Anti-transthyretin antibodies, compositions, kits, methods and uses thereof

Abstract

The present invention relates to anti-transthyretin antibodies, compositions, kits, methods and uses thereof. The present invention provides an anti-transthyretin (TTR) antibody, or an antigen-binding fragment thereof, capable of binding to a mutant, misfolded, misassembled and / or aggregated transthyretin type, or fragment thereof, without substantially recognizing a physiological transthyretin type, the antibody or the antigen binding fragment of the antibody comprises six CDRs of VH and VL variable regions in a variable region or a binding structural domain of the antibody, wherein CDR-H1 comprises an amino acid sequence NYAMH; the CDR-H2 comprises an amino acid sequence IISSYDGNNKYYADSVRG, and the amino acid sequence is shown in the description; the CDR-H3 comprises an amino acid sequence GSGRAARHWFDP (Glutamic Glutamic Region The CDR-L1 comprises an amino acid sequence of TGTSSDVGGYNYVS, and the amino acid sequence of the CDR-L1 is The CDR-L2 comprises an amino acid sequence DVFNRPS; and the CDR-L3 comprises an amino acid sequence SSYTSSVTPHWV (Sequence Sequence Tyr Sequence

Description

[0001] The present application is a divisional application of the invention patent application with application number 201480076058.2 (application number 202010121489.3 of the first division) and application date of December 22, 2014, with the invention name “Antibody-based therapy for transthyretin (TTR) amyloidosis and humanized antibodies thereof”. Technical Field

[0002] The present invention generally relates to antibody-based therapies for transthyretin (TTR) amyloidosis. In particular, the present invention relates to novel molecules that specifically bind to human transthyretin (TTR) and its antigens, in particular human recombinant antibodies and 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 disorders caused by such pathogenic TTR isotypes.

[0003] In addition, the present invention relates to pharmaceutical compositions and diagnostic compositions comprising such binding molecules, antibodies and mimetics thereof, both of which serve as valuable diagnostic tools to identify diseases associated with TTR amyloidosis and as passive vaccine strategies for treating conditions associated with diseases associated with TTR amyloidosis, 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 ligamentous amyloidosis (including carpal tunnel syndrome, rotator cuff tears and lumbar spinal stenosis) and pre-eclampsia.

[0004] In addition, the invention relates to a method for diagnosing a disease or condition caused by a pathogenic TTR isoform, such as misfolded and / or aggregated TTR present in amyloid deposits, wherein the level of the pathological TTR isoform is determined in a bodily fluid sample derived from a subject after administration of an anti-TTR antibody, wherein the presence of the pathological TTR isoform or an altered level thereof (e.g., as determined by the presence of an immune complex of TTR and the anti-TTR antibody) when compared to a control sample obtained prior to administration is indicative of the disease and / or condition. Background Art

[0005] Transthyretin (TTR), formerly known as prealbumin, is a soluble protein of 127 amino acids (NCBI reference sequence: NP_000362.1) that is involved in the transport of thyroxine and retinol in vivo. TTR is secreted by the liver in the blood and by the choroid plexus in the cerebrospinal fluid, and can also be expressed in specific tissues like pancreatic alpha cells or retinal epithelium. TTR synthesis begins during the embryonic period and continues throughout life. It is present in high concentrations in plasma (3.6-7.2 μM) and CSF (0.04-0.4 μM) and normally forms a soluble homotetramer of approximately 55 kDa under physiological conditions.

[0006] Under specific conditions that have not yet been elucidated, which may include acidic pH, oxidative stress, and local factors, the TTR protein adopts an alternative tripartite conformation and becomes toxic.

[0007] The toxicity of misfolded TTR protein was discovered through studies of a rare autosomal dominant neurodegenerative disorder called familial amyloid polyneuropathy (FAP), which affects adults in middle age (Planté-Bordeneuve et al., Lancet Neurol. 10 (2011), 1086-1097). FAP is characterized by progressive sensory, motor and autonomic disorders leading to death ten years after diagnosis. Neuropathy is associated with deposition of amorphous aggregates and amyloid fibrils composed of TTR protein. The Val30Met substitution is the most common mutation causing FAP, especially in areas where the disease is prevalent, such as northern Portugal, but more than 100 different mutations have been identified in the TTR gene; see Table IV below. The pathophysiological mechanism at play is the same for all pathological mutations in that the mutation alters the structural stability of the TTR tetramer, promotes TTR misfolding, and leads to the formation of toxic TTR species (Saraiva et al., Curr. Med. Chem. 19 (2012), 2304-2311).

[0008] Toxicity of TTR 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. This mutation is associated with familial amyloid cardiomyopathy (FAC), in which massive TTR accumulation in the myocardium leads to cardiac weakness and ultimately heart failure (Ruberg et al., Circulation. 126 (2012), 1286-1300).

[0009] Mutations in the TTR protein sequence are not a strict requirement for TTR toxicity, and wild-type TTR proteins are also prone to misfolding and forming toxic aggregates. For example, senile systemic amyloidosis (SSA) is characterized by heart failure and accumulation of wild-type TTR aggregates in the myocardium (Ikeda, Amyloid. 18 Suppl 1 (2011), 155-156; Dungu et al., Heart. 98 (2012), 1546-1554). Wild-type TTR deposits have also been observed in many cases of ligament and tendon inflammation, including carpal tunnel syndrome, rotator cuff tears, and lumbar spinal stenosis (Sueyoshi et al., Hum. Pathol. 42 (2011), 1259-1264; Gioeva et al., Amyloid. 20 (2013), 1-6). Furthermore, TTR amyloidosis has recently been reported in the placentas of mothers with pre-eclampsia (Kalkunte et al., Am. J. Pathol. 183 (2013) 1425-1436).

[0010] Treatments for diseases with TTR amyloidosis are limited and primarily invasive, with treatment primarily for symptoms. In the case of FAP, treatment relies on analgesics for the management of neuropathic pain, on liver transplantation to remove the primary source of the mutated TTR protein, and on treatment with Tafamidis. Tafamidis is a small molecule that binds to the TTR tetramer and stabilizes its conformation. It acts on the dissociation of the TTR tetramer, the rate-limiting step of the misfolding pathway that leads to the formation of toxic TTR species. Tafamidis has been approved for the treatment of FAP in Europe, but not in the United States, and its therapeutic effect is limited, at best, slowing the progression of the disease. There are currently no available treatments that target the misfolded TTR protein.

[0011] In view of the foregoing, novel therapeutic strategies for the effective and safe treatment of diseases associated with TTR amyloidosis are needed.

[0012] This technical problem is solved by the embodiments characterized in the claims and further described and illustrated in the following examples and drawings. Summary of the invention

[0013] The present invention provides anti-transthyretin (TTR) antibodies and equivalent TTR binding molecules for preventing or treating diseases and conditions associated with TTR amyloidosis. More specifically, humanized recombinant antibodies and fragments and derivatives thereof that recognize misfolded, misassembled or aggregated forms of TTR that are useful for treatment are provided.

[0014] Misfolded TTR aggregates are associated with markers of cellular stress, oxidative stress, inflammatory response, and apoptosis many years before the onset of symptoms (Macedo et al., Mol. Med. 13 (2007), 584-91). The body's natural ability to recognize abnormally folded proteins and degrade them is a protective factor, and differences between patients in the ability to clear toxic TTR proteins undoubtedly contribute to age differences in disease onset and rate of disease progression. Supporting this hypothesis, it has been shown that patients who receive liver transplants from FAP donors rapidly develop antibodies to pathogenic TTR proteins (Ando et al., Transplantation. 73 (2002), 751-755), and FAP patients with high antibody titers against mutant TTR proteins have a later onset of disease than patients without such antibodies (Obayashi et al., Clin. Chim. Acta. 419 (2013), 127-131). Furthermore, active immunization against pathogenic TTR conformations was demonstrated to almost completely remove 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, treatment of related diseases using anti-TTR antibodies has not been pursued to date. For example, in international application WO2010 / 030203 a specific isolated mouse monoclonal antibody to TTR has been described and suggested for use in FAP screening, as well as in the study and treatment of related diseases. However, since mouse monoclonal antibodies induce human anti-mouse antibody (HAMA) responses, they are not suitable for human treatment. Therefore, since the international application lapsed and no subsequent developments have been published, it is clear that antibody-based therapeutic approaches have not been pursued. However, to date only diagnostic applications for patients with TTR amyloidosis have been further investigated for anti-TTR antibodies; see, e.g., Phay M. et al., Rejuvenation Res. Oct. 28, 2013. [Electronic version published ahead of print].

[0016] In contrast, the experiments conducted according to the present invention were successful in isolating human monoclonal TTR-specific antibodies that mature in humans and are specific for misfolded, misassembled, mutated and / or aggregated TTR forms and / or fragments thereof. The human subjects and patients from which the B cells (from which the human monoclonal anti-TTR antibodies and cDNAs encoding their variable domains, respectively) were isolated, did not show significant amounts of misfolded TTR and did not have symptoms of diseases associated with pathogenic isoforms. However, in another embodiment of the present invention, the source of the B cells (from which the human monoclonal anti-TTR antibodies and cDNAs encoding their variable domains, respectively) was a patient showing symptoms of diseases and / or disorders associated with TTR amyloidosis. Therefore, it is prudent to expect that the human monoclonal anti-TTR antibodies and derivatives thereof of the present invention, in addition to being non-immunogenic, will show therapeutically beneficial effects in humans.

[0017] Therefore, the present invention relates to human recombinant antibodies, antigen-binding fragments, and similar antigen-binding molecules that can specifically recognize TTR. Unless otherwise indicated, antibodies that "specifically recognize TTR," "antibodies specific for / for TTR," and "anti-TTR antibodies" are intended to specifically, generally, and commonly bind to the native monomeric form of TTR; antibodies that specifically bind to any form of TTR, such as mutant TTR, oligomeric, fibrous and / or non-fibrous TTR. Provided herein are human antibodies that are selective for full-length and / or fragments and / or misfolded, misassembled, and / or polymerized forms of TTR.

[0018] As mentioned above, preferably the anti-TTR antibodies of the invention are recombinant antibodies in which at least one, preferably two or more, preferably all three complementarity determining regions (CDRs), and / or substantially the entire variable region of the variable heavy and / or light chain is encoded by cDNA derived from mRNA obtained from human memory B cells producing anti-TTR antibodies. In a preferred embodiment, the anti-TTR antibodies of any combination of the invention exhibit more than one binding and biological property as demonstrated for the subject antibodies shown in the accompanying examples and figures, preferably more than one binding and biological property as demonstrated for the accompanying exemplary antibodies NI-301.59.F1, NI-301.35G11, and NI-301.37F1.

[0019] In a particularly preferred embodiment of the present invention, the anti-TTR antibody or TTR binding fragment thereof demonstrates the immunological binding properties of the antibody, which is Figure 1 The variable region V H and / or V L Characterized by.

[0020] The antigen-binding fragment of the antibody may be a single-chain Fv fragment, a F(ab') fragment, a F(ab) fragment, and a F(ab') 2 Fragments, or any other antigen binding fragments. In a specific embodiment below, the antibody or fragment thereof is a human IgG isotype antibody. Optionally, the antibody is a human rodent chimeric antibody or a rodentized antibody, such as a murine or murinized, rat or ratized antibody, the rodent version being particularly useful for diagnostic methods and research in animals.

[0021] In addition, the present invention relates to a composition comprising the antibody or active fragment thereof of the present invention, and also to immunotherapeutic and immunodiagnostic methods using such a composition 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 polynucleotides encoding at least one variable region of an immunoglobulin chain of an antibody of the present invention. Preferably, the variable region comprises Figure 1 The variable region 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 mutated, misfolded, misassembled and / or aggregated TTR types.

[0023] Thus, the present invention also includes vectors containing the polynucleotides and host cells transformed therewith, and their use for producing antibodies and equivalent binding molecules specific for TTR. In a further embodiment of the present invention, the antibodies or binding molecules are capable of binding to misfolded, misassembled and / or aggregated forms of TTR or fragments thereof. Means and methods for recombinant production of antibodies and mimetics thereof, as well as screening methods for molecules that may or may not be antibody-competing binding molecules are well known in the art. However, as described herein, particularly with respect to therapeutic applications to humans, the antibodies of the present invention are human antibodies, meaning that there is substantially no immune response to such antibodies with their use, whereas immune responses to such antibodies are observed with chimeric or even humanized antibodies.

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

[0025] In addition, in one embodiment of the present invention, an anti-TTR antibody and a TTR binding molecule comprising at least one CDR of an antibody of the present invention are provided for the preparation of a composition, and the composition is used for in vivo detection of TTR (also referred to as in vivo imaging) or a therapeutic and / or diagnostic agent targeting TTR, particularly a mutated, misfolded, misassembled and / or aggregated TTR type or fragment thereof in humans and animals. The methods and compositions disclosed herein can provide assistance in diseases associated with TTR amyloidosis and characterized, for example, by the occurrence of TTR forms, and can be used to monitor disease progression and provide therapeutic efficacy of treatment to subjects, such as in vivo imaging-related diagnostic methods. Therefore, in one embodiment, an anti-TTR antibody and / or TTR binding molecule of the present invention is provided wherein the in vivo detection (imaging) includes imaging, positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR) optical imaging, or magnetic resonance imaging (MRI).

[0026] It is therefore a particular object of the present invention to provide a method for treating, diagnosing or preventing a disease associated with TTR amyloidosis. The method comprises administering to a subject an effective concentration of a preferably human antibody or antibody derivative, wherein the antibody is targeted to TTR or a fragment thereof, preferably a misfolded, misassembled and / or aggregated TTR form or a fragment thereof.

[0027] In a further aspect, the invention provides peptides having a TTR epitope specifically recognized by the antibodies of the invention, preferably an epitope of misfolded, misassembled and / or aggregated TTR or a fragment thereof. 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, wherein one or more amino acids are substituted, deleted and / or added, with the proviso that the peptide is still recognized by the homologous antibody. As mentioned, such peptides can be used as antigens, i.e. as immunogens, and are therefore useful for eliciting an immune response in a subject and stimulating the production of the antibodies of the invention in vivo. Thus, the peptides of the invention are particularly useful as vaccines.

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

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

[0030] In a preferred embodiment of the invention, particularly when non-human animals are used for testing recombinant humanized antibodies as shown in Example 13 and other anti-TTR antibodies intended for broad use in humans, the level of misfolded and / or aggregated TTR in a sample is determined by determining the complex formed between the anti-TTR antibody and misfolded and / or aggregated TTR, for example by immunoprecipitation with anti-human IgG or anti-idiotypic antibodies.

[0031] With regard to diagnostic aspects, in particular for human subjects and patients, the presence and elevated levels of misfolded and / or aggregated TTR and its complex with anti-TTR antibodies, respectively, indicate TTR amyloid deposition 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 the patient or subject. Thus, the methods of the present invention allow, on the one hand, the identification and determination of diseases associated with TTR amyloidosis in a subject, and, on the other hand, the removal of TTR deposits from a patient, thus also indicating the therapeutic progress of a given treatment and the efficacy of a drug such as an anti-TTR antibody for the treatment of TTR amyloidosis.

[0032] Thus, as demonstrated in Example 13, the anti-TTR antibodies of the invention are capable of binding misfolded and / or aggregated TTR with sufficient affinity to alter the stability of pathological TTR deposits, such as capturing and removing misfolded and / or aggregated TTR from the deposits to body fluids, particularly blood. The specific time interval after administration, i.e., the time frame, after which the level of pathological TTR and anti-TTR antibody complexes are measured and determined by a practitioner, respectively. Typically, the time interval used is no more than one week. In a preferred embodiment, the level of pathological TTR in a sample derived from a patient or subject after administration of an anti-TTR antibody or antigen-binding fragment thereof is determined within less than or equal to 48 hours; see also Example 13.

[0033] The present invention also relates to the use of any anti-TTR antibody and TTR binding molecule in the above methods. However, due to the advantageous properties, in particular because of human origin, the use of the anti-TTR antibodies disclosed herein is preferred. In a preferred embodiment, the antibody exhibits substantially the same binding and biological activity as any of the following antibodies, wherein the antibodies are 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. Anti-TTR antibodies may also be altered to facilitate processing in diagnostic methods involving labeling of the antibodies as described in detail below.

[0034] Further embodiments of the invention will become apparent from the description and the following examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1: Amino acid sequences of the variable regions of 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.

[0036] Framework (FR) and complementarity determining regions (CDR) where CDR is underlined. The Kabat numbering scheme is used (see http: / / www.bioinf.org.uk / abs / ).

[0037] Figure 2 : Binding of polymerized, wild-type and mutant TTR by direct ELISA.

[0038] A, B, C: ELISA plates with aggregated human wild-type TTR (●), aggregated recombinant V30M-TTR and bovine serum albumin (BSA) (■) (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.

[0039] EC 50 Values ​​were estimated by fitting the data points using the least squares method.

[0040] NI-301.59F1: Aggregation of wt-TTR EC50 = 3.0 nM, Aggregation of V30M-TTR EC50 = 15.5 nM

[0041] NI-301.35G11: Aggregation of wt-TTR EC50 = 3.9 nM, Aggregation of V30M-TTR EC50 = 5.0 nM

[0042] NI-301.37F1: Aggregation of wt-TTR EC50 = 0.35 nM, Aggregation of V30M-TTR EC50 = 0.15 nM

[0043] Figure 3 : Specificity for aggregated TTR in dot blot.

[0044] Human wild-type TTR protein in native (1) or aggregated (2) conformation, and aggregated recombinant V30M-TTR protein (3) were deposited on nitrocellulose membranes and incubated with the following antibodies: a) commercially available 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 at 50 nM).

[0045] Figure 4 : Specificity for polymerized TTR in Western blotting.

[0046] Human wild-type TTR protein (300 ng) in native (1) or aggregated (2) conformation, and wild-type mouse liver extract (10 μg total protein) (3) were loaded onto SDS-PAGE gels and processed for Western blotting using the following antibodies: A) commercial rabbit polyclonal anti-TTR antibody (Dako-A0002; 150 ng / mL), B) NI-301.59F1 C) NI-301.35G11 and D) NI-301.37F1 (B, C and D: 50 nM human monoclonal antibodies). To prevent dissociation of high molecular weight aggregates, aggregated TTR samples were cross-linked with glutaraldehyde (1%, 5 minutes) before loading onto the gel.

[0047] Figure 5 : Lack of binding to human plasma TTR in Western blotting.

[0048] 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 blotting using the following antibodies: A) commercial rabbit polyclonal anti-TTR antibody (Dako-A0002; 150 ng / mL), B) secondary antibody only (anti-human IgG-HRP, 1 / 10000 dilution), C) NI-301.35G11 and D) NI-301.37F1 (C and D: human monoclonal antibodies at 50 nM).

[0049] Figure 6 : Lack of binding to human plasma TTR in dot blot.

[0050] Pure wild-type and mutant TTR proteins in native and aggregated conformations and plasma samples from controls, asymptomatic mutation carriers, and FAP patients were deposited on nitrocellulose membranes and incubated with the following antibodies: A) commercially available rabbit polyclonal anti-TTR antibody (Dako-A0002; 150 ng / mL), B) secondary antibody only (anti-mouse IgG2a-HRP, 1 / 10000 dilution), and C) murine chimeric antibody NI-301.mur35G11 (10 nM).

[0051] Samples 1-6: 150 ng 1) aggregated wt-TTR, 2) native wt-TTR, 3) BSA, 4) native V30M-TTR, 5) native L55P-TTR, and 6) native Y78F-TTR.

[0052] Samples 7-18: 2 μL of plasma collected from 7-10) controls (n=4), 11-14) asymptomatic mutation carriers (n=4), and 15-18) FAP patients (n=4).

[0053] Figure 7 : Specifically binds aggregated TTR in solution.

[0054] Human wild-type and recombinant TTR proteins in native or aggregated conformations, and 3 different dilutions of human plasma samples were used for TTR immunoprecipitation (IP) using the following antibodies: A) commercial rabbit polyclonal anti-TTR antibody (Dako-A0002), B) NI-301.35G11 and C) NI-301.37F1. The immunoprecipitated proteins were subjected to SDS-PAGE and detected by Western blotting (WB) using Dako-A0002 antibody (150 ng / mL).

[0055] Lanes 1-2: WB loading control: 300 ng of 1) human wt-TTR, 2) recombinant wt-TTR

[0056] Lanes 3-6: IP of pure TTR proteins: 3) human native wild-type wt-TTR, 4) human aggregated wt-TTR, 5) recombinant native wt-TTR and 6) recombinant aggregated wt-TTR

[0057] Lanes 7-10: IP of human plasma diluted 7) 10-fold, 8) 100-fold, 9) 1000-fold with PBS and 10) PBS alone.

[0058] Figure 8 : Specific binding to TTR in FAP mouse tissues.

[0059] 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 protein deposition in various tissues. Liver and intestinal tissue sections collected from A) FAP mice and B) TTR-KO mice were processed for immunohistochemistry using the following antibodies: 1) commercially available rabbit polyclonal anti-TTR antibody (Dako-A0002; 1 / 1000 dilution), 2) NI-301.35G11 and 3) NI-301.37F1 (2 and 3: 50 nM human monoclonal antibodies).

[0060] Fig. 9 : Specific binding to misfolded TTR deposits, but not native TTR, in human tissues.

[0061] The antibodies were characterized by their ability to bind TTR on skin biopsies from patients with FAP and pancreatic sections from healthy controls: misfolded TTR aggregates that are characteristic of FAP are present in patient skin biopsies, while pancreatic alpha cells show endogenous expression of TTR. Sections were processed for immunohistochemistry using the following antibodies: 1A) commercial rabbit polyclonal anti-TTR antibody (Dako-A0002; 1 / 1000 dilution) 1B) HRP-conjugated anti-rabbit IgG antibody (1 / 125 dilution), 2A) mouse chimeric antibody NI-301.mur35G11 (50 nM), 2B) HRP-conjugated anti-mouse IgG2a antibody (1 / 125 dilution), 3A) NI-301.37F1 (50 nM), and Figure 3 B) HRP-conjugated anti-human IgG (1 / 125 dilution).

[0062] Fig.10 : TTR binding epitope analyzed by peptide scanning.

[0063] Antibody binding epitopes on TTR were determined using this peptide scanning method. In addition to peptides covering the entire sequence of human wild-type TTR (spots 1 to 29), selected TTR mutations were also represented on the membrane (spots 30 to 44). The peptide scanned membrane was incubated with 50 nM of the following antibodies A) NI-301.59F1, B) NI-301.35G11 and C) NI-301.37F1. As summarized in Table D):

[0064] NI-301.59F1 binds EEEFVEGIY (TTR 61-69); NI-301.35G11 binds GELHGLTTEEE (TTR53-63); the L55P mutation prevents antibody binding; and

[0065] NI-301.37F1 binds to WEPFA (TTR 41-45); the E42G mutation prevents antibody binding. To determine the sequence requirements of the epitope, antibodies that bind to the epitope on TTR were further characterized using alanine scanning. The full sequence of the human wild-type TTR protein was represented on the membrane as a set of 151 contiguous peptides of 15 amino acids in length, starting at each amino acid of the TTR protein. For each peptide, the amino acid at position 10 was substituted with alanine, or with glycine or proline when the starting amino acid was alanine. The peptide scanned membranes were incubated with 20 nM of the following antibodies: E) NI-301.59F1, F) NI-301.35G11 and G) NI-301.37F1. As summarized in Table H):

[0066] NI-301.59F1 combined with EEFXEGIY (TTR 62-69).

[0067] NI-301.35G11 binds ELXGLTXE (TTR 54-61).

[0068] NI-301.37F1 binds to WEPFA (TTR 41-45), where X represents an amino acid; substitution of E42 with alanine did not interfere with binding, but substitution with guanine prevented antibody binding as reported in C.

[0069] Fig.11 : The binding kinetics of antibodies to TTR protein in solution were evaluated by surface plasmon resonance.

[0070] The binding kinetics of antibody NI-301.37F1 to TTR protein were determined by surface plasmon resonance (SPR). Antibody NI-301.37F1 was captured on the sensor by means of an anti-human IgG antibody, and a TTR protein solution was flown over the sensor surface in a concentration range of 3.2 to 316 nM. A simple 1:1 binding model was used to fit the data and derive the corresponding association (ka) and dissociation (kd) constants and affinity (KD). The 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.

[0071] Natural wild-type TTR: ka = not determined, kd = not determined, KD>316nM

[0072] Denatured wild-type TTR: ka = 2.1 x 10 4 M -1 Second -1 , kd = 2.6 x 10 -5 Second -1 , KD=1.2nM

[0073] Recombinant TTR-L55P: ka = 3.3 x 10 4 M -1 Second -1 , kd = 4.6 x 10 -5 Second -1 , KD=1.4nM.

[0074] Fig.12 : Chronic treatment with anti-TTR antibodies reduces pathological TTR deposition in a mouse model of FAP.

[0075] Mice with FAP (Tg (6.0hMet30) x muTTR-KO) received weekly intraperitoneal injections of mouse chimeric NI-301.37F1 or isotype control antibody at a dose of 3 mg / kg for 12 weeks. At the end of the treatment period, tissues were collected and the extent of TTR deposition was quantified by immunofluorescence. A) Treatment effect on 7-month-old mice (n = 14-15 mice per group); B) Treatment effect on 17-month-old mice (n = 10 mice per group). Two-tailed, unpaired t-tests were used for comparisons between groups.

[0076] Fig.13 : Antibody binding to pathological TTR deposits in vivo.

[0077] Target engagement was characterized 48 hours after adult FAP mice (7 months old) received a single intraperitoneal injection of antibody NI-301.37F1 at a dose of 30 mg / kg or PBS. Immunofluorescence was used to simultaneously detect the localization of pathological TTR deposits and injected antibodies.

[0078] A, D) Pathological TTR deposits in the kidneys of (A) NI-301.37F1- or (D) PBS-injected mice. B, E) Detection of human antibodies in (B) NI-301.37F1- or (E) PBS-injected mice. C, F) Overlays of images showing (C) colocalization of TTR and NI-301.37F1 and (F) no specific staining.

[0079] Fig.14 : Unstructured detection of misfolded TTR in vivo.

[0080] Adult FAP mice received a single intraperitoneal injection of antibody NI-301.37F1 or isotype control antibody at a dose of 3 mg / kg. Blood samples were collected before (t=0) and 48 hours after antibody injection (t=48 hours). Plasma samples were processed by immunoprecipitation using anti-human IgG antibody and analyzed by Western blotting for detection of: A) conformation-independent anti-TTR polyclonal antibody (Dako A0002, 150 ng / mL), and B) NI 301.37F1 (20 nM). In parallel, plasma samples obtained from uninjected FAP mice were incubated in vitro with antibody NI-301.37F1 before treatment.

