Anti-transthyretin (TTR) binding protein and its use

Abstract

Relates to anti-transthyretin (TTR) binding proteins and their use. Transthyretin amyloidosis is characterized by the progressive deposition of the plasma protein transthyretin in the myocardium, peripheral nerves and / or other tissues, ultimately leading to death from congestive heart failure and polyneuropathy. The present invention provides binding proteins that can specifically bind to isoaspartic acid residues in the transthyretin protein, as well as their therapeutic, diagnostic and prognostic uses.

Description

Technical Field

[0001] The present invention may be included in the pharmaceutical field. In particular, the present invention provides a binding protein that can specifically bind to isoaspartic acid (isoD) residues in transthyretin (TTR) protein, and its use in the prophylactic and therapeutic treatment of transthyretin amyloidosis (ATTR). Furthermore, the present invention provides a prognostic diagnosis method and / or the use of the binding protein in a diagnostic method.

Background Art

[0002] Systemic amyloidosis represents a group of rare diseases that are often underdiagnosed due to their multifaceted symptoms. In this multi-system disease, misfolded protofibril proteins deposit extracellularly in various organs, leading to a continuous loss of function and integrity (Non-Patent Document 1). Transthyretin amyloidosis (ATTR) is characterized by the progressive deposition of the plasma protein transthyretin (TTR) in the myocardium, peripheral nerves, and / or other tissues, ultimately leading to congestive heart failure, polyneuropathy, and death. Typically, patients die 4 to 12 years after diagnosis (Non-Patent Documents 2 and 3). ATTR has two forms: wild-type ATTR (ATTRwt) and hereditary ATTR (hATTR). ATTRwt is often underdiagnosed as a cause of heart failure and mainly affects elderly individuals. On the other hand, hATTR is an autosomal dominant genetic disease caused by mutations in the TTR gene. More than 130 different mutations have been known so far, and the age at disease onset and clinical symptoms vary. Some mutations mainly cause polyneuropathy, while other mutations are cardiomyopathy or result in a mixed phenotype. The most prevalent TTR mutation worldwide is the Val30Met mutation, which usually causes peripheral neuropathy. The second most common genetic mutation is Val122Ile. Here, organ involvement may be limited to the heart (Non-Patent Document 5).

[0003] TTR is a 55 kDa homotetrameric protein that binds to and transports thyroxine and holoretinol-binding protein in cerebrospinal fluid and plasma (Non-Patent Document 6). TTR is mainly produced in the liver. Approximately 5% of the total protein is derived from the choroid plexus and retinal pigment epithelium (Non-Patent Document 7). Factors such as low pH, high temperature, or the presence of mutations in the TTR gene increase the instability of the homotetramer complex. This leads to separation and more rapid release of monomers, which then leads to the aggregation process and the formation of amyloid fibrils (Non-Patent Document 8). Recently, Non-Patent Document 9 elucidated the first cryo-EM structure of human ATTR-V30M fibrils. They showed that fibril formation causes almost complete unfolding of the native conformation and rearrangement of the polypeptide chain into a significantly different conformation. Furthermore, this structure provides valuable insights into the protein regions of the fibrils that can induce protein-protein interactions.

[0004] Therapeutic approaches for ATTR treatment include liver transplantation (Non-Patent Document 10), gene silencing by siRNA (patisiran, Non-Patent Document 11) or antisense oligonucleotides (inotersen, Non-Patent Document 12), and stabilization of the TTR complex in its native folded physiological form (see tafamidis, Non-Patent Document 13; diflunisal, Non-Patent Document 14; epigallocatechin-3-gallate, Non-Patent Document 15). The treatment is designed to prevent misfolding / unfolding, aggregation, and deposition in tissues. Although the development has shown an effective approach to reducing the progression of amyloid deposition, alternative means are needed to remove existing deposits of TTR protein in the tissues of affected patients. Passive immunotherapy, including the administration of monoclonal antibodies (mAbs) against unnaturally folded dissociated monomers and non-native oligomers or amyloid deposits, could be a viable approach to target them for degradation via Fc-mediated phagocytosis.

[0005] As far as we know, all the mAbs developed so far target potential epitopes on the surface of amyloidogenic TTR and are specific to non-native TTR because they are exposed during the pathogenic formation of β-sheet protofibrils. Among them, mAb PRX004 (Non-Patent Document 16) has already achieved the first phase of clinical trials. However, due to the impact of the COVID-19 pandemic, the trial ended early. In Non-Patent Document 17, another mAb, NI006, for use in ATTR cardiomyopathy was developed, and clinical trials including the administration of NI006 are planned.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Summary of the Invention

Problems to be Solved by the Invention

[0007] There is still a need for an effective treatment that can treat and / or prevent ATTR.

Means for Solving the Problems

[0008] Recent progress in the treatment of other amyloidoses may provide guidance for the development of effective therapies for the treatment of ATTR. Donanemab (Barton) is a mAb currently in a Phase 3 clinical trial for the treatment of Alzheimer's disease (AD), highlighting the potential of a tailored therapeutic approach by targeting the pyroglutamic acid form of Aβ, a non-physiological post-translational modification (PTM) (Mintun et al., 2021. N Engl J Med. 384(18):1691-1704). In contrast, little is known about PTMs in TTR that may affect the development of ATTR.

[0009] Based on a comprehensive analysis of the post-translational modifications of Aβ and their functions, some of these may also be inferred for TTR in ATTR. One of these modifications is the formation of isoaspartic acid (isoD) resulting from either hydrolysis from L-aspartatyl or deamidation of L-asparaginyl residues (Shimizu et al., 2000. Arch Biochem Biophys. 381(2):225-34), and the isomerization occurs spontaneously in a 3:1 ratio to L-isoaspartic acid or L-aspartic acid via an L-succinimidyl intermediate. This modification determines the half-life of the protein and significantly changes its structure by introducing an additional methylene group into the protein backbone (Robinson & Robinson, 2001. Proc Natl Acad Sci U S A. 98(3):944-9; Aswad et al., 2000. J Pharm Biomed Anal. 21(6):1129-36; Geiger & Clarke, 1987. J Biol Chem. 262(2):785-94). This potentially affects the solubility, conformation, and function of the protein. Here, the present inventors provide evidence for the formation of isoaspartic acid at position 38 of the TTR protein and the presence of isoD38-TTR in amyloid deposits. The present inventors have developed two monoclonal antibodies, 2F2 and 4D4, that specifically detect this modification in amyloid deposits in human pathological tissues. Native TTR protein is not bound by 2F2 and 4D4. Both antibodies promote the cellular uptake of human isoD 38-modified TTR protofibrils into THP-1 cells by phagocytosis. Thus, the present inventors propose that these antibodies and other binding proteins targeting isoD-modified TTR may be useful for diagnosis and treatment.

[0010] Accordingly, the present invention provides a binding protein that specifically binds to isoD-modified TTR. Also provided are a nucleic acid comprising a sequence encoding the binding protein of the present invention, and a host cell comprising the nucleic acid of the present invention. The present invention also provides a pharmaceutical composition comprising the binding protein, nucleic acid or host cell of the present invention and a pharmaceutically acceptable carrier or diluent. The binding protein, nucleic acid, host cell or pharmaceutical composition of the present invention may be for use as a medicine. In particular, the binding protein, nucleic acid, host cell or pharmaceutical composition of the present invention may be for use in the treatment and / or prevention of ATTR. The present invention also provides a diagnostic and / or prognostic method for detecting the presence of isoD-modified TTR in a sample. The method may be an in vitro or in vivo method. The method may include contacting the sample with the binding protein of the present invention.

[0011] The in vitro method includes an isolated sample. For example, the present invention provides an in vitro diagnostic and / or prognostic method for detecting the presence of isoD-modified TTR in an isolated sample, the method comprising contacting the isolated sample with the binding protein of the present invention and determining whether isoD-modified TTR is present in the isolated sample. The present invention also provides the binding protein of the present invention for use in an in vivo diagnostic and / or prognostic method of ATTR.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0013] Definition The term "affibody" refers to a protein derived from the Z domain of protein A and engineered to bind to a specific target (see Frejd & Kim, 2017. Exp Mol Med. 49(3): e306). As used herein, the term "animal" refers to any non-human multicellular eukaryotic heterotrophic organism. In certain embodiments, the animal is selected from the group consisting of cats, dogs, pigs, ferrets, rabbits, guinea pigs, hamsters, gerbils, horses, rats, mice, cows, sheep, goats, alpacas, camels, donkeys, yaks, giraffes, elephants, meerkats, squirrel monkeys, lions, tigers, kangaroos, komas, bats, monkeys, chimpanzees, gorillas, bears, orangutans, manatees, seals, and tapirs.

[0014] The term "antibody" refers to a molecule that binds to a specific target and contains at least one immunoglobulin domain that is specific therefor. The term includes whole antibodies and any antigen-binding portion thereof (e.g., F(ab’)2, Fab’, Fab, and Fv). The term includes antibodies of horses, humans, mice, rabbits, goats, donkeys, camelids (e.g., llamas, camels, or alpacas), or cartilaginous fish (e.g., sharks), or any antibodies derived therefrom. A normal antibody comprises at least two heavy chains ("HC") and two light chains ("LC") interconnected by disulfide bonds. Each "heavy chain" comprises a "heavy chain variable domain" (abbreviated herein as "VH") and a "heavy chain constant domain" (abbreviated herein as "CH"). The heavy chain constant domain typically comprises three constant domains, namely, CH1, CH2, and CH3. Each "light chain" includes a "light chain variable domain" (abbreviated herein as "VL") and a "light chain constant domain" ("CL"). The light chain constant domain (CL) can be of the kappa or lambda type. The VH and VL domains can be further subdivided into regions of hypervariability called "complementary determining regions" ("CDRs") interspersed with more conserved regions called "framework regions" ("FW"). Each VH and VL is composed of three CDRs and four FWs and is arranged in the following order from the amino terminus to the carboxy terminus: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The present disclosure presents, among other things, VH and VL sequences, and subsequences corresponding to CDR1, CDR2, and CDR3. Thus, one of ordinary skill in the art will understand that the sequences of FW1, FW2, FW3, and FW4 are equally disclosed. For a particular VH, FW1 is the subsequence between the N-terminus of VH and the N-terminus of HCDR1, FW2 is the subsequence between the C-terminus of HCDR1 and the N-terminus of HCDR2, FW3 is the subsequence between the C-terminus of HCDR2 and the N-terminus of HCDR3, and FW4 is the subsequence between the C-terminus of HCDR3 and the C-terminus of VH. Similarly, for a particular VL, FW1 is the subsequence between the N-terminus of VL and the N-terminus of LCDR1, FW2 is the subsequence between the C-terminus of LCDR1 and the N-terminus of LCDR2. FW3 is the subsequence between the C-terminus of LCDR2 and the N-terminus of LCDR3, and FW4 is the subsequence between the C-terminus of LCDR3 and the C-terminus of VL.

[0015] The variable domains of the heavy and light chains include regions that interact with the binding target, and this region that interacts with the binding target is also referred to herein as the "antigen binding site" or "antigen binding site". The constant domain of the antibody can mediate the binding of the antibody to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). Exemplary antibodies of the present disclosure include not only normal antibodies but also divalent fragments thereof such as F(ab’)2 and variants.

[0016] As used herein, the term "antibody" also encompasses full-length polyclonal antibodies, full-length monoclonal antibodies, bivalent antibody fragments (such as F(ab’)2), multispecific antibodies such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and any other modified immunoglobulin molecules containing two antigen-binding sites. Antibodies can be any of the five major classes (isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy-chain constant domains, which are alpha, delta, epsilon, gamma, and mu, respectively. Different classes of immunoglobulins have different well-known subunit structures and three-dimensional conformations. Antibodies can be naked or complexed with other molecules such as therapeutic or diagnostic agents to form immune complexes.

[0017] The term "anticalin" refers to a protein derived from lipocalin that has been engineered to bind to a specific target (see Skerra, 2008. FEBS J. 275(11):2677-83). The term "binding protein" refers to a protein that can specifically bind to a target. Non-limiting examples of binding proteins include antibodies, anticalins, lipobodies, monobodies, scFv, scFab, affibodies, finomers, DARPins, nanobodies, chimeric antigen receptors, chimeric uptake receptors, and peptide aptamers. The binding protein is preferably an antibody. The term "designed ankyrin repeat protein" or "DARPin" refers to a protein derived from ankyrin repeats that has been engineered to bind to a specific target (Plueckthun, 2015. Annu Rev Pharmacol Toxicol. 55:489-511).

