 4, 7 and 9, the human monoclonal anti-TTR antibodies of the present invention, NI-301.59F1, NI-301.35G11 and NI-301.37F1, are preferably characterized in that they specifically bind to pathological misfolded, misassembled or aggregated TTR and do not substantially recognize TTR in its physiological form. Thus, the present invention provides a set of human anti-TTR antibodies with binding properties that are particularly useful for diagnostic and therapeutic purposes. Thus, in one embodiment, the present invention provides antibodies capable of specifically binding to pathological aggregated forms of TTR.

[0133] In one embodiment, the antibody of the present invention exhibits the binding characteristics of the exemplary antibodies NI-301.59F1, NI-301.35G11 and NI-301.37F1 as described in the Examples. The anti-TTR antibody of the present invention preferentially recognizes pathologically altered TTR (e.g., mutated, misfolded, misassembled or aggregated TTR species and fragments thereof, etc.) rather than physiological TTR. Thus, in one embodiment, the antibody of the present invention does not substantially recognize physiological TTR species.

[0134] The term "does not substantially recognize" when used in this application to describe the binding affinity of a group of molecules, including antibodies, fragments thereof, or binding molecules for a specific target molecule, a specific antigen, and / or a specific conformation of said target molecule and / or antigen, means that said group of molecules binds to said molecule, antigen, and / or conformation with a binding affinity that is less than half, less than a third, less than a fourth, less than a fifth, less than a sixth, less than a seventh, less than an eighth, or less than a ninth of the binding affinity of said group of molecules to bind to another molecule, antigen, and / or conformation. Very often, the dissociation constant (KD) is used as a measure of binding affinity. Sometimes, it is the EC50 in a specific assay, such as, for example, an ELISA assay, that is used as a measure of binding affinity. Preferably, the term "does not substantially recognize" as used in the present application means that a molecule of the aforementioned group binds to said molecule, antigen and / or conformation with a binding affinity that is less than 10 times, less than 20 times, less than 50 times, less than 100 times, less than 1000 times, or less than 10,000 times lower than the binding affinity of said molecule of the aforementioned group for binding to another molecule, antigen and / or conformation.

[0135] Additionally or alternatively, the anti-TTR antibodies of the invention bind to disease-causing misfolded, misassembled or aggregated forms of human TTR. In this regard, the binding specificity may be in the range as shown for exemplary antibodies NI-301.59F1, NI-301.35G11 and NI-301.37F1 in Figure 2, respectively Figure 10, i.e., with a half maximal effective concentration (EC50) of about 1 pM to 500 nM for human aggregated TTR and aggregated recombinant TTR as shown for NI-301.59F1 and NI-301.35G11, preferably an EC50 of about 50 pM to 100 nM, most preferably an EC50 of about 1 nM to 20 nM, or an EC50 of about 100 pM to 1 nM for human aggregated TTR and aggregated recombinant TTR as shown for NI-301.37F1.

[0136] Specifically, the anti-TTR antibodies, binding fragments or derivatives thereof have a binding affinity for binding to aggregated wild-type TTR corresponding to an EC50 value of 5 nM or less, and / or a binding affinity for binding to aggregated V30M-TTR corresponding to an EC50 of 20 nM or less, preferably an EC50 of 10 nM or less, and most preferably an EC50 of 1 nM or less (see Example 3 and Figure 2).

[0137] Some antibodies can bind to a wide range of biomolecules (e.g., proteins). As one of skill in the art will understand, the term specific is used herein to indicate that other biomolecules that are not TTR protein or fragments thereof do not significantly bind to an antigen-binding molecule (e.g., one of the antibodies of the present invention). Preferably, the level of binding to a biomolecule other than TTR results in a binding affinity that is at most no more than 20%, no more than 10%, no more than 5%, no more than 2%, or no more than 1% (i.e., less than 5-fold, less than 10-fold, less than 20-fold, less than 50-fold, or less than 100-fold, or any degree less) of the affinity for TTR, respectively (see, e.g., FIG. 2).

[0138] In one embodiment, the anti-TTR antibodies of the invention preferentially bind aggregated forms of TTR, misfolded TTR, misassembled TTR, and / or fragments, derivatives, fibrils, and / or oligomers thereof. In another embodiment, the anti-TTR antibodies of the invention preferentially bind both native TTR and pathologically misfolded, misassembled, or aggregated forms of TTR.

[0139] As mentioned above, amorphous TTR deposits and amyloid TTR deposits can cause various diseases depending on where in the body misfolded TTR species, misassembled TTR species and / or aggregated TTR species or its fragments occur.For example, patients with familial amyloid polyneuropathy (FAP) mainly exhibit TTR deposits in small diameter nerve fibers, and therefore mainly exhibit symptoms such as altered sensory perception and autonomic dysfunction (including gastrointestinal dysfunction or impotence); patients with familial amyloid cardiomyopathy (FAC) or senile systemic amyloidosis (SSA) mainly exhibit TTR deposits in the heart, and therefore mainly exhibit symptoms such as heart failure or cardiac arrhythmia; patients with TTR deposits in the kidney may exhibit renal dysfunction and proteinuria.

[0140] Thus, in one embodiment, the antibodies of the invention are useful for treating familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), senile systemic amyloidosis (SSA), systemic familial amyloidosis, leptomeningeal / central nervous system (CNS) amyloidosis (including Alzheimer's disease), ocular amyloidosis, renal amyloidosis, hyperthyroxinemia, ligament amyloidosis (including carpal tunnel syndrome, rotator cuff tears and lumbar spinal stenosis) and pre-eclampsia, and symptoms thereof.

[0141] The present invention also relates to antibodies or antigen-binding fragments, variants or derivatives thereof, wherein the antibodies are selected from the group consisting of NI-301.59F1, NI-301.35G11, NI-301.37F1, NI-301.2F5, NI-301.28B3, NI-301.119C12, NI-301.5D8, NI-301.9D5, NI-301.104 and NI-301.14C3.

[0142] The present invention further provides a V antibody having, in its variable region, e.g., in the binding domain, any one of the amino acid sequences shown in FIG. H Variable region and / or V L Some such binding molecules, such as antibodies and binding fragments thereof, are exemplified, which may be characterized by comprising at least one complementarity determining region (CDR) of a variable region. The corresponding nucleotide sequences encoding the above variable regions are shown below in Table 2, respectively. H Area and / or V L An exemplary set of CDRs of the above amino acid sequences of the regions are shown in Figure 1. However, as discussed below, the skilled artisan is well aware of the fact that, in addition or alternatively, various CDRs may be used, which differ in their amino acid sequences by one, two, three or more amino acids in the case of CDR2 and CDR3 from the amino acid sequences shown in Figure 1, respectively. Thus, in one embodiment, an antibody or TTR-binding fragment of the invention is provided that comprises in its variable region at least one complementarity determining region (CDR) shown in Figure 1 and / or one or more of the CDRs comprising one or more amino acid substitutions in the CDRs shown in Figure 1.

[0143] In one embodiment, the antibody of the invention has the V H Area and / or V L A region or its V that contains one or more amino acid substitutions. H Area and / or V L Preferably, the antibody of the present invention is characterized by the preservation of the cognate pairing of heavy and light chains as present in human B cells.

[0144] In further embodiments of the present invention, anti-TTR antibodies, TTR binding fragments, synthetic or bioengineered variants thereof can be optimized to have suitable binding affinity to the target and suitable pharmacokinetic properties. Thus, at least one amino acid in the CDR region or in the variable region that is susceptible to a modification selected from the group consisting of glycosylation, oxidation, deamination, peptide bond cleavage, isoaspartic acid formation and / or unpaired cysteine ​​is replaced by a mutant amino acid lacking such an alteration, or at least one carbohydrate moiety is chemically or enzymatically removed or added to the antibody. Various examples for amino acid optimization can be found, for example, in International Patent Application Publications WO2010 / 121140 and WO2012 / 049570. Further modifications to optimize antibody properties are described in Gavel et al., Protein Engineering, 3 (1990), 433-442, and Helenius et al., Annu. Rev. Biochem., 73 (2004), 1019-1049.

[0145] Alternatively, the antibodies of the invention may have a VV domain as shown in Figures 1A-T for binding to TTR. H Area and / or V L The antibody or antigen-binding fragment, derivative or variant thereof competes with at least one of the antibodies having the region.

[0146] The experimental results provided in Figure 2 and Example 3 suggest that some of the anti-TTR antibodies of the invention preferentially bind to disease causing misfolded, misassembled, or aggregated forms of human anti-TTR over physiological forms of the protein. Thus, in one embodiment, the antibodies of the invention preferentially bind to misfolded, misassembled, and / or aggregated forms of human anti-TTR and / or fragments and / or derivatives thereof over physiological forms of the protein.

[0147] The antibody of the present invention may be a human antibody, particularly for therapeutic applications. Alternatively, the antibody of the present invention is a rodent antibody, a rodentized antibody or a rodent-human chimeric antibody, preferably a mouse antibody, a mouse-human chimeric antibody, or a rat antibody, a rat-human chimeric antibody, which is particularly useful for diagnostic methods and research in animals. In one embodiment, the antibody of the present invention is a rodent-human chimeric antibody or a rodentized antibody.

[0148] Moreover, in one embodiment, the chimeric antibodies of the invention, i.e., chimeric antibodies comprising the variable domains of a human antibody (e.g., NI-301.35G11) and the common light and heavy chain constant domains of a mouse, exhibit the binding properties of the exemplary mouse chimeric antibody NI-301.mur35G11 as described in the Examples. Furthermore, the mouse chimeric antibodies of the invention bind with high affinity to human TTR as described in Examples 4 and 8. Preferably, the binding affinity of the chimeric antibodies is similar to their human counterparts.

[0149] In one embodiment, the antibodies of the invention are provided by cultures of single or oligoclonal B cells and culture supernatants containing antibodies produced by the B cells, which are screened for the presence and affinity of anti-TTR antibodies. The screening process includes screening for binding to native monomeric aggregates, fibrillar aggregates or non-fibrillar aggregates, such as oligomers of hTTR derived from synthetic full-length TTR peptide or purified from, for example, human plasma or recombinant expression.

[0150] Additionally or alternatively, the screening process for the presence and affinity of anti-TTR antibodies may include a step of a sensitive Tissue Amyloid Plaque Immunoreactivity (TAPIR) assay, such as that described in International Patent Application Publication No. WO2004 / 095031, the disclosure of which is incorporated herein by reference. Additionally or alternatively, screening of kidney, heart sections for binding to anti-TTR may be performed, as may screening of brain and spinal cord sections, as similarly described in International Patent Application Publication No. WO2008 / 081008.

[0151] As mentioned above, due to their generation in the human immune response, the human monoclonal antibodies of the invention will recognize epitopes that have a particular pathological relevance and that may not be accessible or less immunogenic in the case of, for example, the immunization process for the generation of mouse monoclonal antibodies and in vitro screening of phage display libraries, respectively. It is therefore advisable to specify that the epitopes of the human anti-TTR antibodies of the invention are unique and that there are no other antibodies capable of binding to the epitopes recognized by the human monoclonal antibodies of the invention (see also FIG. 10). Further demonstrating the uniqueness of the antibodies of the present invention is the fact that, as shown in Example 8, the antibodies NI-301.59F1, NI-301.35G11 and NI-301.37F1 of the present invention bind to epitopes specific to misfolded, misassembled and / or aggregated TTR conformations, which, as shown above, have particular pathological relevance and which may not be similarly obtainable by conventional processes for antibody generation, such as, for example, immunization or in vitro library screening.

[0152] Thus, in one embodiment, the invention also extends generally to anti-TTR antibodies and TTR binding molecules that compete with the human monoclonal antibodies of the invention for specific binding to TTR. The invention more specifically relates to antibodies or antigen-binding fragments, variants or derivatives thereof, which specifically bind to the same epitope of TTR as a reference antibody selected from the group consisting of NI-301.59F1, NI-301.35G11, NI-301.37F1 and / or NI-301.12D3.

[0153] Moreover, in one embodiment, the present invention also generally extends to anti-TTR antibodies and / or anti-TTR binding molecules that compete with the human monoclonal antibodies of the present invention for specific binding to misfolded, misassembled, and / or aggregated TTR species or fragments thereof. Thus, the present invention more specifically also relates to antibodies or antigen-binding fragments, variants or derivatives thereof, which specifically bind to the same epitope of misfolded, misassembled, and / or aggregated TTR species or fragments thereof as a reference antibody selected from the group consisting of NI-301.59F1, NI-301.35G11, NI-301.37F1 and / or NI-301.12D3.

[0154] Competition between antibodies 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 TTR). Numerous types of competitive binding assays are known: for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli et al., Methods in Enzymology 9 (1983), 242-253), solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137 (1986), 3614-3619, and Cheung et al., Virology 176 (1990), 546-552), solid-phase direct labeling assays, solid-phase direct labeling sandwich assays (see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988)), I 125Direct labeling RIA on solid phase using a label (see Morel et al., Molec. Immunol. 25 (1988), 7-15, and Moldenhauer et al., Scand. J. Immunol. 32 (1990), 77-82). Typically, such assays involve the use of purified TTR or misfolded, misassembled, or aggregated TTR (e.g., oligomers and / or fibrils bound to a solid surface or to cells bearing either of these), an unlabeled test immunoglobulin, and a labeled reference immunoglobulin (i.e., a human monoclonal antibody of the invention). Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Preferably, the competitive binding assay is performed under conditions as described for the ELISA assay in the accompanying Examples. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes sufficiently close to the epitope bound by the reference antibody due to steric hindrance. Typically, when a competing antibody is present in excess, it will inhibit specific binding of the reference antibody to a common antigen by at least 50% or 75%. Thus, the present invention further relates to an antibody or antigen-binding fragment, variant or derivative thereof, wherein the antibody is NI-301.59F1, The antibody competitively inhibits binding to TTR of a reference antibody selected from the group consisting of NI-301.35G11, NI-301.37F1, NI-305.2F5, NI-301.28B3, NI-301.119C12, NI-301.5D8, NI-301.9D5, NI-301.104F5, NI-301.21F10, NI-301.9G12, NI-301.12D3, NI.301.44E4, NI-301.18C4NI-301.11A10, NI-301.3C9, NI-301.14D8, NI-301.9X4 and / or NI-301.14C3.

[0155] Additionally, the present invention further relates to an antibody or antigen-binding fragment, variant, or derivative thereof, wherein the antibody is selected from the group consisting of NI-301.59F1, NI-301.35G11, NI-301.37F1, NI-305.2F5, NI-301.28B3, NI-301.119C12, NI-301.5D8, NI-301.9D5, NI-301.104F5, NI-301.21F10, NI-301.9G12, NI-301.12D3, NI-301.44E4, A reference antibody selected from the group consisting of NI-301.18C4, NI-301.11A10, NI-301.3C9, NI-301.14D8, NI-301.9X4 and / or NI-301.14C3 competitively inhibits binding to misfolded, misassembled or aggregated TTR species or fragments thereof.

[0156] In another embodiment, the present invention provides an immunoglobulin heavy chain variable region (V H An isolated polypeptide comprising an immunoglobulin heavy chain variable region (V H ), or an isolated polypeptide consisting essentially of an immunoglobulin heavy chain variable region (V H ), comprising a heavy chain variable region V H - at least one of the CDRs or the V of the heavy chain variable region H - at least two of the CDRs are V of a reference heavy chain obtained from an antibody disclosed herein H - Amino acid sequence of CDR1, V H -CDR2 amino acid sequence or V H In the alternative, the present invention provides such an isolated polypeptide, which is at least 80%, 85%, 90% or 95% identical to the amino acid sequence of V. H V H -CDR1 region, V H -CDR2 region and V H - The CDR3 region is the V of a reference heavy chain obtained from an antibody disclosed herein. H - Amino acid sequence of CDR1, V H -CDR2 amino acid sequence or V H- at least 80%, 85%, 90% or 95% identical to the amino acid sequence of CDR3. Thus, according to this embodiment, the heavy chain variable region of the invention comprises a V associated with the group shown in FIG. H -CDR1 polypeptide sequence, V H -CDR2 polypeptide sequence and V H -CDR3 polypeptide sequence. H -CDRs are shown, but other CDR definitions are used, e.g., V as defined by the Chothia system H -CDRs are also included in the present invention and can be readily identified by one of skill in the art using the data presented in FIG.

[0157] In another embodiment, the present invention provides an immunoglobulin heavy chain variable region (V H An isolated polypeptide comprising an immunoglobulin heavy chain variable region (V H ), or an isolated polypeptide consisting essentially of an immunoglobulin heavy chain variable region (V H ), comprising: H -CDR1 region, V H -CDR2 region and V H - the CDR3 region is V H -CDR1 group, V H -CDR2 group and V H -CDR3 group.

[0158] In another embodiment, the present invention provides an immunoglobulin heavy chain variable region (V H An isolated polypeptide comprising an immunoglobulin heavy chain variable region (V H ), or an isolated polypeptide consisting essentially of an immunoglobulin heavy chain variable region (V H ), comprising: H -CDR1 region, V H -CDR2 region and V H - The CDR3 region is any one of V H- V as shown in Figure 1, respectively, except for one, two, three, four, five or six amino acid substitutions in the CDRs. H -CDR1 group, V H -CDR2 group and V H -CDR3 group. In certain embodiments, the amino acid substitutions are conservative.

[0159] In another embodiment, the present invention provides an immunoglobulin light chain variable region (V L An isolated polypeptide comprising an immunoglobulin light chain variable region (V L ), or an isolated polypeptide consisting essentially of an immunoglobulin light chain variable region (V L ), comprising a light chain variable region V L At least one of the CDRs or the V of the light chain variable region L - at least two of the CDRs are V of a reference light chain obtained from an antibody disclosed herein L - Amino acid sequence of CDR1, V L -CDR2 amino acid sequence or V L In the alternative, the present invention provides such an isolated polypeptide, which is at least 80%, 85%, 90% or 95% identical to the amino acid sequence of V. L V L -CDR1 region, V L -CDR2 region and V L - The CDR3 region is the V of a reference light chain obtained from an antibody disclosed herein. L - Amino acid sequence of CDR1, V L -CDR2 amino acid sequence or V L - at least 80%, 85%, 90% or 95% identical to the amino acid sequence of CDR3. Thus, according to this embodiment, the light chain variable region of the invention has a V associated with the polypeptides shown in Figure 1, respectively. L -CDR1 polypeptide sequence, V L -CDR2 polypeptide sequence and V L-CDR3 polypeptide sequence. L -CDRs are shown, but other CDR definitions are used, e.g., V as defined by the Chothia system L -CDRs are also included in the present invention.