[0081] Fig.15 : Specificity of antibodies for aggregated proteins evaluated by ELISA.

[0082] The specificity of the antibodies for TTR protein was evaluated by direct ELISA assays for binding to selected aggregated proteins. Antibody binding was evaluated at 4 nM and 20 nM, and signal intensity was expressed as fold change relative to background levels, with each assay performed in the absence of anti-TTR antibodies.

[0083] AB) Binding of NI-301.37F1 was measured at 20 nM (A) and 4 nM (B).

[0084] CD) Binding of NI-301.44E4 was determined at 20 nM (C) and 4 nM (D). DETAILED DESCRIPTION

[0085] The present invention relates generally to immunological and non-invasive methods for detecting diseases and conditions associated with the presence of pathological, usually mutated and / or misfolded isoforms of transthyretin (TTR). More specifically, the present invention relates to recombinant human monoclonal antibodies and antigen-binding fragments thereof, which have been generated based on sequence information obtained from a selected population of human donors and are capable of binding to such TTR isoforms and their antigens. The recombinant human monoclonal antibodies of the present invention advantageously have the characteristic of specifically binding to misfolded, misassembled, mutated and / or aggregated TTR or fragments thereof, allowing the treatment and / or diagnosis to be targeted to pathologically altered TTR types. Due to their human origin, the recombinant antibodies obtained by the present invention can reasonably be expected to be effective and safe as therapeutic drugs and to be highly specific as diagnostic agents for detecting pathological TTR without giving false positives.

[0086] Furthermore, the antibodies of the invention and their derivatives may be used in combination therapy for patients who are still at risk of developing TTR amyloidosis after organ transplantation (due to, for example, their deposition, for example, genetic mutations in TTR or defects in the production of TTR in the liver). Thus, as a particularly advantageous embodiment, the present invention relates to human monoclonal antibodies and any derivatives thereof as described herein, used alone for the treatment of patients or for the treatment of patients who receive, for example, immunosuppressants or other drugs effective for symptoms associated with TTR amyloidosis after organ transplantation, wherein the antibodies of the invention and any derivatives thereof are designed to be administered simultaneously with the immunosuppressant and / or drugs that inhibit further side effects or to be administered sequentially before or after administration. In this regard, the anti-TTR antibodies and TTR binding fragments of the invention are preferably substantially non-immunogenic to humans. In one embodiment of the invention, a pharmaceutical composition comprising a human monoclonal antibody of the invention or any derivative thereof and one or more immunosuppressants is provided and / or used for symptoms associated with TTR amyloidosis.

[0087] I. Definition

[0088] Unless otherwise indicated, terms used herein have the definitions given in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0 19850673 2.

[0089] It should be noted that the term "a" or "an" entity refers to one / one or more / multiple entities; for example, "an antibody" should be understood to represent one or more antibodies. Thus, the terms "a" (or "a"), "one / one or more / multiples" and "at least one / at least one" can be used interchangeably herein.

[0090] Unless otherwise specifically stated, the term "TTR" is used interchangeably to specifically refer to different forms of transthyretin (TTR). The term "TTR" is also used to generally identify other conformations of TTR, for example, oligomers and / or misfolded, misassembled and / or aggregated forms of TTR. The term "TTR" is also used to collectively refer to all types / species and forms of TTR, such as mutant TTR. The letters added in front of the term TTR are used to indicate the organism from which the specific homolog is derived, for example, hTTR represents human TTR or mTTR represents murine origin. In addition, unless otherwise specified, the numbering system used herein for the amino acid sequence of TTR refers to the mature TTR protein, i.e., the TTR protein secreted by the cell, for example, after the signal peptide is cleaved. This numbering is the numbering used to define TTR mutations found in patients, such as TTR-V30M or TTR-L55P, but is different from the numbering system used for the transthyretin precursor protein sequence (NCBI reference sequence: NP_000362.1). In this regard, positions and substituted amino acids in mutant TTR may be indicated in different but equivalent ways; see, e.g., "TTR-V30M" and "V30M-TTR."

[0091] The anti-TTR antibodies disclosed herein specifically bind to TTR and its epitopes, as well as various conformations of TTR and its epitopes. For example, antibodies disclosed herein specifically bind to pathologically altered TTR or fragments thereof, such as oligomers / fibrils and / or mutated, misfolded, misassembled and / or aggregated forms of TTR or fragments thereof. The terms (pathological) mutated, misfolded, misassembled, aggregated TTR / TTR aggregates are used interchangeably to refer specifically to the above forms. The terms (pathological) "aggregated forms" or "aggregates" used herein describe products that accumulate or cluster due to the wrong / pathological interaction of TTR with each other. These aggregates, aggregates or clustered forms can be, essentially consist of or consist of both TTR and / or TTR fragments, and consist of non-fibrous oligomers and / or fibrous oligomers and their fibrils. As used herein, reference to antibodies that "specifically bind", "selectively bind" or "preferentially bind" to TTR refers to antibodies that do not bind to other unrelated proteins. In one embodiment, the TTR antibodies disclosed herein can bind to TTR or an epitope thereof and show no binding to other proteins at a level greater than about 2 times background. In a preferred embodiment, the antibodies of the invention do not substantially recognize a protein selected from the group consisting of α-synuclein (α-syn), Tau, mediator responsive DNA binding protein 43 (TDP-43), serum amyloid A (SAA), huntingtin (HTT) (see, e.g. Fig.15). An antibody that "specifically binds" or "selectively binds" a TTR conformation refers to an antibody that does not bind to all conformations of TTR, i.e., does not bind to at least one other TTR conformation.

[0092] For example, disclosed herein are antibodies that can preferentially bind to misfolded, misassembled, and / or aggregated forms of TTR both in vitro and in tissues derived from patients with or at risk for developing a disease associated with TTR amyloidosis. Because the sequences of the TTR antibodies of the invention have been obtained from human subjects, the TTR antibodies of the invention may also be referred to as "human autoantibodies" or "humanized antibodies" to emphasize that those antibodies are indeed originally expressed by the subject and are not synthetic constructs generated, for example, by human immunoglobulin expression phage libraries, which heretofore represents a common approach to attempting to provide human-like antibodies.

[0093] The term "peptide" is to be understood as including the terms "polypeptide" and "protein" (which are sometimes used interchangeably herein) within its meaning. Similarly, fragments of proteins and polypeptides are also contemplated and may be referred to herein as "peptides". However, 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 present invention typically has no more than 100 consecutive amino acids of a TTR polypeptide, preferably less than 80 consecutive amino acids, more preferably less than 50 consecutive amino acids, and even more preferably no more than 15 consecutive amino acids.

[0094] Peptides:

[0095] As used herein, the term "polypeptide" is intended to include the singular "polypeptide" as well as the plural "polypeptide", and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also called 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. Therefore, "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 is included in the definition of "polypeptide", and the term "polypeptide" can be used to replace any of these terms or can be interchangeable with any of these terms.

[0096] The term "polypeptide" also means the product of post-expression modification of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation and derivatization by known protecting / blocking groups, proteolytic cleavage (or protein cleavage), or modification by non-naturally occurring amino acids. The polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It may be produced in any manner, including chemical synthesis.

[0097] The polypeptides of the present invention can be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more, or 2000 or more amino acids in size. Polypeptides with a defined three-dimensional structure are referred to as folded, while polypeptides that do not have a defined three-dimensional structure can adopt a large number of different conformations and are referred to as unfolded. As used herein, the term glycoprotein refers to a protein coupled to at least one glycosyl group, which is attached to the protein through an oxygen-containing or nitrogen-containing side chain of an amino acid residue (e.g., a serine residue or an asparagine residue).

[0098] An "isolated" polypeptide or fragment, variant or derivative thereof refers to a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can be removed from its natural or native environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for 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.

[0099] "Recombinant peptide, polypeptide or protein" refers to a peptide, polypeptide or protein produced by recombinant DNA technology (i.e., produced from cells, microorganisms or mammals, transformed with an exogenous recombinant DNA expression construct encoding a fusion protein comprising the desired peptide). Proteins or peptides expressed in most bacterial cultures are generally glycan-free. Proteins or polypeptides expressed in yeast may have a different glycosylation pattern than proteins or polypeptides expressed in mammalian cells.

[0100] Included as polypeptides of the present invention are fragments, derivatives, analogs or variants of the aforementioned polypeptides and any combination thereof. The terms "fragment", "variant", "derivative" and "analog" include peptides and polypeptides having an amino acid sequence sufficiently similar to the amino acid sequence of a natural peptide. The term "sufficiently similar" refers to a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence so that the first and second amino acid sequences have a common domain and / or a common functional activity. For example, amino acid sequences containing 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 common domains are defined herein as sufficiently similar. Preferably, the variant will be sufficiently similar to the amino acid sequence of the preferred peptides of the present invention, in particular sufficiently similar to TTR, variants, derivatives or their respective analogs. Such variants generally retain the functional activity of the peptides of the present invention. Variants include peptides that differ from the native peptide and the WT peptide in amino acid sequence, respectively, by one or more amino acid deletions, additions and / or substitutions. These can be naturally occurring variants as well as artificially designed variants.

[0101] In addition, the terms "fragment", "variant", "derivative" and "analog" when referring to the antibodies or antibody polypeptides of the present invention include any polypeptide that retains at least some of the antigen binding properties of the corresponding naturally-bound molecules, antibodies or polypeptides. In addition to the specific antibody fragments discussed elsewhere herein, the polypeptide fragments of the present invention include proteolytic fragments, as well as deletion fragments. Variants of the antibodies and variants of the antibody polypeptides of the present invention include fragments as described above, and polypeptides having amino acid sequences altered by 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 contain conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of TTR-specific binding molecules such as the antibodies and antibody polypeptides of the present invention are polypeptides that have been altered to exhibit additional properties not found in natural polypeptides. Examples include fusion proteins. Variant polypeptides may also be referred to herein as "polypeptide analogs". As used herein, a "derivative" of a binding molecule or fragment thereof, an antibody or antibody polypeptide refers to a subject polypeptide having one or more residues chemically derived from a reaction of a functional side group. Also included as "derivatives" are those peptides containing one or more naturally-occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can replace proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; homoserine can replace serine; and ornithine can replace lysine.

[0102] Determination of molecular similarity and / or identity :

[0103] "Similarity / similarity / analogy / 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 / analogous to the corresponding amino acid of a second peptide if it is an identical or conservative amino acid substitution. Conservative substitutions include those described in Dayhoff, MO, ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, D.C. (1978), and in Argos, EMBO J. 8 (1989), 779-785. For example, amino acids belonging to one of the following groups represent conservative changes or substitutions: - alanine, proline, glycine, glutamine, asparagine, serine, threonine; - cysteine, serine, tyrosine, threonine; - valine, isoleucine, leucine, methionine, alanine, phenylalanine; - lysine, arginine, histidine; - phenylalanine, tyrosine, tryptophan, histidine; and - aspartic acid, glutamic acid.

[0104] "Similarity / similarity / analogy / similarity" between two polynucleotides is determined by comparing the nucleic acid sequence of one polynucleotide to the sequence of a polynucleotide. A nucleic acid of one polynucleotide is similar / analogous to the corresponding nucleic acid of a second polynucleotide if it is identical or the nucleic acid is part of a coding sequence and the respective triplets contain nucleic acids encoding the same amino acids or for conservative amino acid substitutions.

[0105] The determination of percent identity or similarity between two sequences is preferably achieved 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, which are available at NCBI (http: / / www.ncbi.nlm.nih.gov / blast / Blast.cge).

[0106] Determination of percent identity or similarity is made using standard parameters for the BLASTn program for BLAST polynucleotide searches and the BLASTp program for BLAST protein searches, as recommended on the NCBI web page and in the "BLAST Program Selection Guide" for sequences of a particular length and composition.

[0107] BLAST polynucleotide searches are performed using the BLASTn program. For general parameters, the "Maximum target sequences" box can be set to 100, the "Short query" box can be checked, the "Expectation threshold" box can be set to 1000, and the "Word size" box can be set to 7, as recommended by the NCBI web page for short sequences (less than 20 bases). For longer sequences, the "Expectation threshold" box can be set to 10, and the "Word size" box can be set to 11. For the scoring parameter "Match / mismatch score" can be set to 1, -2, and the "Gap cost" box can be set to linear. For filter and mask parameters, the "Low complexity regions" box can be unchecked, the "Species-specific repeats" box can be unchecked, the "Mask for lookup table only" box can be checked, the "Dust filter settings" can be checked, and the "Mask lowercase letters" box can be unchecked. In general, the "Search for short near exact matches" may be used for this, which provides most of the above settings. More information on this can be found in the "BLAST Program Selection Guide" published on the NCBI web page.

[0108] BLAST protein searches were performed using the BLASTp program. For general parameters, the "Maximum target sequences" box can be set to 100, the "Short query" box can be checked, the "Expectation threshold" box can be set to 10, and the "Word size" box can be set to "3". For scoring parameters, the "Matrix" box can be set to "BLOSUM62", the "Drop cost" box can be set to "Presence: 11 Extension: 1", and the "Adjustment for components" box can be set to "Conditional adjustment for component score matrix". For filter and masking parameters, the "Low complexity regions" box can be unchecked, the "Mask for lookup table only" box can be unchecked, and the "Mask lowercase letters" box can be unchecked.

[0109] Modifications of these two programs, for example, for the length of the sequences searched, were made according to the recommendations in the HTML and PDF versions of the "BLAST Program Selection Guide" published on the NCBI web page.

[0110] Polynucleotides:

[0111] The term "polynucleotide" is intended to include nucleic acids in the singular as well as nucleic acids in the plural, and refers to isolated nucleic acid molecules or constructs, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). Polynucleotides may include conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. "Isolated" nucleic acid or polynucleotide means a nucleic acid molecule, DNA or RNA, that has been taken out of its natural environment. For example, a recombinant polynucleotide contained in a vector of an encoded antibody is considered to be isolated for the purposes of the present invention. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides purified (partially or substantially) in solution. Isolated RNA molecules include RNA transcripts of polynucleotides of the present invention in vivo or in vitro. Polynucleotides or nucleic acids isolated according to the present invention also include these molecules produced synthetically. In addition, polynucleotides or nucleic acids may be or may include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0112] As used herein, "coding region" is a part of a nucleic acid that consists of codons that are translated into amino acids. Although "stop codons" (TAG, TGA or TAA) are not translated into amino acids, they can be considered as part of the coding region, but any flanking 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 present invention may be present in a single polynucleotide construct, for example, on a separate vector, or in a separated polynucleotide construct, for example, on a separated (different) vector. In addition, any vector may contain a single coding region, or may include two or more coding regions, for example, a separate vector may encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, respectively. In addition, the vector, polynucleotide or nucleic acid of the present invention may encode a heterologous coding region, or may be fused or not fused with 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 secretory signal peptides or heterologous functional domains.

[0113] 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 generally contain a promoter and / or other operable transcription or translation control elements associated with one or more coding regions. Operable association is when the coding region of a gene product (such as a polypeptide) is associated with one or more regulatory sequences in such a way that the expression of the gene product is placed under the influence or control of the regulatory sequence. Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" or "operably linked" if the induction of the function of the promoter leads to the transcription of the mRNA encoding the desired gene product, and if the nature of the connection between the two DNA fragments does not interfere with the ability of the expression regulatory sequence to direct the expression of the gene product or the ability of the DNA template to be transcribed. Therefore, if the promoter is able to affect the transcription of the nucleic acid, the promoter region will be operably associated with the nucleic acid encoding the polypeptide. The promoter may be a cell-specific promoter that directs a large amount of DNA transcription only in predetermined cells. In addition to the promoter, other transcription control elements, such as enhancers, regulators, repressors, and transcription termination signals, may be operably associated with polynucleotides that direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.

[0114] 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, but not limited to, promoter and enhancer segments derived from cytomegalovirus (immediate early promoter, combined with intron-A), simian virus 40 (early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes, such as actin, heat shock protein, bovine growth hormone, and rabbit beta-endoglobulin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers, and lymphokine-induced promoters (e.g., promoters induced by interferons or interleukins).

[0115] Similarly, various translation control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly internal ribosome entry sites or IRES, also known as CITE sequences).

[0116] In other embodiments, the polynucleotides of the invention are RNA, for example, in the form of messenger RNA (mRNA).

[0117] The polynucleotides and nucleic acid coding regions of the present invention may be associated with additional coding regions encoding secretory peptides or signal peptides, which direct the secretion of the polypeptides encoded by the polynucleotides of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have signal peptides or secretory leader sequences, which are cut from mature proteins once the export of the growing protein chain across the rough endoplasmic reticulum is initiated. Those of ordinary skill in the art know that polypeptides secreted by vertebrate cells usually have a signal peptide fused to the N-terminus of the polypeptide, which is cut from the complete or "full-length" polypeptide to produce a secretory form or "mature" form of the polypeptide. In certain embodiments, a natural signal peptide, such as an immunoglobulin heavy chain or light chain signal peptide, is used, or a functional derivative of a sequence that retains the ability to direct the secretion of an operably related polypeptide is used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof can be used. For example, the wild-type leader sequence can be replaced by the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0118] As used in the context of the present invention, "binding molecules" primarily refer 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, molecular chaperones such as heat shock proteins (HSPs), and cell-cell adhesion molecules such as cadherins, integrins, C-type lectins, and members of the immunoglobulin (Ig) superfamily. Therefore, for clarity only, and not to limit the scope of the present invention, most of the following examples are discussions of antibodies and antibody-like molecules that represent preferred binding molecules for development of therapeutic and diagnostic agents.

[0119] Antibody:

[0120] The terms "antibody" and "immunoglobulin" are used interchangeably herein. An antibody or immunoglobulin is a binding molecule that comprises at least the variable domain of a heavy chain, and usually at least the variable domains of a heavy chain and a light chain. The structure of basic immunoglobulins 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.

[0121] As will be discussed in more detail below, the term "immunoglobulin" includes various broad types of polypeptides that can be distinguished biochemically. It will be appreciated by those skilled in the art that heavy chains are classified as γ, μ, α, δ or ε, (γ, μ, α, δ or ε), with some subclasses (e.g., γ1-γ4) therein. It is the nature of this chain that determines the "class" of antibodies, IgG, IgM, IgA, IgD or IgE, respectively. Immunoglobulin subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well characterized and are known to confer functional specialization. Modified forms of each of these classes and isotypes are readily identifiable to those skilled in the art according to the disclosure of the present invention, and therefore, are within the scope of the present invention. All immunoglobulin classes are obviously within the scope of the present invention, and the following discussion will primarily relate to the IgG class of immunoglobulin molecules. For IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides having a molecular weight of about 23,000 Daltons, and two identical heavy chain polypeptides having a molecular weight of about 53,000-70,000 Daltons. The four chains are typically connected by disulfide bonds in a "Y" configuration, with the light chains supporting the heavy chains starting at the mouth of the "Y" and continuing through the variable region.

[0122] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be combined with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bound together, and when the immunoglobulin is produced by a hybridoma, B cell, or genetically modified host cell, the "tails" of the two heavy chains are bonded to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain.

[0123] Both the light chain and the heavy chain are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it is understood that the light chain (V L ) and heavy chain (V H ) portion determines antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant region domains increases as they become more distal to 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 include the carboxyl termini of the heavy and light chains, respectively.

[0124] As described above, the variable region allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. L Domain and V H The subunits (or subsets) of the domains, or complementarity determining regions (CDRs), combine to form the variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site present at the end of each arm of the Y. More precisely, the antigen binding site is composed of H and V L Any antibody or immunoglobulin fragment that contains sufficient structure to specifically bind to TTR is referred to interchangeably herein as a "binding fragment" or an "immunospecific fragment."

[0125] In naturally occurring antibodies, antibodies contain six hypervariable regions, sometimes referred to as "complementarity determining regions" or "CDRs" present in each antigen binding domain, which are short, non-contiguous amino acid sequences that are specifically positioned to form the antigen binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment. The "CDRs" are flanked by four relatively conserved "framework" regions or "FRs" that display lower intermolecular variability. The framework regions largely adopt a -fold conformation, and the CDRs form a loop structure that connects, and in some cases forms part of, the -fold structure.

[0126] Therefore, the framework region acts to form a scaffold that provides a CDR in the correct orientation through non-covalent interactions between chains. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDR and framework regions can be easily determined by a person of ordinary skill in the art for any given heavy or light chain variable region, respectively, because they have been accurately defined; see, Sequences of Proteins of Immunological Interest, Kabat, E et al., U.S. Department of Health and Human Services (1983), and Chothia and Lesk, J. Mol. Biol., 196 (1987), 901-917.

[0127] In some cases, there are two or more definitions of a term used and / or accepted in the art, and the definition of the term used herein is intended to include all of these meanings, unless explicitly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the discontinuous antigen binding sites found in the variable regions of heavy and light chain polypeptides. This particular region has been described in Sequences of Proteins of Immunological Interest, Kabat, E et al., U.S. Department of Health and Human Services (1983) and Chothia and Lesk, J. Mol. Biol., 196 (1987), 901-917, which are incorporated herein by reference, wherein the definitions include overlapping or subunits of amino acid residues when compared to each other. However, the application of any definition to refer to the CDR of an antibody or its variants is intended to be within the scope of the term defined and used herein. The appropriate amino acid residues of the CDRs defined by each of the references cited above are listed in Table I below as a comparison. The exact number of residues containing a particular CDR will vary with the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular hypervariable region or CDR of the human IgG subclass of the antibody given the variable region amino acid sequence of the antibody.

[0128] <![CDATA[Table I: CDR Definitions 1 > Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-65 52-58 VH CDR3 95-102 95-102 VLCDR1 24-34 26-32 VLCDR2 50-56 50-52 VLCDR3 89-97 91-96

[0129] 1 The numbering of all CDR definitions in Table I is according to the numbering convention proposed by Kabat et al. (see below).

[0130] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can clearly assign this system "Kabat numbering" to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to a numbering system proposed by Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1983). Unless otherwise indicated, the numbering of specific amino acid residue positions in the antibody or antigen-binding fragment, variant, or derivative thereof of the present invention is based on the Kabat numbering system, which is theoretical, however, and may not be equally applicable to each antibody of the present invention. For example, depending on the position of the first CDR, the following CDR may move in either direction.

[0131] The antibodies or antigen-binding fragments, immunospecific fragments, variants or derivatives thereof of the present invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primate, murine or chimeric antibodies, single chain antibodies, epitope binding fragments, e.g., Fab, Fab' and F(ab') 2 , Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), containing V L or V H Fragments of the Fab domain, fragments produced by Fab expression libraries, 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 in, for example, 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 molecules.

[0132] In one embodiment, the antibody of the invention is not an IgM or a derivative thereof having a pentavalent structure. In particular, in certain applications of the invention, especially therapeutic uses, IgM is less useful than IgG and other bivalent antibodies or corresponding binding molecules, as IgM often exhibits non-specific cross-reactivity and very low affinity due to its pentavalent structure and lack of affinity maturation.

[0133] In a particularly preferred embodiment, the antibody of the invention is not a polyclonal antibody, ie, it consists essentially of one specific antibody type rather than a mixture obtained from a plasma immunoglobulin sample.

[0134] Antibody fragments, including single chain antibodies, may include only the variable region or together with all or part of the following: hinge region, CH1, CH2, and CH3 domains. TTR binding fragments are also included in the present invention, which include any combination of variable regions with hinge region, CH1, CH2, and CH3 domains. The antibodies of the present invention or their immunospecific fragments may be from any animal source, including birds and mammals. Preferably, the antibody is an antibody of a human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken. In another embodiment, the variable region may be derived from a condricthoid in origin, for example, from a shark.

[0135] In one aspect, the antibodies of the invention are human monoclonal antibodies isolated from humans. Optionally, the framework regions of the human antibodies are calibrated and adopted based on relevant human germline variable region sequences in a database; see, for example, Vbase (http: / / vbase.mrc-cpe.cam.ac.uk / ) hosted by the MRC Center for Protein Engineering (Cambridge, UK). For example, amino acids considered to be potential deviations from the true germline sequence may be due to the sequence of PCR primers incorporated during the cloning process. Compared to artificially produced human-like antibodies (e.g., single-chain antibody fragments (scFv) derived from phage display antibody libraries or xenogeneic mice), the human monoclonal antibodies of the invention have the following characteristics: (1) they are obtained using human immune responses rather than animal substitutes, i.e., the antibodies are generated in response to natural TTR-related conformations in humans, (2) they have protected individuals or are at least significant to the presence of TTR, and (3) because the antibodies are human, the risk of cross-reactions against self-antigens is minimized. Thus, according to the present invention, the terms "human monoclonal antibody", "human monoclonal autoantibody", "human antibody" and the like are used to refer to a TTR binding molecule of human origin, i.e., it is isolated from a human cell, such as a B cell or a hybridoma thereof, or its cDNA is directly cloned from mRNA of a human cell, such as a human memory B cell. A human antibody is still "human", i.e., of human origin, even if amino acid substitutions are made within the antibody, for example, to improve binding characteristics.

[0136] In one embodiment, the humanized antibodies of the present invention include heterologous regions compared to naturally occurring antibodies, such as amino acid substitutions in the framework regions, constant regions exogenously fused to variable regions, different amino acids at the C-terminus or N-terminus, etc.

[0137] Antibodies derived from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described below and, for example, in U.S. Pat. No. 5,939,598 to Kucherlapati et al., are designated human-like antibodies to distinguish them from the truly human antibodies of the present invention.

[0138] For example, the pairing of heavy and light chains of human-like antibodies, such as synthetic and semisynthetic antibodies typically isolated from phage display, does not necessarily reflect the original pairing that occurs in original human B cells. Therefore, the acquisition of Fab and scFv fragments from recombinant expression libraries, commonly used in the prior art, can be considered artificial, with all the possible associated effects on immunogenicity and stability.

[0139] In contrast, the present invention provides affinity matured antibodies isolated from selected human subjects, characterized for their therapeutic utility and their tolerability in humans.