[0018] As used herein, the term "effective amount" of an agent, such as a therapeutic agent like an antibody, is an amount sufficient to produce a beneficial or desired result, such as a clinical result. Thus, an "effective amount" depends on the circumstances in which it is being applied. For example, in the context of administering a therapeutic agent for treating ATTR, an effective amount of the agent is an amount sufficient to reduce or decrease TTR deposits, for example, as compared to the response obtained without administration of the agent. The term "effective amount" can be used interchangeably with "effective dose", "therapeutically effective amount", or "therapeutically effective dose". The term "fynomer" refers to a protein derived from the SH3 domain of human Fyn kinase engineered to bind to a specific target (Bertschinger et al., 2007. Protein Eng Des Sel. 20(2):57-68).

[0019] The terms "identical" or "percent identity" in the context of two or more polypeptide or nucleic acid molecule sequences mean that, when compared and aligned for maximum correspondence over a comparison window or specified region using methods known in the art, such as by manual alignment or by using a sequence comparison algorithm by visual inspection, the sequences are the same or have a specified percentage of amino acid residues or nucleotides that are the same over the specified region (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity). For example, preferred algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1977. Nucleic Acids Res. 25:3389 and Altschul et al., 1990. J Mol Biol. 215:403. In certain embodiments, the percent identity is determined over the length of the reference sequence disclosed herein.

[0020] The terms "individual", "patient" or "subject" are used interchangeably in the present application to refer to a human, and are not meant to be limiting in any way. An "individual", "patient" or "subject" can be of any age, sex and physical condition. The terms "isoaspartic acid modified transthyretin", "isoAsp modified transthyretin", "isoAsp modified TTR", "isoD modified transthyretin" and "isoD modified TTR" refer to a transthyretin protein in which an aspartic acid residue has been post-translationally modified to isoaspartic acid (preferably L-isoaspartic acid). An exemplary isoD modified TTR is isoD38 TTR in which the aspartic acid at position 38 of SEQ ID NO: 1 has been post-translationally modified to L-isoaspartic acid. IsoD38 TTR refers to a TTR protein comprising SEQ ID NO: 3. SEQ ID NO: 1 is the following amino acid sequence: GPTGTGESKCPLMVKVLDAVRGSPAINVAVHVFRKAADDTWEPFASGKTSESGELHGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTTAVVTNPKE is an exemplary TTR.

[0021] The term "K D " refers to the dissociation constant. In a preferred embodiment, K D is determined by surface plasmon resonance or isothermal titration calorimetry. Preferably, K D is determined by surface plasmon resonance at 25°C. The term "monobody" refers to a protein derived from a fibronectin type III domain engineered to bind to a specific target (Koide et al., 2013. J Mol Biol. 415(2):393-405). The term "nanobody" refers to a protein comprising a soluble single antigen-binding V domain of a heavy chain, preferably of a camelid (see Bannas et al., 2017. Front Immunol. 8:1603). The term "peptide aptamer" refers to a short 5-20 amino acid residue sequence that can bind to a specific target. Peptide aptamers are usually inserted into the loop region of a stable protein backbone (see Reverdatto et al., 2015. Curr Top Med Chem. 15(12):1082-101).

[0022] As used in this application, the term "prevention" refers to a series of hygienic, pharmacological, surgical, and / or physical means used to prevent the onset and / or progression of a disease and / or symptom. Since the term "prevention" is used to maintain the health of an animal or individual, it includes preventive methods.

[0023] The term "lipobody" refers to a protein derived from a leucine-rich repeat module that has been engineered to bind to a specific target (Lee et al., 2012. PNAS. 109(9): 3299-3304). The term "single-chain antigen-binding fragment" or "scFab" refers to a fusion protein comprising one variable domain and one constant domain of the heavy chain of an antibody and one variable domain and one constant domain of the light chain of an antibody bound thereto, wherein the heavy chain and the light chain are both linked via a short peptide. The term "single-chain variable fragment" or "scFv" refers to a fusion protein comprising the variable domains of the heavy and light chains of an antibody linked to each other by a peptide linker.

[0024] The term "specifically binds" in the context of the present invention refers to the K when the binding protein binds to TTR that does not contain isoD residues D which is at least 10-fold lower (e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200-fold lower) than the K D and is used to describe a binding protein (e.g., an antibody) that binds to isoD-modified TTR. In certain embodiments, the epitope to which the binding protein specifically binds comprises an isoD residue (preferably the isoD residue at position 38). As used herein, the terms "treatment" and "therapy" refer to a series of hygienic, pharmacological, surgical, and / or physical means that are intended to cure and / or alleviate diseases and / or symptoms for the purpose of improving health problems. The terms "treatment" and "therapy" both include preventive and therapeutic methods for maintaining and / or restoring the health of an individual or animal subject. Administration of an appropriate medicament to alleviate and / or cure a health problem, regardless of the origin of the symptoms, disease, and physical impairment, should be construed as a form of treatment or therapy within the context of the present application.

[0025] Binding protein In a first aspect, the present invention provides a binding protein that specifically binds to isoD-modified TTR. In certain embodiments, the binding protein specifically binds to isoD38 TTR. In certain embodiments, the K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 3 (e.g., TTR) D is at least 10-fold lower (e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 350, 400, or 450-fold lower) than the K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 2 (e.g., TTR). More preferably, the K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 3 (e.g., TTR) D is at least 150, 160, 170, 180, 190, or 200-fold lower than the K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 2 (e.g., TTR). D is at least 10-fold lower (e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 350, 400, or 450-fold lower) than the K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 2 (e.g., TTR). More preferably, the K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 3 (e.g., TTR) D is at least 150, 160, 170, 180, 190, or 200-fold lower than the K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 2 (e.g., TTR).

[0026] In certain embodiments, the K of the interaction between the binding protein and SEQ ID NO: 3 D is the K of the interaction between the binding protein and SEQ ID NO: 2 DAt least 10-fold lower (e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 350, 400, or 450-fold lower) than. More preferably, the K of the interaction between the binding protein and SEQ ID NO: 3 D is the K of the interaction between the binding protein and SEQ ID NO: 2 D and is at least 150, 160, 170, 180, 190, or 200-fold lower.

[0027] Preferably, K D is determined by surface plasmon resonance or isothermal titration calorimetry. More preferably, K D is determined by surface plasmon resonance at 25°C. In certain embodiments, K D is determined using SEQ ID NO: 5 with respect to SEQ ID NO: 3 (in other words, K D is determined using a peptide consisting of SEQ ID NO: 3 with a PEG-biotin moiety covalently attached to the N-terminal residue). In certain embodiments, K D is determined using SEQ ID NO: 6 with respect to SEQ ID NO: 2 (in other words, K D is determined using a peptide consisting of SEQ ID NO: 2 with a PEG-biotin moiety covalently attached to the N-terminal residue).

[0028] In certain embodiments, the K of the interaction between the binding protein and SEQ ID NO: 5 D is the K of the interaction between the binding protein and SEQ ID NO: 6 D and is at least 10-fold lower (e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 350, 400, or 450-fold lower). More preferably, the K of the interaction between the binding protein and SEQ ID NO: 5 D is the K of the interaction between the binding protein and SEQ ID NO: 6 D and is at least 150, 160, 170, 180, 190, or 200-fold lower.

[0029] SEQ ID NO:2 is TTR(35-43) and has the following sequence: KAADDTWEP. SEQ ID NO:3 is isoD38 TTR(35-43) and has the following sequence: KAAXDTWEP (wherein X is L-isoAsp). SEQ ID NO:5 is isoD38 TTR(35-43) PEG-biotin and has the following sequence: KAAXDTWEP (wherein X is L-isoAsp, the N-terminal residue is modified with a PEG-biotin moiety, and the C-terminal residue is amidated).

[0030] In certain embodiments, SEQ ID NO:5 is a modified peptide according to formula (I):

[0031]

Chemical formula

[0032] In certain embodiments, SEQ ID NO:6 is a modified peptide according to formula (II)

[0033]

Chemical formula

[0034] In certain embodiments, the K of the interaction between the binding protein and the modified peptide according to formula (I) D is the K of the interaction between the binding protein and the modified peptide according to formula (II) DAt least 10-fold lower (e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 350, 400, or 450-fold lower) than. In a preferred embodiment, the K of the interaction between the binding protein and the modified peptide according to formula (I) D is at least 100-fold lower than the K of the interaction between the binding protein and the modified peptide according to formula (II) D More preferably, the K of the interaction between the binding protein and the modified peptide according to formula (I) D is at least 150, 160, 170, 180, 190 or 200-fold lower than the K of the interaction between the binding protein and the modified peptide according to formula (II) D In a preferred embodiment, the K at 25 °C of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 3 (e.g., TTR) D is less than 200 nM. Preferably, K D is less than 150 nM. More preferably, K D is less than 10 nM. Most preferably, K D is less than 5 nM. Preferably, K D is determined by surface plasmon resonance or isothermal titration calorimetry. More preferably, K D is determined by surface plasmon resonance at 25 °C. In a preferred embodiment, the K at 25 °C of the interaction between the binding protein and SEQ ID NO: 3 D is less than 200 nM. Preferably, K D is less than 150 nM. More preferably, K D is less than 10 nM. Most preferably, K D is less than 5 nM. Preferably, K D is determined by surface plasmon resonance or isothermal titration calorimetry. More preferably, K D is determined by surface plasmon resonance at 25 °C. In one embodiment, K D is determined using SEQ ID NO: 5 against SEQ ID NO: 3 (in other words, K D ​(determined using the peptide consisting of SEQ ID NO: 3 having a PEG-biotin moiety covalently attached to the N-terminal residue).

[0035] In a preferred embodiment, the K of the interaction between the binding protein and SEQ ID NO: 5 at 25°C D is less than 200 nM. Preferably, the K D is less than 150 nM. More preferably, the K D is less than 10 nM. Most preferably, the K D is less than 5 nM. Preferably, the K D is determined by surface plasmon resonance or isothermal titration calorimetry. More preferably, the K D is determined by surface plasmon resonance at 25°C.

[0036] In a preferred embodiment, the K of the interaction between the binding protein and the modified peptide according to formula (I) at 25°C D is less than 200 nM. Preferably, the K D is less than 150 nM. More preferably, the K D is less than 10 nM. Most preferably, the K D is less than 5 nM. Preferably, the K D is determined by surface plasmon resonance or isothermal titration calorimetry. More preferably, the K D is determined by surface plasmon resonance at 25°C.

[0037] In certain embodiments, the binding protein comprises an Fc domain. Exemplary Fc domains can consist of the second and third constant domains (CH2-CH3) of the human IgG1 heavy chain. The Fc domain enables the binding protein to bind to Fc receptors and / or C1q, thereby inducing antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated phagocytosis, and / or activation of the classical complement pathway. In alternative embodiments, the binding protein is a chimeric antigen receptor (see, e.g., Guedan et al., 2018 Mol Ther Methods Clin Dev. 12:145-156) or a chimeric uptake receptor (see, e.g., International Publication No. WO 2018 / 064076). In certain embodiments, the binding protein is capable of inducing phagocytosis of isoD-modified TTR. In certain embodiments, the binding protein is an antibody, scFv, scFab, chimeric antigen receptor, or chimeric uptake receptor.