[0160] In another embodiment, the present invention provides an immunoglobulin light chain variable region (V L An isolated polypeptide comprising an immunoglobulin light chain variable region (V L ), or an isolated polypeptide consisting essentially of an immunoglobulin light chain variable region (V L ), comprising: L -CDR1 region, V L -CDR2 region and V L - the CDR3 region is V L -CDR1 group, V L -CDR2 group and V L -CDR3 group.

[0161] In another embodiment, the present invention provides an immunoglobulin heavy chain variable region (V L An isolated polypeptide comprising an immunoglobulin heavy chain variable region (V L ), or an isolated polypeptide consisting essentially of an immunoglobulin heavy chain variable region (V L ), comprising: L -CDR1 region, V L -CDR2 region and V L - The CDR3 region is any one of V L - V as shown in Figure 1, respectively, except for one, two, three, four, five or six amino acid substitutions in the CDRs. L -CDR1 group, V L -CDR2 group and V L -An isolated polypeptide having a polypeptide sequence which is identical to the CDR3 group. In certain embodiments, the amino acid substitutions are conservative.

[0162] The immunoglobulin or its coding cDNA may be further modified. Thus, in a further embodiment, the method of the present invention comprises any one of the steps of producing a chimeric antibody, a murine antibody, a single chain antibody, a Fab fragment, a bispecific antibody, a fusion antibody, a labeled antibody, or an analog of any one of them. Corresponding methods are known to those skilled in the art and are described, for example, in Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor (1988). When the antibody derivative is obtained by phage display technology, surface plasmon resonance as used in the BIAcore system can be used to increase the efficiency of phage antibodies binding to the same epitope as any one 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 Patent Application Publication WO89 / 09622. Methods for producing humanized antibodies are described, for example, in European Patent Application EP-A10239400 and International Patent Application Publication WO90 / 07861. Another source of antibodies for use according to the present invention are so-called xenogenous antibodies. The general principles for producing xenogenous antibodies (e.g., human-like antibodies) in mice are described, for example, in International Patent Application Publication WO91 / 10741, WO94 / 02602, WO96 / 34096 and WO96 / 33735. As discussed above, the antibodies of the present invention may exist in various forms other than complete antibodies, including, for example, Fv, Fab and F(ab)2, as well as in single chains (see, for example, International Patent Application Publication WO88 / 09344). Thus, in one embodiment, there is provided an antibody of the invention selected from the group consisting of a single chain Fv fragment (scFv), a F(ab') fragment, a F(ab) fragment and a F(ab')2 fragment.

[0163] The various antibodies of the invention or their corresponding immunoglobulin chains can be further modified using conventional techniques known in the art, for example, by using amino acid deletions, amino acid insertions, amino acid substitutions, amino acid additions and / or recombinations and / or any other modifications known in the art, either alone or in combination. Methods for introducing such modifications into the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are widely known to those of skill in the art; see, for example, Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989), NY, and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1994). Modifications of the antibodies of the invention include chemical and / or enzymatic derivatization at one or more of the constituent amino acids, including side chain modifications, including acetylation, hydroxylation, methylation, amidation, and attachment of carbohydrate or lipid moieties and cofactors, backbone modifications, and N- and C-terminal modifications. Similarly, the invention encompasses the production of chimeric proteins comprising the described antibodies or any fragments thereof fused at the amino terminus to a heterologous molecule, such as an immunostimulatory ligand at the carboxyl terminus; for corresponding technical details, see, for example, International Patent Application Publication No. WO 00 / 30680.

[0164] In addition, since it is often recognized that the heavy chain CDR3 (HCDR3) is the region with the greater degree of variability and predominant involvement in antigen-antibody interaction, the present invention encompasses peptides containing binding molecules as described above, such as peptides containing the CDR3 region (particularly the CDR3 of the heavy chain) of the variable region of any one of the antibodies mentioned. Such peptides may be easily synthesized to produce binding agents that are useful according to the present invention, or may be produced by recombinant means. Such methods are well known to those skilled in the art. The peptides can be synthesized, for example, using automated peptide synthesizers that are commercially available. The peptides can also be produced by recombinant techniques by incorporating DNA expressing the peptide into an expression vector and transforming cells with the expression vector to produce the peptide.

[0165] Thus, the present invention is directed to the above-mentioned anti-TTR antibodies and / or antibodies of the present invention capable of binding to mutated, misfolded, misassembled, or aggregated TTR species and / or fragments thereof, and also relates to any binding molecules (e.g., antibodies or binding fragments thereof) exhibiting the described properties, i.e., specifically recognizing TTR and / or mutated, misfolded, misassembled, or aggregated TTR species and / or fragments thereof. Such antibodies and binding molecules can be tested for their binding specificity and binding affinity by ELISA and immunohistochemistry as described herein (see, e.g., Examples). These properties of antibodies and binding molecules can likewise be tested by Western blot.

[0166] Exemplary human antibody NI-301.37F1 showed significant staining of misfolded TTR on sections from FAP patient skin biopsies, but no staining on healthy control pancreas, where pancreatic alpha cells show endogenous expression of TTR (i.e., native TTR) (see Example 8 and Figure 9). Exemplary antibodies of the invention NI-301.35G11 and NI-301.37F1 also gave positive results on FAP mouse tissues showing abnormal TTR deposits in various tissues, including the intestine (see Figure 8). This binding specificity for pathological forms of TTR in human and animal tissues, in addition to the biochemical experiments shown herein (see Figure 10), highlights that the antibodies of the invention can be used in the treatment and diagnosis of diseases associated with TTR amyloidosis, which preferably arise due to the presence of misfolded, misassembled and / or aggregated TTR species and / or fragments, derivatives thereof.

[0167] As an alternative to obtaining immunoglobulins directly from cultures of B cells or B memory cells, these cells can be used as a source of rearranged heavy and light chain loci for subsequent expression and / or genetic engineering. The rearranged antibody genes can be reverse transcribed from the appropriate mRNA to generate cDNA. If desired, the heavy chain constant region can be exchanged for a heavy chain constant region of a different isotype or can be completely removed. The variable regions can be linked to code for a single chain Fv region. Multiple Fv regions can be linked to confer binding capacity to more than one target, or chimeric combinations of heavy and light chains can be used. With the genetic material available, it is straightforward to design analogs as described above that both retain their ability to bind to the desired target. Methods for cloning antibody variable regions and producing 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.

[0168] Once the appropriate genetic material has been obtained and, if desired, modified to code for an analog, the coding sequence (which includes, at a minimum, sequences encoding the heavy and light chain variable regions) can be inserted into an expression system contained in a vector that can be transfected into a standard recombinant host cell. A variety of such host cells may be used; however, for efficient processing, mammalian cells are preferred. Exemplary mammalian cell lines useful for this purpose include, but are not limited to, CHO cells, HEK293 cells, or NSO cells.

[0169] The production of the antibody or analogue is then commenced by culturing the modified recombinant host under culture conditions suitable for the growth of the host cell and the expression of the coding sequence. The antibody is then recovered by isolating the antibody from the culture. The expression system is preferably designed to include a signal peptide so that the resulting antibody is secreted into the medium. However, intracellular production is also possible.

[0170] In accordance with the above, the present invention also relates to a polynucleotide encoding an antibody of the invention or an equivalent binding molecule, and in the case of an antibody, preferably a polynucleotide encoding at least the variable domain of an immunoglobulin chain of the antibody described above. Typically, said variable region encoded by a polynucleotide is the V H and / or V L In one embodiment of the invention, the polynucleotide is a cDNA.

[0171] Those skilled in the art will easily understand that the variable domains of the antibodies having the above variable domains can be used to construct other polypeptides or antibodies of desired specificity and biological function. Thus, the present invention also encompasses polypeptides and antibodies that contain at least one CDR of the variable domains described above and advantageously have substantially the same or similar binding properties as the antibodies described in the appended examples. Those skilled in the art will understand that binding affinity can be enhanced by making amino acid substitutions within the CDRs or within the hypervariable loops (Chothia and Lesk, J. Mol. Biol. 196 (1987), 901-917) that overlap with the 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 described CDRs contain one or more, preferably no more than two, amino acid substitutions. Preferably, an antibody of the invention comprises, in one or both of its immunoglobulin chains, two or all three CDRs of the variable regions as shown in FIG.

[0172] A binding molecule, such as an antibody of the present invention or an antigen-binding fragment, variant, or derivative thereof, can include a constant region that mediates one or more effector functions, as known by those skilled in the art. For example, the C1 component of complement may bind to the constant region of an antibody, thereby activating the complement system. Complement activation is important in opsonizing and lysing cellular pathogens. Complement activation also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity. Furthermore, antibodies bind to receptors on various cells via the Fc region, i.e., by binding of an Fc receptor binding site in the Fc region of the antibody to an Fc receptor (FcR) on the cell. There are several Fc receptors that are specific for different classes of antibodies, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors), and IgM (mu receptors). The binding of antibodies to cell surface Fc receptors elicits a number of important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (this is called antibody-dependent cell-mediated cytotoxicity or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.

[0173] Thus, certain embodiments of the present invention include antibodies or antigen-binding fragments, variants, or derivatives thereof in which at least one or more portions of the constant region domains have been deleted or otherwise altered to provide desired biochemical properties, such as reduced effector function, ability to non-covalently form dimers, increased ability to localize to sites of TTR aggregation and deposition, reduced serum half-life, or increased serum half-life, when compared to a complete unaltered antibody of approximately the same immunogenicity. For example, certain antibodies for use in the diagnostic and treatment methods described herein are domain-deleted antibodies that contain a polypeptide chain similar to an immunoglobulin heavy chain, but lack at least a portion of one or more heavy chain domains. For example, in certain antibodies, an entire domain of the constant region of the modified antibody will be deleted. 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 constant regions (e.g., IgG heavy chain constant regions) that are altered to eliminate glycosylation, referred to elsewhere herein as non-glycosylated or "agly" antibodies. Such "agly" antibodies may be prepared enzymatically and by engineering consensus glycosylation sites in the constant region. Without being bound by theory, it is believed that "agly" antibodies may have an improved profile of safety and stability in vivo. Methods for producing non-glycosylated antibodies with desired effector functions are found, for example, in International Patent Application Publication WO2005 / 018572, which is incorporated by reference in its entirety.

[0174] In certain antibodies or antigen-binding fragments, variants or derivatives thereof described herein, the Fc portion may be mutated to reduce effector functions using techniques known in the art. For example, deletion or inactivation (by point mutation or other means) of the constant region domain may reduce Fc receptor binding of the circulating modified antibody, thereby increasing TTR localization. In other cases, constant region modifications consistent with the present invention may moderate complement binding and thus reduce serum half-life and non-specific association of conjugated cytotoxins. Still other modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties that allow enhanced localization due to increased antigen specificity or antibody flexibility. The resulting physiological profile, bioavailability and other biochemical effects of such modifications, such as TTR localization, biodistribution and serum half-life, may be easily measured and quantified using well-known immunological techniques without undue experimentation.

[0175] In certain antibodies or antigen-binding fragments, variants or derivatives thereof described herein, the Fc portion may, for example, bind to an Fcγ receptor, an LRP or a Thy1 receptor. The antibody may be mutated or exchanged for alternative protein sequences to increase cellular uptake of the antibody by enhancing receptor-mediated endocytosis of the antibody via TTR or by "SuperAntibody Technology," which is said to allow antibodies to be shuttled into living cells without harming them (Expert Opin. Biol. Ther. (2005), 237-241). For example, fusion proteins of the antibody binding region with the cognate protein ligand of a cell surface receptor, or the creation of bispecific or multispecific antibodies with specific sequences that bind to TTR as well as to a cell surface receptor, may be designed using techniques known in the art.

[0176] In certain antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein, the Fc portion may be mutated or exchanged for an alternative protein sequence to increase its blood-brain barrier penetration, or the antibody may be chemically modified.

[0177] Modified forms of the antibodies of the present invention or their antigen-binding fragments, variants or derivatives can be produced from intact precursor or parent antibodies using techniques known in the art. Exemplary techniques are discussed in more detail herein. The antibodies of the present invention or their antigen-binding fragments, variants or derivatives can be produced or manufactured using techniques known in the art. In certain embodiments, the antibody molecules or fragments thereof are "recombinantly produced", i.e., produced using recombinant DNA techniques. Exemplary techniques for producing antibody molecules or fragments thereof are discussed in more detail elsewhere herein.

[0178] The antibodies or antigen-binding fragments, variants or derivatives of the present invention also include derivatives that are modified, for example, by covalently attaching any type of molecule to the antibody such that the covalent attachment does not prevent the antibody from specifically binding to its cognate epitope. For example, but not by way of limitation, antibody derivatives include antibodies that have been modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, and the like. Any of a number of chemical modifications may be made by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, derivatives may contain one or more non-classical amino acids.

[0179] In particularly preferred embodiments, the antibodies, or antigen-binding fragments, variants, or derivatives thereof, of the invention will not induce a deleterious immune response in the animal to be treated, e.g., in a human. In certain embodiments, a binding molecule, e.g., an antibody, or antigen-binding fragment thereof, of the invention, is derived from a patient, e.g., a human patient, and subsequently used in the same species from which the antibody or antigen-binding fragment was derived, e.g., a human, thereby mitigating or minimizing the occurrence of a deleterious immune response.

[0180] Deimmunization can also be used to reduce the immunogenicity of an antibody. As used herein, the term "deimmunization" includes altering an antibody to modify T cell epitopes (see, for example, International Patent Application Publications WO98 / 52976 and WO00 / 34317). For example, the V H Array and V L The sequences are analyzed and a human T cell epitope "map" is generated from each V region showing the location of the epitope in relation to the complementarity determining regions (CDRs) and other important residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed to identify alternative amino acid substitutions that have a low risk of altering the activity of the final antibody. A variety of alternative V regions, including combinations of amino acid substitutions, are analyzed. H Array and V L Sequences are designed and these sequences are then incorporated into various binding polypeptides (e.g., TTR-specific antibodies or immunospecific fragments thereof) for use in the diagnostic and treatment methods disclosed herein, which are then tested for function. Typically, between 12 and 24 variant antibodies are made and tested. The complete heavy and light chain genes, including the modified V regions and human C regions, are then cloned into an expression vector, and the subsequent plasmids are introduced into a cell line for production of the complete antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.

[0181] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant and phage display techniques or a combination thereof. For example, monoclonal antibodies can be produced using a variety of hybridoma techniques, including those known in the art and those taught in, for example, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, NY, 563-681 (1981), which are incorporated by reference in their entirety. The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma techniques. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic or phage clone, and does not refer to the method by which the monoclonal antibody is produced. Thus, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. In certain embodiments, the antibodies of the present invention are derived from human B cells that have been immortalized by transformation with Epstein-Barr virus as described herein.

[0182] In the well-known hybridoma process (Kohler et al., Nature 256 (1975), 495), relatively short-lived, i.e., mortal, lymphocytes obtained from a mammal, e.g., B cells from a human subject as described herein, are fused with an immortal tumor cell line (e.g., a myeloma cell line), thus resulting in hybrid cells, or "hybridomas," that are immortal and capable of producing the genetically encoded antibodies of the B cells. The resulting hybrids are segregated by selection, dilution, and regrowth into single genetic strains, each individual strain containing a specific gene for the formation of a single antibody. They produce antibodies that are homogeneous against the desired antigen, and, in reference to their pure genetic origin, are called "monoclonal."

[0183] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused original myeloma cells.Those skilled in the art will understand that the reagents, cell lines and media for the formation, selection and growth of hybridomas are commercially available from several sources, and standardized protocols are well established.Generally, the culture medium in which the hybridoma cells are growing is assayed for the production of monoclonal antibodies against the desired antigen.The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by in vitro assay, such as, for example, immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) as described herein. After hybridoma cells which produce antibodies of the desired specificity, affinity, and / or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (see, e.g., Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)). It will further be understood that the monoclonal antibodies secreted by the subclones can be separated from the culture medium, ascites fluid, or serum by conventional purification means, such as, for example, protein A, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0184] In another embodiment, lymphocytes can be selected by micromanipulation and variable genes can be isolated. For example, peripheral blood mononuclear cells can be isolated from an immunized or naturally immune mammal (e.g., human) and cultured in vitro for about 7 days. The cultures can be screened for specific IgG that meet the screening criteria. Cells from positive wells can be isolated. Individual Ig-producing B cells can be isolated by FACS or by identifying Ig-producing B cells in a complement-mediated hemolytic plaque assay. Ig-producing B cells can be micromanipulated into a tube and V H Genes and V L The gene can be amplified, for example, using RT-PCR. H Genes and V L The genes can be cloned into an antibody expression vector and transfected into cells (eg, eukaryotic or prokaryotic cells) for expression.

[0185] Alternatively, antibody-producing cell lines may be selected and cultured using techniques that are widely known to those skilled in the art. Such techniques are described in various laboratory manuals and primary publications. In this regard, the techniques suitable for use in the present invention as described below are described in Current Protocols in Immunology, Coligan et al., eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), which is incorporated herein by reference in its entirety, including any supplements.

[0186] Antibody fragments that recognize specific epitopes can be produced by known techniques. For example, Fab and F(ab')2 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')2 fragments). F(ab')2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain. Such fragments are sufficient for use in immunodiagnostic procedures that involve, for example, linking the immunospecific portion of the immunoglobulin to a detection reagent, such as a radioisotope.

[0187] In one embodiment, an antibody of the invention comprises at least one CDR of an antibody molecule. In another embodiment, an antibody of the invention comprises at least two CDRs from one or more antibody molecules. In another embodiment, an antibody of the invention comprises at least three CDRs from one or more antibody molecules. In another embodiment, an antibody of the invention comprises at least four CDRs from one or more antibody molecules. In another embodiment, an antibody of the invention comprises at least five CDRs from one or more antibody molecules. In another embodiment, an antibody of the invention comprises at least six CDRs from one or more antibody molecules. Exemplary antibody molecules comprising at least one CDR that may be included in these subject antibodies are described herein.

[0188] The antibodies of the present invention can be produced by any method known in the art for synthesizing antibodies, in particular, by chemical synthesis or, preferably, by recombinant expression techniques as described herein.