[0140] As used herein, the term "rodentized antibody" or "rodentized immunoglobulin" refers to an antibody comprising one or more CDRs derived from a human antibody of the present invention; and a human framework region comprising amino acid substitutions and / or deletions and / or insertions based on rodent antibody sequences. When referring to rodents, sequences derived from mice and rats are preferably used, wherein antibodies comprising such sequences are referred to as "murine" or "rodentized", respectively. The CDR human immunoglobulin provided is referred to as the "parent" or "acceptor", and the rodent antibody providing the framework changes is referred to as the "donor". Constant regions do not need to be present, but if present, they are generally substantially identical to rodent antibody constant regions, i.e., at least about 85% to 90%, preferably about 95% or more identical. Therefore, in some embodiments, a full-length murine human heavy or light chain immunoglobulin comprises a mouse constant region, a human CDR, and a substantially human framework with some "murine" amino acid substitutions. Typically, a "murine antibody" is an antibody comprising a murine variable light chain and / or a murine variable heavy chain. For example, murinized antibodies would not include typical chimeric antibodies, for example, because the entire variable region of a chimeric antibody is non-murine. Antibodies modified by the "murinization" process that are "murinized" bind to the same antigen as the parent antibody providing the CDRs, and are generally less immunogenic in mice than the parent antibody. The above explanations for "murinized" antibodies are similarly applicable to "rodentized" antibodies, such as "ratized antibodies," in which rat sequences are used instead of murine sequences.

[0141] As used herein, the term "heavy chain portion" includes an amino acid sequence derived from an immunoglobulin heavy chain. The polypeptide comprising the heavy chain portion includes at least one of the following: a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, the binding polypeptide used 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, the polypeptide of the present invention includes a polypeptide chain comprising a CH3 domain. In addition, the binding polypeptide used in the present invention may lack at least one portion of a CH2 domain (e.g., all or part of a CH2 domain). As noted above, those of ordinary skill in the art will appreciate that these domains (eg, heavy chain portions) can be modified such that they differ from the amino acid sequence of a naturally occurring immunoglobulin molecule.

[0142] 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 second polypeptide chain of the multimer. Alternatively, the monomers contained in the heavy chain portion of the present invention are not identical. For example, each monomer may contain a different target binding site, forming, for example, a bispecific antibody or a diabody.

[0143] In another embodiment, the antibodies or antigen-binding fragments, variants or derivatives disclosed herein consist of a single polypeptide chain (eg, scFv) and are to be expressed intracellularly (intracellular antibodies) for potential in vivo therapeutic and diagnostic applications.

[0144] The heavy chain portion of the binding polypeptide used in the diagnosis and treatment methods disclosed herein can be derived from different immunoglobulin molecules. For example, the heavy chain portion of the polypeptide can include a CH1 domain from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain portion can include a hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In another example, the heavy chain portion can include a chimeric hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0145] As used herein, the term "light chain portion" includes an amino acid sequence derived from an immunoglobulin light chain. Preferably, the light chain portion comprises V L or at least one of the CL domains.

[0146] The minimum size of a peptide or polypeptide epitope for an antibody is considered to be about 4 to 5 amino acids. A peptide or polypeptide epitope preferably comprises at least seven, more preferably at least nine, and most preferably at least about 15 to about 30 amino acids. Because a CDR can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids of the epitope included need not be contiguous, and in some cases, may not even be in the same peptide chain. In the present invention, a peptide or polypeptide epitope recognized by an antibody of the present invention comprises a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or about 15 to about 30 consecutive or non-contiguous amino acids of TTR.

[0147] "Specific binding" or "specific recognition", used interchangeably herein, generally refers to a binding molecule, e.g., an antibody, that binds to an epitope via its antigen binding domain, and the binding results in some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is described as "specifically binding" to an epitope when it binds to the epitope more readily via its antigen binding domain than to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which an antibody of interest binds to an epitope. For example, antibody "A" may be considered to have a higher specificity for a given epitope than antibody "B", or antibody "A" may be said to bind to epitope "C" with a higher specificity than a related epitope "D".

[0148] When present, the term "immunological binding property," or other binding property of an antibody to an antigen, in all its grammatical forms, refers to the specificity, affinity, cross-reactivity, and other binding properties of the antibody.

[0149] "Preferentially binds" refers to a binding molecule, such as an antibody, that specifically binds to a certain epitope more readily than to a related, similar, homologous, or analogous epitope. Thus, an antibody that "preferentially binds" to a given epitope is more likely to bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.

[0150] By way of non-limiting example, a binding molecule, such as an antibody, may be considered to preferentially bind a first epitope if it binds with a dissociation constant (K) that is greater than that of the antibody for a second epitope. D ) Smaller K D In another non-limiting example, an antibody can be considered to preferentially bind a first epitope if it is an antibody Kp1 binding site to a second epitope. D In another non-limiting example, an antibody can be considered to preferentially bind a first epitope if it binds to the first epitope with an affinity that is at least one order of magnitude less than that of an antibody to the second epitope.D The first epitope is bound with an affinity at least two orders of magnitude smaller.

[0151] In another non-limiting example, a binding molecule, such as an antibody, can be considered to preferentially bind a first epitope if it binds the first epitope with a dissociation rate (k(off)) that is less than the antibody k(off) for the second epitope. In another non-limiting example, an antibody can be considered to preferentially bind a first epitope if it binds the first epitope with an affinity that is at least one order of magnitude less than the antibody k(off) for the second epitope. In another non-limiting example, an antibody can 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 the antibody k(off) for the second epitope.

[0152] Binding molecules, such as antibodies or antigen-binding fragments, variants or derivatives disclosed herein, can be said to have a molecular mass less than or equal to 5 x 10 -2 Second -1 ,10 -2 Second -1 ,5x l0 -3 Second -1 or l0 -3 Second -1 More preferably, the antibodies of the present invention can be said to bind to TTR or a fragment, variant or specific conformation thereof at a dissociation rate (k(dissociation)) of less than or equal to 5 x 10 -4 Second -1 ,10 -4 Second -1 ,5x 10 -5 Second -1 , or 10 -5 Second -1 5x 10 -6 Second -1 ,10 -6 Second -1 ,5x 10 -7 Second -1 or 10 -7 Second -1 The invention relates to a method for binding TTR or a fragment, variant or specific conformation thereof with a dissociation rate (k(off)).

[0153] A binding molecule, such as an antibody or antigen-binding fragment, variant or derivative disclosed herein, can be said to have a molecular weight greater than or equal to 10 3 M -1 Second -1 ,5x 10 3 M -1 Second -1 ,10 4 M -1 Second -1or 5x 10 4 M -1 Second -1 More preferably, the antibodies of the invention can be said to bind to TTR or a fragment, variant or specific conformation thereof at an association rate (k(binding)) greater than or equal to 10 5 M -1 Second -1 ,5x10 5 M -1 Second -1 ,10 6 M -1 Second -1 ,or 5x 10 6 M -1 Second -1 or 10 7 M -1 Second -1 The binding rate (k(binding)) of the peptide binds to TTR or a fragment, variant or specific conformation thereof.

[0154] A binding molecule, such as an antibody, is said to competitively inhibit binding of a reference antibody to a given epitope if it preferentially binds to the epitope to the extent that it blocks binding of the reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art, for example, by a competition ELISA assay. An antibody can be said to competitively inhibit binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0155] As used herein, the term "affinity" refers to a measure of the strength of binding of an independent epitope to a 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 ed., (1988), pp. 27-28. As used herein, the term "avidity" refers to the overall stability of a complex of a population of immunoglobulins and an antigen, that is, the functional binding strength of an immunoglobulin mixture to an antigen; see, e.g., Harlow, pp. 29-34. Avidity is related to the affinity of an independent immunoglobulin molecule in a population to a specific epitope, and also to the valence of the immunoglobulin to the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen having a highly repetitive epitope structure (e.g., a polymer) 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, for example, Berzofsky et al., "Antibody-Antigen Interactions," 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. General techniques for determining the affinity of an antibody for an antigen include ELISA, RIA, and surface plasmon resonance. The affinity of a particular antibody-antigen interaction measured may be different if measured under different conditions (e.g., salt concentration, pH). Therefore, affinity and other antigen binding parameters (e.g., K) may vary. D ,IC 50 ) is preferably carried out using standardized solutions of antibodies and antigens and a standardized buffer.

[0156] 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 cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of an antibody specific for one antigen to react with a second antigen; a measure of the relatedness between two different antigenic species. Thus, an antibody is cross-reactive if it binds to an epitope other than the one that induced its formation. Cross-reactive epitopes typically contain many of the same complementary structural features as the inducing epitope, and in some cases may actually be more compatible than the original.

[0157] For example, certain antibodies have a certain degree of cross-reactivity because they bind to related but not identical epitopes, e.g., epitopes that are 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% identical to a reference epitope (as calculated using methods known in the art and described herein). An antibody can be said to have little or no cross-reactivity if it does not bind to an epitope that is 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%, less than 50% identical to a reference epitope (as calculated using methods known in the art and described herein). For a certain epitope, an antibody can be considered "highly specific" if it does not bind to any other analog, ortholog, or homolog of that epitope.

[0158] 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 for TTR and / or mutated, misfolded, misassembled, and / or aggregated TTR or fragments thereof. Preferred binding affinities include those having a dissociation constant or Kd value of less than 5 x 10 -2 M, 10 -2 M, 5x 10 -3 M, 10 -3 M, 5x 10 -4 M, 10 -4 M, 5x 10 -5 M, 10 -5 M, 5x 10 -6 M, 10 -6 M, 5x 10 -7 M, 10 -7 M, 5x 10 -8 M, 10 -8 M, 5x 10 -9 M, 10 -9 M, 5x 10 -10 M, 10 -10 M, 5x 10 -11 M, 10 -11 M, 5x 10 -12 M, 10 -12 M, 5x 10 -13 M, 10 -13 M, 5x 10 -14 M, 10 -14 M, 5x 10 -15 M, or 10 -15 The binding affinity of M.

[0159] In one embodiment, the antibodies of the invention have a Kd for different TTR isotypes, as shown in Table V below for exemplary antibodies, i.e., a Kd of >300 nM for wild-type native TTR, and / or a Kd of ≤15 nM for denatured TTR, preferably ≤5 nM, most preferably ≤2 nM, and / or a Kd of ≤35 nM for native TTR-V30M, preferably ≤20 nM, and / or a Kd of ≤150 nM for native TTR-L55P, preferably ≤5 nM, most preferably ≤2 nM.

[0160] As noted above, the subunit structures and three-dimensional structures of the constant regions of the various immunoglobulin classes are known. As used herein, the term "V H The term "CH1 domain" includes the amino terminal variable domain of an immunoglobulin heavy chain, and the term "CH2 domain" includes the domain of the first (most amino terminal) constant region of an immunoglobulin heavy chain. The CH1 domain is adjacent to the V H domain and is amino terminal to the hinge region of the immunoglobulin heavy chain molecule.

[0161] As used herein, the term "CH2 domain" includes a portion of a heavy chain molecule that extends, for example, from about residue 244 to residue 360 ​​of an antibody using a conventional numbering scheme (residues 244 to 360, Kabat numbering system; residues 231-340, EU numbering system; see Kabat EA et al. supra). The CH2 domain is unique in that it is not tightly paired with another domain. Instead, there are two N-linked branched sugar chains between the two CH2 domains of a complete native IgG molecule. It is also documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises about 108 residues.

[0162] As used herein, the term "hinge region" includes the portion of the heavy chain molecule that joins the CH1 domain to the CH2 domain. The hinge region contains about 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 different domains: upper, middle, and lower hinge domains; see Roux et al., J. Immunol. 161 (1998), 4083-4090.

[0163] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a sulfhydryl group that can form a disulfide bond or bridge with a second sulfhydryl 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 disulfide bonds located at positions 239 and 242 corresponding to the Kabat numbering system (positions 226 or 229, EU numbering system).

[0164] As used herein, the terms "connected", "fused" or "fusion" are used interchangeably. These terms refer to the joining of two or more elements or components together by any means, including chemical conjugation or recombinant means. "In-frame fusion" refers to the joining of open reading frames (ORFs) of two or more polynucleotides to form a continuous longer ORF in a manner that maintains the correct translation reading frame of the original ORFs. Therefore, a recombinant fusion protein is a single protein containing two or more segments corresponding to polypeptides encoded by the original ORFs (under natural conditions, the segments are not usually so joined). Although the reading frame is made continuous throughout the fused segments, these segments can be physically or spatially separated by, for example, in-frame linker sequences. For example, the CDRs of an immunoglobulin variable region encoded by a polynucleotide can be fused, in frame, but separated by at least one immunoglobulin framework region or additional CDR region encoded by the polynucleotide, as long as the "fused" CDRs are co-translated as part of a continuous polypeptide.

[0165] As used herein, the term "expression" refers to the process by which a gene produces a biochemical, such as RNA or polypeptide. The process includes any manifestation of the functional presence of a gene in a cell, including, but not limited to, gene knockout, and both temporary expression and stable expression. It includes, but is not limited to, transcription of a gene into messenger RNA (mRNA), transfer RNA (tRNA), short hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and translation of mRNA into a polypeptide. If the final desired product is biochemical, expression includes the production of biochemical and any precursors. The expression of a gene produces a "gene product". As used herein, a gene product can be a nucleic acid, for example, a messenger RNA produced by gene transcription, or a polypeptide translated from a transcript. The gene product described herein further includes a nucleic acid with post-transcriptional modification, for example, polyadenylation, or a polypeptide with post-translational modification, for example, methylation, glycosylation, addition of lipids, binding to other protein subunits, proteolytic cleavage, and the like.

[0166] As used herein, the term "sample" refers to any biological material obtained from a subject or patient. On the one hand, the sample may include blood, peritoneal fluid, cerebrospinal fluid, saliva or urine. In other aspects, the sample may include whole blood, plasma, serum, B cells enriched from blood samples, and cultured cells (e.g., B cells from a subject). The sample may also include a biopsy or tissue sample, including neural tissue. In other aspects, the sample may include a lysate of whole cells and / or the cell. The blood sample may be collected by methods known in the art. On the one hand, the particles may be suspended by vortexing in 200 μL buffer (20 mM Tris, pH.7.5, 0.5% nonan detergent, 1 mM EDTA, 1 mM PMSF, 0.1 M NaCl, IX Sigma protease inhibitors, and IX Sigma phosphatase inhibitors 1 and 2) at 4 ° C. The suspension may be kept on ice for 20 minutes with intermittent vortexing. After spinning at 15,000 x g for 5 minutes at about 4°C, aliquots of the supernatant can be stored at about -70°C.

[0167] disease:

[0168] Unless otherwise indicated, the terms "disorder" and "disease" are used interchangeably herein and include any undesirable physiological change in a subject, animal, isolated organ, tissue, or cell / cell culture.

[0169] Transthyretin (TTR) amyloidosis is a pathophysiological mechanism that plays a role in many different diseases and is characterized by abnormal deposition of TTR protein in various tissues due to changes in the structure (i.e. conformation) of the TTR protein. Misfolded and misassembled TTR protein is toxic and usually occurs as a result of mutations in the TTR gene. The toxicity of misfolded TTR leads to local tissue damage, and accumulation over time may lead to organ dysfunction and even organ failure. There are many types of tissues and organs that are susceptible to TTR amyloidosis, such as the peripheral and autonomic nervous system, heart, leptomeninges, eyes, tendons, ligaments or kidneys. The wide range of tissues that can be affected by TTR amyloidosis is the reason for the diversity of symptoms presented by patients with TTR amyloidosis. In fact, patients with TTR amyloidosis are clinically classified according to the different diseases they suffer from, depending on which tissue or organ is most severely affected by TTR amyloidosis and the corresponding symptoms.

[0170] On this basis, TTR amyloidosis is classified in a neurological form, in which the peripheral and autonomic nervous systems are primarily affected, with patients presenting primarily with pain, paresthesias, muscle weakness, and autonomic dysfunction. There is also a cardiac form of TTR amyloidosis, in which the heart is primarily affected, with patients presenting primarily with postural hypotonia or hypertonia, arrhythmias, and cardiomegaly. These two forms are not mutually exclusive, and many patients present with a combination of both. When TTR amyloidosis affects other tissues, this may result in vitreous opacities, dry eyes or glaucoma, proteinuria, hyperthyroxinemia, carpal tunnel syndrome, or preeclampsia.

[0171] Thus, in one embodiment of the invention, an antibody of the invention, a binding molecule of the invention having substantially the same binding specificity as any one thereof, a polynucleotide, a vector, a cell and / or a polypeptide of the invention is used to prepare a pharmaceutical or diagnostic composition for preventing and / or treating TTR amyloidosis, for monitoring disease progression and / or treatment response, and for diagnosing 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 tear, lumbar spinal stenosis and preeclampsia.

[0172] treat:

[0173] As used herein, the terms "treating" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, in which the object is to prevent or slow (lessen) an undesirable physiological change or condition, such as the development of a cardiac defect. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, a stable (i.e., non-worsening) disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" may also refer to prolonged survival, as compared to expected survival if not receiving treatment. Those in need of treatment include those already having the condition, disorder, or disease, as well as those prone to developing the condition, disorder, or disease or those whose manifestations are to be prevented.

[0174] If not otherwise specified, the terms "drug", "medicine" or "medicament" are used interchangeably herein and shall include, but are not limited to, all (A) articles, drugs and preparations for internal or external use, and any substance or mixture of substances intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease in man or other animals; and (B) articles, drugs and preparations (other than foods) intended to affect the structure or any function of the human body or other animals; and (C) articles intended for use as a component of any article specified in clauses (A) and (B). The terms "drug", "medicine" or "medicament" include complete formulations for preparations for use in man or other animals, containing one or more "agents", "compounds", "substances" or "(chemical) compositions" as well as other pharmaceutically inactive excipients such as fillers, disintegrants, lubricants, glidants, binders or agents to ensure that the "drug", "medicine", or "medicament" is easily transported, disintegrated, deagglomerated, dissolved and bioavailable at the intended target location in the human body or other animals, for example, in the skin, in the stomach or intestine. The terms "agent," "compound," or "substance" are used interchangeably herein and, in a more specific aspect, shall include, but not be limited to, all pharmacologically active agents, i.e., agents that induce a desired biological or pharmacological effect or agents that are studied or tested for their ability to induce a possible pharmacological effect by the methods of the present invention.

[0175] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, such as a human patient, for whom diagnosis, prognosis, prevention, or treatment is desired.

[0176] Drug carrier:

[0177] Pharmaceutically acceptable carriers and routes of administration can be obtained from the corresponding literature known to those skilled in the art. The pharmaceutical compositions of the present invention can be formulated according to methods known in the art; see, for example, Remington: The Science and Practice of Pharmacy, University of Science in Philadelphia, ISBN 0-683-306472, Robinson et al., Vaccine Protocols, Second Edition Humana Press, Totowa, New Jersey, USA, 2003; Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Second Edition, Taylor and Francis Publishers. (2006), ISBN: 0-8493-1630-8. Examples of suitable pharmaceutical carriers are well known in the art, including phosphate buffered solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Compositions comprising such carriers can be formulated by well-known traditional methods. These pharmaceutical compositions can be administered to a subject at a suitable dose. The administration of suitable compositions can be achieved in different ways. Examples include administration of compositions comprising pharmaceutically acceptable carriers by oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods. Aerosols such as nasal spray formulations include purified aqueous or other solutions of active agents with preservatives and isotonic agents. Such preparations are preferably adjusted to a pH and isotonic state consistent with the nasal mucosa. Pharmaceutical compositions for oral administration, such as single domain antibody molecules (e.g., "nanobodies TM ” (“nM antibody TM”)) and the like are also contemplated in the present invention. For example, oral formulations may be in tablet, capsule, powder, liquid or semisolid form. Tablets may contain a solid carrier, such as gelatin or an adjuvant. Formulations for rectal or vaginal administration may be presented as suppositories with suitable carriers; see also O'Hagan et al., Nature Reviews, Drug Discovery 2 (9) (2003), 727-735. Further guidance on formulations suitable for different types of administration may be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pennsylvania, 17th edition (1985) and corresponding updates. For a brief review of drug delivery methods, see Langer, Science 249 (1990), 1527-1533.

[0178] 2. Antibodies of the Present Invention

[0179] The present invention is primarily directed to human anti-TTR antibodies and antigen-binding fragments thereof, which preferably exhibit the immunological binding properties and / or biological properties listed for the antibodies as described in the examples. The human monoclonal antibodies specific for TTR according to the present invention are cloned from a large number of healthy human subjects. However, in another embodiment of the present invention, the human monoclonal anti-TTR antibodies may also be cloned from patients who exhibit symptoms of diseases and / or disorders associated with TTR amyloidosis.

[0180] In the course of experiments conducted according to the present invention, antibodies from cultured human memory B cells present in conditioned medium were evaluated for their ability to bind to TTR and to more than 10 other proteins including bovine serum albumin (BSA). Supernatants of B cells that could only bind to the TTR protein and not to the other proteins in the scan were selected for further analysis, including determination of the antibody species and light chain subclass. The selected B cells were then used for antibody cloning.

[0181] Briefly, this involves extracting messenger RNA from selected B cells, reverse transcription by RT-PCR, amplifying the antibody coding region by PCR, cloning into a plasmid vector and sequencing. The selected human antibodies are then produced by recombinant expression and purification in HEK293 or CHO cells, and subsequently characterized for their ability to bind to human TTR protein. A combination of various tests, such as recombinant expression of the antibodies in HEK293 or CHO cells and subsequent characterization of binding specificity to human TTR protein, as well as their differential binding to pathologically misfolded, misassembled and / or aggregated forms thereof, confirmed that human antibodies with high specificity for TTR and that differentially recognize and selectively bind to pathological aggregated forms of TTR protein (such as TTR fibers) have been cloned for the first time. In some cases, mouse chimeric antibodies were also produced based on the variable domains of the human antibodies of the present invention. These mouse chimeric antibodies showed comparable binding to human antibodies (shown in Figure 6 and Fig. 9 and in Example 4 and Example 8) with the same binding affinity, specificity and selectivity.

[0182] Therefore, the present invention generally relates to recombinant human monoclonal anti-TTR antibodies and binding fragments, derivatives and variants thereof. In one embodiment of the present invention, the antibody is capable of binding to human TTR.

[0183] 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 within human TTR comprising amino acids 61-EEEFVEGIY-69 (SEQ ID NO: 49) as a unique linear epitope recognized by the antibody NI-301.59F1 of the invention, a sequence within human TTR comprising amino acids 53-GELHGLTTEEE-63 (SEQ ID NO: 50) as a unique linear epitope recognized by the antibody NI-301.35G11 of the invention, and a sequence within human TTR comprising amino acids 41-WEPFA-45 (SEQ ID NO: 51) as a unique linear epitope recognized by the antibody NI-301.37F1 of the invention (see Fig.10 and Example 9). Thus, in one embodiment, an antibody of the invention is provided, wherein the antibody 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).

[0184] In this case, as explained in Example 9, by using a panel of 29 consecutive peptides 15 amino acids long and overlapping by 11 amino acids (i.e. the first peptide TTRaa 1-15 ; The second peptide TTRaa 5-19 ; etc.), the binding epitopes of exemplary antibodies NI-301.59F1, NI-301.35G11 and NI-301.37F1 were analyzed, wherein antibodies NI-301.59F1 and 301.35G11 recognized two overlapping peptides, (15 and 16) and (13 and 14), respectively, and antibody NI 301.37F1 recognized three overlapping peptides (9, 10 and 11); see Examples 9 and Fig.10 .

[0185] Therefore, with respect to the amino acid sequence of the mature TTR polypeptide and the corresponding peptide mapping, this means that the antibody NI-301.59F1 that binds to the epitope EEEFVEGIY (SEQ ID NO: 49) is able to recognize the antibody having the amino acid sequence GLTT EEEFVEGIY KV (SEQ ID NO:85) and EEEFVEGIY KVEIDT (SEQ ID NO: 86).

[0186] Likewise, the anti-TTR antibody NI-301.35G11, which binds to the epitope GELHGLTTEEE (SEQ ID NO: 50), can recognize a protein having the amino acid sequence TSES GELHGLTTEEE (SEQ ID NO:87) and GELHGLTTEEE FVEG (SEQ ID NO: 88).

[0187] Similarly, the anti-TTR antibody NI-301.37F1, which binds to the epitope WEPFA (SEQ ID NO: 51), is able to recognize a protein having the amino acid sequence FRKAADDT WEPFA SG (SEQ ID NO:89), ADDT WEPFA SGKTSE (SEQ ID NO:90) and WEPFA SGKTSESGEL (SEQ ID NO:91).

[0188] Thus, the subject antibodies of the invention shown in this example are different from antibodies that recognize any of the mentioned epitopes only with respect to the additional N-terminal and / or C-terminal amino acids. Thus, in a preferred embodiment of the invention, specific binding of an anti-TTR antibody 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) is determined according to Examples 9 and Fig.10 A to D were defined using consecutive peptides 15 amino acids long and overlapping by 11 amino acids.

[0189] In this case, extended epitope mapping performed according to the invention and described in Example 9 using a panel of 151 consecutive peptides 15 amino acids long and overlapping by 14 amino acids (wherein for each peptide the non-alanine amino acid at position 10 was substituted by alanine, but alanine was substituted by glycine or proline) showed that the antibody NI-301.59F1 binds to the epitope EEFXEGIY (TTRaa 62-69 ), and antibody NI-301.35G11 binds to ELXGLTXE (TTRaa 54-61 ), without further sequence requirements being determined to be the epitope of antibody NI-301.37F1. Therefore, in another embodiment, whether a given antibody binds to the same epitope as antibodies NI-301.59F1, NI301.35G11 and NI-301.37F1 is determined according to Example 9 and Fig.10 E to Fig.10 Conducted by H.

[0190] It goes without saying that epitope mapping is important for determining whether a given antibody binds to the Fig.10 The same epitope as the subject antibody shown in the embodiment can also be applied to the subject antibody described in the embodiment Figure 1 Any other anti-TTR antibodies of the invention having variable regions depicted in A to 1T.

[0191] Thus, the present invention relates generally to any anti-TTR antibody and any antibody that binds to a protein shown in the Examples and has at least Figure 1 A-1T any of the CDRs and / or variable heavy chain and light chain regions of the antibody-like molecule described.

[0192] In a further embodiment, the antibody specifically binds to the amino acid sequence GELHGLTTEEE (SEQ ID NO: 50), but not GELHGPTTEEE, corresponding to an epitope of the TTR-L55P mutant, or the antibody specifically binds to the amino acid sequence WEPFA (SEQ ID NO: 51), but not WGPFA, corresponding to an epitope of the TTR-E42G mutant.

[0193] Furthermore, it is not intended to be limited by the Figure 2 , 3, 4, 7 and 9, the human monoclonal NI-301.59F1, NI-301.35G11, and NI-301.37F1 anti-TTR antibodies of the present invention are preferably characterized by specific binding to pathological misfolded, misassembled or aggregated TTR without substantially recognizing physiological forms of TTR. Thus, the present invention provides a group of human anti-TTR antibodies with binding properties 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.