[0038] In preferred embodiments, the binding protein is as follows: (a) a heavy chain variable region (VH) comprising an HCDR3 polypeptide selected from NSYYGMDY (SEQ ID NO: 16) and EDY (SEQ ID NO: 26); and / or (b) a light chain variable region (VL) comprising HQYLSSRT (SEQ ID NO: 13) and QHFWNIPFT (SEQ ID NO: 23). In preferred embodiments, the binding protein comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises the polypeptides of LCDR1, LCDR2, and LCDR3, and the VH comprises the polypeptides of HCDR1, HCDR2, and HCDR3, where (a) LCDR1 is KSSQSVLYSSNQKNYLA (SEQ ID NO: 11), LCDR2 is WASTRES (SEQ ID NO: 12), LCDR3 is HQYLSSRT (SEQ ID NO: 13), HCDR1 is GFTFSSFAMH (SEQ ID NO: 14), HCDR2 is FISSGSNTIYYADTVKG (SEQ ID NO: 15), and HCDR3 is NSYYGMDY (SEQ ID NO: 16); and (b) LCDR1 is RTSGNIRNSLA (SEQ ID NO: 21), LCDR2 is NGKTLAD (SEQ ID NO: 22), LCDR3 is QHFWNIPFT (SEQ ID NO: 23), HCDR1 is GNTFSSRWIE (SEQ ID NO: 24), HCDR2 is EIFPGNGNTNYNEKFKG (SEQ ID NO: 25) and HCDR3 is EDY (SEQ ID NO: 26). Variants of the CDRs shown in SEQ ID NOs: 11-26 are also contemplated by the present invention. Methods for identifying variant CDRs, and thus variant binding proteins, are known in the art (see, for example, WO 2006 / 050491).

[0039] The amino acids of the binding protein may be substituted by amino acids having similar properties (based on size, polarity, hydrophobicity, etc.) to the amino acids being substituted. In other words, the amino acids of the binding protein may be substituted with different amino acids of the same class, where the amino acids are classified as follows: Aromatic: Phe, Tyr, Trp; Non-polar: Leu, Val, Ile, Ala, Met; Aliphatic: Ala, Val, Leu, Ile; Acidic: Asp, Glu; Basic: His, Lys, Arg; Polar: Gln, Asn, Ser, Thr, Tyr; and may be classified as such. In certain embodiments, up to 50% (preferably up to 30%, more preferably up to 20%, even more preferably up to 10%) of the amino acid residues in 1, 2, 3, 4, 5 or 6 (preferably 6) CDRs may be substituted according to the following table.

[0040] [Table 1] The amino acids of the binding protein may be substituted by different amino acids present at the same position in a related binding protein (i.e., a binding protein that can bind to the same target). As used herein, 2F2 and 4D4 are related binding proteins.

[0041] In certain embodiments, the invention encompasses a binding protein variant, wherein the binding protein variant comprises a CDR with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues different from the CDRs of 2F2 (i.e., SEQ ID NOs: 11-16) or 4D4 (i.e., SEQ ID NOs: 21-26).

[0042] In preferred embodiments, the binding protein is as follows: (a) a heavy chain variable region (VH) comprising an HCDR3 polypeptide selected from NSYYGMDY (SEQ ID NO: 16) or a sequence having at least 70% (preferably at least 80%) identity thereto and EDY (SEQ ID NO: 26) or a sequence having at least 65% identity thereto; and / or (b) a light chain variable region (VL) comprising an LCDR3 polypeptide selected from HQYLSSRT (SEQ ID NO: 13) or a sequence having at least 70% (preferably at least 80%) identity thereto and QHFWNIPFT (SEQ ID NO: 23) or a sequence having at least 70% (preferably at least 80%) identity thereto. In preferred embodiments, the binding protein comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises the polypeptides of LCDR1, LCDR2, and LCDR3, and the VH comprises the polypeptides of HCDR1, HCDR2, and HCDR3, wherein (a) LCDR1 is a sequence that is KSSQSVLYSSNQKNYLA (SEQ ID NO: 11) or has at least 70% (preferably at least 90%) identity thereto, LCDR2 is a sequence that is WASTRES (SEQ ID NO: 12) or has at least 70% (preferably at least 80%) identity thereto, LCDR3 is a sequence that is HQYLSSRT (SEQ ID NO: 13) or has at least 70% or at least 80% identity thereto, HCDR1 is a sequence that is GFTFSSFAMH (SEQ ID NO: 14) or has at least 70% (preferably at least 90%) identity thereto, HCDR2 is a sequence that is FISSGSNTIYYADTVKG (SEQ ID NO: 15) or has at least 70% (preferably at least 90%) identity thereto and HCDR3 is a sequence that is NSYYGMDY (SEQ ID NO: 16) or has at least 70% (preferably at least 80%) identity thereto; and (b) selected from the group consisting of: LCDR1 is a sequence that is RTSGNIRNSLA (SEQ ID NO: 21) or has at least 70% (preferably at least 90%) identity thereto, LCDR2 is a sequence that is NGKTLAD (SEQ ID NO: 22) or has at least 70% (preferably at least 80%) identity thereto, LCDR3 is a sequence that is QHFWNIPFT (SEQ ID NO: 23) or has at least 70% (preferably at least 80%) identity thereto, HCDR1 is a sequence that is GNTFSSRWIE (SEQ ID NO: 24) or has at least 70% (preferably at least 90%) identity thereto, HCDR2 is a sequence that is EIFPGNGNTNYNEKFKG (SEQ ID NO: 25) or has at least 70% (preferably at least 90%) identity thereto and HCDR3 is a sequence that is EDY (SEQ ID NO: 26) or has at least 65% identity thereto.

[0043] In a preferred embodiment, the binding protein comprises VL and VH, and the VL and VH are polypeptides selected from the group consisting of: (a) VL of SEQ ID NO: 17 and VH of SEQ ID NO: 18, and (b) VL of SEQ ID NO: 27 and VH of SEQ ID NO: 28. In a preferred embodiment, the binding protein comprises VL and VH, and the VL and VH are: (a) the VL of SEQ ID NO: 17 or a sequence having at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80% or 85%) identity thereto and the VH of SEQ ID NO: 18 or a sequence having at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80% or 85%) identity thereto; (b) the VL of SEQ ID NO: 27 or a sequence having at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80% or 85%) identity thereto and the VH of SEQ ID NO: 28 or a sequence having at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80% or 85%) identity thereto; and (c) the VL of SEQ ID NO: 17 or a sequence having at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80% or 85%) identity thereto and the VH of SEQ ID NO: 28 or a sequence having at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80% or 85%) identity thereto; and (d) the VL of SEQ ID NO: 27 or a sequence having at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80% or 85%) identity thereto and the VH of SEQ ID NO: 18 or a sequence having at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80% or 85%) identity thereto, and is a polypeptide selected from the group consisting of these. In a preferred embodiment, the binding protein comprises VL and VH, and the VL and VH are polypeptides selected from the group consisting of the following: (a) the VL of SEQ ID NO: 17 or a sequence having at least 55% identity thereto and the VH of SEQ ID NO: 18 or a sequence having at least 55% identity thereto; (b) the VL of SEQ ID NO: 27 or a sequence having at least 55% identity thereto and the VH of SEQ ID NO: 28 or a sequence having at least 55% identity thereto; (c) the VL of SEQ ID NO: 17 or a sequence having at least 55% identity thereto and the VH of SEQ ID NO: 28 or a sequence having at least 55% identity thereto; (d) the VL of SEQ ID NO: 27 or a sequence having at least 55% identity thereto and the VH of SEQ ID NO: 18 or a sequence having at least 55% identity thereto. In a preferred embodiment, the binding protein comprises VL and VH, and the VL and VH are polypeptides selected from the group consisting of the following: (a) VL of SEQ ID NO: 17 or a sequence having at least 60% identity thereto and VH of SEQ ID NO: 18 or a sequence having at least 60% identity thereto; (b) VL of SEQ ID NO: 27 or a sequence having at least 60% identity thereto and VH of SEQ ID NO: 28 or a sequence having at least 60% identity thereto; (c) VL of SEQ ID NO: 17 or a sequence having at least 60% identity thereto and VH of SEQ ID NO: 28 or a sequence having at least 60% identity thereto; (d) VL of SEQ ID NO: 27 or a sequence having at least 60% identity thereto and VH of SEQ ID NO: 18 or a sequence having at least 60% identity thereto. In a preferred embodiment, the binding protein comprises VL and VH, and the VL and VH are polypeptides selected from the group consisting of the following: (a) VL of SEQ ID NO: 17 or a sequence having at least 65% identity thereto and VH of SEQ ID NO: 18 or a sequence having at least 65% identity thereto; (b) VL of SEQ ID NO: 27 or a sequence having at least 65% identity thereto and VH of SEQ ID NO: 28 or a sequence having at least 65% identity thereto; (c) VL of SEQ ID NO: 17 or a sequence having at least 65% identity thereto and VH of SEQ ID NO: 28 or a sequence having at least 65% identity thereto; (d) VL of SEQ ID NO: 27 or a sequence having at least 65% identity thereto and VH of SEQ ID NO: 18 or a sequence having at least 65% identity thereto. In a preferred embodiment, the binding protein comprises VL and VH, and the VL and VH are polypeptides selected from the group consisting of the following: (a) VL of SEQ ID NO: 17 or a sequence having at least 70% identity thereto and VH of SEQ ID NO: 18 or a sequence having at least 70% identity thereto; (b) VL of SEQ ID NO: 27 or a sequence having at least 70% identity thereto and VH of SEQ ID NO: 28 or a sequence having at least 70% identity thereto; (c) VL of SEQ ID NO: 17 or a sequence having at least 70% identity thereto and VH of SEQ ID NO: 28 or a sequence having at least 70% identity thereto; (d) VL of SEQ ID NO: 27 or a sequence having at least 70% identity thereto and VH of SEQ ID NO: 18 or a sequence having at least 70% identity thereto. In a preferred embodiment, the binding protein comprises VL and VH, and the VL and VH are polypeptides selected from the group consisting of the following: (a) VL of SEQ ID NO: 17 or a sequence having at least 75% identity thereto and VH of SEQ ID NO: 18 or a sequence having at least 75% identity thereto; (b) VL of SEQ ID NO: 27 or a sequence having at least 75% identity thereto and VH of SEQ ID NO: 28 or a sequence having at least 75% identity thereto; (c) VL of SEQ ID NO: 17 or a sequence having at least 75% identity thereto and VH of SEQ ID NO: 28 or a sequence having at least 75% identity thereto; (d) VL of SEQ ID NO: 27 or a sequence having at least 75% identity thereto and VH of SEQ ID NO: 18 or a sequence having at least 75% identity thereto. In a preferred embodiment, the binding protein comprises VL and VH, and the VL and VH are polypeptides selected from the group consisting of the following: (a) the VL of SEQ ID NO: 17 or a sequence having at least 80% identity thereto and the VH of SEQ ID NO: 18 or a sequence having at least 80% identity thereto; (b) the VL of SEQ ID NO: 27 or a sequence having at least 80% identity thereto and the VH of SEQ ID NO: 28 or a sequence having at least 80% identity thereto; (c) the VL of SEQ ID NO: 17 or a sequence having at least 80% identity thereto and the VH of SEQ ID NO: 28 or a sequence having at least 80% identity thereto; (d) the VL of SEQ ID NO: 27 or a sequence having at least 80% identity thereto and the VH of SEQ ID NO: 18 or a sequence having at least 80% identity thereto. In a preferred embodiment, the binding protein comprises VL and VH, and the VL and VH are polypeptides selected from the group consisting of the following: (a) the VL of SEQ ID NO: 17 or a sequence having at least 85% identity thereto and the VH of SEQ ID NO: 18 or a sequence having at least 85% identity thereto; (b) the VL of SEQ ID NO: 27 or a sequence having at least 85% identity thereto and the VH of SEQ ID NO: 28 or a sequence having at least 85% identity thereto; (c) the VL of SEQ ID NO: 17 or a sequence having at least 85% identity thereto and the VH of SEQ ID NO: 28 or a sequence having at least 85% identity thereto; (d) the VL of SEQ ID NO: 27 or a sequence having at least 85% identity thereto and the VH of SEQ ID NO: 18 or a sequence having at least 85% identity thereto.

[0044] SEQ ID NO: 17 is the VL fragment present in 2F2 and has the following sequence: NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQYLSSRTFGAGTKLELK There is.

[0045] SEQ ID NO: 18 is the VH fragment present in 2F2 and has the following sequence: DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFAMHWVRQAPDKGLEWVAFISSGSNTIYYADTVKGRFTVSRDNPKNTLFLQMTSLRSEDTAMYYCASNSYYGMDYWGQGTSVTVSS is present.