[0189] In one embodiment, the antibody or antigen-binding fragment, variant or derivative thereof of the present invention comprises a synthetic constant region in which one or more domains are partially or completely deleted ("domain deleted antibody"). In a particular embodiment, a compatible modified antibody will comprise a domain deleted construct or variant in which the entire CH2 domain is removed (ΔCH2 construct). For other embodiments, a short connecting peptide may be used in place of the deleted domain to provide the variable region with flexibility and freedom of movement. Those skilled in the art will understand that such constructs are particularly preferred due to the regulatory properties of the CH2 domain on the catabolic rate of the antibody. A domain deleted construct can be obtained using a vector encoding an IgG1 human constant domain (see, for example, International Patent Application Publication Nos. WO02 / 060955 and WO02 / 096948A2). This vector is engineered to provide a synthetic vector that deletes the CH2 domain and expresses a domain deleted IgG1 constant region.

[0190] In certain embodiments, the antibody or antigen-binding fragment, variant, or derivative thereof of the present invention is a minibody. Minibodies can be produced using methods described in the art (see, for example, U.S. Pat. No. 5,837,821 or International Patent Application Publication No. WO94 / 09817).

[0191] In one embodiment, the antibody or antigen-binding fragment, variant or derivative thereof of the present invention comprises an immunoglobulin heavy chain with a few amino acid deletions or substitutions, or even just one amino acid deletion or substitution, so long as it allows the association between the monomeric subunits. For example, a single amino acid mutation in a selected region of the CH2 domain may be sufficient to substantially reduce Fc binding and thereby increase TTR localization. Similarly, it may be desirable to simply delete that portion of one or more constant region domains that controls the effector function to be modulated (e.g., complement binding). Such partial deletion of the constant region may improve selected properties of the antibody (serum half-life), while leaving other desirable functions associated with the constant region domain intact. Moreover, as alluded to above, the constant region of the disclosed antibodies may be synthetic, with one or more amino acid mutations or substitutions that enhance the profile of the resulting construct. In this regard, it may be possible to interfere with the activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the configuration and immunogenicity profile of the modified antibody. Still other embodiments include adding one or more amino acids to the constant region to enhance a desirable property (such as, for example, effector function) or to provide greater binding of a cytotoxin or carbohydrate. In such embodiments, it may be desirable to insert or duplicate specific sequences from a selected constant region domain.

[0192] The present invention also provides antibody molecules as described herein (e.g., V H Area and / or V L Antibodies comprising variants (including derivatives) of the V H Area and / or V L or an antibody molecule as described herein (e.g., H Area and / or V LThe present invention provides an antibody comprising a variant (including derivative) of a reference V region, such that such an antibody or fragment thereof immunospecifically binds to TTR. A variety of standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding the antibody, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions. Preferably, the variant (including derivative) is a variant of a reference V region, such as a nucleotide sequence encoding the V region, which is a nucleotide sequence encoding the V region, and which is a nucleotide sequence encoding the V region, ... H area, V H -CDR1, V H -CDR2, V H -CDR3, V L area, V L -CDR1, V L -CDR2 or V L-encodes less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions or less than 2 amino acid substitutions for CDR3. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind TTR and / or misfolded, misassembled, or aggregated TTR species and / or fragments thereof).

[0193] For example, mutations can be introduced only in the framework regions of an antibody molecule or only in the CDR regions of an antibody molecule. The mutations introduced may be silent or neutral missense mutations, e.g., they may have no or little effect on the antigen-binding ability of the antibody, and in fact, some such mutations do not change the amino acid sequence at all. These types of mutations may be useful for optimizing codon usage or improving antibody production of hybridomas. Codon-optimized coding regions encoding the antibodies of the present invention are disclosed elsewhere herein. Alternatively, non-neutral missense mutations may change the antigen-binding ability of the antibody. Most silent and neutral missense mutations are likely to be located in framework regions, while most non-neutral missense mutations are likely to be located in CDRs, although this is not an absolute requirement. Those skilled in the art will be able to design and test mutant molecules with desired properties, such as no change in antigen-binding activity or changes in binding activity (e.g., improved antigen-binding activity or changes in antibody specificity). Following mutagenesis, the encoded protein may be routinely expressed and the functional and / or biological activity of the encoded protein (e.g., the ability to immunospecifically bind to TTR and / or misfolded, misassembled, or aggregated TTR species and / or fragments thereof) may be determined using the techniques described herein or by routine modification of techniques known in the art.

[0194] III. Polynucleotides Encoding Antibodies A polynucleotide encoding 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 that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA that may be single-stranded or, more typically, double-stranded or may be a mixture of single-stranded and double-stranded regions. In addition, a polynucleotide encoding an antibody or antigen-binding fragment, variant, or derivative thereof may be composed of RNA or DNA, or a triple-stranded region containing both RNA and DNA. A polynucleotide encoding an antibody or antigen-binding fragment, variant, or derivative thereof may also contain one or more modified bases or DNA or RNA backbones that are modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unconventional bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.

[0195] An isolated polynucleotide encoding a non-naturally occurring variant of a polypeptide derived from an immunoglobulin (e.g., an immunoglobulin heavy or light chain portion) can be generated by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the immunoglobulin, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations may be introduced by standard techniques, such as, for example, site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues.

[0196] As is well known, RNA may be isolated from original B cells, hybridoma cells or other transformed cells by standard techniques, such as guanidinium isothiocyanate extraction and precipitation followed by centrifugation or chromatography. If desired, mRNA may be isolated from total RNA by standard techniques, such as chromatography on oligo-dT cellulose. Suitable techniques are well known in the art. In one embodiment, cDNAs encoding the light and heavy chains of the antibody may be made simultaneously or separately using reverse transcriptase and DNA polymerase according to well-known methods. PCR may be initiated by consensus constant region primers or by more specific primers based on published heavy and light chain DNA and amino acid sequences. As discussed above, PCR may also be used to isolate DNA clones encoding the light and heavy chains of the antibody. In this case, the library may be screened by consensus primers or larger homologous probes, such as human constant region probes.

[0197] DNA, typically plasmid DNA, may be isolated from the cells using techniques known in the art and subjected to restriction mapping and sequencing according to standard, well-known techniques, e.g., as detailed in the above references relating to recombinant DNA technology. Of course, the DNA may be synthetic according to the present invention at any point during the isolation process or subsequent analysis.

[0198] In this regard, the present invention also relates to polynucleotides encoding at least the binding domain or variable region of an immunoglobulin chain of an antibody of the present invention. In one embodiment, the present invention relates to a polynucleotide encoding at least the binding domain or variable region of an immunoglobulin heavy chain variable region (V H An isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a heavy chain variable region CDR or a heavy chain variable region VDR. H - at least two of the CDRs are V of a reference heavy chain obtained from an antibody disclosed herein H - Amino acid sequence of CDR1, V H -CDR2 amino acid sequence or V H -CDR3. H V H -CDR1 region, V H -CDR2 region or V H - The CDR3 region is the V of a reference heavy chain obtained from an antibody disclosed herein. H - Amino acid sequence of CDR1, V H -CDR2 amino acid sequence or V H - at least 80%, 85%, 90% or 95% identical to the amino acid sequence of CDR3. Thus, according to this embodiment, the heavy chain variable region of the invention has a V related to the polypeptide sequence shown in FIG. H -CDR1 polypeptide sequence, V H -CDR2 polypeptide sequence or VH -has a CDR3 polypeptide sequence.

[0199] In another embodiment, the present invention provides an immunoglobulin light chain variable region (V L An isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a light chain variable region V L At least one of the CDRs or the V of the light chain variable region L - at least two of the CDRs are V of a reference light chain obtained from an antibody disclosed herein L - Amino acid sequence of CDR1, V L -CDR2 amino acid sequence or V L -CDR3. L V L -CDR1 region, V L -CDR2 region or V L - The CDR3 region is the V of a reference light chain obtained from an antibody disclosed herein. L - Amino acid sequence of CDR1, V L -CDR2 amino acid sequence or V L - at least 80%, 85%, 90% or 95% identical to the amino acid sequence of CDR3. Thus, according to this embodiment, the light chain variable region of the invention has a V related to the polypeptide sequence shown in FIG. L -CDR1 polypeptide sequence, V L -CDR2 polypeptide sequence or V L -has a CDR3 polypeptide sequence.

[0200] In another embodiment, the present invention provides an immunoglobulin heavy chain variable region (V H An isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a nucleic acid encoding V H-CDR1 region, V H -CDR2 region and V H - the CDR3 region is V as shown in Figure 1 H -CDR1 group, V H -CDR2 group and V H -CDR3 group.

[0201] As known in the art, the "sequence identity" between two polypeptides or two polynucleotides is determined by comparing the amino acid sequence or nucleic acid sequence of one polypeptide or polynucleotide to the sequence of a second polypeptide or polynucleotide. As discussed herein, whether any particular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can be determined using methods and computer programs / software known in the art, 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 the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2 (1981), 482-489) to find the best segment of homology 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 according to the present invention, it is understood that the percentage of identity is calculated over the entire length of the reference polypeptide sequence and the parameters are set so as to allow gaps in homology of up to 5% of the total number of amino acids in the reference sequence.

[0202] In a preferred embodiment of the invention, the polynucleotide is selected from the group consisting of anti-TTR antibodies and / or antibodies that recognize misfolded, misassembled, or aggregated TTR species and / or fragments thereof, as shown in Table 2. H Area or V L In one embodiment, the polynucleotide comprises, consists essentially of, or consists of a nucleic acid having a polynucleotide sequence of a region of the light and / or heavy chain. In this regard, one skilled in the art will readily appreciate that the polynucleotide encoding at least the variable domains of the light and / or heavy chains may encode the variable domains of both immunoglobulin chains or only one immunoglobulin chain. Thus, in one embodiment, the polynucleotide encodes the V domain of an anti-TTR antibody that recognizes misfolded, misassembled, or aggregated TTR species and / or fragments thereof, as shown in Table 2. H Area and V L It comprises, consists essentially of, or consists of a nucleic acid having the polynucleotide sequence of the region.

[0203] Table 2: Nucleotide sequences of the VH and VL regions of antibodies that recognize mutated, misfolded, misassembled, or aggregated TTR species and / or fragments thereof. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10]

[0204] Due to the cloning strategy, the amino acid sequences at the N-terminus and C-terminus of the heavy and light chains may potentially contain primer-induced changes in FR1 and FR4, however, this change does not substantially affect the biological activity of the antibody. To provide a consensus human antibody, the nucleotide and amino acid sequences of the initial clones can be aligned and matched with the corresponding human germline variable region sequences in a database; see, for example, Vbase2 as described above. The amino acid sequences of the human antibody are shown in bold when the amino acid sequences at the N-terminus and C-terminus are considered to deviate from the consensus germline sequence, potentially due to PCR primers, and thus have been replaced by primer-induced mutation correction (PIMC) (see Table III). Thus, in one embodiment of the invention, the polynucleotide comprises, consists essentially of, or consists of a nucleic acid having a polynucleotide sequence of a VH as shown in Table III and a corresponding VL region of an anti-TTR antibody as shown in Table II. Table III: V of antibodies recognizing mutated, misfolded, misassembled or aggregated TTR species and / or fragments thereof, showing substitutions with PIMC (bold) H Area and V L Nucleotide sequence of the region. [Table 3]

[0205] The present invention also includes fragments of the polynucleotides of the present invention, as described elsewhere. In addition, polynucleotides encoding fusion polynucleotides, Fab fragments and other derivatives, as described herein, are also encompassed by the present invention.

[0206] Polynucleotides may be generated or manufactured by any method known in the art. For example, if the nucleotide sequence of an antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides, for example as described in Kutmeier et al., BioTechniques 17 (1994), 242, which briefly involves synthesizing overlapping oligonucleotides containing portions of the antibody encoding sequence, annealing and ligating the oligonucleotides, and amplifying the ligated oligonucleotides by PCR.

[0207] Alternatively, a polynucleotide encoding an antibody or antigen-binding fragment, variant, or derivative thereof may be produced from nucleic acid obtained from a suitable source. If a clone containing nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, nucleic acid encoding the antibody may be chemically synthesized or may be prepared from a suitable source, such as an antibody cDNA library, or a cDNA library generated from any tissue or cell that expresses a TTR-specific antibody, such as a hybridoma cell selected to express the antibody, or nucleic acid isolated from such, preferably a polyA + The nucleic acid sequence may be obtained by PCR amplification from a nucleic acid sequence (RNA) using synthetic primers capable of hybridizing to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for a particular gene sequence to identify a cDNA clone from, for example, a cDNA library encoding an antibody. The amplified nucleic acid generated by PCR may then be cloned into a replicable cloning vector using any method well known in the art. Thus, in one embodiment of the invention, cDNAs encoding antibodies, immunoglobulin chains, or fragments thereof are used to produce anti-TTR antibodies.

[0208] Once the nucleotide sequence and corresponding amino acid sequence of an antibody or antigen-binding fragment, variant, or derivative thereof has been determined, the nucleotide sequence may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, such as recombinant DNA techniques, site-directed mutagenesis, PCR, etc., to generate antibodies having different amino acid sequences, e.g., to effect amino acid substitutions, deletions, and / or insertions (see, e.g., the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1990), and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998), both of which are incorporated by reference in their entireties herein).

[0209] IV. Expression of Antibody Polypeptides After the isolated genetic material has been manipulated to provide an antibody or antigen-binding fragment, variant or derivative thereof of the present invention, the polynucleotide encoding the antibody is typically inserted into an expression vector for introduction into a host cell that may be used to produce the desired amount of antibody. Recombinant expression of an antibody or a fragment, derivative or analog thereof (e.g., a heavy or light chain of an antibody that binds to a target molecule) is described herein. Once a polynucleotide encoding an antibody molecule of the present invention or a heavy or light chain of an antibody or a portion thereof (preferably a portion thereof that contains a heavy or light chain variable domain) is obtained, a vector for producing the antibody molecule may be made by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing the antibody coding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. Thus, the present invention provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the present invention or its heavy or light chain or heavy or light chain variable domain operably linked to a pro promoter. Such vectors may contain nucleotide sequences encoding the constant region of the antibody molecule (see, e.g., International Patent Applications WO86 / 05807 and WO89 / 01036, and U.S. Patent No. 5,122,464), and the variable domain of the antibody may be cloned into such a vector for expressing the entire heavy or light chain.

[0210] The term "vector" or "expression vector" is used herein to mean a vector used according to the present invention as a vehicle for introducing a desired gene into a host cell and expressing the gene in the host cell. As known to those skilled in the art, such vectors may be easily selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the present invention include 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, numerous expression vector systems may be used. For example, one class of vectors utilizes DNA elements derived from animal viruses, such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV or MOMLV) or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. In addition, cells that have integrated DNA into their chromosomes may be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers may confer prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics), or resistance to heavy metals (such as copper). Selectable marker genes can be directly linked to the DNA sequences to be expressed or can be introduced into the same cell by cotransformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, and transcriptional promoters, enhancers, and termination signals.

[0211] In a particularly preferred embodiment, the cloned variable region genes are inserted into an expression vector together with the heavy and light chain constant region genes (preferably human genes) as discussed above. This vector contains the cytomegalovirus promoter / enhancer, mouse beta globin major promoter, SV40 origin of replication, bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, dihydrofolate reductase gene and leader sequence. This vector has been found to result in very high levels of antibody expression upon incorporation of the variable and constant region genes, transfection in CHO cells, followed by selection in G418-containing medium, and methotrexate amplification. Of course, any expression vector capable of inducing expression in eukaryotic cells may be used in the present invention. Examples of suitable vectors include, but are not limited to, the plasmids pcDNA3, pHCMV / Zeo, pCR3.1, pEF1 / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAX1 and pZeoSV2, available from Invitrogen (San Diego, Calif.), and the plasmid pCI, available from Promega (Madison, Wis.). In general, screening a large number of transformed cells for those that express suitably high levels of immunoglobulin heavy and light chains is a routine experiment that can be performed, for example, by a robotic system. Vector systems are also taught in U.S. Patent Nos. 5,736,137 and 5,658,570, each of which is incorporated herein by reference in its entirety. This system provides high expression levels, for example, greater than 30 pg / cell / day. Other exemplary vector systems are disclosed, for example, in US Pat. No. 6,413,777.

[0212] In other preferred embodiments, the antibodies or antigen-binding fragments, variants or derivatives thereof of the present invention may be expressed using polycistronic constructs, such as those disclosed in US Patent Publication No. 2003-0157641 A1, which is incorporated herein in its entirety. In these expression systems, multiple gene products of interest (such as heavy and light chains of an antibody) may be provided from a single polycistronic construct. In these systems, internal ribosome entry sites (IRES) are conveniently used to provide relatively high levels of antibodies. A suitable IRES sequence is disclosed in US Patent No. 6,193,980, which is also incorporated herein. Those skilled in the art will appreciate that such expression systems may be used to effectively produce the full range of antibodies disclosed in this application. Thus, in one embodiment, the present invention provides vectors comprising polynucleotides encoding at least the binding domain or variable region of an immunoglobulin chain of an antibody, optionally in combination with polynucleotides encoding the variable regions of other immunoglobulin chains of said binding molecule.

[0213] More generally, once the vector or DNA sequence encoding the monomeric subunits of an antibody has been prepared, the expression vector may be introduced into a suitable host cell. Introduction of the plasmid into the host cell can be accomplished by a variety of techniques well known to those skilled in the art. These include, but are not limited to, transfection, including lipotransfection using, for example, Fugene® or Lipofectamine, protoplast fusion, calcium phosphate precipitation, cell fusion with packaged DNA, microinjection, and infection with intact viruses. Typically, plasmid introduction into the host is performed by standard calcium phosphate co-precipitation methods. The host cells carrying the expression construct are grown under conditions appropriate for the production of light and heavy chains, and assayed for heavy and / or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or fluorescence-activated cell sorter analysis (FACS) and immunohistochemistry.

[0214] The expression vector is transferred to a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the antibody for use in the methods described herein. Thus, the present invention includes a host cell comprising a polynucleotide encoding the antibody of the present invention or its heavy or light chain, or at least its immunoglobulin binding domain or variable region, preferably operably linked to a heterologous promoter. Additionally or alternatively, the present invention also includes a host cell comprising a vector as defined herein above, comprising a polynucleotide encoding at least the binding domain or variable region of an immunoglobulin chain of the antibody, optionally in combination with a polynucleotide encoding the variable region of the other immunoglobulin chain of said binding molecule. In a preferred embodiment for expressing a double-chain antibody, a single vector or multiple vectors encoding both the heavy and light chains may be co-expressed in a host cell for expression of a complete immunoglobulin molecule, as detailed below.