[0194] In one embodiment, the antibodies of the invention exhibit the binding properties of NI-301.59F1, NI-301.35G11, and NI-301.37F1 antibodies as described in the exemplary embodiments. The anti-TTR antibodies of the invention preferentially recognize pathologically altered TTR, such as mutated, misfolded, misassembled and / or aggregated TTR species or fragments thereof, rather than physiological TTR. Thus, in one embodiment, the antibodies of the invention do not substantially recognize physiological TTR species.

[0195] 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, antigen and / or conformation of a target molecule and / or antigen), the term "substantially does not recognize" means that the molecules of the above group bind to the molecules, antigens and / or conformations with a binding affinity that is at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times or 9 times less than the binding affinity of the molecules of the above group to other molecules, antigens and / or conformations. Very often, the dissociation constant (KD) is used as a measure of binding affinity. Sometimes, in a specific assay such as an ELISA assay, EC50 is used as a measure of binding affinity. Preferably, when the term "substantially does not recognize" is used in this application, it means that the molecules of the above group bind to the molecules, antigens and / or conformations with a binding affinity that is at least 10 times, 20 times, 50 times, 100 times, 1000 times or 10000 times smaller than the binding affinity of the molecules of the above group to other molecules, antigens and / or conformations.

[0196] Additionally, or alternatively, the anti-TTR antibodies of the invention bind to disease-causing mutated, misfolded, misassembled, and / or aggregated forms of TTR. In this case, the binding affinity may be as described above. Figure 2 ,corresponding Fig.10 The exemplary NI-301.59F1, NI-301.35G11 and NI-301.37F1 antibodies shown in (i.e., having 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).

[0197] In particular, the anti-TTR antibody, binding fragment or derivative thereof has an affinity corresponding to an EC50 value of ≤5 nM for binding to aggregated wild-type and / or an EC50 value of ≤20 nM, preferably ≤10 nM, most preferably ≤1 nM for binding to aggregated V30M-TTR; see Examples 3 and Figure 2 .

[0198] Some antibodies are capable of binding to a variety of biomolecules, e.g., proteins. As will be understood by those skilled in the art, the term "specific" is used herein to indicate that other biomolecules that are not TTR proteins or fragments thereof do not significantly bind to an antigen binding molecule, e.g., one of the antibodies of the invention. Preferably, the level of binding to a biomolecule that is not TTR results in a binding affinity that is at most only 20% or less, 10% or less, only 5% or less, only 2% or less, or only 1% or less (i.e., at least 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold lower, or any multiple greater than this) of the affinity for TTR, respectively; see, e.g. Figure 2 .

[0199] In one embodiment, the anti-TTR antibodies of the invention preferably bind to 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 preferably bind to native TTR and pathologically misfolded, misassembled or aggregated forms of TTR.

[0200] As mentioned previously, amorphous and amyloid TTR deposits can lead to different diseases, depending on where in the body the misfolded, misassembled and / or aggregated TTR type or fragment thereof occurs. For example, patients with familial amyloid polyneuropathy (FAP) exhibit TTR deposits primarily in small-diameter nerve fibers, and thus primarily present 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) exhibit TTR deposits primarily in the heart, and thus present symptoms such as cardiac dysfunction or arrhythmias; patients with TTR deposits in the kidneys may present with renal dysfunction and proteinuria.

[0201] Thus, in one embodiment, the antibodies of the invention can be used to treat 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), ocular amyloidosis, renal amyloidosis, hyperthyroxinemia, ligamentous amyloidosis (including carpal tunnel syndrome, rotator cuff tear, and lumbar spinal stenosis), and pre-eclampsia and its symptoms.

[0202] The present invention also provides an antibody, or an antigen-binding fragment, variant or derivative thereof, wherein the antibody comprises an antigen-binding domain, and the antigen-binding domain is equivalent to a protein 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-301.9D 5. The antigen binding domain of an antibody of the group consisting of NI-301.104F5, NI-301.21F10, NI-301.9G12, NI-301.12D3, NI-301.44E4, NI-301.18C4, NI-301.11A10, NI-301.3C9, NI-301.14D8, NI-301.9X4 and NI-301.14C3.

[0203] The present invention further exemplifies several binding molecules, such as antibodies and binding fragments thereof, which may include in their variable regions (e.g., binding domains) Figure 1 V of any of the amino acid sequences described in H and / or V L The variable regions are characterized by at least one complementarity determining region (CDR). The corresponding nucleotide sequences encoding the variable regions identified above are set forth in Table II below. H and / or V LAn exemplary set of CDRs of the above amino acid sequences of the region is Figure 1 However, as discussed below, it will be apparent to those skilled in the art that, in addition or alternatively, Figure 1 CDRs having amino acid sequences that differ from the sequences set forth in may be used, with one, two, three or even more amino acid differences in the case of CDR2 and CDR3. Thus, in one embodiment, an antibody or TTR-binding fragment thereof of the invention is provided that comprises in its variable region at least one of Figure 1 The described complementarity determining regions (CDRs) and / or one or more CDRs thereof comprise one or more amino acid substitutions.

[0204] In one embodiment, an antibody of the invention comprises Figure 1 The V depicted H and / or V L Any of the antibodies having an amino acid sequence of the V H and / or V L The region comprises one or more amino acid substitutions. Preferably, the antibodies of the invention are characterized by the preservation of heavy and light chain cognate pairing as present in human B cells.

[0205] In a further embodiment of the invention, anti-TTR antibodies, TTR binding fragments thereof, synthetic or biotechnology-based variants can be optimized to have suitable binding affinity and pharmacokinetic properties for the target. Thus, at least one of the CDRs or variable regions that is susceptible to modification selected from the group consisting of glycosylation, oxidation, deamination, peptide bond cleavage, isoaspartic acid and / or formation of unpaired cysteine ​​is replaced by a mutant amino acid lacking such changes or at least one glycosyl group is deleted or chemically or enzymatically added to the antibody. Examples of amino acid optimization can be found in, for example, international applications WO 2010 / 121140 and WO 2012 / 049570. Additional modifications to optimize antibody performance are described in Gavel et al., Protein Engineering 3 (1990), 433-442 and Helenius et al., Annu. Rev. Biochem. 73 (2004), 1019-1049.

[0206] Alternatively, the antibody of the present invention is an antibody or an antigen-binding fragment, derivative or variant thereof, which has Figure 1 A to Figure 1 V depicted by any of T H and / or V L At least one antibody in the region competes for binding to TTR.

[0207] Figure 2The experimental results provided in Example 3 show that some anti-TTR antibodies of the present invention preferentially bind to the misfolded, misassembled or aggregated form of human anti-TTR protein caused by the disease compared to the physiological form of the protein. In such an embodiment, compared to the physiological TTR, the antibodies of the present invention preferentially recognize the misfolded, misassembled and / or aggregated TTR or its fragment and / or derivative thereof.

[0208] The antibodies of the invention may be human, particularly for therapeutic applications. Alternatively, the antibodies of the invention are rodent, rodentized or chimeric rodent human antibodies, preferably murine, murineized or chimeric murine human antibodies or rat, ratized or chimeric rat human antibodies, which are particularly useful for diagnostic methods and research in animals. In one embodiment, the antibodies of the invention are chimeric rodent human antibodies or rodent antibodies.

[0209] In addition, in one embodiment, chimeric antibodies of the invention (i.e., comprising human antibody variable domains such as NI-301.35G11 and typical murine light and heavy chain constant domains) exhibit binding properties to the exemplary NI-301.mur35G11 mouse chimeric antibody as described in the Examples. Further, mouse chimeric antibodies of the invention bind to human TTR with high affinity, as described in Examples 4 and 8. Preferably, the binding affinity of the chimeric antibodies is similar to that of their human counterparts.

[0210] In one embodiment, the antibodies of the invention are provided by a culture of cultured monoclonal or oligoclonal B cells, and the culture supernatant, which contains antibodies produced by said B cells, is scanned for the presence and affinity of anti-TTR antibodies. The scanning process includes scanning for binding of native monomers, fibrillar or non-fibrillar aggregates (such as oligomers of hTTR derived from synthetic full-length hTTR peptides or purified hTTR, e.g., from human plasma or recombinant expression).

[0211] Additionally or alternatively, the scanning process for the presence and affinity of anti-TTR antibodies may include the steps of a sensitive tissue amyloid plaque immune response (TAPIR) test, such as described in International Application WO2004 / 095031, the disclosure of which is incorporated herein by reference. Additionally or alternatively, scanning for binding of anti-TTR in kidney, heart sections (such as described in brain and spinal cord sections described in similar International Application WO2008 / 081008) may be performed.

[0212] As described above, because human-based immune responses lead to the generation of epitopes, the human monoclonal antibodies of the present invention will recognize epitopes with specific pathological relevance, which are inaccessible or less immunogenic in the immunization process for generating, for example, mouse monoclonal antibodies and in vitro screening of phage display libraries, respectively. Therefore, for the sake of caution, it is stipulated that the epitope of the human anti-TTR antibody of the present invention is unique, and no other antibody that can bind to the epitope recognized by the human monoclonal antibody of the present invention exists; see also Fig.10 Another indication of the uniqueness of the antibodies of the invention is that, as shown in Example 8, the antibodies of the invention NI-301.59F1, NI-301.35G11 and NI-301.37F1 bind to an epitope that is specific for misfolded, misassembled and / or aggregated TTR conformations, which, as shown above, are of specific pathological relevance and cannot be obtained by common processes of antibody generation, such as immunization or in vitro library screening.

[0213] Thus, in one embodiment, the invention also broadly extends to anti-TTR antibodies and TTR binding molecules that compete with the human monoclonal antibodies of the invention that specifically bind to TTR. The invention more specifically relates to an 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 TTR selected from the group consisting of NI-301.59F1, NI-301.35G11, NI-301.37F1, and / or NI-301.12D3.

[0214] Furthermore, in one embodiment, the invention also broadly extends to anti-TTR antibodies and TTR binding molecules that compete with the human monoclonal antibodies of the invention that specifically bind to misfolded, misassembled and / or aggregated TTR or fragments thereof. The invention therefore also more specifically relates to antibodies, or antigen binding fragments, variants or derivatives thereof, wherein the antibodies specifically bind to the same epitope of misfolded, misassembled and / or aggregated TTR forms 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.

[0215] Competition between antibodies is determined by a test in which the test immunoglobulin inhibits specific binding of a reference antibody to a common antigen (e.g., TTR). Many types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay; 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 assay, solid phase direct labeling sandwich assay; see Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor Press (1988); using I 125Label solid phase direct labeling RIA; see Morel et al., Molec.Immunol.25 (1988), 7-15 and Moldenhauer et al., Scand.J.Immunol.32 (1990), 77-82. Typically, such an assay includes the use of purified TTR or misfolded, misassembled or aggregated TTR, such as oligomers and / or fibrils thereof bound to a solid surface or cells having any of them, unlabeled test immunoglobulins and labeled reference immunoglobulins (i.e., human monoclonal antibodies of the present invention). Competitive inhibition is measured by determining the amount of labeling bound to a solid surface or cell in the presence of a test immunoglobulin. Typically, the test immunoglobulin is present in excess. Preferably, the competitive binding assay is performed under conditions as described for ELISA assays in the accompanying examples. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope of the reference antibody and antibodies that bind to adjacent epitopes sufficiently close to the reference antibody binding epitope for steric hindrance. Typically, when a competing antibody is present in excess, it will inhibit specific binding of the reference antibody to the common antigen by at least 50% or 75%. Thus, the present invention further relates to antibodies, or antigen-binding fragments, variants, or derivatives thereof, wherein the antibody competitively inhibits binding of a reference antibody 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, NI-301.18C4, NI-301.11A10, NI-301.3C9, NI-301.14D8, NI-301.9X4, and / or NI-301.14C3 to TTR.

[0216] In addition, the present invention further relates to an antibody, or an antigen-binding fragment, variant or derivative thereof, wherein the antibody competitively inhibits an antigen 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- The binding of the reference antibodies of the group consisting of NI-301.21F10, NI-301.9G12, NI-301.12D3, NI-301.44E4, NI-301.18C4, NI-301.11A10, NI-301.3C9, NI-301.14D8, NI-301.9X4, and / or NI-301.14C3 to misfolded, misassembled and / or aggregated TTR or fragments thereof.

[0217] In another embodiment, the invention provides an isolated polypeptide comprising an immunoglobulin heavy chain variable region (V H ), or essentially an immunoglobulin heavy chain variable region (V H ) or is composed of the variable region of an immunoglobulin heavy chain (V H ) in which the heavy chain variable region V H - at least one of the CDRs or V H - at least two of the CDRs are identical to a reference heavy chain V H -CDR1, V H -CDR2 or V H - the CDR3 amino acid sequences are at least 80%, 85%, 90% or 95% identical. Alternatively, V H V H -CDR1, V H -CDR2 and V H -CDR3 region is identical to the reference heavy chain V H -CDR1, V H -CDR2 or V H -CDR3 amino acid sequences are at least 80%, 85%, 90% or 95% identical. Therefore, according to this embodiment, the heavy chain variable region of the present invention is respectively identical to Figure 1 The group-related V H -CDR1, V H -CDR2 and V H -CDR3 polypeptide sequence. Figure 1 The V defined by the Kabat system is shown H -CDR, other CDR definitions (such as V defined by the Chothia system H-CDR) are also included in the present invention and can be used Figure 1 The data presented in the figure are readily recognized by a person of ordinary skill in the art.

[0218] In another embodiment, the invention provides an isolated polypeptide comprising an immunoglobulin heavy chain variable region (V H ), or essentially an immunoglobulin heavy chain variable region (V H ) or is composed of the variable region of an immunoglobulin heavy chain (V H ),where V H -CDR1, V H -CDR2 and V H -CDR3 region has Figure 1 The V H -CDR1, V H -CDR2 and V H - The polypeptide sequence of the CDR3 group is identical.

[0219] In another embodiment, the invention provides an isolated polypeptide comprising an immunoglobulin heavy chain variable region (V H ), or essentially an immunoglobulin heavy chain variable region (V H ) or is composed of the variable region of an immunoglobulin heavy chain (V H ),where V H -CDR1, V H -CDR2 and V H -CDR3 region has Figure 1 The V H -CDR1, V H -CDR2 and V H - CDR3 groups have identical polypeptide sequences except for any V H - One, two, three, four, five or six amino acid substitutions in the CDRs. In certain embodiments, the amino acid substitutions are conservative.

[0220] In another embodiment, the invention provides an isolated polypeptide comprising an immunoglobulin light chain variable region (V L ), or essentially an immunoglobulin light chain variable region (V L ) or is composed of the immunoglobulin light chain variable region (V L ), wherein the light chain variable region V L - at least one of the CDRs or the variable region V L - at least two of the CDRs are identical to a reference light chain V L -CDR1, V L -CDR2 or V L- the CDR3 amino acid sequences are at least 80%, 85%, 90% or 95% identical. Alternatively, V L V L -CDR1, V L -CDR2 and V L -CDR3 region is identical to the reference light chain V from the antibody disclosed in the present invention L -CDR1, V L -CDR2 or V L -CDR3 amino acid sequences are at least 80%, 85%, 90% or 95% identical. Therefore, according to this embodiment, the light chain variable region of the present invention is respectively identical to Figure 1 The peptides shown in V L -CDR1, V L -CDR2 and V L -CDR3 polypeptide sequence. Figure 1 The V defined by the Kabat system is shown L -CDR, other CDR definitions (such as V defined by the Chothia system L -CDR) are also included in the present invention.

[0221] In another embodiment, the invention provides an isolated polypeptide comprising an immunoglobulin light chain variable region (V L ), or essentially an immunoglobulin light chain variable region (V L ) or is composed of the immunoglobulin light chain variable region (V L ),where V L -CDR1, V L -CDR2 and V L -CDR3 region has Figure 1 The V L -CDR1, V L -CDR2 and V L - The polypeptide sequence of the CDR3 group is identical.

[0222] In another embodiment, the invention provides an isolated polypeptide comprising an immunoglobulin light chain variable region (V L ), or essentially an immunoglobulin light chain variable region (V L ) or is composed of the immunoglobulin light chain variable region (V L ),where V L -CDR1, V L -CDR2 and V L -CDR3 region has Figure 1 The V L -CDR1, V L -CDR2 and VL - CDR3 groups have identical polypeptide sequences except for any V L - One, two, three, four, five or six amino acid substitutions in the CDRs. In certain embodiments, the amino acid substitutions are conservative.

[0223] The immunoglobulin or its encoded cDNA may be further modified. Therefore, in a further embodiment, the method of the present invention comprises any one of the steps of producing a chimeric antibody, a murinized antibody, a single-chain antibody, a Fab fragment, a bispecific antibody, a fusion antibody, a labeled antibody or an analog of any one of these. Corresponding methods are well known to the person skilled in the art and are described, for example, in Antibodies, A Laboratory Manual (eds.) by Harlow and Lane, CSH Press, Cold Spring Harbor (1988). When derivatives of the antibodies are obtained by phage display technology, surface plasmon resonance, as used in the BIAcore system, can be used to increase the efficiency of phage antibodies that bind to the same epitope as any of the antibodies described herein (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). The production of chimeric antibodies is described, for example, in International Application WO 89 / 09622. Methods for producing humanized antibodies are described, for example, in European Application EP-A1 0239. 400 and International Application WO90 / 07861. A further source of antibodies to be used according to the present invention are so-called xenoantibodies. The general principles for producing xenoantibodies such as human-like antibodies in mice are described, for example, in International Applications WO91 / 10741, WO94 / 02602, WO96 / 34096 and WO 96 / 33735. As mentioned above, the antibodies of the present invention may exist in a variety of forms in addition to intact antibodies; including, for example, Fv, Fab and F(ab) 2 , and in a single chain; see, for example, International Application WO88 / 09344. In one embodiment, therefore, there is provided a fragment selected from a single chain Fv fragment (scFv), a F(ab') fragment, a F(ab) fragment and a F(ab') 2 The antibodies of the present invention are composed of a group of fragments.

[0224] The 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, insertions, substitutions, additions, and / or recombination and / or any other modification known in the art, alone or in combination. Methods for introducing such modifications to the DNA sequence underlying the amino acid sequence of the immunoglobulin chain are well known to those skilled in the art; see, for example, Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) New York and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, New York (1994). Modifications of the antibodies of the invention include chemical and / or enzymatic derivatization of one or more of the constituent amino acids, including side chain modifications, backbone modifications, and modifications to the N-terminus and C-terminus, including acetylation, hydroxylation, methylation, amidation, and attachment of carbohydrate compounds or lipid moieties, cofactors, and the like. Likewise, the invention encompasses the production of chimeric proteins comprising the described antibodies or fragments thereof fused at the amino terminus to a heterologous molecule (such as an immunostimulatory ligand at the carboxyl terminus); see, for example, International Application WO 00 / 30680 for details of the corresponding technology.

[0225] In addition, the present invention includes peptides, including those containing binding molecules as described above, for example, containing the CDR3 region of any variable region of the antibody mentioned, particularly the CDR3 of the heavy chain, because it is often observed that the heavy chain CDR3 (HCDR3) is a region with a greater degree of variability and is mainly involved in antigen-antibody interactions. These peptides can be easily synthesized or produced by recombinant methods to produce binding agents useful according to the present invention. Such methods are well known to those of ordinary skill in the art. Peptides can be synthesized, for example, using commercially available automatic peptide synthesizers. Peptides can also be produced by recombinant technology by introducing DNA expressing the peptide into an expression vector and using the expression vector to transform cells to produce peptides.

[0226] Thus, the present invention relates to any binding molecule, such as an antibody or a binding fragment thereof, which refers to the anti-TTR antibody of the present invention and / or an antibody capable of binding to a mutated, misfolded, misassembled and / or aggregated TTR form or fragment thereof, and exhibits the above-mentioned properties, i.e., it specifically recognizes TTR and / or a mutated, misfolded, misassembled and / or aggregated TTR form or fragment thereof. Such antibodies and binding molecules can be assayed for their binding specificity and affinity by ELISA and immunohistochemistry as described herein, see, for example, the Examples. These properties of antibodies and binding molecules can also be assayed by Western blotting (protein blotting).

[0227] The exemplary human antibody NI-301.37F1 showed significant staining of misfolded TTR in skin biopsies from FAP patients, but not in healthy control pancreases (where pancreatic α cells show endogenous expression of TTR, i.e., native TTR (see Examples 8 and Fig. 9 )) No staining. The exemplary antibodies NI-301.35G11 and NI-301.37F1 of the present invention also gave positive results for FAP mouse tissues showing abnormal TTR accumulation in various tissues (including the intestine), see Figure 8 This binding specificity for pathological forms of TTR in human and animal tissues emphasizes that in addition to the biochemical experiments shown herein (see Fig.10 ), the usefulness of the antibodies of the invention in the treatment and diagnosis of diseases associated with TTR amyloidosis (which preferably occur due to the occurrence of misfolded, misassembled and / or aggregated TTR forms and / or fragments, derivatives thereof).

[0228] As an alternative to immunoglobulins obtained directly from cultures of B cells or memory B cells, cells can be used as a source of rearranged heavy and light chain loci for subsequent expression and / or genetic manipulation. Rearranged antibody genes can be reverse transcribed from appropriate mRNA to produce cDNA. If necessary, the heavy chain constant region can be exchanged for a heavy chain constant region of a different isotype or eliminated completely. The variable region can be linked to encode a single-chain Fv region. Multiple Fv regions can be connected to confer binding ability to more than one target, or chimeric heavy and light chain combinations can also be used. Once genetic material is obtained, the design of analogs that retain their binding ability to the desired target as described above is simple and direct. Methods for cloning antibody variable regions and producing recombinant antibodies are well known to those skilled in the art and are described in, for example, Gilliland et al., Tissue Antigens 47 (1996), 1-20; Doenecke et al., Leukemia 11 (1997), 1787-1792.

[0229] Once appropriate genetic material is obtained and, if desired, modified to encode the analogs, the coding sequences, including the coding sequences encoding at least the variable regions of the heavy and light chains, can be inserted into an expression system contained in a vector that can be transfected into standard recombinant host cells. A variety of such host cells can be used; for efficient processing, however, mammalian cells are preferred. Typical mammalian cell lines used for this purpose include, but are not limited to, CHO cells, HEK 293 cells, or NSO cells.

[0230] The antibody or analog is then produced by culturing the modified recombinant host under culture conditions suitable for host cell growth and expression of the coding sequence. The antibody is then isolated from the culture and recovered. The expression system is preferably designed to include a signal peptide so that the produced antibody is secreted into the culture medium; however, intracellular production is also possible.

[0231] According to the above, the present invention also relates to polynucleotides encoding the antibodies or equivalent binding molecules of the present invention, and in the case of antibodies, preferably at least the variable region of the immunoglobulin chain of the above antibodies. Generally, the variable region encoded by the polynucleotide includes the V H and / or V L In one embodiment of the invention, the polynucleotide is a cDNA.

[0232] Those skilled in the art will readily appreciate that the variable domains of antibodies having the above-described variable domains can be used for the construction of other polypeptides or antibodies of desired specificity and biological function. Therefore, the present invention also includes polypeptides and antibodies comprising at least one CDR of the above-described variable domains and advantageously having substantially the same or similar binding properties as the antibodies described in the accompanying embodiments. Those skilled in the art will appreciate that binding affinity can be improved by substitution of amino acids within the CDR or within the hypervariable loops that overlap with the CDR portions defined by Kabat (Chothia and Lesk, J. Mol. Biol. 196 (1987), 901-917); see, for example, Riechmann et al., Nature 332 (1988), 323-327. Therefore, the present invention also relates to antibodies in which one or more of the CDRs comprises one or more, preferably no more than two, amino acid substitutions. Preferably, the antibodies of the present invention comprise two or all three CDRs of the variable region in one or both of their immunoglobulin chains, such as Figure 1 Explained.

[0233] Binding molecules, such as antibodies of the present invention, or antigen-binding fragments, variants or derivatives thereof, as known to those of ordinary skill in the art, may comprise a constant region that mediates one or more effector functions. For example, the binding of the C1 portion of complement to the constant region of an antibody can activate the complement system. Activation of complement is important in the opsonization and lysis of cellular pathogens. Activation of complement also stimulates inflammatory responses and may also be involved in autoimmune hypersensitivity. In addition, antibodies bind to receptors on various cells via the Fc region, by virtue of the Fc receptor binding site on the antibody Fc region that binds to Fc receptors (FcR) on cells. There are many Fc receptors that are specific for different types of antibodies, including IgG (gamma receptors), IgE's (epsilon receptors), IgA's (alpha receptors) and IgM (mu receptors). Binding of antibodies to Fc receptors on the cell surface triggers many 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 (termed antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production.

[0234] Therefore, certain embodiments of the present invention include antibodies, or antigen-binding fragments, variants or derivatives thereof, wherein at least one or more portions of the constant region domain are deleted or altered to provide desired biochemical properties, such as reduced effector function, ability to non-covalent dimerization, improved ability to localize at sites of TTR aggregation and deposition, improved serum half-life or reduced serum half-life when compared to intact, unaltered antibodies with substantially the same immunogenicity. For example, certain antibodies used in the diagnostic and therapeutic methods described herein are domain-deleted antibodies that contain polypeptide chains similar to immunoglobulin heavy chains, but lack at least a portion of one or more heavy chain domains. For example, in certain antibodies, a complete 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 used in the diagnostic and therapeutic methods described herein have a constant region (e.g., an IgG heavy chain constant region) that has been altered to eliminate glycosylation (referred to elsewhere herein as glycosylated or "agly" antibodies). Such "agly" antibodies can be prepared enzymatically or by designing a common glycosylation site in the constant region. While not being bound by theory, it is believed that "agly" antibodies may have improved safety and stability characteristics in vivo. Methods for preparing glycosylated antibodies with desired effector functions can be found, for example, in International Application WO 2005 / 018572, which is incorporated herein by reference.

[0235] In certain antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein, the Fc portion may be mutated using techniques known in the art to reduce effector function. For example, deletion or inactivation of a constant region domain (by point mutation or other means) may reduce binding of Fc receptors to circulating modified antibodies, thereby enhancing TTR localization. In other cases, modifications of the constant region consistent with the present invention may reduce complement binding, thereby reducing serum half-life and nonspecific association of conjugated cytotoxins. However, other modifications of the constant region may be used to modify disulfide bonds or oligosaccharide moieties, which allow enhanced localization due to increased antigen specificity or antibody flexibility. The resulting physiological information, bioavailability, and other biochemical effects of the modification (such as TTR localization, biodistribution, and serum half-life) can be easily determined and quantified using well-known immunological techniques without excessive experimentation.