[0046] SEQ ID NO: 27 is the VL fragment present in 4D4 and has the following sequence: DIQMTQSPASLSASVGETVTITCRTSGNIRNSLAWYRQKQGKSPQLLVFNGKTLADGVPSRFSGSGSGTQYSLKINSLQPEDLGTYYCQHFWNIPFTFGSGTKLEIK is present.

[0047] SEQ ID NO: 28 is the VH fragment present in 4D4 and has the following sequence: QVQLQQSGAELMKPGASVKISCKAPGNTFSSRWIEWVKERPGHGLEWIGEIFPGNGNTNYNEKFKGKATFTADTSSNTAYIQFSSLASEDSGVYYCVSEDYWGQGTLVTVSS is present.

[0048] Preferably, the binding protein is an antibody. Phage display and combinatorial methods for making antibodies are known in the art (e.g., Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No. 92 / 18619; Dower et al. International Publication No. 91 / 17271; Winter et al. International Publication No. 92 / 20791; Markland et al. International Publication No. 92 / 15679; Breitling et al. International Publication No. 93 / 01288; McCafferty et al. International Publication No. 92 / 01047; Garrard et al. International Publication No. 92 / 09690; Ladner et al. International Publication No. 90 / 02809; Fuchs et al. (1991) Bio / Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio / Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137 and Barbas et al. (1991) PNAS 88:7978-7982).

[0049] In one embodiment, the antibody is a fully human antibody (e.g., an antibody made in a mouse that produces an antibody from a human immunoglobulin sequence by genetic engineering) or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat) antibody. Methods for producing rodent antibodies are known in the art. Human monoclonal antibodies can be produced using transgenic mice that have human immunoglobulin genes rather than mouse systems. Using splenocytes from the transgenic mice immunized with the antigen of interest, hybridomas that secrete human mAbs with specific affinity for epitopes derived from human proteins are produced (for example, Wood et al., WO 91 / 00906; Kucherlapati et al., WO 91 / 10741; Lonberg et al., WO 92 / 03918; Kay et al., WO 92 / 03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L.L. et al. 1994 Nature Genet. 7:13-21; Morrison, S.L. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).

[0050] The antibody may be produced in a non-human organism, such as a rat or mouse, in the variable region or a portion thereof, such as a CDR. Chimeric antibodies, CDR-grafted antibodies, and humanized antibodies are within the scope of the present invention. Antibodies that have been modified to reduce human antigenicity, for example, in the variable framework or constant region, after being produced in a non-human organism, such as a rat or mouse, are within the scope of the present invention. Chimeric antibodies may be produced by recombinant DNA techniques well known in the art (see Robinson et al., International Patent Application PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Publication No. 86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449 and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[0051] Humanized or CDR-grafted antibodies have at least one or two, but generally all three, recipient CDRs (of the immunoglobulin heavy and / or light chains) replaced with donor CDRs. The antibody may be replaced with at least a portion of the non-human CDR, or only a portion of the CDR may be replaced with the non-human CDR. It is only necessary to replace the number of CDRs necessary for the binding of the humanized antibody to isoD-modified TTR. Preferably, the donor is a rodent antibody, such as a rat or mouse antibody, and the recipient is a human framework or human consensus framework. Usually, the immunoglobulin that provides the CDR is referred to as the "donor", and the immunoglobulin that provides the framework is referred to as the "recipient". In one embodiment, the donor immunoglobulin is a non-human (e.g., rodent) immunoglobulin. The receptor framework is a naturally occurring (e.g., human) framework or consensus framework, or a sequence that is about 85% or more, preferably 90%, 95%, 99% or more identical thereto.

[0052] As used herein, the term "consensus sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of related sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a protein family, the positions in the consensus sequence are each occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either may be included in the consensus sequence. "Consensus framework" refers to the framework regions in a consensus immunoglobulin sequence.

[0053] Antibodies can be humanized by methods known in the art (see, e.g., Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214 and Queen et al. U.S. Patent No. 5,585,089, U.S. Patent No. 5,693,761 and U.S. Patent No. 5,693,762). Humanized antibodies or CDR-grafted antibodies can be produced by CDR-grafting or CDR-substitution, where one, two, or all of the CDRs of an immunoglobulin chain can be replaced. See, e.g., U.S. Patent No. 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter U.S. Patent No. 5,225,539. Winter describes CDR-grafting methods that can be used to prepare the humanized antibodies of the present invention (British Patent Application 2188638 filed Mar. 26, 1987; Winter U.S. Patent No. 5,225,539). Also, humanized antibodies in which specific amino acids are substituted, deleted, or added are within the scope of the present invention. The criteria for selecting amino acids from the donor are described in U.S. Patent No. 5,585,089, for example, columns 12 to 16 of U.S. Patent No. 5,585,089, for example, columns 12 to 16 of U.S. Patent No. 5,585,089. Other techniques for humanizing antibodies are described in Padlan et al. European Patent Application Publication No. 519596, published December 23, 1992.

[0054] In still other embodiments, the antibody is selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; in particular, there is a heavy chain constant region selected from the (e.g., human) heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. In other embodiments, the antibody has a light chain constant region selected from, for example, the (e.g., human) kappa or lambda light chain constant regions. The constant region can be modified, for example, mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, and / or complement function). In one embodiment, the antibody has effector function and can fix complement. In other embodiments, the antibody does not recruit effector cells and does not fix complement. In other embodiments, the antibody has a reduced or no ability to bind to Fc receptors. For example, it is an isotype or subtype, fragment, or other variant that does not support binding to Fc receptors, for example, the Fc receptor binding region has been mutagenized or deleted. Methods for modifying antibody constant regions are known in the art. Antibodies with modified functions, for example, antibodies with modified affinity for effector ligands such as FcR on cells or the C1 component of complement, can be produced by substituting at least one amino acid residue in the constant portion of the antibody with a different residue (see, for example, European Patent Application Publication No. 388,151, U.S. Patent No. 5,624,821, and U.S. Patent No. 5,648,260). Similar types of modifications that reduce or eliminate these functions can be described when applied to mouse or other species immunoglobulins.

[0055] Antibodies can be derivatized or conjugated to another functional molecule (e.g., another peptide or protein). As used herein, the term "derivatized" antibody molecule is modified. Methods of derivatization include, but are not limited to, the addition of a fluorescent moiety, a radioactive nucleotide, a toxin, an enzyme, or an affinity ligand such as biotin. Accordingly, the antibody molecules of the present invention are intended to include derivatized forms of the antibodies described herein, including immunoadhesion molecules, and other modified forms. For example, an antibody molecule can be functionally linked (by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detectable agent, a cytotoxic agent, a pharmaceutical, and / or a protein or peptide that can mediate the association of the antibody or antibody portion with another molecule (e.g., a streptavidin core region or a polyhistidine tag). One type of derivatized antibody molecule is produced by crosslinking two or more antibodies (of the same or different types, e.g., for making bispecific antibodies). Suitable crosslinking agents include hetero-bifunctional (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homo-bifunctional (e.g., disuccinimidyl suberate) crosslinking agents having two clearly reactive groups separated by a suitable spacer. Such linkers are available from Pierce Chemical Company, Rockford, IL.

[0056] Antibody molecules can complex with other molecular entities, typically labels or agents (e.g., cytotoxic or cytostatic) or portions thereof. Radioisotopes can be used for diagnostic or therapeutic applications. Such radioisotopes include iodine ( 131 I or 125 I), yttrium ( 90 Y), lutetium ( 177 Lu), actinium ( 225 Ac), praseodymium, astatine ( 211 At), rhenium ( 186 Re), bismuth (212 Bi or 213 Bi), indium ( 111 In), technetium ( 99 mTc), phosphorus ( 32 P), rhodium ( 188 Rh), sulfur ( 35 S), carbon ( 14 C), tritium ( 3 H), chromium ( 51 Cr), chlorine ( 36 Cl), cobalt ( 57 Co or 58 Co), iron ( 59 Fe), selenium ( 75 Se), or gallium ( 67 Ga), among others. For example, useful radioisotopes as diagnostic labels include iodine ( 131 I or 125 I), indium ( 111 In), technetium ( 99 mTc), phosphorus ( 32 P), carbon ( 14 C) and tritium ( 3 H), or one or more of the therapeutic isotopes listed above. The present invention provides radio-labeled antibody molecules and methods for labeling them. In one embodiment, a method for labeling an antibody molecule is disclosed. The method includes contacting the antibody molecule with a chelating agent to produce a complex antibody. The complex antibody is radio-labeled with a radioisotope, such as indium-111, yttrium-90, and lutetium-177, to generate a labeled antibody molecule. The antibody molecule can be complexed with a therapeutic agent. The antibody may be labeled. For example, the antibody can be labeled with a biotin molecule, an enzyme, or a fluorophore.

[0057] The exact amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described in Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (the “Kabat” numbering scheme), and Al-Lazikani et al., (1997) JMB 273,927-948 (the “Chothia” numbering scheme). The CDRs defined according to the “Chothia” numbering scheme used herein may also be referred to as “hypervariable loops.” In certain embodiments, the amino acid sequence boundaries of the CDRs are determined using the Kabat numbering scheme. An exemplary application of the Kabat numbering scheme is provided in Figure 3.

[0058] In preferred embodiments, the antibody or antigen-binding fragment thereof comprises two LCs and two HCs, and each LC and each HC is a polypeptide selected from the group consisting of: (a) an LC of SEQ ID NO: 31 and an HC of SEQ ID NO: 32; (b) an LC of SEQ ID NO: 33 and an HC of SEQ ID NO: 34; (c) an LC of SEQ ID NO: 31 and an HC of SEQ ID NO: 34; and (d) an LC of SEQ ID NO: 33 and an HC of SEQ ID NO: 32.

[0059] SEQ ID NO: 31 is the LC present in 2F2 and has the following sequence: NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQYLSSRTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC is as follows.

[0060] Sequence number 32 is the HC present in 2F2, with the following sequence: DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFAMHWVRQAPDKGLEWVAFISSGSNTIYYADTVKGRFTVSRDNPKNTLFLQMTSLRSEDTAMYYCASNSYYGMDYWGQGTSVTVSSATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPGK There is.

[0061] Sequence number 33 is the LC present in 4D4, with the following sequence: DIQMTQSPASLSASVGETVTITCRTSGNIRNSLAWYRQKQGKSPQLLVFNGKTLADGVPSRFSGSGSGTQYSLKINSLQPEDLGTYYCQHFWNIPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC There is.

[0062] Sequence number 34 is the HC present in 4D4, with the following sequence: QVQLQQSGAELMKPGASVKISCKAPGNTFSSRWIEWVKERPGHGLEWIGEIFPGNGNTNYNEKFKGKATFTADTSSNTAYIQFSSLASEDSGVYYCVSEDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK is present. In a preferred embodiment, the antibody is a monoclonal antibody.

[0063] Nucleic acid In a second aspect, the present invention provides a nucleic acid comprising a sequence encoding the binding protein of the present invention. In a preferred embodiment, the sequence is codon-optimized to ensure high expression of the fusion protein. Methods for optimizing the codons of gene constructs are known in the art (Quax et al., 2015. Mol Cell. 59(2):149-61). In a preferred embodiment, the nucleic acid is contained in a vector. The vector can be a cloning vector, an expression vector, a transfer vector or a viral vector. In certain embodiments, the nucleic acid is a modified mRNA molecule suitable for in vivo expression of the binding protein. The mRNA can be delivered using lipid nanoparticles (Hou et al., 2021. Nat Rev Mater. 6(12):1078-1094).

[0064] In certain embodiments, the nucleic acid is SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 29 and SEQ ID NO: 20; or SEQ ID NO: 19 and SEQ ID NO: 30.

[0065] SEQ ID NO: 19: Aacattatgatgacgcagtcgccatcatctctggctgtgtctgcaggagaaaaggtcactatgagctgtaagtccagtcagagtgttttatacagttcaaatcagaagaattacttggcctggtaccagcagaaaccagggcagtctcctaaactgctgatctactgggcatccactagggaatctggtgtccctgatcgcttcacaggcagtggatctgggacagattttactctcaccatcagcagtgtacaagctgaagacctggcagtttattactgtcatcaatacctctcctcgcgcacgttcggtgctgggaccaagctggagctgaag is a nucleic acid sequence encoding the VL fragment of 2F2.