[0215] A host cell may be co-transfected with two expression vectors of the present invention, where the first vector encodes a heavy chain-derived polypeptide and the second vector encodes a light chain-derived polypeptide. These two vectors may contain the same selection marker that allows for equal expression of heavy and light chain polypeptides. Alternatively, only one vector may be used that encodes both heavy and light chain polypeptides. In such a situation, the light chain is advantageously placed before the heavy chain to avoid an excess of non-toxic heavy chain (see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad. Sci. USA 77 (1980), 2197). The coding sequences for heavy and light chains may comprise cDNA or genomic DNA.

[0216] As used herein, "host cell" refers to a cell that has a vector constructed using recombinant DNA technology and that encodes at least one heterologous gene. In the description of the process for isolating an antibody from a recombinant host, the terms "cell" and "cell culture" are used interchangeably to indicate the source of the antibody, unless otherwise clearly specified. In other words, recovery of polypeptide from "cell" can mean either from whole cells that have been centrifuged, or from the cell culture that contains both the medium and the suspended cells.

[0217] A variety of host-expression vector systems may be utilized to express the antibody molecules for use in the methods described herein. Such host-expression systems represent vehicles in which a coding sequence of interest may be produced and subsequently purified, but also cells which, when transformed or transfected with the appropriate nucleotide coding sequence, express the antibody molecule of the invention in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors containing the antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., Cauliflower Mosaic Virus, CaMV; Tobacco Mosaic Virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequences; or mammalian cell systems (e.g., COS cells, CHO cells, NSO cells, BLK cells, 293 cells, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). Preferably, bacterial cells, such as E. coli, and more preferably eukaryotic cells, especially for the expression of the complete recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells, such as Chinese hamster ovary (CHO) cells, in conjunction with vectors, such as the major immediate early gene promoter element from human cytomegalovirus, are efficient expression systems for antibodies (see, e.g., Foecking et al., Gene 45 (1986), 101; Cockett et al., Bio / Technology 8 (1990), 2).

[0218] The host cell line used for protein expression is often of mammalian origin; those skilled in the art will be able to determine the specific host cell line that is best suited for the desired gene product expressed in the host cell line.Exemplary host cell lines include, but are not limited to, CHO (Chinese hamster ovary), DG44 and DUXB11 (Chinese hamster ovary line, DHFR-deficient), HELA (human cervical cancer), CVI (monkey kidney line), COS (derivative of CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, WI38, R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney line), SP2 / O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cell), RAJI (human lymphocyte) and 293 (human kidney). Particularly preferred are CHO and 293 cells. Host cell lines are typically available from commercial services, such as the American Tissue Culture Collection, or from published literature.

[0219] In addition, a host cell line may be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.

[0220] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express antibody molecules may be engineered. Rather than using expression vectors containing viral origins of replication, host cells may be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selection markers. After introducing the foreign DNA, engineered cells may be grown in rich medium for 1-2 days and then switched to selection medium. The selection marker in the recombinant plasmid confers resistance to selection and also allows the cells to stably integrate the plasmid into their chromosomes and grow to form foci that can be subsequently cloned and expanded into cell lines. This method may be conveniently used to engineer cell lines that stably express antibody molecules.

[0221] A number of selection systems may be used, including, but not limited to, the thymidine kinase gene of herpes simplex virus (Wigler et al., Cell 11 (1977), 223), the hypoxanthine-guanine phosphoribosyltransferase gene (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48 (1992), 202), and the adenine phosphoribosyltransferase gene (Lowy et al., Cell 22 (1980), 817) can be used in tk- cells, hgprt- cells, or aprt- cells, respectively. Antimetabolite resistance can also be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77 (1980), 357; O'Hare et al., Proc. Natl. Acad. Sci. USA 78 (1981), 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78 (1981), 2072); neo, which confers resistance to the aminoglycoside G-418 (Goldspiel et al., Clinical Pharmacy 12 (1993), 488-505; Wu and Wu, Biotherapy 3 (1991), 87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32 (1993), 573-596; Mulligan, Science 260 (1993), 926-932; and Morgan and Anderson, Ann. Rev. Biochem. 62 (1993), 191-217; TIB TECH 11 (1993), 155-215; and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30 (1984), 147).Various methods generally known in the art 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 also 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 by reference in their entireties.

[0222] The expression level of an antibody molecule can be increased by vector amplification (for a review 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 an antibody expression vector system is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region will be associated with the antibody gene, production of the antibody will also increase (see Crouse et al., Mol. Cell. Biol. 3 (1983), 257).

[0223] In vitro production can be scaled up to give large amounts of the desired polypeptide. Various techniques for mammalian cell culture under tissue culture conditions are known in the art, including homogeneous suspension culture, e.g., in airlift reactors or continuous stirred tank reactors, or immobilized or entrapped cell culture, e.g., in hollow fibers, microcapsules, agarose microbeads or ceramic cartridges. If necessary and / or desired, the solution of the polypeptide can be purified by conventional chromatographic methods, e.g., gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose, or (immuno) affinity chromatography, e.g., after the preferential biosynthesis of the synthetic hinge region polypeptide, or before or after the HIC chromatography step described herein.

[0224] Genes encoding the antibodies of the present invention or their antigen-binding fragments, variants or derivatives can also be expressed in non-mammalian cells, such as bacterial cells or insect cells or yeast cells or plant cells. Bacteria that readily incorporate nucleic acids include members of the Enterobacteriaceae family, such as Escherichia coli or strains of the Salmonella genus; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. It will be further understood that when expressed in bacteria, the heterologous polypeptide typically becomes part of inclusion bodies. The heterologous polypeptide must be isolated, purified and then assembled into a functional molecule. If a tetravalent form of the antibody is desired, the subunits will self-assemble into a tetravalent antibody (see, for example, International Patent Application Publication WO02 / 096948).

[0225] In bacterial systems, a number of expression vectors may be conveniently selected depending on the intended use for which the antibody molecule is expressed. For example, vectors directing high level expression of fusion protein products that are easily purified may be desirable when large quantities of such proteins are to be produced for the generation of pharmaceutical compositions of the antibody molecule. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2 (1983), 1791) (wherein the antibody coding sequence may be individually ligated into the vector in frame with the lacZ coding region, resulting in the production of a fusion protein); pIN 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 may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to a matrix of glutathione-agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0226] In addition to prokaryotes, eukaryotic microorganisms may also be used. Saccharomyces cerevisiae, i.e., common baker's yeast, is the most commonly used of eukaryotic microorganisms, although many other strains are commonly available (e.g., Pichia pastoris). For expression in Saccharomyces, for example, the plasmid YRp7 (Stinchcomb et al., 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 TRP1 gene, which provides a selection marker for mutant strains of yeast lacking the ability to grow in tryptophan (e.g., ATCC No. 44076 or PEP4-1 (Jones, Genetics 85 (1977), 12)). In that case, the presence of the trp1 lesion as a characteristic of the yeast host cell genome provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0227] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is typically used as a vector to express foreign proteins. The virus is grown in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0228] Once the antibody molecules of the invention are recombinantly expressed, the complete antibodies of the invention, their dimers, individual light and heavy chains or other immunoglobulin forms can be purified according to standard procedures in the art, including, for example, by chromatography (e.g., ion exchange, affinity, particularly for a specific antigen followed by protein A and size fractionation column chromatography), centrifugation, differential solubility (e.g., ammonium sulfate precipitation), or any other standard technique for purification of proteins (see, e.g., Scopes, "Protein Purification", Springer Verlag, NY (1982)). Alternatively, a preferred method for increasing the affinity of the antibodies of the invention is disclosed in US Patent Application Publication No. 2002-0123057 A1. Thus, in one embodiment, the invention also relates to a method for preparing an anti-TTR antibody, or an antibody recognizing a mutated, misfolded, misassembled or aggregated TTR species and / or fragments thereof, or an immunoglobulin chain thereof, comprising: (a) a method according to the present invention comprising a polynucleotide or vector as defined hereinabove Cultivating a host cell as defined hereinabove, and (b) isolating the antibody or immunoglobulin chain thereof from the culture. The present invention provides a method comprising:

[0229] Furthermore, in one embodiment, the present invention also relates to an antibody or an immunoglobulin chain thereof encoded by a polynucleotide as defined hereinabove, or an antibody or an immunoglobulin chain thereof obtainable by said method for preparing an anti-TTR antibody or an immunoglobulin chain thereof.

[0230] V. Fusion Proteins and Conjugates In certain embodiments, the antibody polypeptide comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, the single chain Fv antibody fragments of the invention may comprise a flexible linker sequence or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label (e.g., fluorescent, radioactive, enzymatic, nuclear magnetic, heavy metal, etc.)).

[0231] The antibody polypeptide of the present invention may comprise, consist essentially of, or consist of a fusion protein.A fusion protein is, for example, a chimeric molecule that comprises an immunoglobulin TTR binding domain with at least one target binding site and at least one heterologous portion, i.e., a portion that is not naturally linked in nature.These amino acid sequences may be normally present in separate proteins that are combined in the fusion polypeptide, or may be normally present in the same protein, but are placed in a new arrangement in the fusion polypeptide.Fusion proteins may be produced, for example, by chemical synthesis, or by producing and translating a polynucleotide that encodes peptide regions in the desired relationship.

[0232] The term "heterologous," when applied to a polynucleotide or polypeptide, means that the polynucleotide or polypeptide is derived from a different entity than that of the remainder of the entity to which it is being compared. For example, as used herein, a "heterologous polypeptide" to be fused to an antibody, or antigen-binding fragment, variant, or analog thereof, is derived from a non-immunoglobulin polypeptide of the same species, or an immunoglobulin or non-immunoglobulin polypeptide of a different species. As discussed in more detail elsewhere herein, the antibodies, or antigen-binding fragments, variants, or derivatives thereof, of the invention may further be recombinantly fused at the N-terminus or C-terminus to heterologous polypeptides, or may be chemically conjugated (including covalent and non-covalent conjugation) to polypeptides or other compositions. For example, antibodies may be recombinantly fused or conjugated to molecules that are useful as labels in detection assays, and to effector molecules such as heterologous polypeptides, drugs, radionuclides or toxins (see, e.g., International Patent Application Publications WO 92 / 08495, WO 91 / 14438, WO 89 / 12624, U.S. Pat. No. 5,314,995 and European Patent Application EP 0 396 387).

[0233] The antibodies or antigen-binding fragments, variants or derivatives of the present invention may be composed of amino acids linked together by peptide bonds or modified peptide bonds (i.e., peptide isosteres) and may contain amino acids other than the 20 genetically encoded amino acids. Antibodies may be modified by natural processes (e.g., post-translational processing, etc.) or by chemical modification techniques well known in the art. Such modifications are well described in basic textbooks, more detailed monographs, and a vast research literature. Modifications may be made anywhere in the antibody, including the peptide backbone, the amino acid side chains and the amino or carboxyl termini, or to moieties such as carbohydrates. It will be understood that the same type of modification may be present in the same or varying degrees at several sites in a given antibody. Also, a given antibody may contain many types of modifications. Antibodies may be branched, for example, as a result of ubiquitination, or may be cyclic, with or without branching. Cyclic antibodies, branched antibodies, and branched cyclic antibodies may result from posttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of a flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination (see, e.g., Proteins-Structure And Molecular Properties, TECreighton, W. H. Freeman and Company, New York, 1999). New York, 2nd ed. (1993); Posttranslational Covalent Modification Of Proteins, ed. B. C. Johnson, Academic Press, New York, pp. 1-12 (1983); Seifter et al., Meth. Enzymol. 182 (1990), 626-646; Rattan et al., Ann. NY Acad. Sci. 663 (1992), 48-62).

[0234] The present invention also provides a fusion protein comprising an antibody or antigen-binding fragment, variant, or derivative thereof and a heterologous polypeptide. In one embodiment, the fusion protein of the present invention comprises the V H Any one or more amino acid sequences of the V region or the V LIn another embodiment, the fusion protein for use in the diagnostic and treatment methods disclosed herein comprises, consists essentially of, or consists of a polypeptide having one or more amino acid sequences of any one or more of the V domains or fragments or variants thereof and a heterologous polypeptide sequence. H - any one, two or three amino acid sequences of the CDR or a fragment, variant or derivative thereof, or the V of the antibody L In one embodiment, the fusion protein comprises, consists essentially of, or consists of a polypeptide having the amino acid sequence of any one, two, three, or four of the V-CDRs or fragments, variants, or derivatives thereof and a heterologous polypeptide sequence. H In another embodiment, the fusion protein comprises a polypeptide having the amino acid sequence of at least one V-CDR3 or a fragment, derivative or variant thereof and a heterologous polypeptide sequence, such that the fusion protein specifically binds to TTR. H The amino acid sequence of the region and at least one V L Preferably, the fusion protein comprises a polypeptide having the amino acid sequence of the V domain or a fragment, derivative or variant thereof and a heterologous polypeptide sequence. H Area and V L The region corresponds to a single source antibody (or scFv or Fab fragment) that specifically binds TTR. In yet another embodiment, the fusion protein for use in the diagnostic and treatment methods disclosed herein comprises the V H Any one, two, three or more amino acid sequences of the CDRs and the V L The present invention also includes a polypeptide having one, two, three or more amino acid sequences of any one of the CDRs or fragments or variants thereof and a heterologous polypeptide sequence. H -CDR or V L- two, three, four, five, six or more of the CDRs correspond to a single source antibody (or scFv fragment or Fab fragment) of the invention. Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention.

[0235] Exemplary fusion proteins reported in the literature include fusions of the following: T-cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84 (1987), 2936-2940); CD4 (Capon et al., Nature 337 (1989), 525-531; Traunecker et al., Nature 339 (1989), 68-70; Zettmeissl et al., DNA Cell Biol. USA 9 (1990), 347-353; and Byrn et al., Nature 344 (1990), 667-670); L-selectin (a homing receptor) (Watson et al., J. Cell. Biol. 110 (1990), 2221-2229; and Watson et al., Nature 349(1991), 164-167);CD44(Aruffo et al., Cell 61(1990), 1303~1313); CD28 and B7 (Linsley et al., J.Exp.Med.173(1991), 721~730); CTLA-4 (Lisley et al., J.Exp.Med.174(1991), 561~569); CD22 (Stamenković et al., Cell 66(1991), 1133-1144); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88 (1991), 10535-10539; Lesslauer et al., Eur. J. Immunol. 27 (1991), 2883-2886; and Peppel et al., J. Exp. Med. 174 (1991), 1483-1489 (1991)); and IgE receptor a (Ridgway and Gorman, J. Cell. Biol. 115 (1991), Abstract no: 1448).

[0236] As discussed elsewhere herein, the antibodies of the present invention or their antigen-binding fragments, variants or derivatives may be fused to heterologous polypeptides to increase the in vivo half-life of the polypeptide or for use in immunoassays using methods known in the art. For example, in one embodiment, PEG can be conjugated to the antibodies of the present invention to increase their half-life in vivo (see, e.g., Leong et al., Cytokine, 16 (2001), 106-119; Adv. in Drug Deliv. Rev. 54 (2002), 531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512).

[0237] Moreover, the antibodies or antigen-binding fragments, variants or derivatives thereof of the present invention can be fused to a marker sequence, such as a peptide to facilitate their purification or detection. In a preferred embodiment, the marker amino acid sequence is a hexa-histidine peptide (HIS), of which many are commercially available, such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311). As described, for example, in Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86 (1989), 821-824, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags that are useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37 (1984), 767), GST, c-myc and "flag" tags (see, e.g., Bill Brizzard, BioTechniques 44 (2008), 693-695, for a review of epitope tagging technology, and Table 1 therein on page 694 which lists the most common epitope tags that can be used in the present invention, the subject matter of which is hereby expressly incorporated by reference).

[0238] Fusion proteins can be prepared using methods well known in the art (see, for example, U.S. Patent Nos. 5,116,964 and 5,225,538). The exact site at which the fusion is made may be empirically selected to optimize the secretion or binding properties of the fusion protein. DNA encoding the fusion protein is then transfected into a host cell for expression, which is carried out as described herein above.

[0239] The antibody of the present invention may be used in unconjugated form or may be conjugated to at least one of various molecules, for example, to improve the therapeutic properties of the molecule, to facilitate target detection, or for imaging or treatment of patients.The antibody of the present invention or its antigen-binding fragment, variant or derivative may be labeled or conjugated either before or after purification, when purification is performed.In particular, the antibody of the present invention or its antigen-binding fragment, variant or derivative may be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent or PEG.

[0240] Conjugates that are immunotoxins containing conventional antibodies have been widely described in the art. The toxins may be coupled to the antibodies by conventional coupling techniques or immunotoxins containing protein toxin moieties can be produced as fusion proteins. The antibodies of the invention can be used in corresponding methods to obtain such immunotoxins. Exemplary of such immunotoxins are those described by Byers, Seminars Cell.Biol.2 (1991), 59-70 and Fanger, Immunol.Today 12 (1991), 51-54.

[0241] Those skilled in the art will understand that conjugates can also be assembled using various techniques depending on the agent selected to be conjugated. For example, conjugates with biotin are prepared, for example, by reacting a TTR-binding polypeptide with an activated ester of biotin (such as biotin N-hydroxysuccinimide ester). Similarly, conjugates with fluorescent markers may be prepared in the presence of a coupling agent (such as those listed herein) or by reaction with an isothiocyanate, preferably fluorescein isothiocyanate. Conjugates of the antibody of the present invention or its antigen-binding fragment, variant or derivative are prepared in a similar manner.

[0242] The present invention further encompasses the antibody of the present invention or its antigen-binding fragment, variant or derivative conjugated to a diagnostic or therapeutic agent. The antibody can be used diagnostically, for example, to reveal the presence of TTR amyloidosis and indicate the risk of developing a disease associated with misfolded, misassembled or aggregated TTR, to monitor the onset or progression of such a disease (i.e., to show the appearance of or associated with misfolded, misassembled or aggregated TTR), or as part of a clinical testing procedure, for example, to reveal the efficacy of a given treatment and / or prevention therapy. Thus, in one embodiment, the present invention relates to an antibody that is detectably labeled. Furthermore, in one embodiment, the present invention relates to an antibody that is conjugated to a drug. Detection can be facilitated by coupling the antibody or its antigen-binding fragment, variant or derivative to a detectable substance. The detectable substance or label can generally be an enzyme, a heavy metal (preferably gold), a dye (preferably a fluorescent or luminescent dye) or a radioactive label. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomography techniques, and non-radioactive paramagnetic metal ions (see, e.g., U.S. Pat. No. 4,741,900 for metal ions which 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, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, 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; examples of suitable radioactive materials are125 I, 131 I, 111 In or 99 Tc. Thus, in one embodiment, the invention provides a detectably labeled antibody, wherein the detectable label is selected from the group consisting of an enzyme, a radioisotope, a fluorophore, and a heavy metal.