[0236] In certain antibodies, or antigen-binding fragments, variants or derivatives thereof described herein, the Fc portion may be mutated or exchanged for alternative protein sequences to improve cellular uptake of the antibody by enhancing receptor-mediated endocytosis of the antibody via Fcγ receptors, LRP or Thy1 receptors or by "super antibody technology" (said to allow antibodies to be shuttled into living cells without harming them, Expert Opin. Biol. Ther. (2005), 237-241). For example, the generation of bispecific or multispecific antibodies that are fusion proteins of antibody binding regions and cognate protein ligands of cell surface receptors or have specific sequences that bind to TTR and cell surface receptors can be designed using techniques known in the art.

[0237] 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, or the antibody may be chemically modified to improve its blood-brain barrier penetration.

[0238] Modified forms of the antibodies, or antigen-binding fragments, variants, or derivatives described herein can be prepared from entire precursor or parent antibodies using techniques known in the art. Exemplary techniques are discussed in more detail herein. The antibodies, or antigen-binding fragments, variants, or derivatives described herein can be prepared or produced using techniques known in the art. In certain embodiments, the antibody molecule or fragment thereof is "recombinantly produced," i.e., produced using recombinant DNA technology. Exemplary techniques for preparing antibody molecules or fragments thereof are discussed in more detail elsewhere herein.

[0239] The antibodies, or antigen-binding fragments, variants or derivatives thereof of the present invention also include modified derivatives, for example, by covalently linking any type of molecule to the antibody so that the covalent linking does not prevent the antibody from specifically binding to its cognate epitope. For example, but not limited to, the antibody derivatives include antibodies that have been modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolysis, connection to cellular ligands or other proteins. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the derivatives may contain one or more non-classical amino acids.

[0240] In a particularly preferred embodiment, the antibodies, or antigen-binding fragments thereof, variants or derivatives described herein, including modified derivatives, do not cause a harmful immune response in the animal to be treated, such as in humans. In certain embodiments, the binding molecules, such as antibodies or antigen-binding fragments thereof of the present invention, are derived from a patient, such as a human patient, and are subsequently used in the same species from which they are derived, such as humans, to reduce or minimize the occurrence of a harmful immune response.

[0241] Deimmunization can also be used to reduce the immunogenicity of an antibody. As used herein, the term "deimmunization" includes alterations of an antibody to modify T cell epitopes; see, for example, International Applications WO 98 / 52976 and WO 00 / 34317. For example, V from a starting antibody H and V L The sequences are analyzed and human T cell epitopes are "mapped" from each V region showing the localization of the epitope relative to the complementarity determining regions (CDRs) and other key residues in 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 batch of alternative V H and V L Sequences are designed, including combinations of amino acid substitutions, and these sequences are then introduced into a batch of binding polypeptides, e.g., TTR-specific antibodies or immunospecific fragments thereof, used in the diagnostic and therapeutic methods disclosed herein, which are then used for functional testing. Typically, between 12 and 24 variant antibodies are generated and tested. The complete heavy and light chain genes containing the modified V regions and human C regions are then cloned into expression vectors, and the plasmids are then introduced into cell lines for production of the whole antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the best variants are determined.

[0242] Monoclonal antibodies can be prepared using a variety of techniques known in the art (including the use of hybridomas, recombinant and phage display techniques or combinations thereof). For example, it can be prepared using hybridoma techniques including those known in the art and taught (e.g., Harlow et al., in Antibodies: A Laboratory Manual ("Antibodies: Laboratory Manual") Cold Spring Harbor Laboratory Press, 2nd edition (1988); Hammerling et al., in Monoclonal Antibodies and T-Cell Hybridomas ("Monoclonal Antibodies and T-Cell Hybridomas") Elsevier, New York, 563-681 (1981)), which are incorporated herein by reference. The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to antibodies derived from a single clone, including any eukaryotic, prokaryotic, or phage clones, rather than the method for their production. Therefore, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. In certain embodiments, antibodies of the invention are derived from human B cells that have been immortalized by transformation with Epstein-Barr virus, as described herein.

[0243] In the well-known hybridoma process (Kohler et al., Nature 256 (1975), 495), relatively short-lived or dying lymphocytes derived from mammals (e.g., B cells derived from human subjects as described herein) are fused with immortalized tumor cell lines (e.g., myeloma cell lines) to produce hybrid cells or "hybridomas" that are both immortalized and capable of producing antibodies encoded by the genes of the B cells. The resulting hybrids are separated into separate genetic strains by selection, dilution, and regeneration with each individual cell strain containing specific genes for the formation of a single antibody. They produce antibodies that are homologous to the desired antigen and are referred to as "monoclonal" in reference to their pure genetic lineage relationship.

[0244] The hybridoma cells thus prepared are inoculated and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. It will be appreciated by those skilled in the art that reagents, cell lines and culture medium for the formation, selection and growth of hybridomas are commercially available from many sources, and standardized protocols have been well established. Generally, the culture medium in which the hybridoma cells are grown is determined for the production of monoclonal antibodies against desired antigens. The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation such as described herein, and the in vitro test of radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) is determined. After hybridoma cells are identified as producing antibodies of the desired specificity, affinity and / or activity, 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 (1986), 59-103. It will also be appreciated that the monoclonal antibodies secreted by the subclones can be isolated from the culture medium, ascites fluid or serum by conventional purification methods (such as protein A, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography).

[0245] In another embodiment, lymphocytes can be selected by micromanipulation, and variable genes are isolated. For example, peripheral blood mononuclear cells can be isolated from immunized or naturally immune mammals (e.g., humans) and cultured in vitro for about 7 days. The culture can be screened for specific IgG that meets the screening criteria. Cells derived from positive wells can be isolated. Individual B cells that produce Ig can be isolated by FACS or by identifying them in a complement-mediated hemolytic plaque assay. B cells that produce Ig can be micromanipulated into a tube, and V H and V L The gene can be amplified using, for example, RT-PCR. H and V L The gene can be cloned into an antibody expression vector and transfected into cells (eg, eukaryotic or prokaryotic cells) for expression.

[0246] Alternatively, the cell line that produces antibody can be selected and cultivated using the technology that is well-known to those skilled in the art. Such technology is described in various laboratory manuals and major publications. In this regard, the technology that is suitable for the present invention as described below is described in Current Protocols in Immunology (" Modern Immunological Methods "), compiled by the people such as Coligan, Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), which is incorporated herein by reference.

[0247] Antibody fragments that recognize specific epitopes can be generated 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 F(ab') 2 The fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain. Such fragments are sufficient for use, for example, in immunodiagnostic procedures involving coupling the immunospecific portion of the immunoglobulin to a detection reagent (such as a radioisotope).

[0248] In one embodiment, an antibody of the present invention comprises at least one CDR of an antibody molecule. In another embodiment, an antibody of the present invention comprises at least two CDRs from one or more antibody molecules. In another embodiment, an antibody of the present invention comprises at least three CDRs from one or more antibody molecules. In another embodiment, an antibody of the present invention comprises at least four CDRs from one or more antibody molecules. In another embodiment, an antibody of the present invention comprises at least five CDRs from one or more antibody molecules. In another embodiment, an antibody of the present invention comprises at least six CDRs from one or more antibody molecules. An exemplary antibody molecule comprising at least one CDR may be included in a subject antibody described herein.

[0249] The antibodies of the present invention may be prepared by any method known in the art for the synthesis of antibodies, in particular by chemical synthesis or preferably by recombinant expression techniques as described herein.

[0250] In one embodiment, the antibodies, or antigen-binding fragments, variants or derivatives thereof of the present invention comprise a synthetic constant region in which one or more domains are partially or completely deleted ("domain-deleted antibodies"). In certain embodiments, compatible modified antibodies will comprise a domain-deleted construct or variant in which the entire CH2 domain has been removed (CH2 construct). For other embodiments, a short connecting peptide can replace the deleted domain to provide flexibility and freedom of movement of the variable region. Those skilled in the art will appreciate that such constructs are particularly preferred because of the regulatory properties of the CH2 domain for the rate of antibody catabolism. Domain-deleted constructs can be obtained with vectors encoding human IgG1 constant regions, see, e.g., International Applications WO02 / 060955 and WO02 / 096948A2. The vector is designed to delete the CH2 domain and provide a synthetic vector for expressing a domain-deleted IgG1 constant region.

[0251] In certain embodiments, the antibodies, or antigen-binding fragments, variants or derivatives thereof of the invention are minibodies. Minibodies can be prepared using methods described in the art, see, e.g., U.S. Pat. No. 5,837,821 or International Patent Application WO 94 / 09817.

[0252] In one embodiment, the antibody, or antigen-binding fragment, variant or derivative thereof of the present invention comprises an immunoglobulin heavy chain with some or even single amino acid substitutions or deletions, as long as it allows association between monomer subunits. For example, a mutation of a single amino acid in a selected region of the CH2 domain may be sufficient to substantially reduce Fc binding and thereby increase the localization of TTR. Similarly, it may be desirable to simply delete portions of one or more constant region domains that control effector functions (e.g., complement binding) for regulation. This partial deletion of the constant region may improve the selected properties (serum half-life) of the antibody while leaving other desired functions associated with the subject constant region domain undamaged. In addition, as mentioned above, the constant region of the disclosed antibody may be synthesized by one or more amino acid substitutions or mutations, which improve the properties of the resulting construct. In this regard, it is possible to destroy the activity provided by the conserved binding site (e.g., Fc binding), while substantially maintaining the structural and immunogenic characteristics of the modified antibody. However, other embodiments include adding one or more amino acids to the constant region to improve the desired properties (e.g., effector functions), or to provide more cytotoxins or carbohydrate compounds to be connected. In such embodiments, it may be desirable to insert or replicate specific sequences derived from selected constant region domains.

[0253] The invention also provides a method comprising, consisting essentially of, or consisting of an antibody molecule (e.g., V H Area and / or V LThe antibody or fragment thereof immunospecifically binds to TTR. Standard techniques well known to those skilled in the art can be used to introduce mutations in 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 mutations are expressed relative to the reference V H Area, V H -CDR1, V H -CDR2, V H -CDR3, V L Area, V L -CDR1, V L -CDR2 or V L -CDR3, the variant (including derivative) 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. A "conservative amino acid substitution" is one 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 with similarly charged side chains 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, for example, by saturation mutagenesis, and the resulting mutants can be used to scan for biological activity to identify mutants that retain activity (e.g., the ability to bind to TTR and / or misfolded, misassembled or aggregated TTR forms and / or fragments thereof).

[0254] For example, it is possible to introduce mutations only in the framework region of the antibody molecule or only in the CDR region. The mutation introduced can be a silent or neutral missense mutation, for example, there is no or little effect on the ability of the antibody to bind to the antigen, and indeed some such mutations do not change any amino acid sequence. These types of mutations may be useful for optimizing the use of codons, or the antibody production of improved hybridomas. Codon-optimized coding regions encoding antibodies of the present invention are disclosed elsewhere herein. Alternatively, non-neutral missense mutations can change the ability of antibodies to bind to antigens. The positions of most silent mutations and neutral missense mutations may be in the framework region, while the positions of most non-neutral missense mutations may be in the CDR, 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 change in binding activity (e.g., changes in antigen binding activity or antibody specificity). Once mutagenesis has occurred, the encoded protein can be routinely expressed and the function and / or biological activity of the encoded protein (e.g., the ability to immunospecifically bind to at least one epitope of TTR and / or mutated, misfolded, misassembled and / or aggregated TTR or fragments thereof) can be determined using the techniques described herein or by routine modification techniques known in the art.

[0255] 3. Polynucleotide-encoded antibodies

[0256] The polynucleotide encoding the antibody, or its Fab, variant or derivative can be composed of any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA, or modified RNA or DNA. For example, the polynucleotide encoding the antibody or its Fab, variant or derivative can 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 (RNA that is a mixture of single-stranded and double-stranded regions), comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single-stranded and double-stranded regions. In addition, the polynucleotide encoding the antibody or its Fab, variant or derivative can be composed of a triple-stranded region comprising RNA or DNA or both RNA and DNA. The polynucleotide encoding the antibody or its Fab, variant or derivative can also contain one or more modified bases or DNA or RNA backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and rare bases such as inosine. Various modifications may be made to DNA and RNA; thus, "polynucleotide" includes chemically, enzymatically, or metabolically modified forms.

[0257] The isolated polynucleotide encoding a non-natural variant of a polypeptide from an immunoglobulin (e.g., an immunoglobulin heavy chain portion or a light chain portion) can be created 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 can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues.

[0258] As is well known, RNA can be separated by standard techniques from original B cells, hybridoma cells or from other transformed cells, such as guanidine isothiocyanate extraction and precipitation followed by centrifugation or column chromatography. If necessary, mRNA can be separated from total RNA by standard techniques (such as in oligo dT cellulose column chromatography). Suitable technology is familiar in the art. In one embodiment, the cDNA encoding the light chain and heavy chain of an antibody can be prepared simultaneously or separately using reverse transcriptase and DNA polymerase according to known methods. PCR can be started by common constant region primers or by more specific primers based on published heavy chain and light chain DNA and amino acid sequences. As mentioned above, PCR can also be used to separate DNA clones encoding antibody light chain and heavy chain. In this case, the library can be screened with universal primers or larger homologous probes (such as human constant region probes).

[0259] DNA, typically plasmid DNA, can be isolated from cells using techniques known in the art and rigorously mapped and sequenced according to standard well-known techniques that have been elaborated in detail (e.g., the aforementioned references involving recombinant DNA technology). Of course, DNA can be synthesized at any point in the isolation process or subsequent analysis according to the present invention.

[0260] In this context, the present invention also relates to polynucleotides encoding at least the binding domain or variable region of the immunoglobulin chain of the antibodies of the present invention. In one embodiment, the present invention provides polynucleotides comprising, consisting essentially of, or consisting of a polynucleotide encoding an immunoglobulin heavy chain variable region (V H ) wherein at least one CDR of the heavy chain variable region or at least two V H -CDRs from the reference heavy chain V H -CDR1, V H -CDR2 or V H - the CDR3 amino acid sequences are at least 80%, 85%, 90% or 95% identical. Alternatively, V H V H -CDR1, V H -CDR2 and V H-CDR3 region is identical to the reference heavy chain V H -CDR1, V H -CDR2 or V H -CDR3 amino acid sequence is at least 80%, 85%, 90% or 95% identical. Figure 1 The polypeptide sequence shown in V H -CDR1, V H -CDR2 and V H - CDR3 polypeptide sequence.

[0261] In another embodiment, the present invention provides a method comprising, consisting essentially of, or consisting of a sequence encoding an immunoglobulin light chain variable region (V L ) nucleic acid, wherein at least one V L - At least two V CDRs or light chain variable regions L -CDRs from the reference light chain V L -CDR1, V L -CDR2 or V L - the CDR3 amino acid sequences are at least 80%, 85%, 90% or 95% identical. Alternatively, V L V L -CDR1, V L -CDR2 and V L -CDR3 region is identical to the reference light chain V from the antibody disclosed in the present invention L -CDR1, V L -CDR2 and V L -CDR3 amino acid sequence is at least 80%, 85%, 90% or 95% identical. Therefore, according to this embodiment, the light chain variable region of the present invention has the same Figure 1 The polypeptide sequence shown in V L -CDR1, V L -CDR2 and V L - CDR3 polypeptide sequence.

[0262] In another embodiment, the present invention provides a method comprising, consisting essentially of, or consisting of a sequence encoding an immunoglobulin heavy chain variable region (V H ) is an isolated polynucleotide composed of a nucleic acid, wherein V H -CDR1, V H -CDR2 and V H -CDR3 region has Figure 1 The V H -CDR1, V H -CDR2 and V H- The same polypeptide sequence in the CDR3 region.

[0263] As is known in the art, "sequence identity" between two polypeptides or two polynucleotides is determined by comparing the amino acid or nucleic acid sequence of one polypeptide or polynucleotide to the sequence of a second polypeptide or polynucleotide. When 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 homology fragment between two sequences. When using BESTFIT or any other sequence comparison / calibration program to determine whether a particular sequence has, for example, 95% identical with a reference sequence according to the present invention, the parameters are set, of course, so that the percentage of identity is calculated over the full length of the reference polypeptide sequence, and so that the highest 5% homology gap / gap of the total amino acid in the reference sequence is allowed.

[0264] In a preferred embodiment of the present invention, the polynucleotide comprises, consists essentially of, or consists of V with an anti-TTR antibody and / or an antibody that recognizes misfolded, misassembled or aggregated TTR forms or fragments thereof as described in Table II. H or V L In this regard, it will be readily appreciated by those skilled in the art that the polynucleotide encoding at least the variable domains of the light chain and / or heavy chain may encode the variable domains of both chains or only one chain of an immunoglobulin. In one embodiment, the polynucleotide thus comprises, consists essentially of, or consists of a V domain of an anti-TTR antibody that recognizes misfolded, misassembled or aggregated TTR and / or fragments thereof as described in Table II. H or V L The nucleic acid composition of the polynucleotide sequence of the region.

[0265] Table II: V of antibodies recognizing mutant misfolded, misassembled or aggregated TTR forms and / or fragments thereof H and V L Polynucleotide sequence of the region

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272] 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, which however do not substantially affect the biological activity of the antibody. To provide a common human antibody, the nucleotide and amino acid sequences of the original clones may be aligned and adjusted according to the relevant human germ cell line variable region sequences in the database (see, e.g., Vbase2, as described above). The amino acid sequences of the human antibodies are shown in bold, when the amino acids at the N-terminus and C-terminus are considered to potentially deviate from the common germ cell line sequence due to PCR primers and are therefore replaced by primer-induced mutation correction (PIMC), see Table III. Therefore, in one embodiment of the present 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 polynucleotide sequence of the corresponding VL region of an anti-TTR antibody as shown in Table II.

[0273] Table III: V of antibodies recognizing mutated, misfolded, misassembled and / or aggregated TTR forms and / or fragments thereof H and V L The nucleotide sequence of the region is shown to be substituted by PIMC (bold).

[0274]

[0275] 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 contemplated by the present invention.

[0276] The polynucleotides can be produced or manufactured by any method known in the art. For example, if the nucleotide sequence of an antibody is known, the polynucleotides encoding the antibody can be assembled from chemically synthesized oligonucleotides, such as described in Kutmeier et al., BioTechniques 17 (1994), 242, which in brief involves the synthesis of overlapping oligonucleotides containing portions of the coding antibody sequence, annealing and connection of those oligonucleotides, and then amplification of the connected oligonucleotides by PCR.

[0277] Alternatively, polynucleotides encoding antibodies or antigen-binding fragments, derivatives or variants thereof can be generated from nucleic acids derived 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 can be chemically synthesized or generated from a suitable source (e.g., an antibody cDNA library or a cDNA library generated from, or nucleic acid, preferably poly A + RNA, isolated from any tissue or cell expressing a TTR-specific antibody, such as a hybridoma cell selected to express the antibody) is obtained by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence, or by cloning using an oligonucleotide probe that is specifically recognized by a particular gene sequence (e.g., a cDNA clone derived from a cDNA library encoding the antibody). The amplified nucleic acid produced by PCR can then be cloned into a replicable cloning vector using any method known in the art. Thus, in one embodiment of the invention, a cDNA encoding an antibody, immunoglobulin chain, or fragment thereof is used to generate an anti-TTR antibody.

[0278] Once the nucleotide sequence and corresponding amino acid sequence of an antibody, or antigen-binding fragment, variant or derivative thereof, is determined, the nucleotide sequence can be manipulated to generate antibodies with different amino acid sequences, e.g., to generate amino acid substitutions, deletions and / or insertions, using methods known in the art for manipulation of nucleotide sequences (e.g., recombinant DNA techniques, site-directed mutagenesis, PCR, etc., see, e.g., as described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1990) and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1998), both of which are incorporated herein by reference).

[0279] 5. Expression of Antibody Peptides

[0280] After the regulation of the isolated genetic material for providing the antibody of the present invention, or its antigen-binding fragment, variant or derivative, the polynucleotide encoding the antibody is usually inserted into an expression vector for introduction into a host cell that can be used to produce the desired amount of antibody. The recombinant expression of antibodies or their fragments, derivatives or analogs (e.g., heavy or light chains of antibodies that are bound to target molecules) is described herein. Once the polynucleotides encoding the antibody molecules of the present invention or the heavy or light chains of antibodies, or parts thereof (preferably containing heavy or light chain variable domains), the vectors used to produce antibody molecules can be prepared by recombinant DNA techniques using techniques well known in the art. Therefore, methods for preparing proteins by expressing polynucleotides containing nucleotide sequences encoding antibodies are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcription and translation control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Therefore, the present invention provides a reproducible vector comprising a nucleotide sequence encoding the antibody molecules of the present invention, or its heavy or light chains, or heavy or light chain variable domains, that can be regulated to be connected to a promoter. Such vectors may include nucleotide sequences encoding the constant region of an antibody molecule (see, e.g., International Applications WO 86 / 05807 and WO 89 / 01036; and U.S. Pat. No. 5,122,464), and the variable domain of an antibody may be cloned into such a vector for expression of an entire heavy or light chain.

[0281] The term "vector" or "expression vector" is used herein to refer to a vector that is used as a vehicle for introducing and expressing a desired gene in a host cell according to the present invention. As known to those skilled in the art, such a vector can be easily selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, a vector compatible with the present invention will contain a selective marker, promote the cloning of the desired gene and promote appropriate restriction sites for the ability to enter eukaryotic or prokaryotic cells and / or replicate in eukaryotic or prokaryotic cells. For the purposes of the present invention, many expression vector systems can be used. For example, a class of vectors utilizes DNA elements 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 a polycistronic system with an internal ribosome binding site. In addition, cells with DNA integrated into their chromosomes can be selected by introducing one or more markers that allow the selection of transfected host cells. The marker can provide prototrophy to auxotrophic hosts, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. The selective marker gene can be directly connected to the DNA sequence and expressed, or imported into the same cell by co-transformation. Other elements for optimal synthesis of mRNA may also be required. These elements may include signal sequences, splicing signals, and transcriptional promoters, enhancers, and termination signals.

[0282] In a particularly preferred embodiment, as discussed above, the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (preferably human). The vector contains a cytomegalovirus promoter / enhancer, a mouse beta globulin major promoter, an SV40 origin of replication, a bovine growth hormone polyadenylation sequence, neomycin phosphotransfer exon 1 and exon 2, a dihydrofolate reductase gene and a leader sequence. The vector has been found to result in very high levels of expression of antibodies based on the variable and constant region genes being incorporated, transfected in CHO cells, followed by selection with G418 containing medium and methotrexate amplification. Of course, any expression vector capable of initiating expression in eukaryotic cells may be used in the present invention. Examples of suitable vectors include, but are not limited to, 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, California) and plasmid pCI (available from Promega, Madison, Wisconsin). In general, screening large numbers of transformed cells expressing appropriately 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. Patents 5,736,137 and 5,658,570, each of which is incorporated herein by reference. The system provides high expression levels, for example, >30 pg / cell / day. Other exemplary vector systems are disclosed, for example, in U.S. Patent 6413777.

[0283] In other preferred embodiments, the antibodies of the present invention, or their Fabs, variants or derivatives can be expressed using polycistronic constructs such as disclosed in U.S. Patent Application 2003-0157641A1 and incorporated herein. In these expression systems, multiple gene products of interest (e.g., heavy and light chains of antibodies) can be prepared by a single polycistronic construct. These systems advantageously use internal ribosome entry sites (IRES) to provide relatively high levels of antibodies. Compatible IRES sequences are disclosed in U.S. Patent 6193980, which are also incorporated herein. Those skilled in the art will appreciate that such expression systems can be used to effectively produce the full range of antibodies disclosed in this application. Therefore, in one embodiment, the invention provides a vector comprising a polynucleotide encoding at least a binding domain or variable region of an antibody immunoglobulin chain, optionally in combination with a polynucleotide of a variable region of other immunoglobulin chains of the binding molecule encoded.

[0284] More generally, once the vector or DNA sequence encoding the antibody monomer subunit has been prepared, the expression vector can be introduced into an appropriate host cell. Introduction of the plasmid into the host cell can be accomplished by various techniques known to those skilled in the art. These include, but are not limited to, including the use of lipofection (e.g. The expression construct is expressed in a variety of ways, such as by transfection of liposomes (e.g., plasmids), natural plastid fusion, calcium phosphate precipitation, cell fusion enveloped DNA, microinjection, and infection with intact viruses. Typically, the plasmid is introduced into the host by standard calcium phosphate coprecipitation. The host cells carrying the expression construct are grown under conditions suitable for producing light and heavy chains, and heavy and / or light chain protein synthesis is measured. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence activated cell sorting analysis (FACS), immunohistochemistry, and the like.

[0285] The expression vector is transferred to the host cell by conventional techniques, and then the transfected cells are cultured by conventional techniques to produce the antibody used in the method described herein. Therefore, the present invention includes a host cell containing polynucleotides encoding the antibody of the present invention, or its heavy chain or light chain, or at least the binding domain of its immunoglobulin or the variable region, and the polynucleotides are preferably regulated to be connected to a heterologous promoter. In addition or alternatively, the present invention also includes a host cell containing a polynucleotide encoding at least the binding domain or the variable region of the antibody immunoglobulin chain as defined above, optionally in combination with polynucleotides encoding the variable region of other immunoglobulin chains of the binding molecule. In a preferred embodiment for expressing double-chain antibodies, a single vector or multiple vectors encoding both heavy and light chains can be co-expressed in a host cell for expressing a whole immunoglobulin molecule, as described in detail below.

[0286] Host cells can be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. Both vectors may contain the same selectable marker, which enables equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used to encode both heavy and light chain polypeptides. In this case, the light chain is advantageously placed before the heavy chain to avoid excess toxic free heavy chain; see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad. Sci. USA 77 (1980), 2197. The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

[0287] As used herein, "host cell" refers to a cell carrying a vector constructed using recombinant DNA technology and encoding at least one heterologous gene. In the description of the process of isolating antibodies from a recombinant host, the terms "cell" and "cell culture" are used interchangeably to represent the source of the antibody, unless explicitly specified. In other words, recovering polypeptides from "cells" may mean recovering intact cells from spin-downs, or from cell cultures containing culture media and suspended cells.

[0288] A variety of host-expression vector systems can be used to express the antibody molecules used in the methods described herein. Such host-expression systems represent vectors by which the coding sequence of interest can be produced and subsequently purified, but also represent cells that can express the antibody molecules of the invention in situ when transformed or transfected with the appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms, such as bacteria (e.g., Escherichia coli, Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus CaMV; tobacco mosaic virus TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, NS0, BLK, 293, 3T3 cells) carrying recombinant expression constructs containing promoters from mammalian cell genomes (e.g., metallothionein promoters) or promoters from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). Preferably, bacterial cells such as E. coli, more preferably eukaryotic cells, especially for the expression of whole recombinant antibody molecules, are used for the expression of recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, conjugated with vectors such as the major intermediate early gene promoter element from human cytomegalovirus, are effective expression systems for antibodies; see, for example, Foecking et al., Gene 45 (1986), 101; Cockett et al., Bio / Technology 8 (1990), 2.