[0066] SEQ ID NO: 20: gatgtgcagctggtggagtctgggggaggcttagtgcagcctggagggtcccggaaactctcctgtgcagcctctggattcactttcagtagctttgcaatgcactgggttcgtcaggctccagacaaggggctggagtgggtcgcattcattagtagtggcagtaataccatctactatgcagacacagtgaagggccgattcaccgtctccagagacaatcccaagaacaccctgttcctgcaaatgaccagtctaaggtctgaggacacggccatgtattactgtgcaagtaattcgtactatggtatggactactggggtcaaggaacctcagtcaccgtctcctca is a nucleic acid sequence encoding the VH fragment of 2F2.

[0067] Sequence number 29: gacatccagatgactcagtctccagcctccctatctgcatctgtgggagaaactgtcaccatcacatgtcgaacaagtgggaatattcgcaattcattagcatggtatcgacagaaacagggaaaatcccctcagctcctggtctttaatggcaaaactttagcagatggtgtgccatcaaggttcagtggcagtggatcaggaacacaatattctctcaagatcaacagcctgcagcctgaagatttagggacttattactgtcaacatttttggaatattccattcacgttcggctcggggacaaagttggaaataaaa is a nucleic acid sequence encoding the VL fragment of 4D4. Sequence number 30: caggttcagctgcagcagtctggagctgagctgatgaagcctggggcctcagtgaagatatcctgcaaggctcctggcaacacattcagtagcaggtggatagagtgggtaaaggagaggcctggacatggccttgagtggattggagagatttttcctggaaatggtaatactaactacaatgagaagttcaagggcaaggccacattcactgcagatacatcctccaacacagcctatatacaattcagcagcctggcatctgaggactctggcgtctattactgtgtatctgaagactattggggccaaggtaccctggtcaccgtctcctca is a nucleic acid sequence encoding the VH fragment of 4D4.

[0068] In certain embodiments, the nucleic acid is sequence number 35 and sequence number 36; sequence number 37 and sequence number 38; sequence number 35 and sequence number 38; or sequence number 37 and sequence number 36.

[0069] SEQ ID NO: 35: Aacattatgatgacgcagtcgccatcatctctggctgtgtctgcaggagaaaaggtcactatgagctgtaagtccagtcagagtgttttatacagttcaaatcagaagaattacttggcctggtaccagcagaaaccagggcagtctcctaaactgctgatctactgggcatccactagggaatctggtgtccctgatcgcttcacaggcagtggatctgggacagattttactctcaccatcagcagtgtacaagctgaagacctggcagtttattactgtcatcaatacctctcctcgcgcacgttcggtgctgggaccaagctggagctgaagcgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgttag It is a nucleic acid sequence encoding the LC of 2F2.

[0070] SEQ ID NO: 36: is a nucleic acid sequence encoding the HC of 2F2.

[0071] SEQ ID NO: 37: Gacatccagatgactcagtctccagcctccctatctgcatctgtgggagaaactgtcaccatcacatgtcgaacaagtgggaatattcgcaattcattagcatggtatcgacagaaacagggaaaatcccctcagctcctggtctttaatggcaaaactttagcagatggtgtgccatcaaggttcagtggcagtggatcaggaacacaatattctctcaagatcaacagcctgcagcctgaagatttagggacttattactgtcaacatttttggaatattccattcacgttcggctcggggacaaagttggaaataaaacgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgttag is a nucleic acid sequence encoding the LC of 4D4. SEQ ID NO: 38: It is a nucleic acid sequence encoding the HC of 4D4. In certain embodiments, the sequences belonging to the heavy and light chains of the antibodies of the invention are encoded on one or two nucleic acid molecules.

[0072] Host cell In a third aspect, the invention provides a host cell comprising the nucleic acid of the invention. In certain embodiments, the host cell is used in a method for producing a binding protein of the invention, which comprises culturing the host cell under conditions suitable for protein expression and then isolating the binding protein. In certain embodiments, the host cell is an immune cell (e.g., a T cell or a natural killer cell), and the binding protein (e.g., a chimeric antigen receptor or a chimeric uptake receptor) is expressed on the surface of the cell.

[0073] Pharmaceutical composition In a fourth aspect, the invention provides a pharmaceutical composition comprising a binding protein, nucleic acid or host cell of the invention and a pharmaceutically acceptable carrier or diluent. As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable diluent" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutical active substances is well known in the art. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed and, without limiting the scope of the present invention, further include buffering agents; preservatives; co-solvents; antioxidants (including ascorbic acid and methionine); chelating agents such as EDTA; biodegradable polymers such as polyesters; salt-forming counterions such as sodium, polyhydric sugar alcohols, etc.; organic sugars or sugar alcohols such as alanine, glycine, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinositol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol, etc.; sulfur-containing reducing agents such as urea, glutathione, thiocctic acid, sodium thioglycolate, thioglycerol, [α]-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; and hydrophilic polymers such as polyvinylpyrrolidone. The pharmaceutical compositions described herein may also contain other substances. Such substances include, but are not limited to, cryoprotective substances, lyoprotectants, surfactants, bulking agents, antioxidants, and stabilizers. In certain embodiments, the pharmaceutical compositions may be lyophilized. As used herein, the term "cryoprotectant" includes agents that are preferentially excluded from the surface of proteins and provide stability to the active ingredient against stress induced by freezing. Cryoprotectants can also provide protection during primary and secondary drying and long-term product storage. Non-limiting examples of cryoprotectants include sugars such as sucrose, glucose, trehalose, mannitol, mannose, and lactose; polymers such as dextran, hydroxyethyl starch, and polyethylene glycol; surfactants such as polysorbate (e.g., PS-20 or PS-80); and amino acids such as glycine, arginine, leucine, and serine. Generally, cryoprotectants that exhibit low toxicity in biological systems are used.

[0074] In one embodiment, a lyoprotectant is added to the pharmaceutical composition described herein. As used herein, the term "lyoprotectant" includes agents that provide an amorphous glassy matrix, bind to the surface of the active ingredient via hydrogen bonds, and replace water molecules removed during the drying process, thereby providing stability to the active ingredient during the lyophilization or dehydration process (primary and secondary lyophilization cycles). This is useful for minimizing the degradation of the product during the lyophilization cycle and improving long-term product stability. Non-limiting examples of lyoprotectants include sugars such as sucrose or trehalose; amino acids such as sodium glutamate, amorphous glycine, or histidine; methylamines such as betaine; lyotropic salts such as magnesium sulfate; polyhydric alcohols with three or more hydroxyl groups, such as polyols like glycerin, erythritol, glycerol, arabinitol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; pluronic (registered trademark); and combinations thereof. The amount of lyoprotectant added to the pharmaceutical composition is generally an amount such that no unacceptable amount of the strain is degraded when the pharmaceutical composition is lyophilized.

[0075] In certain embodiments, the pharmaceutical composition includes a bulking agent. As used herein, the term "bulking agent" includes agents that do not directly interact with the pharmaceutical and provide the structure of the lyophilized product. The bulking agent results in a pharmaceutical-grade cake and can also impart useful qualities by modifying the collapse temperature, providing protection from freeze-thaw, and enhancing stock stability during long-term storage. Non-limiting examples of bulking agents include mannitol, glycine, lactose, and sucrose. The bulking agent may be crystalline (such as glycine, mannitol, or sodium chloride) or amorphous (such as dextran, hydroxyethyl starch), and is generally used in an amount of 0.5% to 10% in the formulation.

[0076] Other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), may be included in the pharmaceutical compositions disclosed herein so long as they do not adversely affect the desired characteristics of the pharmaceutical compositions. As used herein, the term "pharmaceutically acceptable carrier" means any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutical active substances is well known in the art. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and further include buffering agents; preservatives; co-solvents; antioxidants such as ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (such as Zn-protein complexes); biodegradable polymers such as polyesters; salt-forming counterions such as sodium; polyhydric sugar alcohols; amino acids such as alanine, glycine, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinositol, galactose, galactitol, glycerol, cyclitols (such as inositol), polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thiocctic acid, sodium thioglycolate, thioglycerol, [α]-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; and hydrophilic polymers such as polyvinylpyrrolidone.

[0077] The pharmaceutical compositions can be prepared for oral, sublingual, buccal, intravenous, intramuscular, subcutaneous, intraperitoneal, conjunctival, rectal, transdermal, intrathecal, topical, and / or inhalation-mediated administration. In a preferred embodiment, the pharmaceutical composition may be a solution suitable for intravenous, intramuscular, conjunctival, transdermal, intraperitoneal, and / or subcutaneous administration. In an alternative embodiment, the pharmaceutical composition may be a gel or solution suitable for intrathecal administration.

[0078] The pharmaceutical composition may further contain common excipients and carriers known in the art. In the case of a solid pharmaceutical composition, for example, conventional non-toxic solid carriers including pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. can be used. In the case of an injectable solution, the pharmaceutical composition may further contain a cryoprotectant, a lyoprotectant, a surfactant, a bulking agent, an antioxidant, a stabilizer, and a pharmaceutically acceptable carrier. For aerosol administration, the pharmaceutical composition is generally supplied in a finely divided form together with a surfactant and a propellant. The surfactant must, of course, be non-toxic and is generally soluble in the propellant. Representative examples of such agents are esters or partial esters of fatty acids containing 6 to 22 carbon atoms (e.g., caproic acid, octanoic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric acid, and oleic acid) with aliphatic polyhydric alcohols or their cyclic anhydrides. Mixed esters, such as mixed or natural glycerides, may be used. If desired, carriers such as lecithin for nasal delivery can also be included. In the case of suppositories, conventional binders and carriers may include, for example, polyalkylene glycols or triglycerides.

[0079] Therapeutic use In a fifth aspect, the present invention provides the binding protein, nucleic acid, host cell, or pharmaceutical composition of the present invention for use as a medicament. In a sixth aspect, the present invention provides the binding protein, nucleic acid, host cell, or pharmaceutical composition of the present invention for use in the treatment and / or prevention of transthyretin amyloidosis (ATTR). In certain embodiments, the binding proteins of the present invention that bind to and can remove or sequester isoD-modified TTR in biological fluids and tissues are useful for the prevention and / or treatment of conditions associated with the formation of TTR-containing aggregates and / or amyloid. The binding protein of the present invention is thought to induce clearance of aggregated TTR, oligomeric TTR, or monomeric TTR. Clearance is achieved, for example, by targeting aggregated oligomers or monomers of TTR for phagocytosis.

[0080] The binding protein, nucleic acid, host cell, or pharmaceutical composition of the present invention may be used in a method for the treatment and / or prevention of ATTR, wherein a therapeutically effective amount of the binding protein is administered to a subject in need thereof. Suitable patients for treatment include individuals at risk of ATTR but without symptoms, as well as patients currently showing symptoms. A patient may be treated in the pre-ATTR stage. In certain embodiments, ATTR is hereditary ATTR or wild-type ATTR. Examples of hereditary ATTR include familial amyloid cardiomyopathy (FAC), familial amyloid polyneuropathy (FAP), and central nervous system selective amyloidosis (CNSA). Examples of wild-type ATTR include senile systemic amyloidosis (SSA) and senile cardiac amyloidosis (SCA). In certain embodiments, ATTR is Portuguese-type amyloidosis. Portuguese-type amyloidosis (transthyretin amyloidosis, ATTR V30M) is the most common form of systemic hereditary amyloidosis that is inherited in an autosomal dominant mode. This disease, also known as familial amyloid polyneuropathy type I (FAP-I), is caused by a mutant transthyretin (TTR) protein synthesized in the liver. Only one amino acid substitution of valine at position 30 of TTR to methionine (TTR V30M) was found in patients with Portuguese disease (Lobato, 2003. J Nephrol. 16(3):438-42).