[0243] An antibody or antigen-binding fragment, variant, or derivative thereof can also be detectably labeled by coupling to a chemiluminescent compound. The presence of the chemiluminescent-tagged 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, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0244] One way in which an antibody or antigen-binding fragment, variant, or derivative thereof can be detectably labeled is by linking the antibody or antigen-binding fragment, variant, or derivative thereof to an enzyme and using the linked product 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. (eds.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla. (1980); Ishikawa, E. et al. (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo (1981). The enzyme, once bound to the antibody, will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorometric or visual means. Enzymes that can be used to detectably label antibodies include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In addition, detection can be achieved by colorimetric methods using a chromogenic substrate for the enzyme. Detection may also be achieved by visual comparison of the extent of enzymatic reaction of the substrate in comparison to similarly prepared standards.

[0245] Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling an antibody or its antigen-binding fragment, variant, or derivative, the antibody can be detected 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 incorporated herein by reference). Radioisotopes can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.

[0246] Antibodies, or antigen-binding fragments, variants, or derivatives thereof, may also be coupled to fluorescence-emitting metals (e.g., those of the lanthanide series). 152 Metals can be detectably labeled using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0247] Techniques for conjugating various moieties to antibodies or antigen-binding fragments, variants, or derivatives thereof are well known; see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56, Alan R. Liss, Inc. (1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analytical Chemistry and Biochemistry," in See, "Radiolabeled Antibodies in Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press, pp. 303-16 (1985); and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev., 62 (1982), 119-158.

[0248] As mentioned, in certain embodiments, the binding molecule, for example, the binding polypeptide, for example, the antibody or its immunospecific fragment, can be conjugated with a component that enhances the stability or efficacy.For example, in one embodiment, PEG can be conjugated to the binding molecule of the present invention to increase its half-life in vivo.Leong et al., Cytokine, 16 (2001), 106; Adv.in Drug Deliv.Rev.54 (2002), 531; or Weir et al., Biochem.Soc.Transactions 30 (2002), 512.

[0249] VI. Compositions and Methods of Use The present invention relates to a composition comprising the above-mentioned TTR binding molecule (e.g., the antibody or antigen-binding fragment thereof or derivative or variant thereof) or the polynucleotide, vector, cell or peptide of the present invention, as defined hereinabove. In one embodiment, the composition of the present invention is a pharmaceutical composition and further comprises a pharma- ceutically acceptable carrier. Furthermore, the pharmaceutical composition of the present invention may comprise an additional agent (e.g., interleukin or interferon, etc.) depending on the intended use of the pharmaceutical composition. For use in treating a disease or disorder that shows the appearance of mutated TTR, misfolded TTR, misassembled TTR or aggregated TTR or is associated with mutated TTR, misfolded TTR, misassembled TTR or aggregated TTR (e.g., TTR amyloidosis, etc.), the additional agent may be selected from the group consisting of small organic molecules, anti-TTR antibodies, and combinations thereof. Thus, in certain preferred embodiments, the present invention relates to the use of a TTR binding molecule, such as an antibody or antigen-binding fragment thereof of the invention, or a binding molecule having substantially the same binding specificity as any one of them, a polynucleotide, vector or cell of the invention, for the preparation of a pharmaceutical or diagnostic composition for the prophylactic and therapeutic treatment of a disease or disorder associated with TTR amyloidosis, for monitoring in a subject the progression of a disease or disorder associated with TTR amyloidosis or the response to a treatment for TTR amyloidosis, or for determining a subject's risk for developing a disease or disorder associated with TTR amyloidosis.

[0250] Thus, in one embodiment, the present invention relates to a method for treating a disease or disorder characterized by abnormal accumulation and / or deposition of TTR and / or misfolded, misassembled, aggregated TTR in affected systems and organs (e.g., the peripheral nervous system, the autonomic nervous system, the central nervous system, the gastrointestinal system, the vascular system (especially the pia mater), the lymphatic system (especially the lymph nodes), the musculoskeletal system (especially the tendons and ligaments), the heart, the eyes, the kidneys, the lungs, the skin, the tongue, the thyroid gland, and the bladder, etc.), comprising administering to a subject in need thereof a therapeutically effective amount of any one of the above-described TTR-binding molecules, antibodies, polynucleotides, vectors, cells, or peptides of the present invention.

[0251] A particular advantage of the therapeutic approach of the present invention lies in the fact that the recombinant antibodies of the present invention are derived from B cells or memory B cells obtained from healthy human subjects who, for example, have asymptomatic mutations, show the appearance of aggregated TTR, or have no signs or symptoms of diseases associated with aggregated TTR, and therefore, with some probability, it is possible to prevent clinically evident diseases associated with misfolded TTR, misassembled TTR, mutated TTR, and / or aggregated TTR, or to reduce the risk of the appearance of such clinically evident diseases or disorders, or to delay the onset or progression of such clinically evident diseases or disorders. Typically, the antibodies of the present invention have also been successfully optimized for selectivity and efficacy in high affinity binding to the target TTR molecule by somatic mutation, i.e., somatic alteration of the variable region of the antibody.

[0252] The realization that such cells are not activated in vivo, for example in the human body, by related or other physiological proteins or cell structures in the sense of autoimmunological or allergic reactions is also of great medical importance, since this means a considerably increased probability of successfully surviving the clinical trial stage. That is to say, the efficiency, acceptability and tolerability have already been demonstrated in at least one human subject prior to the preclinical and clinical development of a prophylactic or therapeutic antibody. Thus, the human anti-TTR antibody of the present invention can be expected to significantly increase its clinical chances of success, both due to its target structure-specific efficiency as a therapeutic agent and due to its reduced possibility of side effects.

[0253] The invention also provides pharmaceutical and diagnostic packs or kits, respectively, comprising one or more containers filled with one or more of the above-described components (e.g., anti-TTR antibodies, binding fragments, derivatives or variants thereof, polynucleotides, vectors, cells and / or peptides of the invention). Such containers may be accompanied by a notice in a form prescribed by a governmental authority regulating the manufacture, use or sale of pharmaceutical or biological products (provided that such notice reflects approval by the authority of manufacture, use or sale for human administration). Additionally or alternatively, the kits include reagents and / or instructions for use in a suitable diagnostic assay. The compositions, e.g., kits, of the present invention are, of course, particularly suitable for risk assessment, diagnosis, prevention and treatment of diseases or disorders associated with the presence of mutated, misfolded, misassembled and / or aggregated TTR, and are specifically applicable for treating disorders commonly characterized by TTR amyloidosis, including diseases and / or disorders such as familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), senile systemic amyloidosis (SSA), systemic familial amyloidosis, leptomeningeal / central nervous system (CNS) amyloidosis (including Alzheimer's disease), TTR-related ocular amyloidosis, TTR-related renal amyloidosis, TTR-related hyperthyroxinemia, TTR-related ligament amyloidosis (including carpal tunnel syndrome, rotator cuff tear and lumbar spinal stenosis), and pre-eclampsia.

[0254] 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) (University of Sciences in Philadelphia, ISBN 0-683-306472)). Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solution, water, emulsions (e.g., oil / water emulsions, etc.), various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to a subject in a suitable dose. The administration of suitable compositions can be carried out by various methods, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, topical or intradermal administration, or by spinal or brain delivery. Aerosol formulations, such as nasal spray formulations, include purified aqueous or other solutions of the active agent with preservatives and isotonicity agents. Such formulations are preferably adjusted to a pH and isotonicity state compatible with the nasal mucosa. Formulations for rectal or vaginal administration may be presented as a suppository with a suitable carrier.

[0255] Dosage regimens will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any patient will depend on many factors, including the patient's size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health, and other drugs being administered concomitantly. A typical dosage may be, for example, in the range of 0.001 μg to 1000 μg (or a range of nucleic acids for expression or inhibition of expression in this range); however, dosages less than or greater than this exemplary range are contemplated, taking into account, among other things, the factors mentioned above. In general, dosages can be, for example, in the range of about 0.0001 to 100 mg / kg of host body weight, more usually 0.01 to 5 mg / kg (e.g., 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 2 mg / kg, etc.). For example, dosages can be 1 mg / kg body weight or 10 mg / kg body weight, or in the range of 1-10 mg / kg, preferably at least 1 mg / kg. Doses intermediate in the above ranges are also contemplated within the scope of the invention. Subjects can be administered such doses daily, every other day, weekly, or according to any other schedule determined by empirical analysis. Exemplary treatments involve administration in multiple dosages over an extended period of time, for example, at least six months. Further exemplary treatment regimes involve administration once every two weeks, or once a month, or once every three to six months. Exemplary dosage schedules include 1-10 mg / kg or 15 mg / kg on consecutive days, 30 mg / kg on every other day, or 60 mg / kg on a weekly basis. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered is within the ranges indicated. Progress can be monitored by periodic evaluation. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers (such as those based on Ringer's dextrose). Preservatives and other additives may also be present (such as antimicrobials, antioxidants, chelating agents, and inert gases). Furthermore, the pharmaceutical composition of the present invention may contain additional agents (such as dopamine or psychopharmacological drugs), depending on the intended use of the pharmaceutical composition.

[0256] Moreover, in a preferred embodiment of the present invention, for example, if the pharmaceutical composition of the present invention comprises a TTR antibody or its binding fragment, derivative or variant for passive immunization, the pharmaceutical composition may be formulated as a vaccine. As mentioned in the background section, misfolded TTR species, misassembled TTR species, mutated TTR species and / or aggregated TTR species and / or their fragments or derivatives are the main causes of TTR amyloidosis. Therefore, it is sensible to expect that passive immunization with the human anti-TTR antibodies of the present invention and equivalent TTR binding molecules will help to avoid some of the adverse effects of the active immunization therapy concept and result in reduced aggregation of TTR. Thus, the anti-TTR antibodies and equivalents of the present invention will be particularly useful as vaccines for the prevention or amelioration of diseases or disorders that indicate the presence of or are caused by aggregated TTR, such as, for example, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), senile systemic amyloidosis (SSA), systemic familial amyloidosis, leptomeningeal / central nervous system (CNS) amyloidosis (including Alzheimer's disease), TTR-related ocular amyloidosis, TTR-related renal amyloidosis, TTR-related hyperthyroxinemia, TTR-related ligament amyloidosis (including carpal tunnel syndrome, rotator cuff tear and lumbar spinal stenosis), and pre-eclampsia.

[0257] In one embodiment, it may be beneficial to use recombinant Fab fragments (rFab) and single chain fragments (scFv) of the antibodies of the invention, since they may penetrate cell membranes more easily. For example, Robert et al., Protein Eng. Des. Sel. (2008) (Oct 16); S1741-0134 (previously published online) describes the use of chimeric recombinant Fab fragments (rFab) and single chain fragments (scFv) of monoclonal antibody WO-2, which recognizes an epitope in the N-terminal region of Aβ. The engineered fragments (i) were able to prevent amyloid fibrillization, (ii) were able to disaggregate preformed Aβ1-42 fibrils, and (iii) were able to inhibit Aβ1-42 oligomer-mediated neurotoxicity in vitro as efficiently as intact IgG molecules. Recognized advantages of using small Fab and scFv engineered antibody formats that lack effector functions include more efficient crossing of the blood-brain barrier and minimizing the risk of inducing inflammatory side effects. Furthermore, in addition to retaining the binding specificity of full-length antibodies, scFv and single-domain antibodies can be expressed as single genes and expressed intracellularly in mammalian cells as intracellular antibodies, with the potential for altered folding, interaction, modification, or subcellular localization of their targets (for review, see e.g., Miller and Messer, Molecular Therapy, 12 (2005), 394-401).

[0258] In a different approach, Muller et al., Expert Opin. Biol. Ther. (2005), 237-241, describe a technology platform (so-called "SuperAntibody Technology") that is said to enable antibodies to be shuttled into living cells without damaging them. Such cell-penetrating antibodies open up new diagnostic and therapeutic scopes. The term "TransMabs" has been coined for these antibodies.

[0259] In further embodiments, it may be desirable to co-administer or sequentially administer other antibodies that are useful for treating diseases or disorders associated with the appearance of mutated, misfolded, misassembled, and / or aggregated TTR.In one embodiment, such additional antibodies are included in the pharmaceutical composition of the present invention.Examples of antibodies that can be used to treat subjects include, but are not limited to, antibodies that target CD33, SGLT2, IL-6, and IL-1.

[0260] In further embodiments, co-administration or sequential administration of other agents useful for treating diseases or disorders associated with misfolded TTR, misassembled TTR, mutated TTR and / or aggregated TTR may be desirable. In one embodiment, such additional agents are included in the pharmaceutical composition of the present invention. Examples of agents that can be used to treat a subject include, but are not limited to, the following agents: agents that stabilize TTR tetramers, such as tafamidis, meglumine, diflusinal, doxycycline with ursodeoxycholic acid, and the like; anti-inflammatory agents, such as diflusinal, corticosteroids, 2-(2,6-dichloranilino)phenylacetic acid (diclofenac), iso-butyl-propanoic-phenolic acid, and the like; diuretics, epigallocatechin gallate, melphalan hydrochloride, dexamethasone, bortezomib, bortezomib-melphalan, bortezomib-dexamethasone, melphalan-dexamethasone, bortezomib-melphalan-dexamethasone; antidepressants, antipsychotics, neuroleptics, antidementia agents (e.g., the NMDA receptor antagonist memantine), acetylcholinesterase inhibitors (e.g., donepezil HCl, rivastigmine, galantamine), glutamate antagonists and other nootropics blood pressure medications (e.g., dihydralazine, methyldopa), cytostatics, glucocorticoids, angiotensin-converting enzyme (ACE) inhibitors; anti-inflammatory agents, or any combination thereof.Examples of drugs that may be used to treat or prevent organ rejection after clinical organ transplantation include a group of drugs that cause weakening of the immune system, i.e., immunosuppressants, including, for example, calcineurin inhibitors, such as cyclosporine and tacrolimus, proliferation inhibitors, such as mTOR inhibitors, including everolimus and sirolimus (rapamycin), as well as antimetabolites, such as, but not limited to, azathioprine, mycophenolate, mofetil / MMF and mycophenolic acid, and corticosteroids, such as cortisone and cortisol, as well as synthetic substances, such as prednisone or prednisolone, may be used. In addition, various antibodies can be used: for example, anti-IL2 receptor monoclonal antibodies (e.g., basiliximab, daclizumab), as well as anti-CD3 monoclonal antibodies (e.g., muromonab-CD3), and polyclonal compositions, such as antithymocyte globulin (ATG); and glucagon-like peptide-1 (GLP-1) receptor agonists (see, for example, Noguchi et al., Acta Med. Okayama, 60 (2006), and International Patent Application Publication WO2012 / 088157). In addition, additional drugs may include drugs for preventing and / or treating infections and other side effects after organ transplantation, including valganciclovir, cytomegalie-immunoglobulin, ganciclovir, amphotericin B, pyrimethamine, ranitidine, ramipril, furosemide, benzbromarone. Thus, in one embodiment, a composition is provided that further comprises an additional agent that is useful for treating TTR amyloidosis and / or is useful in treating or preventing organ rejection, for example, after liver transplantation in clinical practice.Various examples of other agents that may be used simultaneously with the pharmaceutical composition of the present invention are described in the art (see, for example, International Patent Application Publication No. WO2009005672, WO2010128092, WO2012088157, or European Patent Application EP11158212.8).

[0261] A therapeutically effective dose or amount refers to that amount of active ingredient that is sufficient to ameliorate the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED 50 (the dose that is therapeutically effective in 50% of the population) and LD 50 (the dose that is lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, which is the LD 50 / ED 50 It can be expressed as a ratio of

[0262] From the above, it is clear that the present invention encompasses any use of TTR binding molecules and / or fragments thereof comprising at least one CDR of the above antibodies, in particular any use of such molecules for the diagnosis and / or treatment of diseases or disorders associated with mutated, misfolded, misassembled or aggregated TTR species and / or fragments thereof as described above, such as TTR amyloidosis. Preferably, said binding molecule is an antibody of the present invention or an immunoglobulin chain thereof. In addition, the present invention relates to various anti-idiotypic antibodies of any one of the mentioned antibodies described hereinabove. These are antibodies or other binding molecules that bind to unique antigenic peptide sequences located in the variable region of the antibody near the antigen binding site, and are useful, for example, for detecting anti-TTR antibodies in a sample obtained from a subject. In one embodiment, the invention relates to an antibody as defined hereinabove and below, or a TTR-binding molecule having substantially the same binding specificity as any one of said antibodies, a polynucleotide, vector or cell as defined herein, or a pharmaceutical composition or diagnostic comprising any one of them, for use in the prophylactic treatment, therapeutic treatment and / or monitoring the progression of or response to treatment of a disease or disorder associated with TTR. A composition is provided, preferably wherein the disorder is selected from the group comprising familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), senile systemic amyloidosis (SSA), systemic familial amyloidosis, leptomeningeal / central nervous system (CNS) amyloidosis (including Alzheimer's disease), TTR-related ocular amyloidosis, TTR-related renal amyloidosis, TTR-related hyperthyroxinemia, TTR-related ligament amyloidosis (including carpal tunnel syndrome, rotator cuff tear and lumbar spinal stenosis), and pre-eclampsia. The above diseases or disorders will be referred to as disorders associated with TTR amyloidosis.

[0263] In another embodiment, the present invention relates to a diagnostic composition comprising any one of the above-described TTR-binding molecules, antibodies, antigen-binding fragments, polynucleotides, vectors or cells of the present invention, and optionally suitable means for detection, such as reagents conventionally used in immunological or nucleic acid-based diagnostic methods. The antibodies of the present invention are suitable for use in, for example, immunoassays in which the antibodies can be utilized in liquid phase or can be bound to a solid phase carrier. Examples of immunoassays in which the antibodies of the present invention can be utilized are competitive and non-competitive immunoassays, either in direct or indirect formats. Examples of such immunoassays are radioimmunoassays (RIA), sandwich assays (immunometric assays), flow cytometry assays and Western blot assays. The antigens and antibodies of the present invention can be bound to many different carriers and can be used to isolate cells that specifically bind to them. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses, polyacrylamide, agarose and magnetite. The nature of the carrier can be either soluble or insoluble for the purpose of the present invention.Many different labels and labeling methods are known to those skilled in the art.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 hereinabove).