[0289] Host cell lines used for protein expression are usually of mammalian origin; the art of human origin is considered to have the ability to preferentially determine which are the most suitable specific host cell lines for the expression of the desired gene product. Exemplary host cell lines include, but are not limited to, CHO (Chinese hamster ovary), DG44 and DUXB11 (Chinese hamster ovary cell lines, DHFR negative), HELA (human cervical carcinoma), CVI (monkey kidney cell line), COS (derivative of CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, WI38, R1610 (Chinese hamster fibroblasts) BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney cell line), SP2 / O (mouse myeloma) P3X63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes) and 293 (human kidney). CHO and 293 cells are particularly preferred. Host cell lines are commonly available from commercial services, the American Tissue Culture Collection, or from published literature.

[0290] In addition, host cell strains can be selected to regulate the expression of inserted sequences, or to modify and process those of the desired gene products in a desired specific manner. 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 post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure the correct modification and processing of the exogenous proteins expressed. For this purpose, eukaryotic host cells with cellular machinery for the primary transcription, glycosylation, and appropriate phosphorylation of gene products can be used.

[0291] For the long-term, high-yield production of recombinant proteins, stable expression is preferred.For example, the cell line of stably expressed antibody molecules can be designed.Host cells can be transformed with DNA and selective markers controlled by suitable expression control elements (for example, promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.), rather than using expression vectors containing viral replication sources.After introducing foreign DNA, the cells of design can be grown in enriched medium for 1-2 days, then transferred to selective medium.The selective marker in the recombinant plasmid gives resistance to selection, and allows cells to stably integrate plasmids into their chromosomes and grow to form the focus / foci that can in turn be cloned and expanded into cell lines.This method can be advantageously used to design the cell line of stably expressed antibody molecules.

[0292] A number of selection systems can be used, including but not limited to herpes simplex virus thymidine kinase (Wigler et al., Cell 11 (1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48 (1992), 202), and adenine phosphoribosyltransferase (Lowy et al., Cell 22 (1980), 817) genes can be used in tk-, hgprt- or aprt- cells, respectively. In addition, antimetabolite resistance can be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77 (1980), 357; O'Hare et al., Proc. Natl. Acad. Sci. USA 78 (1981), 1527); gpt, which confers resistance to mycophenolic acid (Mulligan and 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 hygromycin resistance (Santerre et al., Gene 30 (1984), 147).Methods generally known and useful in the field of recombinant DNA technology are described in Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, New York (1990); and in Chapters 12 and 13 of Dracopoli et al., eds., Current Protocols in Human Genetics, John Wiley & Sons, New York (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated herein by reference.

[0293] The expression level of antibody molecules can be increased by vector amplification. For a review, see Bebbington and Hentschel, "The Use of Gene Amplification Vectors for Expression of Cloned Genes in Mammalian Cells Based on DNA Cloning", Academic Press, New York, Volume 3, (1987). When the marker in the vector system expressing the antibody is amplifiable, increasing the level of inhibitor in the host cell culture will increase the copy number of the marker gene. Because the amplified region is associated with the antibody gene, the amount of antibody produced will also increase; see Crouse et al., Mol. Cell. Biol. 3 (1983), 257.

[0294] In vitro preparation allows for scale-up to provide large quantities of the desired polypeptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include homogeneous suspension cultures, such as in airlift reactors or continuous stirred reactors, or immobilized or embedded cell cultures, such as in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and / or desired, solutions of the polypeptide may be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose or (immuno) affinity chromatography, for example following biosynthesis of the preferred synthetic hinge region polypeptides, or before or after the HIC chromatography step described herein.

[0295] The genes encoding the antibodies, or antigen-binding fragments, variants or derivatives thereof of the invention may also be expressed in non-mammalian cells such as bacteria or insect or yeast or plant cells. Bacteria that can readily take up nucleic acids include members of the Enterobacteriaceae family, such as E. coli or Salmonella strains; Bacillaceae, such as B. 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. Where a tetravalent form of the antibody is desired, the subunits will then self-assemble into a tetravalent antibody; see, for example, International Application WO02 / 096948.

[0296] In bacterial systems, a number of expression vectors may be advantageously selected, depending on the intended use of the antibody molecule being expressed. For example, when large quantities of such proteins are to be produced, vectors that direct the expression of high levels of fusion protein products that are easily purified may be desirable for the production 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), in which the antibody coding sequence can be separately linked to a vector designed with (in frame with) the lacZ coding region for fusion protein production; pIN vectors (Inouye and Inouye, Nucleic Acids Res. 13 (1985), 3101-3109; Van Heeke and Schuster, J. Biol. Chem. 24 (1989), 5503-5509); and the like. pGEX vectors can also be used to express exogenous polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to a glutathione-agarose bead matrix (followed by elution in the presence of free glutathione). pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0297] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae or common baker's yeast is the most commonly used of the eukaryotic microorganisms, although many other strains are generally available, for example, Pichia pastoris. For expression in Saccharomyces, for example, 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 selective marker for yeast mutants such as ATCC No. 44076 or PEP4-1 (Jones, Genetics 85 (1977), 12) that lack the ability to grow in tryptophan. The presence of trp1 damage as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth under conditions lacking tryptophan.

[0298] In insect systems, Autographa californicanuclear polyhedrosis virus (AcNPV) is often used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. Antibody coding sequences can be cloned separately into non-essential regions of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0299] Once the antibody molecules of the invention are recombinantly expressed, the whole antibodies, dimers thereof, individual light and heavy chains or other immunoglobulin forms of the invention may be purified according to standard procedures in the art, including, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly by affinity for the specific antigen following protein A, and size exclusion column chromatography), centrifugation, differential solubility, such as ammonium sulfate precipitation, or by any other standard technique for purifying proteins; see, e.g., Scopes, Protein Purification, Springer Verlag, New York, (1982). Alternatively, preferred methods for improving the affinity of the antibodies of the invention are disclosed in U.S. Patent Publication No. 2002-0123057A1. In one embodiment, therefore, the invention also provides a method for preparing an anti-TTR antibody or an antibody or immunoglobulin chain thereof that recognizes a mutated, misfolded, misassembled and / or aggregated TTR form or fragment thereof, the method comprising:

[0300] (a) culturing a host cell as defined above, the cell comprising a polynucleotide or a vector as defined above; and

[0301] (b) isolating the antibody or immunoglobulin chain thereof from the culture.

[0302] Furthermore, in one embodiment, the present invention also relates to an antibody or an immunoglobulin chain thereof encoded by the polynucleotide defined above or obtainable by the method for preparing an anti-TTR antibody.

[0303] 5. Fusion proteins and conjugates

[0304] In certain embodiments, the antibody polypeptide comprises an amino acid sequence or one or more parts that are not usually associated with an antibody. Exemplary modifications are described in more detail below. For example, a single-chain Fv antibody fragment of the present invention may comprise a flexible linking sequence, or may be modified to add a functional part (e.g., PEG, a drug, a toxin, or a marker such as fluorescence, radioactivity, an enzyme, nuclear magnetism, a heavy metal, etc.).

[0305] The antibody polypeptides of the present invention may comprise, consist essentially of, or consist of a fusion protein. A fusion protein is a chimeric molecule comprising, for example, an immunoglobulin TTR binding domain having at least one target binding site and at least one heterologous portion (i.e., a portion that is not naturally associated therewith in nature). The amino acid sequences may normally be present in separate proteins that are brought together in the fusion polypeptide, or they may normally be present in the same protein but are placed in a new arrangement in the fusion polypeptide. Fusion proteins can be created, for example, by chemical synthesis, or by creating and translating polynucleotides (in which peptide regions are encoded in a desired relationship).

[0306] The term "heterologous" as applied to a polynucleotide or polypeptide means that the polynucleotide or polypeptide is derived from an entity that is distinct from the remaining entity to which it is compared. For example, as used herein, a "heterologous polypeptide" to be fused to an antibody or antigen-binding fragment, derivative, variant or analog thereof is a non-immunoglobulin polypeptide from the same species, or an immunoglobulin or non-immunoglobulin polypeptide from a different species. As discussed in more detail elsewhere herein, the antibodies, or antigen-binding fragments, variants or derivatives thereof of the present invention can be further recombinantly fused to a heterologous polypeptide at the N-terminus or C-terminus, or chemically conjugated (including covalent and non-covalent conjugation) to a polypeptide or other composition. For example, an antibody can be recombinantly fused or conjugated to a molecule that can be used as a marker in a detection assay and to an effector molecule such as a heterologous polypeptide, drug, radionuclide or toxin; see, for example, International Application No. WO92 / 08495; WO91 / 14438; WO89 / 12624; U.S. Pat. No. 5,314,995; and European Patent Application No. EP 0 396 387.

[0307] The antibodies of the present invention, or their antigen-binding fragments, variants or derivatives can be composed of amino acids connected to each other by peptide bonds or modified peptide bonds (i.e., peptide isosteres), and can contain other amino acids in addition to the 20 gene-encoded amino acids. Antibodies can be modified by natural processes, such as post-translational processing, or by chemical modification techniques known in the art. Such modifications are fully described in basic textbooks and more detailed monographs as well as a large number of research documents. Modifications can occur in any part of the antibody, including peptide backbones, amino acid side chains and amino or carboxyl ends, or in parts such as sugars. It should be understood that the same type of modification can be present in several sites of a given antibody to the same or different extents. In addition, a given antibody can contain many types of modifications. Antibodies can be branched, for example, as a result of ubiquitination, and they can be cyclic, with or without branches. Cyclic, branched and branched cyclic antibodies can be obtained from natural processes after translation, or can be prepared by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of 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 a 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, T.Creighton, W.H. Freeman and Company, New York, 2nd ed., (1993); Posttranslational Covalent Modification Of Proteins, BC Johnson (ed.), Academic Press, New York, (1983) 1-12; Seifter et al., Meth. Enzymol. 182 (1990), 626-646; Rattan et al., Ann. NY Acad. Sci. 663 (1992), 48-62).

[0308] The present invention also provides a fusion protein comprising an antibody, or an antigen binding fragment, variant or derivative thereof and a heterologous polypeptide. In one embodiment, the fusion protein of the present invention comprises, consists essentially of, or consists of an antibody of the present invention or any one or more V Hor a polypeptide having the amino acid sequence of the antibody of the present invention or any one or more V L In another embodiment, the fusion protein used in the diagnosis and treatment methods disclosed herein comprises, consists essentially of, or consists of any one, two, or three V regions of an antibody. H -CDR amino acid sequence of a polypeptide, or a fragment, variant or derivative thereof, or any one, two or three V L -CDR amino acid sequence of polypeptide, or its fragment, variant or derivative, and heterologous polypeptide sequence. In one embodiment, the fusion protein comprises the antibody V of the present invention. H -CDR3 of the present invention, or a fragment, derivative or variant thereof, and a heterologous polypeptide sequence, wherein the fusion protein specifically binds to TTR. In another embodiment, the fusion protein comprises at least one V H The amino acid sequence of the region and having at least one V L The fusion protein may be a polypeptide having an amino acid sequence of the amino acid region of the fusion protein, or a fragment, derivative or variant thereof, as well as a heterologous polypeptide sequence. H and V L The region corresponds to a single source antibody (or scFv or Fab fragment) that specifically binds to TTR. However, in another embodiment, the fusion protein used in the diagnostic and therapeutic methods disclosed herein includes any one, two, three or more V regions of an antibody. H -The amino acid sequence of the CDR and any one, two, three or more V L -CDR amino acid sequence, or a fragment, variant or derivative thereof, as well as a heterologous polypeptide sequence. Preferably, two, three, four, five, six or more V H -CDR or V L - CDRs correspond to the single-source antibodies (or scFv or Fab fragments) of the present invention. Nucleic acid molecules encoding these fusion proteins are also included in the present invention.

[0309] Exemplary fusion proteins reported in the literature include fusions of T cell receptors (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 (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 (Stamenkovic et al., Cell 66 (1991), 1133-1144); TNF receptor (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 (IgE receptor) (Ridgway and Gorman, J. Cell. Biol. 115 (1991), Abstract No. 1448).

[0310] As discussed elsewhere herein, the antibodies of the invention, or antigen-binding fragments, variants or derivatives thereof, can 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 invention to increase their in vivo half-life; 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.

[0311] In addition, the antibody of the present invention or its Fab, variant or derivative 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), such as the marker provided in the pQE vector (QIAGEN, 9259 Eaton Street, Chatsworth, California, 91311), etc., many of which are commercially available. As described in Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86 (1989), 821-824, for example, hexa-histidine provides a convenient purification of the fusion protein. Other peptide tags used for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope from the influenza hemagglutinin protein (Wilson et al., Cell 37 (1984), 767), GST, C-mycand "flag" tag; see, e.g., Bill Brizzard, BioTechniques 44 (2008) 693-695 for a review of epitope tagging technology, and Table 1 on page 694 lists the most common epitope tags used in the present invention, the subject matter of which is incorporated by reference.

[0312] Fusion proteins can be prepared using methods known in the art; see, for example, U.S. Patents 5,116,964 and 5,225,538. The precise site at which the fusion occurs can be selected empirically to optimize the secretion or binding characteristics of the fusion protein. The DNA encoding the fusion protein is then transfected into a host cell for expression, which is performed as described above.

[0313] The antibodies of the present invention can be used in an unconjugated form, or can be coupled to at least one of a variety of molecules, for example, to enhance the therapeutic properties of the molecule, to facilitate target detection, or for imaging or treatment of patients. When purified, the antibodies of the present invention, or their antigen-binding fragments, variants or derivatives can be labeled or conjugated before or after purification. In particular, the antibodies of the present invention, or their antigen-binding fragments, variants or derivatives can be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.

[0314] Conjugates of immunotoxins comprising conventional antibodies have been extensively described in the art. The toxins can be coupled to the antibodies by conventional coupling techniques, or immunotoxins containing protein toxin portions can be prepared as fusion proteins. The antibodies of the invention can be used in a corresponding manner to obtain such immunotoxins. Representative immunotoxins of this type are those described by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and Fanger, Immunol. Today 12 (1991), 51-54.

[0315] Those skilled in the art will appreciate that conjugates may also be assembled using a variety of techniques, depending on the selected reagent to be conjugated. For example, a conjugate with biotin is prepared by, for example, reacting a TTR binding polypeptide with an activated ester of biotin (e.g., 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 with fluorescein-isothiocyanate. Conjugates of antibodies, or antigen-binding fragments, variants, or derivatives thereof of the present invention are prepared in a similar manner.

[0316] The invention further includes an antibody of the invention, or an antigen-binding fragment, variant or derivative thereof, conjugated to a diagnostic or therapeutic agent. The antibody can be used for diagnosis, for example, to demonstrate the presence of TTR amyloidosis, to indicate the risk of developing a disease or condition associated with misfolded, misassembled or aggregated TTR, to monitor the development or progression of such a disease (i.e., a disease that shows the occurrence of misfolded, misassembled or aggregated TTR, or a disease associated with misfolded, misassembled or aggregated TTR), or as part of a clinical testing procedure to, for example, determine the efficacy of a given treatment and / or prevention regimen. In one embodiment, the invention thus relates to an antibody that is detectably labeled. Furthermore, in one embodiment, the invention relates to an antibody that is linked to a drug. Detection can be facilitated by coupling the antibody, or antigen-binding fragment, variant or derivative thereof, 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 used in various positron emission tomographs, and nonradioactive paramagnetic metal ions; see, e.g., U.S. Pat. No. 4,741,900, for metal ions that can be conjugated to antibodies used as diagnostics according to the 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, dichlorofluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol (aminophthalhydrazide); examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable radioactive materials include 125 I, 131 I, 111 In or 99 Tc. Thus, in one embodiment, the present 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.

[0317] The antibody or its antigen-binding fragment, derivative or variant can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent labeled antibody is then determined by detecting the presence of luminescence generated during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol (aminophthalhydrazide), isoluminol, theromatic acridinium esters, imidazoles, acridinium salts and oxalate esters.

[0318] One of the ways in which an antibody or antigen-binding fragment, derivative or variant thereof can be detectably labeled is to link the antibody or antigen-binding fragment, derivative or variant thereof to an enzyme and use the linked product in an enzyme immunoassay (EIA) (Voller, A., The Enzyme Linked Immunosorbent Assay (ELISA) Microbiological Associates Quarterly Publication, Walkersville, Maryland, Diagnostic Horizons 2 (1978), 1-7; Voller et al., J. Clin. Pathol. 31 (1978), 507-520; Butler, Meth. Enzymol. 73 (1981), 482-523; Maggio, ed., Enzyme Immunoassay, CRC Press, Boca Raton, Florida, (1980); Ishikawa et al., eds., Enzyme Immunoassay, Kgaku et al., Enzyme Immunoassay, Shoin, Tokyo (1981). The enzyme bound to the antibody will react with a suitable substrate, preferably a chromogenic substrate, in such a way as to produce a detectable chemical moiety, for example by spectrophotometry, fluorescence or by visible (light) means. Enzymes that can be used to detectably label the antibody 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, the detection can be accomplished by a colorimetric method in which a chromogenic substrate for the enzyme is used. Detection can also be accomplished by visual comparison of the extent of enzymatic reaction with a similarly prepared standard comparison substrate.

[0319] Detection can also be achieved using any of a variety of other immunoassays. For example, by radiolabeling an antibody, or an antigen-binding fragment, variant, or derivative thereof, it is possible to detect the antibody using a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, (March, 1986), which is incorporated herein by reference). The radioisotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or an autoradiographic apparatus.

[0320] Antibodies or antigen-binding fragments, derivatives or variants thereof may also be used, for example 152 Antibodies can be detectably labeled with fluorescent emitting metals of Eu or other lanthanides. These metals can be attached to antibodies using metal chelating groups such as diethylenetriacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0321] The 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 Reisfeld et al., ed., Monoclonal Antibodies And Cancer Therapy, pp. 243-56 (Alan R. Liss Ltd. (1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (Second Edition), Robinson et al. (eds.), Marcel Dekkery Ltd., (1987) pp. 623-53; Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications (Monoclonal Antibodies '84: Biological and Clinical Applications), Pinchera et al., eds., (1985) pp. 475-506; Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy (Analysis, Results and Prospects of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy), in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al., eds., Academic Press (1985) pp. 303-16, and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62 (1982), pp. 119-158.

[0322] As mentioned, in certain embodiments, a moiety that enhances the stability or efficacy of a binding molecule, such as a binding polypeptide, such as an antibody or immunospecific fragment thereof, can be conjugated. For example, in one embodiment, PEG can be conjugated to a binding molecule of the 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.

[0323] VI. Composition and method of use

[0324] The present invention relates to a composition comprising a TTR binding molecule as described above, for example, an antibody of the invention, or an antigen binding fragment, variant or derivative thereof, or a polynucleotide, vector, cell or peptide as defined above. In one embodiment, the composition of the invention is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition of the invention may comprise an additional agent such as an interleukin or an interferon, depending on the intended use of the pharmaceutical composition. For the treatment of diseases and disorders that show the occurrence of mutated, misfolded, misassembled or aggregated TTR (e.g., TTR amyloidosis) or diseases and disorders associated with mutated, misfolded, misassembled or aggregated TTR (e.g., TTR amyloidosis), the additional agent may be selected from the group consisting of small organic molecules, anti-TTR antibodies and combinations thereof. Thus, in a particularly preferred embodiment, the invention relates to the use of a TTR binding molecule, e.g., an antibody or antigen-binding fragment thereof of the invention, or a binding molecule having substantially the same binding specificity as any of the same, a polynucleotide, vector or cell of the invention, for the preparation of a pharmaceutical or diagnostic composition for preventing and treating a disease or condition associated with TTR amyloidosis, monitoring the progression of a disease or condition associated with TTR amyloidosis or a subject's response to a treatment for TTR amyloidosis or determining a subject's risk of developing a disease or condition associated with TTR amyloidosis.

[0325] Thus, in one embodiment, the invention relates to a method of treating a disease or disorder characterized by abnormal accumulation and / or deposition of TTR and / or mutated, misfolded, misassembled or aggregated TTR in affected systems and organs, such as the peripheral nervous system, autonomic nervous system, central nervous system, gastrointestinal system, vascular system, in particular the leptomeninges, lymphatic system, in particular lymph nodes, musculoskeletal system, in particular tendons and ligaments, heart, eyes, kidneys, lungs, skin, tongue, thyroid and bladder, the method comprising administering to a subject in need thereof a therapeutically effective amount of any of the aforementioned TTR binding molecules, antibodies, polynucleotides, vectors, cells or peptides of the invention.

[0326] A particular advantage of the therapeutic methods 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 of healthy human subjects who have no signs or symptoms of disease, such as carrying asymptomatic mutations and / or mutations, showing the occurrence of aggregated TTR or associated with aggregated TTR, and are therefore, with a certain probability, capable of preventing clinically manifested diseases associated with misfolded, misassembled, mutated, and / or aggregated TTR, or reducing the risk of occurrence of clinically manifested diseases or conditions, or delaying the onset or progression of clinically manifested diseases or conditions. Typically, the antibodies of the present invention have also been successfully optimized for selectivity and effectiveness with respect to high affinity binding to the target TTR molecule by means of somatic maturation, i.e., by somatic variation of the variable regions of the antibodies.

[0327] The knowledge that such cells have not been activated in vivo (e.g., in humans) in the sense of an autoimmune or allergic reaction by means of related or other physiological proteins or cell structures is of great medical importance, as it means a significantly increased chance of successfully passing the clinical trial phase. It can be said that efficacy, acceptability and tolerability have been demonstrated in at least one human subject prior to preclinical and clinical development of a prophylactic or therapeutic antibody. Therefore, it is expected that the human anti-TTR antibodies of the present invention, both their target structure-specific efficacy as therapeutic agents and their reduced chance of side effects, significantly increase their likelihood of clinical success.

[0328] The present invention also provides pharmaceutical and diagnostic packaging or kits comprising one or more containers filled with one or more of the above ingredients, such as anti-TTR antibodies, binding fragments, derivatives or variants thereof, polynucleotides, vectors, cells and / or peptides of the present invention. Associated with such containers may be a notice issued by a governmental agency regulating the production, use or sale of drugs or biological products, which reflects approval for human administration by the agency regulating production, use or sale. Additionally or alternatively, the kit contains reagents and / or instructions for use in appropriate diagnostic assays. The compositions, e.g., kits of the invention are of course particularly suitable for risk assessment, diagnosis, prevention and treatment of diseases or conditions associated with the presence of mutated, misfolded, misassembled and / or aggregated TTR, and are particularly suitable for treating a wide range of conditions with TTR amyloidosis, including, 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 ligamentous amyloidosis (including carpal tunnel syndrome, rotator cuff tears and lumbar spinal stenosis) and pre-eclampsia.

[0329] The pharmaceutical compositions of the present invention can be formulated according to methods known in the art; see, for example, Remington: The Science and Practice of Pharmacy, the University of Sciences in Philadelphia, ISBN 0-683-306472. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline, water, emulsions (such as oil / water emulsions), various types of wetting agents, sterile solutions, and the like. Compositions including such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to a subject at a suitable dose. Administration of suitable compositions can be achieved in different ways, such as by intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, topical or intradermal administration or spinal or brain delivery. Aerosol formulations such as nasal spray formulations contain purified aqueous or other solutions of active agents with preservatives and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucosa. Formulations for rectal or vaginal administration can be presented as suppositories with suitable carriers.

[0330] The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage of any patient depends on many factors, including the patient's size (weight, size), body surface area, age, specific compound to be administered, sex, administration time and route, general health, and other drugs given simultaneously. Typical dosages can be, for example, within the range of 0.001 to 1000 micrograms (or within this range of nucleic acid expression or inhibition of expression); however, it is conceivable that the dosage is lower or higher than this exemplary range, especially considering the aforementioned factors. Generally, the dosage range can be, for example, from about 0.0001 to 100 mg / kg by host body weight, more generally 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, the dosage can be 1 mg / kg body weight or 10 mg / kg body weight or within the range of 1-10 mg / kg, preferably at least 1 mg / kg. Dosages in the middle of the above ranges are also meant to be within the scope of the present invention. Subjects can be given such doses every day, every other day, every week, or according to any other schedule determined by empirical analysis. Exemplary treatment requires the administration of multiple doses over an extended period of time, for example, at least six months. Other exemplary treatment regimens require administration once every two weeks or once a month or once every 3 to 6 months. Exemplary dosage regimens include 1-10 mg / kg or 15 mg / kg for several consecutive days, 30 mg / kg every other day or 60 mg / kg per week. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, and the dose of each antibody administered in this case falls within the range shown. Progress can be monitored by regular assessment. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / 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, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases. In addition, the pharmaceutical compositions of the present invention may contain other agents such as dopamine or psychopharmacological drugs, depending on the intended use of the pharmaceutical composition.

[0331] Furthermore, in a preferred embodiment of the present invention, the pharmaceutical composition can be formulated as a vaccine, for example, if the pharmaceutical composition of the present invention comprises a TTR antibody or antigen-binding fragment, derivative or variant thereof for passive immunization. As mentioned in the background section, misfolded, misassembled, mutated and / or aggregated TTR forms or fragments or derivatives thereof are the main triggers of TTR amyloidosis. Therefore, it is prudent to expect that passive immunization using the human anti-TTR antibodies and equivalent TTR binding molecules of the present invention will help avoid several adverse effects of the active immunotherapy concept and lead to a reduction in TTR aggregation. Therefore, the anti-TTR antibodies of the invention and their equivalents of the invention will be particularly useful as vaccines for preventing or ameliorating diseases or conditions that show the presence of aggregated TTR or are caused by aggregated TTR, 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 ligamentous amyloidosis (including carpal tunnel syndrome, rotator cuff tears and lumbar spinal stenosis) and preeclampsia.