[0081] Exemplary dosage ranges of the binding protein can be about 0.1 - 20, or 0.5 - 5 mg per kg body weight (e.g., 0.5, 1, 2, 3, 4, or 5 mg / kg body weight), or 10 - 1500 mg as a fixed dose. The dosage depends on the patient's condition and, if any, the response to previous treatment for 1 - 42 nt, whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic, among other factors. The compositions of the present invention can be administered at dosages daily, every other day, weekly, every other week, monthly, quarterly, or according to any other schedule determined by empirical analysis. Exemplary treatments require multiple doses over a long period, for example, at least 6 months. Further exemplary treatment regimens require administration once every two weeks, or once a month, or once every 3 - 6 months. The composition can be administered via a peripheral route. Routes of administration include topical, intravenous, oral, subcutaneous, intra - arterial, intracranial, intrathecal, intraperitoneal, intranasal, or intramuscular. The route of administration of the antibody can be intravenous or subcutaneous. Intravenous administration can be, for example, an infusion over a period such as 30 - 90 minutes. The injection is most usually performed in the muscles of the arm or leg. In certain embodiments, the agent (e.g., binding protein, nucleic acid, host cell, or pharmaceutical composition) is directly injected into a specific tissue where deposits have accumulated.

[0082] The binding protein, nucleic acid, host cell, or pharmaceutical composition of the present invention can be co - administered with other agents effective for the treatment or prevention of the disease being treated. The agent can include siRNA that inhibits the expression of TTR, or Vyndaqel, which is a stabilizer of TTR in tetramer formation.

[0083] The efficacy of the treatment can be monitored by determining the level of TTR deposits in the subject by positron emission tomography (PET) before and after administration of the binding protein, nucleic acid, host cell, or pharmaceutical composition disclosed herein.

[0084] Diagnosis isoD-modified TTR was detected in extracted human ATTR-V30M protofibrils. Without being bound to a particular theory, this indicates that isoD in TTR is a biomarker suitable for the diagnosis and prognosis of ATTR. In a seventh aspect, the invention provides an in vitro diagnostic and / or prognostic method for detecting the presence of isoD-modified TTR in an isolated sample. In certain embodiments, the isolated sample is derived from a patient suspected of having ATTR. In certain embodiments, the method comprises the following: (i) contacting the isolated sample with a binding protein of the invention, and (ii) determining whether isoD-modified TTR is present in the isolated sample. In certain embodiments, the method further comprises determining the ratio of isoD-modified TTR to total TTR. Total TTR can be measured, for example, using an antibody that binds specifically to TTR without distinguishing between different forms of TTR. An exemplary antibody capable of detecting total TTR is "TTR, Rabbit, Polyclonal" (Biozol product number: CSB-PA041819) sold by Biozol.

[0085] In an eighth aspect, the invention provides a binding protein of the invention for use in a method for in vivo diagnosis and / or prognosis of ATTR. In certain embodiments, the method for diagnosis and / or prognosis comprises administering the binding protein to an individual. In certain embodiments, the binding protein is labeled with a detectable moiety (e.g., a radiolabel), and by this method, TTR protofibrils can be localized.

[0086] In preferred embodiments, the binding protein is derivatized in any manner conventionally discussed. For example, an antibody can be fused to hydrogen peroxide and used in an ELISA to diagnose and / or predict ATTR. Sandwich immunoassays are common in the art. There are many articles available. For example, Cox KL, Devanarayan V, Kriauciunas A, et al. Immunoassay Methods. 2012 May 1 [Updated 2014 Dec 24]. In: Sittampalam GS, Coussens NP, Brimacombe K, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004. Available from https: / / www.ncbi.nlm.nih.gov / books / NBK92434 / . In a preferred embodiment, the sandwich immunoassay is a direct or indirect ELISA. Compositions and methods for producing binding proteins In a ninth aspect, the invention provides a method for producing a binding protein (e.g., a binding protein of the invention) comprising immunizing an animal (e.g., a mouse) with a polypeptide comprising SEQ ID NO: 3. In a tenth aspect, the invention provides a method for selecting a binding protein (e.g., a binding protein of the invention) comprising contacting a population of binding proteins with a polypeptide comprising SEQ ID NO: 3. In an eleventh aspect, the invention provides a polypeptide comprising SEQ ID NO: 3 (e.g., an isolated polypeptide). In certain embodiments, the isolated polypeptide comprising SEQ ID NO: 3 can be used to produce and / or select a binding protein (e.g., a binding protein of the invention). In certain embodiments, the invention provides a container comprising a polypeptide comprising SEQ ID NO: 3 (e.g., an isolated polypeptide).

[0087] Items of the invention The invention also includes the following items that can be combined with any embodiment or aspect disclosed in this application. [1] A binding protein that specifically binds to isoaspartic acid-modified transthyretin. [2] The binding protein according to item [1], wherein the isoaspartic acid-modified transthyretin contains SEQ ID NO: 3 and / or the binding protein specifically binds to an epitope containing an isoaspartic acid (preferably L-isoaspartic acid) residue. [3] The K of the interaction between the binding protein and SEQ ID NO: 5 D is at least 10 times lower than the K of the interaction between the binding protein and SEQ ID NO: 6 D and, optionally, the K of the interaction between the binding protein and SEQ ID NO: 5 D is at least 200 times lower than the K of the interaction between the binding protein and SEQ ID NO: 6 D The binding protein according to item [1] or [2]. [4] The K of the interaction between the binding protein and a polypeptide containing SEQ ID NO: 3 (e.g., TTR) D is at least 10 times lower than the K of the interaction between the binding protein and a polypeptide containing SEQ ID NO: 2 (e.g., TTR) D and, optionally, the K of the interaction between the binding protein and a polypeptide containing SEQ ID NO: 3 (e.g., TTR) D is at least 200 times lower than the K of the interaction between the binding protein and a polypeptide containing SEQ ID NO: 2 (e.g., TTR) D The binding protein according to any one of items [1] to [3]. [5] The K of the interaction between the binding protein and SEQ ID NO: 3 D is at least 10 times lower than the K of the interaction between the binding protein and SEQ ID NO: 2 D and, optionally, the K of the interaction between the binding protein and SEQ ID NO: 3 D is at least 200 times lower than the K of the interaction between the binding protein and SEQ ID NO: 2 D The binding protein according to item [4]. [6] The K of the interaction between the binding protein and a polypeptide containing SEQ ID NO: 3 (e.g., TTR) at 25°C D is less than 200 nM. The binding protein according to any one of items [1] to [5]. [7] The K of the interaction between the binding protein and SEQ ID NO: 3 at 25°C D is less than 200 nM, and the binding protein according to item [6]. [8] The K of the interaction between the binding protein and SEQ ID NO: 5 at 25°C D is less than 200 nM, and the binding protein according to any one of items [1] to [7].

[0088] [9] The following: (a) A heavy chain variable region (VH) comprising the following: (i) A sequence having at least 70% (preferably at least 80%) identity with NSYYGMDY (SEQ ID NO: 16), and (ii) An HCDR3 polypeptide selected from a sequence having at least 65% identity with EDY (SEQ ID NO: 26); and / or (b) A light chain variable region (VL) comprising the following: (i) A sequence having at least 70% (preferably at least 80%) identity with HQYLSSRT (SEQ ID NO: 13), and (ii) An LCDR3 polypeptide selected from a sequence having at least 70% (preferably at least 80%) identity with QHFWNIPFT (SEQ ID NO: 23), and the binding protein according to any one of items [1] to [8].

[10] Comprising VL and VH, wherein the VL comprises the polypeptides of LCDR1, LCDR2 and LCDR3, and the VH comprises the polypeptides of HCDR1, HCDR2 and HCDR3, where (a) LCDR1 is a sequence having at least 70% (preferably at least 90%) identity with KSSQSVLYSSNQKNYLA (SEQ ID NO: 11), LCDR2 is a sequence having at least 70% (preferably at least 80%) identity with WASTRES (SEQ ID NO: 12), LCDR3 is a sequence having at least 70% or at least 80% identity with HQYLSSRT (SEQ ID NO: 13), HCDR1 is a sequence having at least 70% (preferably at least 90%) identity with GFTFSSFAMH (SEQ ID NO: 14), HCDR2 is a sequence having at least 70% (preferably at least 90%) identity with FISSGSNTIYYADTVKG (SEQ ID NO: 15), and HCDR3 is a sequence having at least 70% (preferably at least 80%) identity with NSYYGMDY (SEQ ID NO: 16); and (b) The binding protein according to any one of items [1] to [9], wherein LCDR1 is a sequence having at least 70% (preferably at least 90%) identity with RTSGNIRNSLA (SEQ ID NO: 21), LCDR2 is a sequence having at least 70% (preferably at least 80%) identity with NGKTLAD (SEQ ID NO: 22), LCDR3 is a sequence having at least 70% (preferably at least 80%) identity with QHFWNIPFT (SEQ ID NO: 23), HCDR1 is a sequence having at least 70% (preferably at least 90%) identity with GNTFSSRWIE (SEQ ID NO: 24), HCDR2 is a sequence having at least 70% (preferably at least 90%) identity with EIFPGNGNTNYNEKFKG (SEQ ID NO: 25), and HCDR3 is a sequence having at least 65% identity with EDY (SEQ ID NO: 26), and is selected from the group consisting of.

[11] Comprising VL and VH, wherein the VL comprises the polypeptides of LCDR1, LCDR2 and LCDR3, and the VH comprises the polypeptides of HCDR1, HCDR2 and HCDR3, wherein (a) LCDR1 is KSSQSVLYSSNQKNYLA (SEQ ID NO: 11), LCDR2 is WASTRES (SEQ ID NO: 12), LCDR3 is HQYLSSRT (SEQ ID NO: 13), HCDR1 is GFTFSSFAMH (SEQ ID NO: 14), HCDR2 is FISSGSNTIYYADTVKG (SEQ ID NO: 15), and HCDR3 is NSYYGMDY (SEQ ID NO: 16); (b) The binding protein according to any one of items [1] to

[10] , wherein LCDR1 is RTSGNIRNSLA (SEQ ID NO: 21), LCDR2 is NGKTLAD (SEQ ID NO: 22), LCDR3 is QHFWNIPFT (SEQ ID NO: 23), HCDR1 is GNTFSSRWIE (SEQ ID NO: 24), HCDR2 is EIFPGNGNTNYNEKFKG (SEQ ID NO: 25), and HCDR3 is EDY (SEQ ID NO: 26).

[12] The binding protein according to any one of items [1] to

[11] , comprising VL and VH, wherein the VL and VH are: (a) VL of SEQ ID NO: 17 or a sequence having at least 50% identity thereto and VH of SEQ ID NO: 18 or a sequence having at least 50% identity thereto; (b) VL of SEQ ID NO: 27 or a sequence having at least 50% identity thereto and VH of SEQ ID NO: 28 or a sequence having at least 50% identity thereto; (c) a polypeptide selected from the group consisting of VL of SEQ ID NO: 17 or a sequence having at least 50% identity thereto and VH of SEQ ID NO: 18 or a sequence having at least 50% identity thereto.

[0089]

[13] The binding protein according to any one of items [1] to

[12] , which is an antibody.

[14] A nucleic acid comprising a sequence encoding the binding protein according to any one of items [1] to

[13] .

[15] A host cell comprising the nucleic acid according to item

[14] .

[16] A pharmaceutical composition comprising the binding protein according to any one of items [1] to

[13] , the nucleic acid according to

[14] , or the host cell according to

[15] , and a pharmaceutically acceptable carrier or diluent.

[17] The binding protein according to any one of items [1] to

[13] , the nucleic acid according to item

[14] , the host cell according to item

[15] , or the pharmaceutical composition according to item

[16] for use as a medicine.

[18] The binding protein according to any one of items [1] to

[13] , the nucleic acid according to item

[14] , the host cell according to item

[15] , or the pharmaceutical composition according to item

[16] for use in the treatment and / or prevention of transthyretin amyloidosis.

[0090]

[19] An in vitro diagnostic and / or prognostic method for detecting the presence of isoaspartic acid-modified transthyretin in an isolated sample, comprising the following: (i) Contacting the isolated sample with the binding protein according to any one of items [1] to

[20] , and (ii) Determining whether isoaspartic acid-modified transthyretin is present in the isolated sample A method comprising.

[21] The binding protein according to any one of items [1] to

[13] for use in an in vivo diagnostic and / or prognostic method for transthyretin amyloidosis.

[22] A binding protein that specifically binds to isoaspartic acid-modified transthyretin.

[23] The binding protein according to item

[22] , wherein isoaspartic acid-modified transthyretin contains SEQ ID NO: 3 and / or the binding protein specifically binds to an epitope containing an isoaspartic acid residue.