[0264] According to further embodiments, the TTR binding molecules, in particular the antibodies of the present invention, may also be used in a method for diagnosing a disorder in an individual by obtaining a body fluid sample, which may be a blood sample, a plasma sample, a serum sample, a lymph sample or any other body fluid sample (such as a saliva sample or a urine sample), from the individual to be tested, and contacting the body fluid sample with the antibody of the present invention under conditions that allow the formation of an antibody-antigen complex. The level of such complex is then determined by methods known in the art, and a level significantly greater than that formed in a control sample indicates disease in the individual to be tested. In the same manner, the specific antigen to which the antibody of the present invention binds may also be used. Thus, the present invention relates to an in vitro immunoassay comprising a binding molecule, for example, an antibody of the present invention or an antigen-binding fragment thereof.

[0265] In a further embodiment of the invention, the TTR binding molecules (particularly antibodies) of the invention may also be used in a method for diagnosing a disease or disorder in an individual by obtaining a biopsy from the individual being tested, which may be a biopsy of the skin, salivary gland, hair root, heart, colon, nerve, subcutaneous fat, or a biopsy from any affected organ.

[0266] In this context, the present invention also relates to a means specifically designed for this purpose.For example, an antibody-based array may be used, and the array is loaded with, for example, the antibody of the present invention that specifically recognizes TTR or its equivalent antigen-binding molecule.The design of immunoassays on microarrays is summarized in Kusnezow et al., Mol.Cell Proteomics 5 (2006), 1681-1696.Therefore, the present invention also relates to a microarray that is loaded with the TTR-binding molecule identified according to the present invention.

[0267] In one embodiment, the present invention relates to a method for diagnosing a disease or disorder associated with mutated, misfolded, misassembled and / or aggregated TTR species and / or fragments thereof in a subject, comprising determining the presence of TTR and / or misfolded, misassembled or aggregated TTR in a sample obtained from the subject to be diagnosed using at least one antibody of the present invention, a TTR-binding fragment thereof, or a TTR-binding molecule having substantially the same binding specificity as any one of them, wherein the presence of pathologically mutated, misfolded, misassembled or aggregated TTR is indicative for TTR amyloidosis, and wherein an increase in the level of pathologically misfolded, misassembled or aggregated TTR compared to physiological TTR levels is indicative for the progression of TTR amyloidosis in the subject.

[0268] The subject to be diagnosed may be asymptomatic or preclinical for the disease.Preferably, the control subject has a disease associated with misfolded TTR, misassembled TTR or aggregated TTR (e.g., familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC) or senile systemic amyloidosis (SSA)), where the similarity between the level of pathologically misfolded TTR, misassembled TTR or aggregated TTR and the reference standard indicates that the subject to be diagnosed has TTR amyloidosis or is at risk of developing TTR amyloidosis. Alternatively or additionally, as a second control, the control subject does not have TTR amyloidosis, but in this case, the difference between the physiological TTR level and / or the misfolded, misassembled or aggregated TTR level and the reference standard indicates that the diagnosed subject has TTR amyloidosis or is at risk of developing TTR amyloidosis.Preferably, the diagnosed subject and the control subject are age-matched.The sample to be analyzed may be any body fluid suspected of containing pathologically misfolded, misassembled or aggregated TTR, for example, blood, plasma, serum, urine, peritoneal fluid, saliva or cerebrospinal fluid (CSF).

[0269] Physiological TTR levels and / or levels of misfolded, misassembled or aggregated TTR may be assessed by any suitable method known in the art, including, for example, analyzing TTR by one or more techniques selected from Western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting (FACS), two-dimensional gel electrophoresis, mass spectrometry (MS), matrix-assisted laser desorption / ionization-time of flight MS (MALDI-TOF), surface-enhanced laser desorption / ionization-time of flight (SELDI-TOF), high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), multidimensional liquid chromatography (LC) followed by tandem mass spectrometry (MS / MS), and laser densitometry. Preferably, said in vivo imaging of TTR comprises scintigraphy, positron emission tomography (PET), single photon emission computed tomography (SPECT), near infrared (NIR) optical imaging or magnetic resonance imaging (MRI).

[0270] In further embodiments, the present invention relates to diagnosing TTR amyloidosis, monitoring the treatment of the disease, and determining the diagnostic or therapeutic utility of anti-TTR drugs without the use of tissue and biopsies, i.e., non-invasively.

[0271] Usually, the concentration of TTR aggregates and / or misfolded TTR that can be detected in body fluids (e.g., plasma) is very low, and therefore the diagnosis of TTR amyloidosis is laborious and time-consuming. In particular, the diagnosis of TTR amyloidosis disease is a difficult and lengthy process. This is because various diseases show very similar signs and symptoms, and as a result, the formal diagnosis of familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC) and senile systemic amyloidosis (SSA) typically requires the taking of tissue biopsies and the identification of TTR amyloid deposits by complex histological staining techniques. Because tissue biopsies are very small and TTR amyloid deposits are dispersed, the histological determination of TTR amyloidosis typically involves a high frequency of false-negative results and delays for patients.

[0272] However, according to the present invention, it has surprisingly been shown that after only one administration of the subject anti-TTR antibody, it is possible to measure aggregates and / or misfolded TTR bound to the anti-TTR antibody in the blood (see Example 13 and Figure 14). Thus, due to the supposedly unique property of the anti-TTR antibody of the present invention, which removes TTR from amyloidogenic TTR deposits and transports it into the blood, a novel method for diagnosing disorders associated with misfolded TTR, mutated TTR and / or aggregated TTR in patients or subjects has been developed, where such a method has the potential to replace tissue biopsy and histological analysis in the diagnostic process of TTR amyloid diseases. This method relies on the use of an antibody specific for the pathological conformation of the TTR protein, where the antibody is injected into the patient and used to probe for the presence of misfolded and / or aggregated TTR protein anywhere in the patient's body. Some time after antibody infusion in the patient, for example after 2 days, a blood sample is taken and used to detect whether the antibody has captured and dissociated the misfolded and / or aggregated TTR particles from the TTR deposits. The main advantage of this method compared to histology relates to the fact that the anti-TTR antibody is directly injected into the patient, allowing its circulation through every tissue and organ via the blood circulation, and that misfolded and / or aggregated TTR protein deposits are detected regardless of their localization.

[0273] Thus, in a further aspect, the present invention relates to a method for diagnosing a disease associated with TTR amyloidosis, comprising assaying the level of misfolded TTR and / or aggregated TTR in a sample from a subject after administration of an anti-TTR antibody to the subject, whereby the presence or elevated level of misfolded TTR and / or aggregated TTR in the sample from the subject, compared to a control (such as a sample obtained from the subject before administration of the anti-TTR antibody), indicates a disease associated with TTR amyloidosis. Furthermore, as shown in Example 13, this novel method is also useful for characterizing anti-TTR drugs and the course of treatment of TTR amyloidosis, respectively, so that this novel method of the present invention is also intended for monitoring the treatment of a disease with an anti-TTR antibody or for revealing the diagnostic or therapeutic utility of an anti-TTR antibody. In this regard, those skilled in the art will recognize that the method of the present invention is not limited to examining the therapeutic utility and therapeutic efficacy of anti-TTR antibodies, but is also applicable to other types of anti-TTR drugs that can degrade TTR amyloid deposits. For example, an anti-TTR antibody of the present invention may be administered in conjunction with another anti-TTR drug, and the levels of misfolded TTR and / or aggregated TTR in a sample from a previously treated subject are compared to a control obtained from the subject before both the anti-TTR antibody and the anti-TTR drug were administered, but only after anti-TTR antibody treatment.

[0274] In one preferred embodiment of the present invention, particularly when non-human animals are used to test recombinant human-derived antibodies as exemplified in Example 13, but generally when other anti-TTR antibodies intended for use in humans, the level of misfolded TTR and / or aggregated TTR in the sample is assayed by measuring the complex formed between the anti-TTR antibody and misfolded TTR and / or aggregated TTR, for example, by immunoprecipitation with an anti-human IgG antibody or an anti-idiotypic antibody. Alternatively, a second anti-TTR antibody may be used that recognizes TTR at an epitope substantially different from that of the drug candidate anti-TTR antibody, thereby binding to the complex formed by the drug candidate anti-TTR antibody and TTR, and thus allowing its presence to be detected, for example, by ELISA or immunoprecipitation.

[0275] With regard to diagnostic aspects, particularly for human subjects and patients, the presence and elevated levels of misfolded TTR and / or aggregated TTR and its complex with anti-TTR antibodies, respectively, indicate the presence of TTR amyloid deposits in the human body, for example in the heart, peripheral nervous system (PNS), eyes, muscles, gastrointestinal tract, kidneys, vascular system and central nervous system (CNS) of a patient or subject. Thus, the method of the present invention allows, on the one hand, the identification and determination of diseases associated with TTR amyloidosis in the subject's body, and, on the other hand, the removal of TTR deposits from the patient's body (which also indicates the therapeutic progress of a given treatment and the efficacy of a TTR amyloidosis-specific drug, such as, for example, an anti-TTR antibody).

[0276] Thus, as demonstrated in Example 13, the anti-TTR antibodies of the present invention can bind to misfolded and / or aggregated TTR with sufficient affinity to alter the stability of pathological TTR deposits, e.g., to capture misfolded and / or aggregated TTR from the deposits and remove it into body fluids, particularly blood.

[0277] The anti-TTR antibody to be used according to the methods of the present invention may be any TTR antibody specific for a pathological conformation of TTR, i.e., specific for misfolded TTR, mutated TTR and / or aggregated TTR. However, in a preferred embodiment, the anti-TTR antibody utilized in the tissue-free method is an anti-TTR antibody or TTR binding molecule of the present invention as described herein and exemplified in the Examples.

[0278] In this regard, the anti-TTR antibodies may be modified, e.g., conjugated to a detectable label, as described for any of the other embodiments herein above. In addition, various immunoassays (e.g., Western blots, dot blots, (sandwich) ELISAs, etc.) known in the art and described for other diagnostic methods and uses based on the anti-TTR antibodies and peptides of the invention may be adapted to the novel TTR amyloid assay of the invention.

[0279] As shown in Example 13 and Figure 14 using a TTR amyloid assay, it could be shown that the anti-TTR antibodies of the present invention can capture and dissociate misfolded TTR and / or aggregated TTR from TTR amyloid deposits, and the corresponding immune complexes can be measured in samples of body fluids, specifically the patient's or subject's blood (see Example 13 and Figure 14). Thus, in one embodiment of the present invention, the anti-TTR antibodies can bind to misfolded TTR and / or aggregated TTR with sufficient affinity to alter the net efflux of misfolded TTR and / or aggregated TTR from, for example, the heart, peripheral nervous system (PNS), eye, muscle, gastrointestinal tract, kidney, vasculature and central nervous system (CNS).

[0280] A bodily fluid sample (preferably blood or CSF from the subject) containing captured and dissociated misfolded TTR and / or aggregated TTR bound to the anti-TTR antibody is obtained at a specified time interval after administration. This specified time interval after administration is typically less than one week. In a preferred embodiment, this time interval after administration of the anti-TTR antibody is 48 hours or less.

[0281] As mentioned above, the above-mentioned tissue-free method can also be utilized to determine the success of a treatment, i.e., by measuring the misfolded and / or aggregated TTR species captured by the anti-TTR antibody before and after treatment. Thus, in further or additional embodiments, the tissue-free method of the present invention may further comprise comparing the level of misfolded and / or aggregated TTR in a sample of bodily fluid to a sample obtained from the subject before the anti-TTR antibody is administered. Thus, in one embodiment, the method of the present invention is used to determine the effectiveness of a treatment of TTR amyloidosis or to monitor the progression of a disease or disorder associated with pathological TTR in a patient or subject.

[0282] As mentioned, the subject samples utilized in the above described methods can be obtained before or after administration of anti-TTR antibodies. However, samples can also be obtained from medical facilities or practicing physicians, as well as other institutions that can obtain clinical samples from subjects. The facilities, physicians, etc. can administer anti-TTR antibodies to the subject and take appropriate samples for use in the above described methods, as well as monitor and / or treat the patient, i.e., by changing the dosage, administration time, administration frequency of the antibody, by interrupting the treatment, by replacing or combining the anti-TTR antibody with at least another anti-TTR antibody or therapeutic agent. The level of TTR can be evaluated by any suitable method known in the art. Various suitable methods are described below and in International Patent Application Publication WO2013 / 066818, the disclosure of which is incorporated herein by reference.

[0283] In one embodiment of the present invention, an antibody of the present invention or a TTR binding molecule having substantially the same binding specificity of any one of said antibodies, a polynucleotide, vector or cell as defined hereinabove, or a pharmaceutical or diagnostic composition comprising any one of them, is provided for use in the preventive, therapeutic treatment of a disease or disorder associated with TTR and / or in monitoring the progression or response to treatment of such a disease or disorder.Thus, in general, the present invention also relates to a method of diagnosing or monitoring the progression of a disease or disorder associated with TTR (such as, for example, TTR amyloidosis) in a subject, comprising revealing the presence of TTR in a sample from said subject to be diagnosed using at least one antibody of the present invention or a TTR binding molecule having substantially the same binding specificity of any one of said antibodies, with the proviso that in this case the presence of mutated, misfolded, misassembled or aggregated TTR species or fragments thereof is indicative for said disease or disorder.In one embodiment, a method of diagnosing or monitoring the progression of TTR amyloidosis in a subject is provided, comprising determining the presence of mutated, misfolded, misassembled or aggregated TTR species and / or fragments thereof in a sample from the subject to be diagnosed using at least one antibody of the invention or a TTR binding molecule having substantially the same binding specificity as any one of said antibodies, wherein the presence of mutated, misfolded, misassembled or aggregated TTR species and / or fragments thereof is indicative of pre-symptomatic, prodromal or clinical TTR amyloidosis, and wherein an increase in the levels of TTR oligomers, TTR aggregates or TTR fibrils, in comparison to physiological TTR levels or in comparison to a reference sample derived from a healthy control subject or a control sample from the same subject, is indicative of the progression of pre-clinical, prodromal or documented TTR amyloidosis. It will be appreciated by those skilled in the art that in one embodiment, the method may also be used to diagnose or monitor the progression of any other disease or disorder from the group of disorders associated with TTR as defined herein above.

[0284] As indicated above, the antibodies of the present invention, their fragments, and molecules of the same binding specificity as the antibodies and their fragments of the present invention may be used in vitro as well as in vivo, and diagnostic as well as therapeutic applications may be carried out. Thus, in one embodiment, the present invention also relates to a TTR-binding molecule comprising at least one CDR of the antibody of the present invention for preparing a composition for the in vivo detection of TTR in the human or animal body, or a composition for targeting a therapeutic and / or diagnostic agent to TTR in the human or animal body. Potential therapeutic and / or diagnostic agents may be selected from the non-exhaustive list of therapeutic agents and potential labels useful in the treatment of TTR amyloidosis, as indicated hereinabove. With regard to in vivo imaging, in one preferred embodiment, the present invention provides said TTR-binding molecule comprising at least one CDR of an antibody of the present invention, wherein said in vivo imaging comprises positron emission tomography (PET), single photon emission computed tomography (SPECT), near infrared (NIR) optical imaging or magnetic resonance imaging (MRI). In a further embodiment, the present invention also provides said TTR-binding molecule comprising at least one CDR of an antibody of the present invention for use in a method for diagnosing or monitoring the progression of a disease or disorder associated with TTR in a subject, as defined herein above, or said molecule for preparing a composition for the in vivo imaging method specified above.

[0285] VII. Peptides containing aggregation-specific TTR epitopes In a further embodiment, the present invention relates to a peptide having an epitope of TTR specifically recognized by any of the antibodies of the present invention. Preferably, such a peptide comprises or consists of an amino acid sequence as shown in SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 as a unique linear epitope recognized by the antibody or a modified sequence thereof in which one or more amino acids are substituted, deleted and / or added, the peptide being recognized by any of the antibodies of the present invention, preferably the antibody NI-301.59F1, the antibody NI-301.35G11, the antibody NI-301.37F1 or the antibody NI-301.12D.

[0286] In one embodiment of the present invention, such peptides may be used to diagnose or monitor a disease or disorder associated with misfolded, misassembled or aggregated TTR species and / or fragments thereof in a subject, such as TTR amyloidosis, comprising the step of determining the presence of an antibody that binds to the peptide in a biological sample of the subject, and used to diagnose such a disease in the subject by measuring the level of an antibody that recognizes said peptide of the present invention and comparing the measurement result to the level found in a healthy subject of comparable age and sex. Thus, in one embodiment, the present invention relates to a method for diagnosing TTR amyloidosis in a subject, which is indicative of presymptomatic or clinical FAP and / or FAC, comprising determining the presence of an antibody that binds to a peptide as defined above in a biological sample of the subject. According to this method, an elevated level of the measured antibodies specific for said peptides of the invention is indicative for diagnosing presymptomatic or clinical FAP and / or FAC in said subject, or for diagnosing any other disease or condition from the group of disorders related to TTR as defined hereinabove in said subject. Moreover, since the peptides of the invention contain epitopes of therapeutically effective antibodies of human origin, such peptides can of course be used as antigens, i.e. as immunogens to induce an immune response in a subject and stimulate the production of the antibodies of the invention in vivo. The peptides of the invention may be formulated in arrays, kits and compositions (such as, for example, vaccines), respectively, as described hereinabove.In this regard, the present invention also relates to a kit useful in diagnosing TTR amyloidosis or monitoring its progression, comprising at least one antibody of the invention or a TTR binding molecule having substantially the same binding specificity as any one of said antibodies, a polynucleotide, a vector or a cell and / or a peptide, respectively, as defined herein above, together with, where necessary, reagents and / or instructions for use.

[0287] These and other embodiments are disclosed and encompassed by the description and examples of the present invention. Further literature on any one of the materials, methods, uses and compounds for use according to the present invention can be retrieved from public libraries and databases, for example, using electronic devices. For example, the public database "Medline" can be utilized (this database is provided by the National Center for Biotechnology Information and / or the National Library of Medicine of the National Institutes of Health). Further databases and web addresses are known to those skilled in the art, such as the database of the European Bioinformatics Institute (EBI), which is part of the European Molecular Biology Laboratory (EMBL), and can also be obtained using Internet search engines. A summary of patent information in biotechnology and a survey of relevant original sources of patent information useful for retrospective searching and current awareness is given in Berks, TIBTECH 12 (1994), 352-364.