[0332] In one embodiment, it may be beneficial to use recombinant Fab (rFAB) and single chain fragments (scFv) of the antibodies of the invention, which may more easily penetrate cell membranes. For example, Robert et al., Protein Eng. Des. Sel. (2008); S1741-0134, published online in advance, describe the use of chimeric recombinant Fab (rFAB) and single chain fragments (scFv) of monoclonal antibody WO-2, which recognize an epitope in the N-terminal region of Aβ. The engineered fragments are able to (1) prevent amyloid fibrillization, (2) decompose preformed Aβ1-42 fibers and (3) inhibit Aβ1-42 oligomer-mediated neurotoxicity in vitro, as effectively as whole IgG molecules. The perceived advantages of using small Fab and scFv engineered antibody structures lacking effector functions include more efficient passage through the blood-brain barrier and minimizing the risk of triggering inflammatory side effects. In addition, in addition to the fact that scFvs and single domain antibodies retain the binding specificity of full-length antibodies, they can be expressed as a single gene and expressed in mammalian cells as intracellular antibodies, which have the potential to alter the folding, interaction, modification, or subcellular localization of their targets; see review, e.g., Miller and Messer, Molecular Therapy 12 (2005), 394-401.

[0333] In a different approach, Muller et al., Expert Opin. Biol. Ther. (2005), 237-241, describe a technology platform, the so-called "super antibody technology", which is said to enable antibodies to be shuttled into living cells without harming them. Such cell-penetrating antibodies open new windows for diagnosis and treatment. The term "TransMabs' has been coined for these antibodies.

[0334] In further embodiments, co-administration or sequential administration of other antibodies for treating diseases or conditions associated with the occurrence of mutated, misfolded, misassembled and / or aggregated TTR may be desirable. In one embodiment, additional antibodies are included in the pharmaceutical compositions of the invention. Examples of antibodies that can be used to treat a subject include, but are not limited to, antibodies targeting CD33, SGLT2, IL-6, and IL-1.

[0335] In further embodiments, co-administration or sequential administration of other agents for treating diseases or conditions associated with mutant, misfolded, misassembled and / or aggregated TTR may be desirable. In one embodiment, the additional agent is included in the pharmaceutical composition of the invention. Examples of agents that can be used to treat a subject include, but are not limited to: agents that stabilize TTR tetramers, such as Tafamidis, methylglucamine, diflusinal, doxycycline and ursodeoxycholic acid; anti-inflammatory agents such as diflusinal, corticosteroids, 2-(2,6-dichloroanilino)phenylacetic acid (diclofenac), isobutylpropionic acid-phenolic acid (ibuprofen); diuretics, epicatechin esters, melphalan hydrochloride, dexamethasone, bortezomib, bortezomib-melphalan, bortezomib-dexamethasone Methazone, melphalan-dexamethasone, bortezomib-dexamethasone-melphalan; antidepressants, antipsychotics, neuroleptics, antidementia drugs (such as NMDA-receptor antagonist memantine), acetylcholinesterase inhibitors (such as donepezil, HCI, rivastigmine, galantamine), glutamate antagonists and other nootropics antihypertensive drugs (such as dihydralazine, methyldopa), cytostatics, glucocorticoids, angiotensin converting enzyme (ACE) inhibitors; anti-inflammatory agents or any combination thereof. Examples of agents that can be used to treat or prevent organ rejection after clinical organ transplantation include, but are not limited to, a group of agents that cause a weakening of the immune system, i.e., immunosuppression, including, for example, calcineurin inhibitors, such as cyclosporine and tacrolimus, expansion inhibitors such as mTOR inhibitors, including everolimus and sirolimus (rapamycin), and antimetabolites, such as azathioprine, mycophenolate mofetil / MMF and mycophenolic acid, as well as corticosteroids such as cortisone, cortisol, and synthetic substances such as prednisone or prednisolone can all be used. In addition, antibodies may be used, such as anti-IL2 receptor monoclonal antibodies (e.g., basiliximab, daclizumab) and anti-CD3 monoclonal antibodies (e.g., muromonab CD3), and polyclonal compositions such as anti-thymocyte globulin (ATG); and glucagon-like peptide-1 (GLP-1) receptor agonists (see, e.g., Noguchi et al., Acta Med.Okayama, 60 (2006), and International Application WO2012 / 088157). In addition, additional agents may include agents for the prevention and or treatment of infection 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 further comprising an additional agent for treating TTR amyloidosis and / or treating or preventing organ rejection, e.g., following liver transplantation, is provided. Examples of other agents that can be used with the pharmaceutical compositions of the invention are described in the art; see, e.g., International Applications WO2009005672, WO2010128092, WO2012088157 or European Application EP11158212.8.

[0336] The therapeutically effective dose or therapeutically effective amount refers to the amount of active ingredient sufficient to improve the symptoms or conditions. The efficacy and toxicity of such compounds can be determined by standard pharmaceutical methods in cell culture or experimental animals, for example, ED 50 (the dose that is therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio LD 50 / ED 50 .

[0337] From the above, it is evident that the present invention encompasses any use of a TTR binding molecule and / or fragment thereof comprising at least one CDR of the above-mentioned antibodies, in particular for the diagnosis and / or treatment of a disease or condition associated with a mutated, misfolded, misassembled and / or aggregated TTR form or fragment thereof as mentioned above, such as TTR amyloidosis. Preferably, the binding molecule is an antibody of the present invention or an immunoglobulin chain thereof. Furthermore, the present invention relates to anti-idiotypic antibodies to any of the antibodies described above. These are antibodies or other binding molecules that bind to unique antigenic peptide sequences of the variable region of an antibody located near the antigen binding site, and they are useful, for example, for the detection of anti-TTR antibodies in a sample obtained from a subject. In one embodiment, the invention thus provides an antibody as defined above and below, or a TTR binding molecule having substantially the same binding specificity as any of them, a polynucleotide, vector or cell as defined herein, or a pharmaceutical or diagnostic composition comprising any of them for use in the prophylactic treatment, therapeutic treatment and / or monitoring the progression or response to treatment of a disease or condition associated with TTR, preferably wherein the disease is selected from the group comprising, 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 ligamentous amyloidosis (including carpal tunnel syndrome, rotator cuff tear and lumbar spinal stenosis) and pre-eclampsia. The above group of diseases or conditions will be referred to as the group of conditions associated with TTR amyloidosis.

[0338] In another embodiment, the present invention relates to a diagnostic composition comprising any of the above-mentioned TTR binding molecules, antibodies, antigen-binding fragments, polynucleotides, vectors, cells and / or peptides of the present invention and optionally suitable means for detection such as reagents conveniently used in immunological or nucleic acid-based diagnostic methods. The antibodies of the present invention are, for example, suitable for use in immunoassays, in which they can be used in liquid phase or bound to a solid phase support. Examples of immunoassays using the antibodies of the present invention are competitive and non-competitive immunoassays in direct or indirect form. Examples of such immunoassays are radioimmunoassays (RIA), sandwich (immunoassays), flow cytometry, and Western blot assays. The antigens and antibodies of the present invention can be bound to many different carriers and used to isolate cells that specifically bind thereto. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses, polyacrylamide, agarose and magnetite. For the purposes of the present invention, the nature of the carrier can be either soluble or insoluble. There are many different labels and labeling methods well known to those of ordinary skill 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 above.

[0339] In a further embodiment, TTR binding molecules, in particular antibodies of the invention, can also be used in a method for diagnosing a disease or condition in an individual by obtaining a body fluid sample (which can be a blood sample, a plasma sample, a serum sample, a lymph sample or any other body fluid sample, such as a saliva or urine sample) from the test individual and contacting the body fluid sample with the antibody of the invention under conditions that allow the formation of an antibody-antigen complex. The level of this complex is then determined by methods known in the art, and a significantly higher level than that formed in a control sample indicates a disease or condition in the test individual. In the same manner, antigens specifically bound by the antibodies of the invention can also be used. Therefore, the present invention relates to an in vitro immunoassay comprising a binding molecule (e.g., an antibody of the invention or an antigen-binding fragment thereof).

[0340] In a further embodiment of the invention, TTR binding molecules, particularly antibodies of the invention, may also be used in a method for diagnosing a disease or condition in an individual by obtaining a biopsy sample (which may be a skin, salivary gland, hair root, heart, colon, nerve, subcutaneous fat biopsy sample, or a biopsy sample from any affected organ) from the test individual.

[0341] In this context, the present invention also relates to means specifically designed for this purpose. For example, antibody-based arrays may be used, which are, for example, loaded with antibodies or equivalent antigen binding molecules of the present invention that specifically recognize TTR. The design of microarray immunoassays is summarized in Kusnezow et al., Mol. Cell Proteomics 5 (2006), 1681-1696. Therefore, the present invention also relates to microarrays loaded with TTR binding molecules identified according to the present invention.

[0342] In one embodiment, the invention relates to a method for diagnosing a disease or condition associated with a mutated, misfolded, misassembled and / or aggregated TTR form and / or fragment thereof in a subject, the method comprising using at least one antibody of the invention, a TTR binding fragment thereof, or a TTR binding molecule having substantially the same binding specificity as any of the foregoing to determine the presence of TTR and / or misfolded, misassembled or aggregated TTR in a sample from a subject to be diagnosed, wherein the pathological mutated, misfolded, misassembled or aggregated TTR is indicative of TTR amyloidosis, and an increased level of the pathological mutated, misfolded, misassembled or aggregated TTR compared to the level of physiological TTR is indicative of progression of TTR amyloidosis in said subject.

[0343] The subject to be diagnosed may be asymptomatic or latent to the disease. Preferably, the control subject has a disease associated with misfolded, misassembled or aggregated TTR, such as familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC) or senile systemic amyloidosis (SSA), wherein the similarity between the level of pathologically mutated, misfolded, misassembled 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 in addition as a second control, the control subject does not have TTR amyloidosis, wherein the difference between the level of physiological TTR and / or misfolded, misassembled 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. Preferably, the subject to be diagnosed and the control subject are age matched. The sample to be analyzed can be any body fluid suspected of containing pathologically misfolded, misassembled or aggregated TTR, such as blood, plasma, serum, urine, peritoneal fluid, saliva or cerebrospinal fluid (CSF).

[0344] The level of physiological TTR and / or pathological misfolded, misassembled or aggregated TTR can be assessed by any suitable method known in the art, including, for example, analyzing TTR by one or more techniques selected from the following: Western blotting, 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 mass spectrometry (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, the in vivo imaging of TTR includes imaging, positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR) light imaging or magnetic resonance imaging (MRI).

[0345] In another aspect, the invention relates to the diagnosis of TTR amyloidosis, monitoring treatment of the disease and the diagnostic or therapeutic use of anti-TTR drugs in a method that does not require tissue and does not require biopsy tissue samples (ie, non-invasively).

[0346] Typically, the concentrations of TTR aggregates and / or misfolded TTR that can be detected in body fluids (e.g., plasma) are very low, and thus the diagnosis of TTR amyloidosis is difficult and time-consuming. In particular, the diagnosis of TTR amyloidosis is a difficult and lengthy process, as the various diseases present very similar symptoms and signs, such that the formal diagnosis of familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), and senile systemic amyloidosis (SSA) generally requires the collection of tissue biopsy samples and the identification of TTR amyloid deposits by means of complex histological staining techniques. Because the tissue biopsy samples are very small and the TTR amyloid deposits are dispersed, histological determination of TTR amyloidosis is often associated with a high frequency of false-negative results and delays for patients.

[0347] However, according to the present invention, it was surprisingly shown that the detection of aggregated and / or misfolded TTR bound to anti-TTR antibodies in the blood is possible after a single administration of anti-TTR antibodies to a subject; see Examples 13 and Fig.14. Therefore, due to the unique properties of the anti-TTR antibodies of the present invention to remove TTR from amyloid TTR deposits and transport it into the blood, a completely new method for diagnosing conditions associated with misfolded, mutated, and / or aggregated TTR in a patient or subject is developed, which has the potential to replace tissue biopsy and histological analysis in the diagnosis of TTR amyloid diseases. The method relies on the use of antibodies specific for the pathological conformation of the TTR protein, which are injected into the patient and used as a probe for the presence of misfolded and / or aggregated TTR protein anywhere in the patient's body. After the antibody is injected into the patient, after a short delay, for example 2 days, a blood sample is obtained and used to detect whether the antibody has captured and separated misfolded and / or aggregated TTR particles from TTR deposits. The main advantage of this method compared to histological methods is related to the direct injection of the anti-TTR antibody into the patient, where the blood circulation allows it to circulate through each tissue and organ and detect misfolded and / or aggregated TTR protein deposits independently of their location.

[0348] Thus, in a further aspect, the present invention relates to a method for diagnosing a disease associated with TTR amyloidosis, comprising determining the level of misfolded and / or aggregated TTR in a sample from a subject after administration of an anti-TTR antibody to the subject, wherein the presence of misfolded and / or aggregated TTR or an increased level thereof in the sample of the subject as 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. In addition, since the novel method is also useful for characterizing anti-TTR drugs and treatment processes for TTR amyloidosis, respectively, as shown in Example 13, the novel method of the present invention is also applicable to monitoring treatment of a disease using an anti-TTR antibody or determining the diagnostic or therapeutic use of a TTR antibody. In this context, one skilled in the art will recognize that the method of the present invention is not limited to studying the therapeutic applications and efficacy of anti-TTR antibodies, but is also applicable to other types of anti-TTR drugs capable of degrading TTR amyloid deposits. For example, an anti-TTR antibody of the invention can be administered in conjunction with other anti-TTR drugs, and the level of misfolded / or aggregated TTR in a sample from a subject who has been treated is compared to a control obtained from the subject before administration of both the anti-TTR antibody and the anti-TTR drug, but only after anti-TTR antibody treatment.

[0349] In a preferred embodiment of the invention, particularly when non-human animals are used for testing recombinant humanized antibodies as described in Example 13, and other anti-TTR antibodies intended for use in humans, generally, the level of misfolded and / or aggregated TTR in a sample is determined by determining the complex formed between the anti-TTR antibody and misfolded and / or aggregated TTR, such as by immunoprecipitation using anti-human IgG or anti-idiotypic antibodies. Alternatively, a second anti-TTR antibody that recognizes TTR at an epitope that is substantially different from the epitope of the candidate drug anti-TTR antibody can be used so as to bind to the complex of the candidate drug anti-TTR antibody and TTR and thereby detect its presence, such as by ELISA or immunoprecipitation.

[0350] With regard to diagnostic aspects, in particular for human subjects and patients, the presence and elevated levels of misfolded 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 the patient or subject. Thus, the methods of the present invention allow, on the one hand, the identification and determination of diseases associated with TTR amyloidosis in a subject, and, on the other hand, the removal of TTR deposits from a patient, thus also indicating the therapeutic progress of a given treatment and the efficacy of a drug specific for TTR amyloidosis, such as an anti-TTR antibody.

[0351] Thus, as demonstrated in Example 13, the anti-TTR antibodies of the invention are able to bind misfolded and / or aggregated TTR with sufficient affinity to alter the stability of pathological TTR deposits, such as capturing misfolded and / or aggregated TTR and removing misfolded and / or aggregated TTR from the deposits to body fluids, particularly blood.

[0352] The anti-TTR antibody used in accordance with the methods of the invention can be any anti-TTR antibody that is specific for a pathological conformation of TTR (i.e., misfolded, mutated, and / or aggregated TTR). However, in a preferred embodiment, the anti-TTR antibody used in the tissue-free method is an anti-TTR antibody or TTR binding molecule of the invention described herein and illustrated in the Examples.

[0353] In this regard, the anti-TTR antibodies may be modified and, for example, linked to a detectable label as described in any of the other embodiments described above. In addition, immunoassays such as 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.

[0354] As in Example 13 and Fig.14 As shown, it can be demonstrated that the anti-TTR antibodies of the present invention are capable of capturing and separating misfolded and / or aggregated TTR from TTR amyloid deposits, and the corresponding immune complexes can be measured in a body fluid sample, in particular the blood of a patient or subject; see Examples 13 and Fig.14 Thus, in one embodiment of the invention, an anti-TTR antibody can bind misfolded and / or aggregated TTR with sufficient affinity to alter the net efflux of misfolded and / or aggregated TTR from, for example, the heart, peripheral nervous system (PNS), eye, muscle, gastrointestinal tract, kidney, vascular system, and central nervous system (CNS).

[0355] A body fluid sample, preferably blood or cerebrospinal fluid (CSF) obtained from a subject (in which misfolded and / or aggregated TTR bound to the anti-TTR antibody to be captured and separated is present), is obtained at a specific time interval after administration. The specific time interval after administration is usually less than one week. In a preferred embodiment, the time interval after administration of the anti-TTR antibody is less than or equal to 48 hours.

[0356] As mentioned above, the tissue-free methods described above can also be used to determine the success of treatment by measuring the misfolded and / or aggregated TTR-type capture captured by the anti-TTR antibody before and after treatment. Therefore, in a further or additional embodiment, the tissue-free method of the present invention further comprises comparing the level of misfolded and / or aggregated TTR in the body fluid sample with the level of misfolded and / or aggregated TTR in the sample obtained from the subject before the administration of the anti-TTR antibody. Therefore, in one embodiment, the method of the present invention is used to determine the effect of treating TTR amyloidosis or to monitor the progression of a disease or condition associated with pathological TTR in a patient or subject.

[0357] As mentioned, the sample of the subject used in the above methods can be obtained before or after the administration of the anti-TTR antibody. However, the sample can also be obtained from a medical facility or a practicing physician, as well as other research institutions from which clinical samples of the subject can be obtained. The facility or physician, etc. can not only perform the administration of the anti-TTR antibody to the subject and the collection of suitable samples for the above methods, but also monitor and / or treat the patient, i.e., replace or combine the anti-TTR antibody with at least another anti-TTR antibody or therapeutic agent by changing the amount, time, frequency of administration of the antibody, interrupting treatment. The level of TTR can be assessed by any suitable method known in the art. Suitable methods are described below and in International Application WO2013 / 066818, the disclosure of which is hereby incorporated by reference herein.

[0358] In one aspect of the invention, an antibody of the invention, a TTR binding molecule having substantially the same binding specificity as any of the above, a polynucleotide, a vector or a cell as defined above, or a pharmaceutical or diagnostic composition comprising any of the above, is provided for use in prophylactic treatment, therapeutic treatment, and / or monitoring the progression of, or response to treatment of, a disease or condition associated with TTR. Thus, in general, the invention relates to a method for diagnosing or monitoring the progression of a disease or condition associated with TTR (e.g., TTR amyloidosis) in a subject, the method comprising using at least one antibody of the invention, or a TTR binding molecule having substantially the same binding specificity as any of the above, to determine the presence of TTR in a sample from a subject to be diagnosed, wherein the presence of a mutated, misfolded, misassembled or aggregated TTR species or fragments thereof is indicative of the disease or condition. In one embodiment, a method of diagnosing or monitoring the progression of TTR amyloidosis in a subject is provided, the method comprising determining the presence of mutated, misfolded, misassembled or aggregated TTR and / or fragments thereof in a sample from a 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 of the antibodies, wherein the presence of mutated, misfolded, misassembled or aggregated TTR and / or fragments thereof is indicative of presymptomatic, prodromal or clinical TTR amyloidosis, and an increase in the level of TTR oligomers, aggregates or fibrils compared to physiological TTR levels, or compared to a reference sample from a healthy control subject, or compared to a control sample from the same subject is indicative of the progression of presymptomatic, prodromal or established TTR amyloidosis. It will be appreciated by any skilled artisan that in one embodiment the method is used to diagnose or monitor the progression of any other disease or condition from the group of TTR-related conditions defined above.

[0359] As described above, the antibodies, fragments thereof, and molecules having the same binding specificity as the antibodies and fragments thereof of the present invention can be used not only in vitro but also in vivo, and diagnostic and therapeutic applications can also be performed. Therefore, 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, the TTR binding molecule being used to prepare a composition for in vivo detection / imaging or a composition for targeting therapeutic and / or diagnostic agents to TTR in the human or animal body. Potential therapeutic and / or diagnostic agents can be selected from the non-exhaustive enumeration of therapeutic agents and potential markers for treating TTR amyloidosis shown above. In terms of in vivo imaging, in a preferred embodiment, the present invention provides the TTR binding molecule comprising at least one CDR of the antibody of the present invention, wherein the in vivo imaging comprises positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR) light 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 or said molecule for the preparation of a composition for the above-mentioned specific in vivo imaging method, for use in a method of diagnosing or monitoring the progression of a TTR-related disease or disorder in a subject as defined above.

[0360] VII. Peptides with Aggregation-Specific TTR Epitopes

[0361] In a further aspect, the present invention relates to a peptide having a TTR epitope 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 in SEQ ID NO:51, as a unique linear epitope recognized by an antibody or a modified sequence thereof, wherein one or more amino acids are substituted, deleted and / or added, wherein the peptide is recognized by any of the antibodies of the present invention, preferably by antibodies NI-301.59F1, NI-301.35G11, NI-301.37F1, or NI-301.12D3.

[0362] In one embodiment of the invention, such peptides can be used to diagnose or monitor a disease or condition associated with a mutated, misfolded, misassembled or aggregated TTR type and / or fragment thereof, such as TTR amyloidosis, in a subject, comprising the step of determining the presence of antibodies that bind to the peptide in a biological sample of said subject, and for diagnosing such a disease in said subject by determining the level of antibodies that recognize said peptide of the invention and comparing it with the level obtained in healthy subjects of comparable age and sex. Thus, in one embodiment, the invention relates to a method for diagnosing TTR amyloidosis indicative of presymptomatic or clinical FAP and / or FAC in a subject, comprising the step of determining the presence of antibodies that bind to said peptide as described above in a biological sample of said subject. According to this method, an elevated level of antibodies specific for said peptide of the invention as determined is indicative for diagnosing said subject presymptomatic or clinical FAP and / or FAC or for diagnosing said subject any other disease or condition of the group of diseases associated with TTR as defined above. In addition, since the peptides of the invention contain epitopes of therapeutically effective antibodies from humans, such peptides can of course be used as antigens, i.e. immunogens for eliciting an immune response in a subject and stimulating the in vivo production of the antibodies of the invention. The peptides of the invention may be formulated as arrays, kits and compositions, such as vaccines, respectively, as described above. In this regard, the invention also relates to a kit for use in diagnosing or monitoring the progression of TTR amyloidosis, the kit comprising at least one antibody of the invention or a TTR binding molecule, polynucleotide, vector or cell having any substantially the same binding specificity and / or peptide, respectively as defined above, together with optional reagents and / or instructions for use.

[0363] These and other embodiments are disclosed and included in the specification and embodiments of the present invention. Any document about the material used according to the present invention, method, purpose, and compound can be retrieved from public libraries and databases, using, for example, electronic devices. For example, the public database "Medline" can be utilized, which is sponsored by the National Center for Biotechnology Information (NCBI) and / or the National Library of Medicine (NLM.NIH) of the National Institutes of Health. Further databases and website addresses, such as those of the European Bioinformatics Institute (EBI), which are known to those skilled in the art as a part of the European Molecular Biology Laboratory (EMBL), can also be obtained using an Internet search engine. An overview of biotechnology patent information useful for retrospective retrieval and current cognition and a survey of patent information related resources are provided in Berks, TIBTECH 12 (1994), 352-364.

[0364] The above disclosure mainly describes the present invention. Unless otherwise stated, the terms used herein are as defined 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 in full in this specification. A complete bibliographic reference can be found at the end of the specification before the claims. The contents of all cited references (including literature references, granted patents, published patent applications, such as cited throughout this application, including background technology sections and manufacturer's instructions, guidelines, etc.) are incorporated herein by reference; however, it is not acknowledged that any document cited is indeed prior art for the present invention. A more complete understanding can be obtained by referring 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.

[0366] Example 1: Isolation and identification of anti-TTR antibodies

[0367] Human antibodies targeting TTR and / or mutant, misfolded, misassembled and / or aggregated TTR forms and / or fragments thereof are identified using the methods described in International Application WO 2008 / 081008, the disclosure of which is incorporated herein by reference with modifications. In particular, human wild-type TTR protein obtained by purification from human plasma and wild-type and mutant TTR proteins obtained by recombinant expression are used for the identification of antibodies targeting TTR in native and misfolded aggregated conformations. The misfolded aggregated conformation is generated in vitro under acidic conditions using a method similar to that described by Colon W. et al., Biochemistry, 31 (1992), 8654-8660 (with minor modifications).

[0368] Example 2: Determination of antibody sequences

[0369] The amino acid sequences of the variable regions of the anti-TTR antibodies identified above were determined based on their mRNA sequences, see Figure 1. In short, live B cells of selected non-immortal memory B cell cultures are harvested. Subsequently, mRNA derived from cells producing selective anti-TTR antibodies is extracted and converted into cDNA, and the sequence encoding the variable region of the antibody is amplified by PCR, cloned into a plasmid vector and sequenced. In short, primer combinations representing all sequence families of the human immunoglobulin germline library (list) are used for the amplification of leader peptides, V-segments and J-segments. The first round of amplification is performed at the 5'-end using a leader peptide-specific primer and at the 3'-end using a constant region-specific primer (Smith et al., Nat Protoc. 4 (2009), 372-384). For heavy chains and kappa light chains, the second round of amplification is performed at the 5'-end using a V-segment-specific primer and at the 3'-end using a J-segment-specific primer. For lambda light chains, a second round of 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).

[0370] Identification of antibody clones with the desired specificity is performed by rescreening for recombinant expression of intact antibodies on ELISA. Recombinant expression of intact human IgG1 antibodies is achieved by inserting variable heavy and light chain sequences "in the correct reading frame" into expression vectors that are complementary to the variable region sequences with sequences encoding a leader peptide at the 5'-end and encoding appropriate constant region sequences at the 3'-end. For this purpose, primers containing restriction sites are designed to facilitate cloning of variable heavy and light chain sequences into antibody expression vectors. Heavy chain immunoglobulins are expressed by inserting the in-frame immunoglobulin heavy chain RT-PCR product into a heavy chain expression vector with a signal peptide and a human or mouse immunoglobulin γ1 constant domain. κ light chain immunoglobulins are expressed by inserting the in-frame κ light chain RT-PCR product into a light chain expression vector that provides a signal peptide and a human κ light chain immunoglobulin constant domain. Lambda light chain immunoglobulins are expressed by inserting the lambda light chain RT-PCR product in frame into a lambda light chain expression vector that provides a signal peptide and human or murine lambda light chain immunoglobulin constant domains.

[0371] Functional recombinant monoclonal antibodies are obtained when HEK 293 or CHO cells (or any other suitable receptor cell line of human or mouse origin) are co-transfected with an Ig heavy chain expression vector and a kappa or lambda Ig light chain expression vector. Recombinant human monoclonal antibodies are then purified from the conditioned medium using a standard protein A column purification. Recombinant human monoclonal antibodies can be produced indefinitely using transiently or stably transfected cells. Cell lines producing recombinant human monoclonal antibodies can be established by directly using an Ig expression vector or by re-cloning the Ig variable region into a different expression vector. Derivatives such as F(ab), F(ab) 2 scFv and scFv can also be generated from these Ig variable regions.