[24] The K of the interaction between the binding protein and SEQ ID NO: 5 D is at least 10 times lower than the K of the interaction between the binding protein and SEQ ID NO: 6 D , and in some cases, the K of the interaction between the binding protein and SEQ ID NO: 5 D is at least 200 times lower than the K of the interaction between the binding protein and SEQ ID NO: 6 D The binding protein according to item

[22] or

[23] .

[25] The K of the interaction between the binding protein and SEQ ID NO: 5 at 25°CD The binding protein according to any one of items

[22] to

[24] , wherein D is less than 200 nM.

[0091]

[26] The following: (a) A heavy chain variable region (VH) comprising the following: (i) A sequence having at least 70% (preferably at least 80%) identity with NSYYGMDY (SEQ ID NO: 16), and (ii) An HCDR3 polypeptide selected from a sequence having at least 65% identity with EDY (SEQ ID NO: 26); and / or (b) A light chain variable region (VL) comprising the following: (i) A sequence having at least 70% (preferably at least 80%) identity with HQYLSSRT (SEQ ID NO: 13), and (ii) An LCDR3 polypeptide selected from a sequence having at least 70% (preferably at least 80%) identity with QHFWNIPFT (SEQ ID NO: 23), The binding protein according to any one of items

[22] to

[25] , comprising .

[27] Comprising VL and VH, wherein the VL comprises the polypeptides of LCDR1, LCDR2 and LCDR3, and the VH comprises the polypeptides of HCDR1, HCDR2 and HCDR3, where (a) LCDR1 is a sequence having at least 70% (preferably at least 90%) identity with KSSQSVLYSSNQKNYLA (SEQ ID NO: 11), LCDR2 is a sequence having at least 70% (preferably at least 80%) identity with WASTRES (SEQ ID NO: 12), LCDR3 is a sequence having at least 70% or at least 80% identity with HQYLSSRT (SEQ ID NO: 13), HCDR1 is a sequence having at least 70% (preferably at least 90%) identity with GFTFSSFAMH (SEQ ID NO: 14), HCDR2 is a sequence having at least 70% (preferably at least 90%) identity with FISSGSNTIYYADTVKG (SEQ ID NO: 15), and HCDR3 is a sequence having at least 70% (preferably at least 80%) identity with NSYYGMDY (SEQ ID NO: 16); and (b) The binding protein according to any one of items

[22] to

[26] , wherein LCDR1 is a sequence having at least 70% (preferably at least 90%) identity with RTSGNIRNSLA (SEQ ID NO: 21), LCDR2 is a sequence having at least 70% (preferably at least 80%) identity with NGKTLAD (SEQ ID NO: 22), LCDR3 is a sequence having at least 70% (preferably at least 80%) identity with QHFWNIPFT (SEQ ID NO: 23), HCDR1 is a sequence having at least 70% (preferably at least 90%) identity with GNTFSSRWIE (SEQ ID NO: 24), HCDR2 is a sequence having at least 70% (preferably at least 90%) identity with EIFPGNGNTNYNEKFKG (SEQ ID NO: 25), and HCDR3 is a sequence having at least 65% identity with EDY (SEQ ID NO: 26), and is selected from the group consisting of:

[28] Comprising VL and VH, wherein the VL comprises the polypeptides of LCDR1, LCDR2 and LCDR3, and the VH comprises the polypeptides of HCDR1, HCDR2 and HCDR3, wherein (a) LCDR1 is KSSQSVLYSSNQKNYLA (SEQ ID NO: 11), LCDR2 is WASTRES (SEQ ID NO: 12), LCDR3 is HQYLSSRT (SEQ ID NO: 13), HCDR1 is GFTFSSFAMH (SEQ ID NO: 14), HCDR2 is FISSGSNTIYYADTVKG (SEQ ID NO: 15), and HCDR3 is NSYYGMDY (SEQ ID NO: 16); (b) The binding protein according to any one of items

[22] to

[27] , wherein LCDR1 is RTSGNIRNSLA (SEQ ID NO: 21), LCDR2 is NGKTLAD (SEQ ID NO: 22), LCDR3 is QHFWNIPFT (SEQ ID NO: 23), HCDR1 is GNTFSSRWIE (SEQ ID NO: 24), HCDR2 is EIFPGNGNTNYNEKFKG (SEQ ID NO: 25), and HCDR3 is EDY (SEQ ID NO: 26).

[29] The binding protein according to any one of items

[22] to

[28] , which comprises VL and VH, and the VL and VH are (a) VL of SEQ ID NO: 17 or a sequence having at least 50% identity thereto and VH of SEQ ID NO: 18 or a sequence having at least 50% identity thereto; (b) VL of SEQ ID NO: 27 or a sequence having at least 50% identity thereto and VH of SEQ ID NO: 28 or a sequence having at least 50% identity thereto; (c) a polypeptide selected from the group consisting of VL of SEQ ID NO: 17 or a sequence having at least 50% identity thereto and VH of SEQ ID NO: 18 or a sequence having at least 50% identity thereto.

[30] The binding protein according to any one of items

[22] to

[29] , which is an antibody.

[31] A nucleic acid comprising a sequence encoding the binding protein according to any one of items

[22] to

[30] .

[32] A host cell comprising the nucleic acid according to item 31.

[33] The binding protein according to any one of items

[22] to

[30] , the nucleic acid according to item

[31] , or the host cell according to item

[32] , for use as a medicament.

[34] A binding protein according to any one of items

[22] to

[30] , a nucleic acid according to item

[31] , or a host cell according to item

[32] , for use in the treatment and / or prevention of transthyretin amyloidosis.

[0092]

[35] An in vitro diagnostic and / or prognostic method for detecting the presence of isoaspartic acid-modified transthyretin in an isolated sample, comprising: (i) contacting the isolated sample with a binding protein according to any one of items

[22] to

[30] , and (ii) determining whether isoaspartic acid-modified transthyretin is present in the isolated sample. A method comprising the above.

[36] A binding protein according to any one of items

[22] to

[30] , for use in an in vivo diagnostic and / or prognostic method for transthyretin amyloidosis.

Examples

[0093] Preparation and screening of monoclonal antibodies against isoAsp38 TTR The aim of this study was to generate monoclonal antibodies that react with isoAsp38-TTR and its shorter peptides containing isoaspartic acid at position 38 (e.g., SEQ ID NO: 3), but not with the same molecule having aspartic acid at position 38 (e.g., SEQ ID NO: 2). A peptide containing amino acids 35 to 43 of human TTR, with isoaspartic acid at position 38 instead of aspartic acid, and having an additional artificial C-terminal amino acid for facilitating coupling (i.e., KAAXDTWEPC, where X is L-isoAsp) (SEQ ID NO: 4) was synthesized by Peptide&Elephant (Berlin, Germany). The peptide was covalently bound to keyhole limpet hemocyanin via a C-terminal cysteine residue. For the production of monoclonal antibodies, 8-week-old female BALB / c mice were used. The mice were immunized subcutaneously with a 1:1 emulsion of KLH-conjugated TTR peptide and Freund's complete adjuvant (VWR), and then boosted with another 95 μg in incomplete Freund's adjuvant 44 days and 73 days later. A final immunogen boost was performed daily starting 4 days before fusion. Antibody titers were confirmed by enzyme-linked immunosorbent assay (ELISA). Homogenized spleen cells were fused to SP2 / 0 myeloma cells using the PEG method (Koehler & Milstein, 1975. Nature. 256(5517):495-7). The cells were immediately plated onto prepared feeder cell plates in HAT medium and screened 10 days later. Hybridomas that specifically bind to isoD38-TTR(35~43)-PEG-biotin (SEQ ID NO: 5) but not to TTR(35~43)-PEG-biotin (SEQ ID NO: 6) were selected using indirect ELISA and surface plasmon resonance (SPR). After selecting stable antibody-producing hybridomas, they were cloned by limiting dilution and further single cell isolation to ensure monoclonal nature of the hybridomas. Hybridoma cell clones were grown and cultured in T175 flasks. The supernatants were collected and sterile filtered (0.22 μm, TPP) prior to purification. For affinity purification of the mAb, each supernatant was mixed with an equal volume of binding buffer (40 mM Na2HPO4, 150 mM NaCl, pH 7.0) and applied to a pre-equilibrated Protein G Sepharose Fast Flow column (GE Healthcare Life Sciences). Elution of the bound antibody was performed using 40 mM Na2HPO4 and 2 M KSCN (pH 7.0). The recovered fractions were dialyzed against PBS (138 mM NaCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 3 mM KCl, pH 7.0) at 4 °C overnight.

Example

[0094] Investigation of antibody specificity by dot blot analysis Dot blot analysis was performed to obtain general findings regarding the specificity and cross-reactivity of the antibody obtained in Example 1. Method Recombinant expressed TTR proteins (TTRwt (SEQ ID NO: 1), TTR-V30M (SEQ ID NO: 7), or TTR-V122I (SEQ ID NO: 8)), 0.5 μg of ATTR protofibrils, or 0.5 μg of newly solubilized Aβ(1-42) (SEQ ID NO: 9) or aggregated Aβ(1-42) (SEQ ID NO: 10) with confirmed isoAsp content were spotted onto nitrocellulose membranes. The membranes were blocked in blocking solution (5% (w / v) non-fat dry milk + 0.05% (v / v) Tween-20 (Carl Roth) in TBS) for 1 hour. Antibodies 2F2, 4D4 or polyclonal anti-TTR antibody (Biozol product number B.CSB-PA041819) were diluted to 1 μg / mL in blocking solution and incubated overnight at 4 °C. Subsequently, alkaline phosphatase-conjugated anti-mouse or anti-rabbit IgG antibodies (abcam) were used for detection in combination with the chromogenic substrates BCIP and NBT (Carl Roth). SEQ ID NO: 7 is TTR-V30M and has the following sequence: GPTGTGESKCPLMVKVLDAVRGSPAINVAMHVFRKAADDTWEPFASGKTSESGELHGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTTAVVTNPKE There is. SEQ ID NO: 8 is TTR-V122I and has the following sequence: GPTGTGESKCPLMVKVLDAVRGSPAINVAVHVFRKAADDTWEPFASGKTSESGELHGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTTAVITNPKE There is. SEQ ID NO: 9 is Aβ(1-42) and has the following sequence: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA There is. SEQ ID NO: 10 is isoD7-Aβ(1-42) and has the following sequence: DAEFRHXSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (wherein X is L-isoAp) There is.

[0095] Results As shown in Figure 1, monomeric proteins (lanes 1-3) recombinantly expressing TTRwt, TTR-V30M, and TTR-V122I were not identified by 2F2 and 4D4, indicating the absence of isoaspartic acid residues at position 38. However, 2F2 and 4D4 recognized aggregated one-year-old samples of recombinant TTR-derived protofibrils (lanes 4-6). Furthermore, isoD38 TTR species were observed in extracted protofibrils of human heart tissue from a patient with pathological ATTR-V30M, detected by 2F2 and 4D4 antibodies (lane 7). In contrast, total TTR pAb did not distinguish between recombinant monomeric TTR variants and aggregated aged TTR protofibrils, either produced in vitro or extracted from human tissue (column 3). Both TTR mAbs did not show reactivity against other isoaspartic acid-containing amyloid protofibrils (isoD7-Aβ) or their monomeric peptides (lanes 8 and 9).