[0288] The above disclosure generally describes the present invention. Unless otherwise stated, terms as used herein are given the definitions as provided in the Oxford Dictionary of Biochemistry and Molecular Biology (Oxford University Press, 1997, revised in 2000 and reprinted in 2003, ISBN 0 19 850673 2). Several documents are cited throughout the text of this specification. Full bibliographic citations may be found at the end of the specification immediately preceding the claims. The contents of all cited references (including literature references, issued patents, published patent applications, and manufacturer's specifications, instructions, etc. as cited throughout this application specification) are hereby expressly incorporated by reference; however, no admission is made that any document cited is in fact prior art with respect to the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided herein for illustrative purposes only and are not intended to limit the scope of the present invention. EXAMPLES

[0289] Example 1: Isolation and characterization of anti-TTR antibodies Various human-derived antibodies targeting TTR and / or mutated, misfolded, misassembled and / or aggregated TTR species and / or fragments thereof were identified using the method described in International Patent Application Publication WO2008 / 081008, the disclosure of which is incorporated herein by reference, with modifications. Specifically, human wild-type TTR protein obtained by purification from human plasma, and wild-type and mutant TTR proteins obtained by recombinant expression were used in both native and misfolded-aggregated conformations to identify TTR-targeting antibodies. Misfolded-aggregated conformers were generated in vitro under acidic conditions using a procedure similar to that described in Colon W. et al., Biochemistry, 31 (1992), 8654-8660, with minor modifications.

[0290] Example 2: Antibody Sequencing The amino acid sequences of the variable regions of the above identified anti-TTR antibodies were determined based on their mRNA sequences (see FIG. 1). Briefly, viable B cells of the selected non-immortalized memory B cell cultures were collected. Subsequently, mRNA from the cells producing the selected anti-TTR antibodies was extracted, converted to cDNA, and sequences encoding the variable regions of the antibodies were amplified by PCR, cloned into a plasmid vector, and sequenced. Briefly, primer combinations representing all sequence families of the human immunoglobulin germline repertoire were used for the amplification of leader peptides, V segments, and J segments. The first round of amplification was performed using a leader peptide-specific primer at the 5' end and a constant region-specific primer at the 3' end (Smith et al., Nat Protoc., 4 (2009), 372-384). For the heavy and kappa light chains, the second round of amplification was performed using a V segment-specific primer at the 5' end and a J segment-specific primer at the 3' end. For the lambda light chain, a second amplification was performed using a V segment-specific primer at the 5' end and a C region-specific primer at the 3' end (Marks et al., Mol. Biol., 222 (1991), 581-597; de Haard et al., J. Biol. Chem., 26 (1999), 18218-18230).

[0291] Identification of antibody clones with the desired specificity was performed by rescreening in ELISA when the complete antibody was recombinantly expressed. Recombinant expression of a complete human IgG1 antibody was achieved when the variable heavy and variable light chain sequences were inserted "in frame" into an expression vector in which the variable region sequences were complemented at the 5' end by a sequence encoding a leader peptide and at the 3' end by a sequence encoding the appropriate constant domain. To that end, the primers contained restriction sites designed to facilitate cloning of the variable heavy and variable light chain sequences into the antibody expression vector. Heavy chain immunoglobulins were expressed by in frame insertion of the RT-PCR product of the immunoglobulin heavy chain into a heavy chain expression vector with a signal peptide and the constant domain of human or mouse immunoglobulin gamma 1. Kappa light chain immunoglobulins were expressed by in frame insertion of the RT-PCR product of the kappa light chain into a light chain expression vector providing a signal peptide and the constant domain of human kappa light chain immunoglobulin. Lambda light chain immunoglobulins were expressed by inserting the lambda light chain RT-PCR product in frame into a lambda light chain expression vector providing the signal peptide and constant domains of human or mouse lambda light chain immunoglobulin.

[0292] Functional recombinant monoclonal antibodies were obtained when Ig heavy chain expression vector and kappa Ig light chain expression vector or lambda Ig light chain expression vector were co-transfected into HEK293 cells or CHO cells (or any other suitable recipient cell line of human or murine origin). Recombinant human monoclonal antibodies were then purified from the conditioned medium using standard protein A column purification. Recombinant human monoclonal antibodies can be produced in unlimited quantities using either transiently or stably transfected cells. Cell lines producing recombinant human monoclonal antibodies can be established either by using the Ig expression vector as is or by recloning the Ig variable regions into different expression vectors. Derivatives such as F(ab), F(ab)2 and scFv can also be made from these Ig variable regions.

[0293] Framework and complementarity determining regions were determined by comparison with reference antibody sequences available in databases (e.g., Abysis (http: / / www.bioinf.org.uk / abysis / )) and these regions were annotated using the Kabat numbering scheme (http: / / www.bioinf.org.uk / abs / ). The following antibody sequences are identified: NI-301.59F1, NI-301.35G11, NI-301.37F1, NI-301.2F5, NI-301.28B3, NI-301.119C12, NI-301.5D8, NI-301.9D5, NI-301.104F5, NI-301.21F10, NI-301.26F10, NI-301.25F10, NI-301.26F10, NI-301.27F10, NI-301.28F3, NI-301.29F10, NI-301.36F10, NI-301.37F1, NI-301.28B3, NI-301.119C12, NI-301.5D8, NI-301.9D5, NI-301.104F5, NI-301.21F10, NI-301.27F10, NI-301.28F3, NI-301.29 ... The amino acid sequences of the variable regions of each of the subject antibodies NI-301.9G12, NI-301.12D3, NI-301.37F1-PIMC, NI-301.44E4, NI-301.18C4, NI-301.11A10, NI-301.3C9, NI-301.14D8, NI-301.9X4 and NI-301.14C3 are shown in Figures 1A to 1T.

[0294] Below, the high affinity of the subject antibodies for the misfolded-aggregated TTR conformation and the substantial lack of binding to the native wild-type TTR conformation (thus revealing a strong selectivity for mutant TTR, misfolded TTR, misassembled TTR and / or aggregated TTR) is illustratively illustrated for the NI-301.59.F1, NI-301.35G11 and NI-301.37F1 antibodies. However, preliminary experiments with other subject antibodies suggest substantially the same preferential binding to mutant TTR, misfolded TTR, misassembled TTR and / or aggregated TTR over physiological TTR species, as do the NI-301.59.F1, NI-301.35G11 and NI-301.37F1 antibodies.

[0295] Example 3: Binding affinity and EC50 determination of anti-TTR antibodies using direct ELISA The ability of antibodies to bind to TTR and / or misfolded, misassembled and / or aggregated forms of TTR was evaluated by direct ELISA assay at various antibody concentrations. This allows the half-maximal effective concentration (EC50), which is commonly used as a surrogate for antibody binding affinity, to be determined for each antibody in this assay (see FIG. 2). Briefly, ELISA plates (high-binding, clear polystyrene, half-area, flat bottom) were coated with misfolded-aggregated human wild-type TTR, misfolded-aggregated recombinant V30M-TTR (both prepared as described in Example 1) and bovine serum albumin (BSA) at a concentration of 10 μg / ml in phosphate-buffered saline (PBS) for 1 hour at 37° C., followed by blocking with a solution of 2% BSA and 0.1% Tween-20 in PBS (PBS-T) for 1 hour at room temperature (RT). Antibodies against TTR were diluted in PBS at 11 different concentrations ranging from 4 nM to 400 nM and incubated in the ELISA plate overnight at 4° C. After three washes with PBS-T, the ELISA plate was incubated with HRP-conjugated human IgG-specific secondary antibody (1 / 4000 dilution) for 1 h at RT. After three washes with PBS-T, the ELISA reaction was developed with TMB for exactly 10 min at RT and quantified by measuring the optical density at 450 nm (OD450nm).

[0296] Exemplary antibodies NI-301.59F1, NI-301.35G11, and NI-301.37F1 showed strong binding to misfolded-aggregated wild-type and mutant TTR but not to control BSA (see Figures 2A-C). The EC50 of the antibodies was then determined by fitting the data by nonlinear regression using least squares to estimate the antibody binding affinity under these conditions.

[0297] Exemplary antibodies NI-301.59F1, NI-301.35G11 and NI-301.37F1 exhibited high affinity for misfolded-aggregated human wild-type TTR, corresponding to EC50s of 3.0 nM, 3.9 nM and 0.35 nM, respectively. These exemplary antibodies also exhibited high affinity for misfolded-aggregated recombinant mutant V30M-TTR, corresponding to EC50s of 15.5 nM, 5.0 nM and 0.15 nM, respectively.

[0298] Example 4: Binding selectivity of anti-TTR antibodies using dot blots To evaluate the binding selectivity of TTR antibodies and / or their fragments for native or misfolded, misassembled and / or aggregated TTR conformations, human wild-type TTR protein in native or misfolded-aggregated conformation and recombinant V30M-TTR protein in misfolded-aggregated conformation were diluted in PBS at four different concentrations and deposited on nitrocellulose membranes by vacuum filtration. The membranes were briefly dried (10 min), blocked with 3% milk in PBS-T for 1 h at RT, and subsequently incubated with anti-TTR antibodies overnight at 4°C. After three washes with PBS-T for 5 min at RT, the membranes were incubated with the appropriate secondary antibodies (HRP-conjugated; 1 / 10000 dilution) for 1 h at RT. After three washes with PBS-T, the membranes were developed with luminol and the signal intensity was quantified by measuring the luminescence.

[0299] The exemplary commercially available anti-TTR antibodies bound to the native TTR conformation as well as to the misfolded-aggregated TTR conformation with similar affinity, thereby revealing that the exemplary commercially available anti-TTR antibodies have no binding selectivity for native TTR or misfolded, misassembled, and / or aggregated TTR conformations (see FIG. 3A). In contrast, the exemplary antibodies NI-301.59.F1, NI-301.35G11, and NI-301.37F1 bound only to the misfolded-aggregated TTR conformation with high affinity and showed no binding to the native TTR conformation at all, thereby revealing strong selectivity for misfolded, misassembled, and / or aggregated TTR (FIG. 3B2, C2, D2). Accordingly, antibodies NI-301.35G11 and NI-301.37F1 also demonstrated strong binding to misfolded-aggregated recombinant V30M-TTR protein, as shown in Figure 3, C3 and D3.

[0300] To further characterize antibody binding selectivity, various TTR preparations, including wild-type and mutant native and misfolded-aggregated conformations, and a panel of 12 human plasma samples were similarly processed for analysis by dot blot using a mouse chimeric anti-TTR antibody and an HRP-conjugated anti-mouse IgG2a secondary antibody for detection (Figure 6). The commercially available antibody showed strong binding to all TTR preparations, including wild-type and mutant native and misfolded-aggregated TTR preparations, and was able to detect TTR in all human plasma samples. This further reveals that there is no selectivity for native or aggregated conformations (see FIG. 6A). In contrast, the exemplary mouse chimeric antibody NI-301.mur35G11 showed very strong binding to the misfolded-aggregated wild-type TTR sample (FIG. 6C1), as well as strong binding to mutant V30M-TTR protein (FIG. 6C4) and mutant Y78F-TTR protein (FIG. 6C6). However, the NI-301.mur35G11 antibody did not bind to TTR in human plasma samples. This further demonstrates the strong selectivity of NI-301.mur35G11 for mutated, misfolded, misassembled and / or aggregated TTR proteins.

[0301] Example 5: Binding affinity and binding selectivity of anti-TTR antibodies using Western blot The binding affinity and binding selectivity of anti-TTR antibodies were evaluated by Western blot (see FIG. 4). Briefly, human wild-type TTR protein (300 ng) in native or misfolded-aggregated conformation and wild-type mouse liver extract (10 μg total protein) were loaded onto SDS-PAGE gels and transferred to nitrocellulose membranes using a semi-dry transfer system. The membranes were then blocked with 2% BSA in PBS-T for 1 h at RT and incubated overnight at 4° C. with anti-TTR antibodies diluted in blocking buffer. After 4 washes with PBS-T for 5 min at RT, the membranes were incubated with the appropriate secondary antibodies (HRP-conjugated; 1 / 10000 dilution in blocking buffer) for 1 h at RT. After three washes with PBS-T and one final wash in PBS, the membranes were developed with luminol and the signal intensity was quantified by measuring the luminescence. Immediately before use, the misfolded-aggregated TTR samples were subjected to cross-linking with glutaraldehyde (1%, 5 min, 37°C) to prevent dissociation of TTR aggregates during the preparation process for SDS-PAGE. In contrast, the native TTR samples were not cross-linked before use, resulting in almost complete dissociation of TTR tetramers (which are the native TTR conformation under physiological conditions) into monomers and dimers.

[0302] The commercially available anti-TTR antibody showed very strong binding to TTR monomer and TTR dimer in human native TTR samples (Figure 4A1) and similarly strong binding to cross-linked misfolded-aggregated TTR samples (Figure 4A2), thereby revealing no selectivity for native or misfolded, misassembled and / or aggregated TTR conformations. In contrast, the exemplary antibodies NI-301.59F1, NI-301.35G11 and NI-301.37F1 showed very strong binding to cross-linked misfolded-aggregated TTR samples (FIG. 4B2, C2, D2), but no binding to TTR monomer and TTR dimer in human native TTR samples (FIG. 4B1, C1, D1), thereby revealing strong selectivity for misfolded, misassembled and / or aggregated TTR conformations over native TTR conformations.

[0303] In addition, the commercial anti-TTR antibody and the exemplary anti-TTR antibody had very low levels of binding to proteins contained in mouse liver extracts (A3, B3, C3, D3 in FIG. 4). Considering the large amount of liver protein used for the experiment and the large antibody concentration relative to their binding affinity, this indicates that the exemplary antibody has a remarkable specificity for TTR and does not bind significantly to other proteins. Furthermore, the exemplary antibody does not appear to bind to mouse TTR protein, which is contained at high levels in mouse liver extracts, indicating that the exemplary antibody shows specificity for human misfolded TTR protein. However, the epitope of antibody NI-301.37F1 is present in rat and mouse TTR proteins. Therefore, the primary amino acid sequence of this epitope is not necessarily critical for detecting misfolded TTR, but may be critical for the conformation.

[0304] To further characterize the antibody binding capacity or absence of antibody binding capacity to native TTR protein, the exemplary antibodies were evaluated for their ability to bind to TTR protein contained in human plasma samples using the same Western blot technique described hereinabove (FIG. 5). The only technical difference was that the top part of the gel at about 25-30 kDa was removed and only the bottom part of the gel was used for protein transfer to nitrocellulose membrane. This was to remove the heavy and light chains of human antibodies present in high concentrations in plasma samples, which could potentially interfere with the analysis.

[0305] In contrast to the commercially available antibody used as a reference, the exemplary antibodies NI-301.35G11 and NI-301.37F1 did not detect any human TTR protein in human plasma samples, thereby demonstrating binding selectivity for TTR conformations that were not present in the analyzed samples under these conditions.

[0306] Example 6: Binding selectivity of anti-TTR antibodies in solution using immunoprecipitation To further confirm the binding selectivity of the anti-TTR antibodies of the present invention, human wild-type and recombinant TTR proteins in native and misfolded-aggregated conformations and human plasma samples at three different dilutions in PBS were used for TTR immunoprecipitation (IP). Briefly, protein A-coated magnetic particles were incubated with anti-TTR antibodies diluted in the manufacturer's binding buffer at RT for 30 min. The antibody / protein A complex was collected and incubated with TTR preparations and human plasma samples at 4°C overnight. After washing, the antibody / protein A complex was resuspended in SDS loading buffer, heated at 90°C for 5 min, and processed for Western blot analysis.

[0307] As shown in Figure 7, the exemplary TTR antibodies NI-301.35G11 and NI-301.37F1, in contrast to the commercially available TTR antibody (Dako A0002), did not show any binding to plasma samples (Figure 7A7-9, B7-9, C7-9), as well as to native wild-type and recombinant TTR samples (Figure 7B3, C3, B5, C5). However, strong binding was assessed in samples in which misfolded-aggregated forms of TTR were present (Figure 7B4, C4, B6, C6).

[0308] These results demonstrate that exemplary antibodies NI-301.35G11 and NI-301.37F1 can bind to misfolded, misassembled and / or aggregated TTR conformations in solution and exhibit remarkable selectivity for these conformations.

[0309] Example 7: Binding to pathological TTR aggregates in FAP mouse tissues Exemplary anti-TTR antibodies were evaluated by immunohistochemistry (IHC) for their ability to bind pathological and non-pathological TTR proteins as present in tissues from transgenic mice expressing exclusively human V30M-TTR protein, but not mouse TTR protein, hereafter referred to as FAP mice. These antibodies were also evaluated for non-specific binding in tissues from TTR knockout (TTR-KO) mice that do not express any TTR protein. The corresponding transgenic and knockout mouse lines were originally generated and described by Professor Suichiro Maeda (Kohno K. et al., American Journal of Pathology, 140(4)(1997), 1497-1508). Briefly, immunohistochemistry was performed on paraffin-embedded mouse sections cut into 3-5 μm thick sections. Sections were first dewaxed, rehydrated, and treated with 3% H2O2 in methanol for 20 min at RT. Blocking buffer (PBS+5% serum (horse / goat)+4% BSA) was applied for 1 h at RT and replaced by anti-TTR antibody diluted in PBS for overnight incubation at 4° C. After 3 washes in PBS, sections were successively incubated with appropriate biotinylated secondary antibodies (anti-human IgG, anti-rabbit IgG, diluted 1 / 125 in PBS, incubated 1 h at RT) and avidin-HRP detection system (diluted 1 / 125 in PBS, incubated 1 h at RT). The reaction was developed with diaminobenzidine for exactly 15 min at RT. Tissue sections were counterstained with hemalun for 1 min at RT, dehydrated in a graded ethanol series and coverslipped.

[0310] As shown in Figure 8, a commercially available TTR antibody (Dako A0002) produced strong staining in liver and intestinal sections of FAP mice, but no staining in the corresponding TTR KO sections (Figure 8, 1A, 1B). Exemplary TTR antibody NI-301.35G11 produced similar patterns and intensity of staining in both liver and intestinal sections of FAP mice (Figure 8, 2A). In contrast, exemplary antibody NI-301.37F1 produced strong staining only in intestinal sections, but not in liver sections of FAP mice (Figure 8, 3A). This indicates that the NI-301.37F1 antibody binds only to pathological (i.e., non-physiological) TTR aggregates that accumulate over time in the gastrointestinal tract of FAP mice, and not to TTR in its native conformation as synthesized by the liver.