[0372] Framework and complementarity determining regions were determined by comparison with reference antibody sequences available in databases such as Abysis (http: / / www.bioinf.org.uk / abysis / ) and annotated using the Kabat numbering scheme (http: / / www.bioinf.org.uk / abs / ). Subject 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. The amino acid sequences of the variable regions of 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, including indications of framework (FR) and complementarity determining regions (CDR), are shown in Figure 1 A-1T.

[0373] Hereinafter, the high affinity of the subject antibodies for misfolded aggregated TTR conformations and the lack of binding to native wild-type TTR conformations (thereby demonstrating high selectivity for mutant, misfolded, misassembled and / or aggregated TTR) are exemplified for antibodies NI-301.59.F1, NI-301.35G11, and NI-301.37F1. However, preliminary experiments for other subject antibodies indicate preferential binding to mutant, misfolded, misassembled and / or aggregated TTR substantially identical to the physiological TTR species, like antibodies NI-301.59.F1, NI-301.35G11, and NI-301.37F1.

[0374] Example 3: Using direct ELISA and EC50 Determination of binding affinity of anti-TTR antibodies

[0375] The ability of the antibodies to bind TTR and / or misfolded, misassembled and / or aggregated forms of TTR was evaluated by direct ELISA assay at different antibody concentrations. This allowed the determination of the half maximal effective concentration (EC) of each antibody in this assay. 50 ), which is a commonly used proxy for antibody binding affinity, see Figure 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 (all 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 2% BSA and 0.1% Tween 20 in PBS (PBS-T) for 1 hour at room temperature. Antibodies against TTR were diluted in PBS to 11 different concentrations from 4 to 400 nM and incubated in the ELISA plates overnight at 4°C. After washing three times with PBS-T, the ELISA plates were incubated with HRP-conjugated human IgG-specific secondary antibodies at room temperature for 1 hour (1 / 4000 dilution). After washing three times with PBS-T, the ELISA reactions were developed with TMB for exactly 10 minutes at room temperature and quantified by measuring the optical density at 450 nm (OD450 nm).

[0376] Exemplary antibodies NI-301.59F1, NI-301.35G11, and NI-301.37F1 exhibited strong binding to misfolded, aggregated wild-type and mutant TTR, but not to control BSA, see Figure 2 A-2C. The EC50 of the antibody was then determined by fitting the data with nonlinear regression using the least squares method to estimate the binding affinity of the antibody under these conditions.

[0377] Exemplary antibodies NI-301.59F1, NI.35G11, and NI-301.37F1 exhibit high affinity for misfolded and aggregated human wild-type TTR with EC50s of 3.0 nM, 3.9 nM, and 0.35 nM, respectively. Exemplary antibodies also exhibit high affinity for misfolded and aggregated recombinant mutant V30M-TTR with EC50s of 15.5 nM, 5.0 nM, and 0.15 nM, respectively.

[0378] Example 4: Binding selectivity of anti-TTR antibodies using dot blot

[0379] To evaluate the binding selectivity of TTR antibodies and / or fragments thereof to native or misfolded, misassembled and / or aggregated TTR conformations, native or misfolded aggregated human wild-type TTR protein and misfolded aggregated recombinant V30M-TTR protein were diluted in PBS to 4 different concentrations and deposited on nitrocellulose membranes by vacuum filtration. The membranes were briefly dried (10 minutes) and blocked with 3% milk in PBS-T for 1 hour at room temperature, followed by incubation with anti-TTR antibodies overnight at 4°C. After washing three times with PBS-T for 5 minutes at room temperature, the membranes were incubated with appropriate secondary antibodies (HRP-conjugated; 1 / 10000 dilution) at room temperature for 1 hour. After washing three times with PBS-T, the membranes were developed with luminol and the signal intensity was quantified by measuring luminescence.

[0380] Exemplary commercially available anti-TTR antibodies bind to native and misfolded, aggregated TTR conformations with similar affinity, demonstrating a lack of binding selectivity for native or misfolded, misassembled and / or aggregated TTR conformations, see Figure 3 A. In contrast, exemplary antibodies NI-301.59.F1, NI-301.35G11, and NI-301.37F1 bound only misfolded aggregated TTR conformations with high affinity and did not show binding to native TTR conformations, thereby demonstrating high selectivity for misfolded, misassembled, and / or aggregated TTR ( Figure 3 B2, C2, D2). Accordingly, antibodies NI-301.35G11 and NI-301.37F1 also showed strong binding to misfolded and aggregated recombinant V30M-TTR protein, such as Figure 3 As shown in C3 and D3.

[0381] To further characterize the selectivity of antibody binding, various TTR preparations, including wild-type and mutant, native and misfolded aggregated conformations, and a pool of 12 human plasma samples were similarly processed for dot blot analysis using a murine chimeric anti-TTR antibody and an HRP-conjugated anti-mouse IgG2a secondary antibody for detection ( Figure 6 ).

[0382] 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 demonstrates the lack of selectivity for native or aggregated conformations, see Figure 6 A. In contrast, the exemplary murine chimeric antibody NI-301.mur35G11 exhibited inhibition of misfolded, aggregated, wild-type TTR samples ( Figure 6 C1), and the mutant V30M-TTR protein ( Figure 6C 4) and mutant Y78F-TTR protein ( Figure 6 C6). However, the NI-301.mur35G11 antibody did not bind TTR in human plasma samples. This further demonstrates the high selectivity of NI-301.mur35G11 for mutant, misfolded, misassembled and / or aggregated TTR proteins.

[0383] Example 5: Binding Specificity and Selectivity of Anti-TTR Antibodies Using Western Blotting

[0384] The binding specificity and selectivity of anti-TTR antibodies were evaluated by means of Western blotting, see Figure 4 Briefly, human wild-type TTR protein (300 ng) in native or misfolded aggregated conformations, and wild-type mouse liver extract (10 μg total protein) were loaded on SDS-PAGE gels and transferred to nitrocellulose membranes using a semi-dry transfer system. The membranes were then blocked with PBS-T containing 2% BSA for 1 h at room temperature and incubated overnight at 4°C with anti-TTR antibodies diluted in blocking buffer. After four washes with PBS-T for 5 min at room temperature, the membranes were incubated with appropriate secondary antibodies (HRP-conjugated; diluted 1 / 10000 in blocking buffer) for 1 h at room temperature. After three washes with PBS-T and a final wash in PBS, the membranes were developed with luminol and the signal intensity was quantified by measuring luminescence. Shortly before use, misfolded aggregated TTR samples were submitted to cross-linking with glutaraldehyde (1%, 5 min, 37°C) to prevent the dissociation of TTR aggregates during preparation for SDS-PAGE. In contrast, native TTR samples were not cross-linked prior to use, resulting in almost complete dissociation of the TTR homotetramer (which is the native TTR conformation under physiological conditions) into monomers and dimers.

[0385] Commercially available anti-TTR antibodies showed very strong binding to TTR monomers and dimers of human native TTR samples ( Figure 4 A1), and similarly strong binding to cross-linked misfolded aggregated TTR samples ( Figure 4 A2), thereby demonstrating a lack of selectivity for native or misfolded, misassembled and / or aggregated TTR conformations. In contrast, exemplary anti-TTR antibodies NI-301.59F1, NI-301.35G11, and NI-301.37F1 exhibited very strong binding to cross-linked misfolded, aggregated TTR samples ( Figure 4 B2, C2, D2), but did not bind to TTR monomers and dimers of natural human TTR samples ( Figure 4B1, C1, D1), thereby demonstrating strong selectivity for misfolded, misassembled and / or aggregated TTR conformations compared to native TTR conformations.

[0386] In addition, commercially available and exemplary anti-TTR antibodies have very low binding levels to proteins contained in mouse liver extracts ( Figure 4 A3, B3, C3, D3). Given the high content of liver protein used for the experiment and the high antibody concentration relative to its affinity, this shows that the exemplary antibody has a clear specificity for TTR and does not significantly bind to other proteins. In addition, it was shown that the exemplary antibody does not 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 the epitope may not necessarily be decisive for the detection of misfolded TTR, but rather the conformation.

[0387] To further characterize the binding ability of the antibodies to native TTR protein, or the lack thereof, the binding ability of the exemplary antibodies to TTR protein contained in human plasma samples was evaluated using the same Western blotting method as described above ( Figure 5 ). The only technical difference is that the upper part of the gel is trimmed at approximately 25-30 kDa and only the lower part of the gel is used for protein transfer to nitrocellulose membrane. This is to remove the high concentration of human antibody heavy and light chains present in plasma samples, which may interfere with the analysis.

[0388] In contrast to the commercially available antibodies used as reference, the exemplary antibodies NI-301.35G11 and NI-301.37F1 did not detect TTR protein contained in human plasma samples at all, thus demonstrating selectivity for a TTR conformation that was not present in the samples analyzed under these conditions.

[0389] Example 6: Binding selectivity of anti-TTR antibodies in solution using immunoprecipitation

[0390] To further verify the binding selectivity of the anti-TTR antibodies of the present invention, native or misfolded aggregated human wild-type TTR proteins and recombinant TTR proteins and human plasma samples were diluted in PBS at 3 different concentrations for TTR immunoprecipitation (IP). Briefly, protein A-coated magnetic beads were incubated with anti-TTR antibodies diluted in the manufacturer's binding buffer at room temperature for 30 minutes. The antibody / protein A complex was recovered 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 minutes, and processed for Western blot analysis.

[0391] like Figure 7 As shown, in contrast to the commercially available TTR antibody Dako A0002, the exemplary TTR antibodies NI-301.35G11 and NI-301.37F1 showed no binding to this plasma sample ( Figure 7 A7-9, B7-9, C7-9), and no binding to natural wild-type and recombinant TTR samples ( Figure 7 However, strong binding was assessed in samples in which misfolded, aggregated forms of TTR were present ( Figure 7 B4, C4, B6, C6).

[0392] These results demonstrate that exemplary antibodies NI-301.35G11 and NI-301.37F1 are able to bind misfolded, misassembled and / or aggregated TTR conformations in solution and display high selectivity for these conformations.

[0393] Example 7: Pathological TTR aggregates bound to FAP mouse tissues

[0394] The ability of exemplary anti-TTR antibodies to bind to pathological and non-pathological TTR proteins present in tissues of transgenic mice expressing only human V30M-TTR protein but not mouse TTR protein (hereinafter referred to as FAP mice) was evaluated by immunohistochemistry (IHC). The non-specific binding of these antibodies to tissues of TTR knockout (TTR-KO) mice that do not express any TTR protein was also evaluated. The corresponding transgenic and knockout mouse strains were originally prepared 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 tissues cut into 3-5 μm thick sections. The sections were initially dewaxed and rehydrated and lysed with 3% H at room temperature. 2 O 2 The sections were treated with methanol solution for 20 minutes. Blocking buffer (PBS + 5% serum (horse / goat) + 4% BSA) was applied for 1 hour at room temperature and replaced with anti-TTR antibody diluted in PBS and incubated overnight at 4°C. After washing 3 times in PBS, the sections were incubated sequentially with appropriate biotinylated secondary antibodies (anti-human IgG, anti-rabbit IgG diluted 1 / 125 in PBS, incubated for 1 hour at room temperature) and avidin-HRP detection system (diluted 1 / 125 in PBS, incubated for 1 hour at room temperature). The reaction was developed with diaminobenzidine at room temperature for exactly 15 minutes. Tissue sections were counterstained with hematoxylin alum (hemalun) for 1 minute at room temperature, dehydrated and coverslipped in a liter ethanol series.

[0395] like Figure 8 As shown, the commercially available TTR antibody Dako A0002 produced strong staining in liver and intestinal sections of FAP mice, but did not produce any staining in the corresponding TTR KO sections ( Figure 8 1A, 1B). The exemplary TTR antibody NI-301.35G11 was expressed in liver and intestinal sections of FAP mice ( Figure 8 2A) produced staining of similar pattern and intensity. In contrast, the exemplary antibody NI-301.37F1 produced strong staining only in the intestinal sections of FAP mice, but not in the liver sections ( Figure 8 3A). This suggests that the NI-301.37F1 antibody binds only to pathological (i.e., nonphysiological) TTR aggregates that accumulate over time in the gastrointestinal tract of FAP mice, and not to TTR in its native conformation that is synthesized by the liver.

[0396] In addition, the two exemplary antibodies NI-301.35G11 and NI-301.37F1 did not produce any staining in liver and intestinal tissue sections of TTR-KO mice ( Figure 8 2B, 3B). Given the high antibody concentrations used in this experiment with respect to the antibody binding affinity, the lack of staining in TTR-KO sections indicates a high binding specificity for the TTR protein.

[0397] Example 8: Binding selectivity to misfolded, misassembled and / or aggregated TTR deposits in human tissues

[0398] The antibodies of the invention were also evaluated for their ability to bind to pathological TTR deposits in human tissue. Skin biopsies from FAP patients and pancreatic tissue sections from healthy individuals were processed for immunohistochemistry using the same methods described in Example 7 (see above). The skin biopsy sample was selected for this experiment because it contains significant amounts of pathological TTR amyloid deposits. In contrast, pancreatic tissue was used in this experiment because pancreatic alpha cells express TTR at higher levels.

[0399] like Fig. 9 As shown, the commercially available antibody Dako A0002 is effective against pathological TTR deposits in the skin and native TTR in pancreatic α cells ( Fig. 9 1A) showed the same intensity of staining. Similarly, the exemplary murine chimeric antibody NI-301.mur35G11 produced the same intensity of staining for pathological TTR deposits in the skin and native TTR in pancreatic α cells (Figure 9 1B). In contrast, antibody NI-301.37F1 only stained pathological TTR deposits in the skin, but not native TTR in pancreatic α cells (Figure 9 1B). Fig. 93A). This result indicates that NI-301.37F1 is highly selective for pathological TTR deposits, including mutant, misfolded, misassembled, and / or aggregated TTR conformations, by IHC.

[0400] Presented on the board ( Fig. 9 The "secondary antibody only" control in 1B, 2B, and 3B) shows tissue staining that occurs in the absence of primary antibody. Figure 2 B) or very low levels (1B, 3B), indicating that the staining observed in panels 1A, 2A, and 3A is indeed specific for the corresponding primary antibodies.

[0401] Example 9: Evaluation of Binding Epitopes of TTR Antibodies

[0402] 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 plate of 29 sequential peptides 15 amino acids long and 11 amino acids overlapping, covalently bound to the membrane. Additional peptides including selected mutations were also mapped on the membrane. The membrane was blocked in Roti blocking buffer overnight at 4°C, first incubated with anti-TTR antibodies diluted in blocking buffer for 2 hours at room temperature, and then incubated with HRP-conjugated anti-human IgG antibodies for 45 minutes at room temperature (diluted 1 / 20000 in TBS). The reactions were developed with luminol and imaged by luminescence.

[0403] Antibody NI-301.59F1 recognizes positions 15, 16 and 44 corresponding to the sequence 61-EEEFVEGIY-69 (SEQ ID NO: 49) of the complete human wild-type TTR, see Fig.10 A. Antibody NI-301.35G11 recognizes positions 13, 14, 42 and 44 corresponding to the sequence 53-GELHGLTTEEE-63 (SEQ ID NO: 50) of intact human wild-type TTR, see Fig.10 B. However, antibody NI-301.35G11 does not recognize site 43, indicating that the antibody cannot bind to sequence 53-GELHGPTTEEE-63 corresponding to the L55P-TTR variant. Antibody NI 301.37F1 recognizes sites 9, 10, 11, 38, and 40 corresponding to sequence 41-WEPFA-45 (SEQ ID NO: 51) of intact human wild-type TTR, see Fig.10 C. However, antibody NI-301.37F1 did not recognize point 43, indicating that the antibody could not bind to the sequence 41-WGPFA-4 corresponding to the E42G-TTR variant.

[0404] To improve the determination of the binding epitope of the exemplary antibodies NI-301.59F1, NI301.35G11, and NI-301.37F1, the entire TTR amino acid sequence was analyzed using a plate of 151 sequential peptides 15 amino acids long and 14 amino acids overlapping, covalently bound to the membrane. For each peptide, the amino acid at position 10 was substituted with alanine for non-alanine, while alanine was substituted with glycine or proline. The membrane was blocked in Roti blocking buffer overnight at 4°C, first incubated with anti-TTR antibody diluted in blocking buffer for 2 hours at room temperature, and then incubated with HRP-conjugated anti-human IgG antibody for 45 minutes at room temperature (diluted 1 / 20000). The reaction was developed with luminol and imaged by luminescence.

[0405] The antibody NI-301.59F1 recognizes only sites 77 and 83, indicating that E61 and V65 are dispensable for 59F1 binding, whereas E62, E63, F64, E66, G67, I68, and Y69 are all essential for antibody binding. The exact contribution of K70 is a matter of interpretation: Fig.10 The strong antibody binding to peptide 44 shown in A clearly indicates that the absence of K70 at the C-terminal position does not prevent antibody binding; however, Fig.10 In a subsequent experiment shown in E, substitution of K70A at position 10 of peptide prevented antibody binding. These seemingly contradictory results indicate 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.

[0406] Antibody NI-301.35G11 recognizes sites 68, 71, 72, 73, 74 and 75, indicating that G53 is dispensable for 35G11 binding, whereas E54, L55, G57 and L58 are essential for antibody binding. The binding pattern of 35G11 also suggests that the presence of either E61 or E62 is essential for antibody binding. The exact contribution of T59 and T60 could not be determined in this experiment, but it is assumed that the presence of one of the two tyrosines is essential for antibody binding. Taken together, the binding properties of NI-301.35G11 in alanine scanning suggest that the antibody recognizes the sequence 54ELXGLTXE 61 (SEQ ID NO: 59), where X can include all known amino acids, see Fig.10 F.

[0407] Antibody NI-301.37F1 binds to spots 50, 52, 55, 56, and 58-62 on the alanine scan membrane, but not to spots 51, 53, 54, and 57. This suggests that W41, P43, F44, and A45 are essential for antibody binding. Combined with earlier observations, mutation E42G abolishes antibody binding ( Fig.10C), these results indicate that NI-301.37F1 binds to the sequence 41WEPFA-45 (SEQ ID NO: 60), see Fig.10 G.

[0408] Example 10: Determination of Antibody Binding Characteristics by Surface Plasmon Resonance

[0409] The binding properties of the antibodies to various water-soluble TTR preparations were determined by surface plasmon resonance (SPR) using a BioRad Proteon XPR36 instrument, see Table V.

[0410] SPR analysis was performed on a BioRad ProteOn XPR36 equipped with a GLM sensor chip. Anti-human antibodies against the Fcγ domain were covalently coupled to the detection surface and saturated with the antibody of interest. Wild-type and mutant TTR proteins in native and misfolded conformations were diluted in HBS-T buffer to a concentration range of 3.2 to 316 nM. Antibody-antigen binding was analyzed during 180 seconds and dissociation during 600 seconds. The Langmuir binding model (simple 1:1 binding) was used to fit the data and obtain association (ka) and dissociation (kd) constants, as well as affinity (KD).

[0411] Anti-human IgG-Fcγ antibodies were covalently coated on the detection surface and used to capture human TTR-specific antibodies. The antibodies were probed with 4 different TTR preparations, including native and misfolded aggregated wild-type TTR, and native V30M and L55P TTR mutants, all prepared at concentrations ranging from 3.2 to 316 nM in HBS-T buffer (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4). Misfolded aggregated wild-type TTR was prepared by acid denaturation in acetate buffer (50 mM acetate HCl, 100 mM KCl, 1 mM EDTA, pH 3.0) at 65°C for 80 min, followed by buffer exchange with HBS-T. 59F1, 35G11, and 37F1 showed linear binding and dissociation characteristics that best fit the Langmuir model.

[0412] The results showed that the three antibodies bound with high affinity to V30M- and L55P-TTR variants, as well as misfolded, aggregated wild-type TTR preparations in solution. In contrast, these exemplary antibodies did not bind to native wild-type TTR in solution.

[0413] Thus, the results indicate that NI-301.37F1 binds to misfolded human wild-type TTR protein with high affinity in solution, with a KD of 1.2 nM, while not binding to the same protein in its native conformation. A similar binding affinity (KD = 1.4 nM) has been determined for the mutant TTR-L55P protein.

[0414] Table V: Binding characteristics of antibodies determined by surface plasmon resonance.

[0415]

[0416] Example 11: Passive immunization of human Val30Met TTR transgenic mice displaying tissue TTR deposits with chimeric human-mouse recombinant anti-TTR antibodies leads to removal of deposits

[0417] Passive immunization was performed similarly to that described in international application WO 2010 / 030203, in particular Example 3, the disclosure of which is incorporated herein by reference, and the disclosures of Kohno et al., Am. J. Pathol. (1997), 1497-1508 and Sousa et al., Am. J. Pathol. (2002), 1935-48, which are incorporated herein by reference.

[0418] Briefly, monoclonal antibodies were administered intraperitoneally weekly for 12 weeks at a dose of 3 mg / kg to 7-month-old and 17-month-old FAP mice, which are transgenic for the human Val30Met-TTR allele and knocked out the murine TTR gene (Kohno et al., (1997), supra). Five days after the last dose, the animals were sacrificed and various tissues were collected and fixed in paraformaldehyde solution and embedded in paraffin. 3-5 μm sections were cut and processed for immunohistochemistry using the commercial anti-TTR antibody described above. A standard immunofluorescence method was used, which was very similar to that shown in Example 7, with the only difference being that a fluorescent secondary antibody was used for detection. The surface of the tissue invaded by TTR deposits was quantified and expressed as a percentage of the total tissue area. Statistical analysis of treatment effects was performed using a two-tailed, unpaired t-test.

[0419] This transgenic mouse strain recapitulates key pathological mechanisms common to TTR amyloid diseases including TTR tetramer dissociation and misfolding of TTR monomers into a toxic and insoluble amyloid conformation. Like FAP patients, these transgenic mice typically present with age-dependent TTR deposition. Evaluation of therapeutic efficacy was studied in two groups of transgenic mice that were 7 and 17 months of age at the start of treatment; age was important for TTR deposition, which invaded many gastrointestinal tissues. Notably, passive immunization with the exemplary antibody NI-301.37F1 was associated with a statistically significant reduction in the surface of tissues invaded by TTR deposits when treatment was started at 7 months of age, see Fig.12 A. In aged mice, treatment had a similar effect, resulting in an almost significant reduction in TTR deposition, see Fig.12 B.

[0420] Example 12: Humanized, recombinant anti-TTR antibodies bind to pathological TTR deposits in vivo

[0421] To determine whether human recombinant anti-TTR antibodies can bind to pathological TTR deposits in vivo, 7-month-old adult FAP mice were injected intraperitoneally with antibody NI-301.37F1 at a dose of 30 mg / kg or with PBS for comparison. After 48 hours, these mice were submitted to transcardiac perfusion and tissues were processed for histological analysis. Rabbit polyclonal anti-TTR antibodies were used in combination with fluorescently labeled anti-rabbit IgG antibodies to detect pathological TTR deposits, while the location (localization) of the injected antibody NI-301.37F1 was detected using fluorescently labeled anti-human IgG antibodies. In particular, immunoprecipitation was performed as follows.

[0422] NI-301.37F1 and isotype control antibodies from mouse plasma samples were immunoprecipitated for 2 hours at room temperature using protein A / G coupled magnetic beads (Pierce#88803) loaded with anti-human IgG antibodies (Jackson Immunoresearch#709-005-098). After washing 3 times with PBS-T, the sample was eluted from the magnetic beads, neutralized with 0.2M glycine buffer (pH2.5), neutralized with 1M Tris hydrochloric acid (pH 8.0), mixed with LDS loading buffer (Life technologies #NP0007), and heated at 90°C for 10 minutes. The sample was then loaded onto a 4-12% bis-tris gel (Lifetechnologies #WG1403A) and run at 200V in MOPS running buffer for 40 minutes. After the protein was transferred to the nitrocellulose membrane, TTR protein was detected using a conformation-independent TTR antibody (Dako #A0002, 150 ng / ml) or antibody NI-301.37F1 (20 nM) in combination with HRP-conjugated protein A (Life technologies #10-1023, 1 / 10000 dilution) and luminescence imaging.

[0423] like Fig.13 As described, in vivo target ...

Claims

1. An anti-transthyretin (TTR) antibody or an antigen-binding fragment thereof, which is capable of binding to mutated, misfolded, misassembled and / or aggregated transthyretin species or fragments thereof, while substantially not recognizing physiological transthyretin species, in, The antibody or antigen-binding fragment thereof comprises six CDRs of the VH and VL variable regions in its variable region or binding domain: CDR-H1 includes the amino acid sequence NYAMH; CDR-H2 includes the amino acid sequence IISYDGNNKYYADSVRG; CDR-H3 includes the amino acid sequence GSGRAARHWFDP; CDR-L1 includes the amino acid sequence TGTSSDVGGYNYVS; CDR-L2 comprises the amino acid sequence DVFNRPS; and CDR-L3 comprises the amino acid sequence SSYTSSVTPHWV.

2. An anti-transthyretin (TTR) antibody or antigen-binding fragment thereof, which is capable of binding to mutated, misfolded, misassembled and / or aggregated transthyretin species or fragments thereof, while not substantially recognizing physiological transthyretin species, in, The antibody or antigen-binding fragment thereof comprises six CDRs of the VH and VL variable regions in its variable region or binding domain: CDR-H1 includes the amino acid sequence SSNFYWG; CDR-H2 includes the amino acid sequence AIYSSGNTYYNPSLKS; CDR-H3 includes the amino acid sequence HSCSSASCYPPGFWFDP; CDR-L1 includes the amino acid sequence RASQTVSYNLA; CDR-L2 includes the amino acid sequence GASTRAT; as well as CDR-L3 comprises the amino acid sequence QQYNNWPPWT.

3. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. The polynucleotide according to claim 3, in, The polynucleotide is cDNA.

5. A vector comprising the polynucleotide according to claim 3. A host cell comprising the polynucleotide according to claim 3 or a vector comprising the polynucleotide.

7. Use of the polynucleotide according to claim 3, the vector according to claim 5 or the host cell according to claim 6 in producing an anti-TTR antibody or an antigen-binding fragment thereof.

8. A method for preparing an anti-TTR antibody or an antigen-binding fragment thereof, the method comprising (a) culturing the host cell according to claim 6; and (b) isolating the antibody from the culture.

9. A composition comprising the antibody or antigen-binding fragment thereof according to claim 1 or 2 and a carrier.

10. A kit for diagnosing or monitoring TTR amyloidosis in a subject, the kit comprising at least one antibody or antigen-binding fragment thereof according to claim 1 or 2, a vector according to claim 5 or a host cell according to claim 6 and instructions for use thereof.

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