[0311] In addition, both exemplary antibodies NI-301.35G11 and NI-301.37F1 did not produce any staining in liver and intestinal tissue sections from TTR-KO mice (FIGS. 8B, 3B). Given the high antibody concentrations used in this experiment relative to the antibody's binding affinity, this lack of staining in TTR-KO sections indicates that binding specificity is high for the TTR protein.

[0312] Example 8: Binding selectivity for misfolded, misassembled and / or aggregated TTR deposits in human tissues The antibodies of the present invention were also evaluated for their ability to bind to pathological TTR deposits in human tissue. Sections of skin biopsies from FAP patients and sections of pancreatic tissue from healthy individuals were processed for immunohistochemistry using the same procedure as described in Example 7 (above). Skin biopsies were selected for this experiment because they contain significant amounts of pathological TTR amyloid deposits. In contrast, pancreatic tissue was used in this experiment because pancreatic alpha cells express high levels of TTR.

[0313] As shown in FIG. 9, a commercially available antibody (Dako A0002) revealed equally strong staining of pathological TTR deposits in skin and native TTR in pancreatic alpha cells (FIG. 9, 1A). Similarly, the exemplary mouse chimeric antibody NI-301.mur35G11 resulted in equally strong staining of both pathological TTR deposits in skin and native TTR in pancreatic alpha cells (FIG. 9, 1B). In contrast, antibody NI-301.37F1 only stained pathological TTR deposits in skin, but not native TTR in pancreatic alpha cells (FIG. 9, 3A). This result reveals that NI-301.37F1 is highly selective by IHC for pathological TTR deposits, including mutated TTR, misfolded TTR, misassembled TTR and / or aggregated TTR conformations.

[0314] The "secondary antibody only" control conditions shown in Panels 1B, 2B, and 3B of Figure 9 demonstrate tissue staining that occurs in the absence of primary antibodies. The absence (2B) or very low levels (1B, 3B) of staining indicates that the staining seen in Panels 1A, 2A, and 3A is indeed specific for the corresponding primary antibodies.

[0315] Example 9: Evaluation of the binding epitope of TTR antibodies To determine the binding epitopes of the exemplary antibodies NI-301.59F1, NI301.35G11 and NI-301.37F1, the entire TTR amino acid sequence was analyzed using a panel of 29 consecutive peptides with a length of 15 amino acids and overlap of 11 amino acids covalently attached to the membrane. Additional peptides containing selected mutations were also plotted on the membrane. The membrane was blocked overnight at 4°C in Roti blocking buffer, and first incubated with anti-TTR antibody diluted in blocking buffer at RT for 2 hours, then incubated with anti-human IgG antibody conjugated with HRP for 45 minutes at RT (1 / 20000 dilution in TBS). The reaction was developed with luminol and imaged by luminescence.

[0316] Antibody NI-301.59F1 recognizes spots 15, 16 and 44, which correspond to the sequence 61-EEEFVEGIY-69 (SEQ ID NO: 49) in full-length human wild-type TTR (see FIG. 10A). Antibody NI-301.35G11 recognizes spots 13, 14, 42 and 44, which correspond to the sequence 53-GELHGLTTEEE-63 (SEQ ID NO: 50) in full-length human wild-type TTR (see FIG. 10B). However, antibody NI-301.35G11 does not recognize spot 43, indicating that this antibody cannot bind to the sequence 53-GELHGPTTEEE-63, which corresponds to the L55P-TTR mutant. Antibody NI-301.37F1 recognizes spots 9, 10, 11, 38 and 40, which correspond to the sequence 41-WEPFA-45 (SEQ ID NO:51) in full-length human wild-type TTR (see FIG. 10C). However, antibody NI-301.37F1 does not recognize spot 43, indicating that this antibody cannot bind to the sequence 41-WGPFA-45, which corresponds to the E42G-TTR mutant.

[0317] To refine the determination of the binding epitopes of the exemplary antibodies NI-301.59F1, NI301.35G11 and NI-301.37F1, the entire TTR amino acid sequence was analyzed using a panel of 151 consecutive peptides with a length of 15 amino acids and an overlap of 14 amino acids covalently attached to the membrane. For each peptide, the amino acid at position 10 was replaced by alanine for amino acids other than alanine, whereas alanine was replaced by glycine or proline. The membrane was blocked overnight at 4°C in Roti blocking buffer and first incubated with anti-TTR antibody diluted in blocking buffer for 2 hours at RT, then incubated with anti-human IgG antibody conjugated with HRP for 45 minutes at RT (1 / 20000 dilution). The reaction was developed with luminol and imaged by luminescence.

[0318] Antibody NI-301.59F1 only recognizes spots 77 and 83, indicating that E61 and V65 are not required for 59F1 binding, whereas E62, E63, F64, E66, G67, I68 and Y69 are required for antibody binding. The exact contribution of K70 is a matter of interpretation: the strong antibody binding to peptide 44 shown in FIG. 10A clearly indicates that the absence of K70 at the C-terminal position does not prevent antibody binding; however, in a subsequent experiment shown in FIG. 10E, a K70A substitution at position 10 in this peptide prevented antibody binding. These seemingly contradictory results suggest that NI-301.59F1 binds to a specific conformation of the amino acid sequence 62-EEFXEGIY-69 (SEQ ID NO:58; where X can be any amino acid).

[0319] Antibody NI-301.35G11 recognizes spots 68, 71, 72, 73, 74 and 75, indicating that G53 is not required for 35G11 to bind, whereas E54, L55, G57 and L58 are required for antibody binding. 35G11 binding pattern also indicates that the presence of E61 or E62 is required for antibody binding. Although the exact contribution of T59 and T60 could not be clarified in this experiment, it is assumed that the presence of one of these two tyrosines is required for antibody binding. In summary, the NI-301.35G11 binding profile in alanine scan indicates that this antibody recognizes the sequence 54ELXGLTXE61 (SEQ ID NO:59, where X can include all known amino acids) (see FIG. 10F).

[0320] Antibody NI-301.37F1 binds to spots 50, 52, 55, 56 and 58-62 on the alanine sca...

Claims

1. An anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, which binds to a TTR epitope having the amino acid sequence WEPFA (SEQ ID NO:51) and does not bind to the corresponding E42G mutant.

2. capable of binding to mutated, misfolded, misassembled and / or aggregated TTR species or fragments thereof; The human-derived anti-transthyretin (TTR) antibody does not substantially recognize physiological TTR species; In the variable region or binding domain, V Ｈ Variable Regions and V Ｌ It has six complementarity determining regions (CDRs) in the variable region, The human-derived anti-transthyretin (TTR) antibody or antigen-binding fragment thereof according to claim 1, which is selected from the group consisting of the following (A) to (R): (A) an anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence NYAMH; CDR-H2 consisting of the amino acid sequence IISYDGNNKYYADSVRG; CDR-H3 consisting of the amino acid sequence GSGRAARHWFDP; CDR-L1 consisting of the amino acid sequence TGTSSDVGGYNYVS; CDR-L2 consisting of the amino acid sequence DVFNRPS; and CDR-L3 consisting of the amino acid sequence SSYTSSVTPHWV (B) an anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence SSNFYWG; CDR-H2 consisting of the amino acid sequence AIYSSGNTYYNPSLKS; CDR-H3 consisting of the amino acid sequence HSCSSASCYPPGFWFDP; CDR-L1 consisting of the amino acid sequence RASQTVSYNLA; CDR-L2 consisting of the amino acid sequence GASTRAT; and CDR-L3 consisting of the amino acid sequence QQYNNWPPWT (C) an anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence SGVYYWS; CDR-H2 consisting of the amino acid sequence YISNTGNTYYNPSLKS; CDR-H3 consisting of the amino acid sequence EYCSGGNCYSRFYYYMDV; CDR-L1 consisting of the amino acid sequence TGSSSNIGAGYGVH; CDR-L2 consisting of the amino acid sequence GDNNRPS; and CDR-L3 consisting of the amino acid sequence QSYDTTLSGSRV (D) an anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence AYYWN; CDR-H2 consisting of the amino acid sequence EVSHGGSSNYSPSLRG; CDR-H3 consisting of the amino acid sequence GSPVVLPGARFDP; CDR-L1 consisting of the amino acid sequence SYNLVS; CDR-L2 consisting of the amino acid sequence EVNKRPS; and CDR-L3 consisting of the amino acid sequence CSYAGSTKV (E) an anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence SGGYYWS; CDR-H2 consisting of the amino acid sequence FIYYTGNTYYNPSLKS; CDR-H3 consisting of the amino acid sequence DCSGGSCPESYFDS; CDR-L1 consisting of the amino acid sequence RASQSVRSFLA; CDR-L2 consisting of the amino acid sequence DASKRAT; and CDR-L3 consisting of the amino acid sequence QRTNWPPHLT (F) An anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence SYGMH; CDR-H2 consisting of the amino acid sequence VIWFDGSNKYYADSVKG; CDR-H3 consisting of the amino acid sequence DGIATYADY; CDR-L1 consisting of the amino acid sequence RASQSVRSYLA; CDR-L2 consisting of the amino acid sequence GASNRAT; and CDR-L3 consisting of the amino acid sequence QQRSNWPIT (G) An anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence SLSSYYMS; CDR-H2 consisting of the amino acid sequence TINPGGSEKSYVDSVKG; CDR-H3 consisting of the amino acid sequence PRYCTSGGCYFDN; CDR-L1 consisting of the amino acid sequence TATNSDVGDYKSVS; CDR-L2 consisting of the amino acid sequence DVGRRPS; and CDR-L3 consisting of the amino acid sequence CIYVGRSSV (H) an anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence SGYYWG; CDR-H2 consisting of the amino acid sequence SMYHSGRTYYNPSLKS; CDR-H3 consisting of the amino acid sequence GFDTSGSHRPLSTDY; CDR-L1 consisting of the amino acid sequence SGSSSNIGNNYVS; CDR-L2 consisting of the amino acid sequence DNNKRPS; and CDR-L3 consisting of the amino acid sequence GTWDSSLSAYV (I) An anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence NYGMH; CDR-H2 consisting of the amino acid sequence VIWSDGSDKYYADSVEG; CDR-H3 consisting of the amino acid sequence EPSSTWAFDY; CDR-L1 consisting of the amino acid sequence TGTSSDVGGYNLVS; CDR-L2 consisting of the amino acid sequence EDIKGPS; and CDR-L3 consisting of the amino acid sequence CSYAGTGTLV (J) An anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence SYAMI; CDR-H2 consisting of the amino acid sequence GISGSGSTTYYADSVKG; CDR-H3 consisting of the amino acid sequence GAWEIPTYFDN; CDR-L1 consisting of the amino acid sequence RASQSIRNNLA; CDR-L2 consisting of the amino acid sequence GASTRAT; and CDR-L3 consisting of the amino acid sequence QQYNNWPPTWT (K) An anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence IYAMT; CDR-H2 consisting of the amino acid sequence TITSGGVSIYYADSIKG; CDR-H3 consisting of the amino acid sequence DGNCDETSCYLRGMDV; CDR-L1 consisting of the amino acid sequence SGSRSDIGSKLVS; CDR-L2 consisting of the amino acid sequence DTYKRPS; and CDR-L3 consisting of the amino acid sequence GSWGNSENFYYV (L) an anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence SRSYY; CDR-H2 consisting of the amino acid sequence SIYYSGSTLYNPSLKS; CDR-H3 consisting of the amino acid sequence MGEGGRDY; CDR-L1 consisting of the amino acid sequence RASQSISSWLA; CDR-L2 consisting of the amino acid sequence DASSLER; and CDR-L3 consisting of the amino acid sequence QHYNGYSRT (M) An anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence RGDFYWS; CDR-H2 consisting of the amino acid sequence YIYSTGDVYYNPSLKS; CDR-H3 consisting of the amino acid sequence EGQYCSGGSCYPEY; CDR-L1 consisting of the amino acid sequence SGDNLGHKFTC; CDR-L2 consisting of the amino acid sequence QDHKRPS; and CDR-L3 consisting of the amino acid sequence QAWAFPYVV (N) An anti-transthyretin (TTR) antibody or an antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence NYWMS; CDR-H2 consisting of the amino acid sequence NIKEDGSDRYYVDSVKG; CDR-H3 consisting of the amino acid sequence IVGVIPSADPYYLDS; CDR-L1 consisting of the amino acid sequence TGTSLNIGTYNLIS; CDR-L2 consisting of the amino acid sequence EGNRRPP; and CDR-L3 consisting of the amino acid sequence CSFAGRVSLV (O) An anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence SHYWN; CDR-H2 consisting of the amino acid sequence SIYHSGSTNYNPSLKS; CDR-H3 consisting of the amino acid sequence DYYYYMDV; CDR-L1 consisting of the amino acid sequence SGDALPDKYAY; CDR-L2 consisting of the amino acid sequence KDSERPS; and CDR-L3 consisting of the amino acid sequence KSADSSGTYWV (P) An anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence SHYMS; CDR-H2 consisting of the amino acid sequence IIYSGGGTYYADSVKG; CDR-H3 consisting of the amino acid sequence IYRSGNTGYSYDY; CDR-L1 consisting of the amino acid sequence SGDKLGSKYAC; CDR-L2 consisting of the amino acid sequence EDKKRPS; and CDR-L3 consisting of the amino acid sequence QAWDSSTSHVV (Q) An anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence SYAMS; CDR-H2 consisting of the amino acid sequence SISGSGDTTKYTDSVKG; CDR-H3 consisting of the amino acid sequence DGSGRIDPFAL; CDR-L1 consisting of the amino acid sequence RSSRSLVYSDGNIYLN; CDR-L2 consisting of the amino acid sequence KVSNRDS; and CDR-L3 consisting of the amino acid sequence MQGTHWPRT (R) an anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin, comprising: CDR-H1 consisting of the amino acid sequence NYWMS; CDR-H2 consisting of the amino acid sequence NINQDSEKYYVDSVKG; CDR-H3 consisting of the amino acid sequence DRYCSGGRCSRGNNWFDP; CDR-L1 consisting of the amino acid sequence RASQSVRSNLA; CDR-L2 consisting of the amino acid sequence GASTRAT; and CDR-L3 consisting of the amino acid sequence QQYNNWPPYT.

3. 2. The human-derived anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of claim 1, wherein the human-derived anti-transthyretin (TTR) antibody or antigen-binding fragment thereof is a human or human-derived antibody.

4. the human-derived anti-transthyretin (TTR) antibody or antigen-binding fragment thereof is a chimeric or rodentized antibody or antigen-binding fragment thereof; The human-derived anti-transthyretin (TTR) antibody or antigen-binding fragment thereof according to claim 1, wherein the human-derived anti-transthyretin (TTR) antibody or antigen-binding fragment thereof is selected from the group consisting of a chimeric mouse-human antibody, a murine antibody, a chimeric rat-human antibody, and a rat antibody.

5. 2. The human-derived anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of claim 1, wherein the human-derived anti-transthyretin (TTR) antibody or antigen-binding fragment thereof is a chimeric mouse-human NI-301.mur35G11 clone.

6. The human-derived anti-transthyretin (TTR) antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antigen-binding fragment is a single-chain Fv fragment (scFv), an F(ab') fragment, an F(ab) fragment, or an F(ab')2 fragment.

7. A polynucleotide encoding the human anti-transthyretin (TTR) antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. The polynucleotide of claim 7, wherein the polynucleotide is a cDNA.

9. A vector comprising the polynucleotide of claim 7 or 8.

10. A host cell comprising a polynucleotide according to claim 7 or 8 or a vector according to claim 9.

11. 1. A method for preparing an anti-FAP antibody or antigen-binding fragment thereof, comprising: (a) culturing the host cell of claim 10; and (b) isolating the antibody from the culture. A method comprising:

12. An antibody or antigen-binding fragment thereof obtainable by the method according to claim 11.

13. The human-derived anti-transthyretin (TTR) antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the human-derived anti-transthyretin (TTR) antibody or antigen-binding fragment thereof has a detectable label or is conjugated to a drug.

14. 14. The human-derived anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of claim 13, wherein the detectable label is selected from the group consisting of an enzyme, a radioisotope, a fluorophore and a heavy metal.

15. An anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin according to any one of claims 1 to 6 and 13, A composition comprising:

16. The composition of claim 15, wherein the composition is a pharmaceutical composition and the carrier is a pharma- ceutically acceptable carrier.

17. The composition of claim 15, wherein the composition is a diagnostic composition.

18. 1. A kit for use in diagnosing or monitoring a patient with TTR amyloidosis, comprising: At least one anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of human origin according to any one of claims 1 to 6 and 13, A kit having instructions for use.

19. 19. The kit of claim 18, wherein the patient has or is suspected of having a disease selected from the group consisting of familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), senile systemic amyloidosis (SSA), systemic familial amyloidosis, leptomeningeal / central nervous system (CNS) amyloidosis, TTR-related ocular amyloidosis, TTR-related renal amyloidosis, TTR-related hyperthyroxinemia, TTR-related ligament amyloidosis, rotator cuff tear, lumbar spinal stenosis, and pre-eclampsia.

20. for use in the prophylactic or therapeutic treatment of TTR amyloidosis in a patient, The human-derived anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of any one of claims 1 to 6 and 13, wherein the human-derived anti-transthyretin (TTR) antibody or antigen-binding fragment thereof is administered to the patient.

21. 21. The human-derived anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of claim 20, wherein the patient has a disease selected from the group consisting of familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), senile systemic amyloidosis (SSA), systemic familial amyloidosis, leptomeningeal / central nervous system (CNS) amyloidosis, TTR-related ocular amyloidosis, TTR-related renal amyloidosis, TTR-related hyperthyroxinemia, TTR-related ligament amyloidosis, rotator cuff tear, lumbar spinal stenosis, and preeclampsia.

22. said leptomeningeal / central nervous system (CNS) amyloidosis is Alzheimer's disease; 22. The human-derived anti-transthyretin (TTR) antibody or antigen-binding fragment thereof of claim 21, wherein the TTR-associated ligament amyloidosis is carpal tunnel syndrome.

23. 18. The composition of claim 15 or 17, for use in in vivo detection, targeting or imaging therapeutic and / or diagnostic agents to FTR in the human or animal body.

24. 24. The composition of claim 23, wherein the in vivo imaging comprises scintigraphy, positron emission tomography (PET), single photon emission computed tomography (SPECT), near infrared (NIR) optical imaging or magnetic resonance imaging (MRI).

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