Example

[0096] Determination of the K of immobilized 2F2 or 4D4 with TTR(35-43) or isoD38-TTR(35-43) by surface plasmon resonance (SPR) analysis D Value determination Method The antibody binding kinetics against TTR(35~43)-PEG-biotin (SEQ ID NO: 5) and isoD38-TTR(35~43)-PEG-biotin (SEQ ID NO: 6) were analyzed using SPR on a BIAcore 3000 instrument at 25°C. For this purpose, the surface of a CM5 sensor chip (Cytiva) was modified with goat anti-mouse antibody using NHS / EDC chemistry. The carboxymethylated dextran surface of the sensor chip was mixed with 0.1M N-hydroxysuccinimide (NHS) and 0.4M N-ethyl-N'-(dimethylaminopropyl)carbodiimide hydrochloride (EDC) at a ratio of 1:1 and activated to immobilize anti-mouse IgG. EDC / NHS was applied to the sensor chip at a flow rate of 20 μL / min for 7 minutes. Goat anti-mouse IgG was diluted to 50 μg / mL with 10 mM sodium acetate (pH 5.5) and injected at a flow rate of 30 μL / min for a contact time of 2×4 minutes. 1M ethanolamine pH 8.5 was applied at a flow rate of 10 μL / min for a contact time of 2×7 minutes for inactivation. Approximately 20,000 RU of goat anti-mouse antibody was immobilized on the chip surface. Antibodies 2F2 and 4D4 were captured using an antibody dilution of 25 μg / mL in HBS-EP buffer and a flow rate of 2 μL / min to achieve a level of approximately 2000 RU for both antibodies on the chip surface. In the case of the 4D4 antibody, multiplex cycle kinetics analysis was performed by applying 10 nM to 100 μM of isoD38-TTR(35-43)-PEG-biotin (SEQ ID NO: 5) and 1 μM to 200 μM of TTR(35-43)-PEG-biotin (SEQ ID NO: 6) respectively. For the 2F2 antibody, five consecutive injections of 2 nM to 512 nM of isoD38-TTR(35-43)-PEG-biotin (SEQ ID NO: 5) and 512 nM to 131 μM of TTR(35-43)-PEG-biotin (SEQ ID NO: 6) were applied. The antibody binding kinetics to isoD38-TTR(28-50) (SEQ ID NO: 39) were analyzed at 25 °C using SPR on a Bruker SPR-24 Pro instrument. For this purpose, approximately 4000 RU of antibodies 2F2 and 4D4 were immobilized on an IgG-Capture Sensor (Bruker) using an antibody dilution of 10 μg / mL in HBS-EP buffer and a flow rate of 5 μL / min. For both antibodies, isoD38-TTR(28-50) (SEQ ID NO: 39) at 0.5 nM to 50 nM was applied for single injection cycle kinetic analysis. SEQ ID NO: 39 is isoD38-TTR(28-50), with the following sequence: VAVHVFRKAAXDTWEPFASGKTS (wherein X is L-isoAsp, the N-terminal residue is acetylated, and the C-terminal residue is amidated).

[0097] Results The dissociation constants for the binding of 2F2 and 4D4 to either isoD38-TTR(35-43)-PEG-biotin (SEQ ID NO: 5), TTR(35-43)-PEG-biotin (SEQ ID NO: 6), or isoD38-TTR(28-50) (SEQ ID NO: 39) are shown in Table 1. Both TTR mAbs showed high selectivity for isoaspartic acid-containing TTR(35-43)-peptide (SEQ ID NO: 5) and TTR(28-50)-peptide (SEQ ID NO: 39). In comparison, the dissociation constant of the unmodified peptide TTR(35-43) (SEQ ID NO: 6) was 233-fold (4D4) and 464-fold (2F2) higher compared to isoD38-TTR(35-43)-PEG-biotin (SEQ ID NO: 5), and 1078-fold (4D4) and 317-fold (2F2) higher compared to isoD38-TTR(28-50)-peptide (SEQ ID NO: 39). A higher dissociation constant value means lower binding affinity.

[0098]

Table 2

Example

[0099] Phagocytosis and degradation of TTR protofibrils by phagocytosis using isoD38-TTR mAbs 2F2 and 4D4 To analyze whether antibodies 2F2 and 4D4 can induce Fc-mediated phagocytosis of TTR protofibrils, the inventors established an in vitro phagocytosis assay using macrophage-like THP-1 cells. THP-1 cells are commonly used to evaluate the Fc-mediated phagocytosis induction ability of monoclonal antibodies (see, for example, Gogesch et al., 2021. Int J Mol Sci. 22(16): 8947, section 2.4.2). Method A sample containing cardiac extract human ATTR-V30M protofibrils or 1 mg / mL native recombinant TTRwt (SEQ ID NO: 1) was amine-coupled with pHrodo Red dye (Thermo Fisher, see, for example, Miksa et al., 2009. J Immunol Methods. 342(1-2):71-7), and a protein:dye ratio of 10:1 was applied and stained at room temperature for 1 hour. In the case of native TTRwt, excess pHrodo Red dye was removed by diafiltration using a 5 kDa molecular weight cut-off (Merck). For amyloid protofibrils, after three centrifugation steps at 17,000×g for 10 minutes, they were resuspended in PBS to remove excess free dye label. For stimulation, 2×10 5 5 Human monocyte THP-1 cells were cultured in cell culture medium (RPMI, 10% low IgG FBS) and differentiated for 3 days using 200 nM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich). After this initial stimulation, the PMA-containing medium was replaced with fresh RPMI + 10% low IgG FBS for an additional 2 days. A 20 μg / mL aliquot of pHrodo-labeled TTR (monomeric cultured TTRwt or extracted human TTR-V30M protofibrils) was pre-incubated separately in cell culture medium at 37°C for 1 hour with 40 μg / mL. After adding 2×10 5 5 individual PMA-treated THP-1 cells, the tissue culture was incubated for 3 hours. The cells were washed three times with PBS to remove antigen-antibody complexes. The degree of phagocytosis was monitored by flow cytometry using a FACSCalibur (Becton Dickinson). Forward and side scatter light were used to identify cell populations and measure cell size and granularity. Red fluorescence emitted by the cells was excited at 560 nm and recorded at 585 nm. The remaining cells were transferred to a glass chamber slide and processed by image processing using a Keyence BZ-X800 with bright-field and fluorescence microscopy.

[0100] Results The extracted human ATTR-V30M protofibrils or recombinant monomeric TTRwt as a control were covalently conjugated to the pH-sensitive dye pHrodo-red. At physiological pH, the fluorescence of this dye is minimal, but it increases strongly in acidic environments such as endocytic vesicles. Thus, the cellular uptake of pHrodo-labeled proteins can be monitored. After treating pHrodo-labeled TTR with two antibodies, 2F2 and 4D4, or isotype controls, THP-1 cells were added. Cellular uptake was visualized by bright-field and fluorescence microscopy (Figure 2A), measured by flow cytometry, and presented as mean fluorescence intensity (MFI, Figure 2B). The fluorescence of either TTRwt monomer treated with the isoD 38-TTR-specific antibody or the isotype control was low. In contrast, after treating ATTR-V30M protofibrils with the 2F2 or 4D4 antibody, the fluorescence increased significantly, indicating antibody-mediated phagocytosis of TTR protofibrils. These data demonstrated that targeting isoD38 results in clearance of TTR protofibrils via antibody-dependent cell phagocytosis. The therapeutic goal is to improve patient survival if TTR amyloid burden can be reduced (Non-Patent Document 17). That is, these data indicated that the binding proteins disclosed herein are surely suitable for the treatment of transthyretin amyloidosis.

Claims

1. This binding protein specifically binds to isoaspartic acid-modified transthyretin, in which the 38th aspartic acid molecule in SEQ ID NO: 1 is post-translationally modified to L-isoaspartic acid.

2. The binding protein according to claim 1, wherein the isoaspartate-modified transthyretin comprises SEQ ID NO: 3, and / or the binding protein specifically binds to an epitope containing an isoaspartate residue.

3. The K of the interaction between the binding protein and SEQ ID NO: 5, determined by surface plasmon resonance at 25°C. Ｄ However, the K of the interaction between the binding protein and SEQ ID NO: 6 is determined by surface plasmon resonance at 25°C. Ｄ At least 10 times lower than that, Optionally, the K of the interaction between the binding protein and SEQ ID NO: 5, determined by surface plasmon resonance at 25°C. Ｄ However, the K of the interaction between the binding protein and SEQ ID NO: 6 is determined by surface plasmon resonance at 25°C. Ｄ The binding protein according to claim 1, which is at least 200 times lower.

4. The K of the interaction between the binding protein and SEQ ID NO: 5, determined by surface plasmon resonance at 25°C. Ｄ The binding protein according to claim 1, wherein the mass is less than 200 nM.

5. below: (a) Heavy chain variable region (VH) which is as follows: (i) NSYYGMDY (Sequence ID 16) or a sequence that is at least 70% or at least 80% identical thereto, (ii) EDY (Sequence ID 26) or a sequence that is at least 65% identical thereto comprising an HCDR3 polypeptide selected from; and / or (b) Light chain variable region (VL) which is as follows: (i) HQYLSSRT (SEQ ID NO: 13) or a sequence that is at least 70% or at least 80% identical thereto, (ii) comprising an LCDR3 polypeptide selected from QHFWNIPFT (SEQ ID NO: 23) or a sequence having at least 70% or at least 80% identity therewith, The binding protein according to claim 1, comprising:

6. A compound comprising VL and VH, wherein VL is a polypeptide of LCDR1, LCDR2, and LCDR3, and VH is a polypeptide of HCDR1, HCDR2, and HCDR3, and the following: (a) LCDR1 is KSSQSVLYSSNQKNYLA (SEQ ID NO: 11) or a sequence that is at least 70% or at least 90% identical thereto; LCDR2 is WASTRES (SEQ ID NO: 12) or a sequence that is at least 70% or at least 80% identical thereto; LCDR3 is HQYLSSRT (SEQ ID NO: 13) or a sequence that is at least 70% or at least 80% identical thereto; HCDR1 is GFTFSSFAMH (SEQ ID NO: 14) or a sequence that is at least 70% or at least 90% identical thereto; HCDR2 is FISSGSNTIYYADTVKG (SEQ ID NO: 15) or a sequence that is at least 70% or at least 90% identical thereto; and HCDR3 is NSYYGMDY (SEQ ID NO: 16) or a sequence that is at least 70% or at least 80% identical thereto; and (b) LCDR1 is RTSGNIRNSLA (SEQ ID NO: 21) or a sequence that is at least 70% or at least 90% identical thereto, LCDR2 is NGKTLAD (SEQ ID NO: 22) or a sequence that is at least 70% or at least 80% identical thereto, LCDR3 is QHFWNIPFT (SEQ ID NO: 23) or a sequence that is at least 70% or at least 80% identical thereto, HCDR1 is GNTFSSRWIE (SEQ ID NO: 24) or a sequence that is at least 70% or at least 90% identical thereto, HCDR2 is EIFPGNGNTNYNEKFKG (SEQ ID NO: 25) or a sequence that is at least 70% or at least 90% identical thereto, and HCDR3 is EDY (SEQ ID NO: 26) or a sequence that is at least 65% identical thereto. A binding protein according to claim 5, selected from the group consisting of the following.

7. A compound comprising VL and VH, wherein VL is a polypeptide of LCDR1, LCDR2, and LCDR3, and VH is a polypeptide of HCDR1, HCDR2, and HCDR3, and the following: (a) LCDR1 is KSSQSVLYSSNQKNYLA (SEQ ID NO: 11), LCDR2 is WASTRES (SEQ ID NO: 12), LCDR3 is HQYLSSRT (SEQ ID NO: 13), HCDR1 is GFTFSSFAMH (SEQ ID NO: 14), HCDR2 is FISSGSNTIYYADTVKG (SEQ ID NO: 15), and HCDR3 is NSYYGMDY (SEQ ID NO: 16); and (b) LCDR1 is RTSGNIRNSLA (SEQ ID NO: 21), LCDR2 is NGKTLAD (SEQ ID NO: 22), LCDR3 is QHFWNIPFT (SEQ ID NO: 23), HCDR1 is GNTFSSRWIE (SEQ ID NO: 24), HCDR2 is EIFPGNGNTNYNEKFKG (SEQ ID NO: 25), and HCDR3 is EDY (SEQ ID NO: 26). A binding protein according to claim 6, selected from the group consisting of the following.

8. The binding protein according to any one of claims 1 to 7, comprising VL and VH, wherein VL and VH are polypeptides selected from the group consisting of: (a) VL of SEQ ID NO: 17 or a sequence having at least 50% identity thereto and VH of SEQ ID NO: 18 or a sequence having at least 50% identity thereto; (b) VL of SEQ ID NO: 27 or a sequence having at least 50% identity thereto and VH of SEQ ID NO: 28 or a sequence having at least 50% identity thereto; (c) VL of SEQ ID NO: 17 or a sequence having at least 50% identity thereto and VH of SEQ ID NO: 28 or a sequence having at least 50% identity thereto; (d) VL of SEQ ID NO: 27 or a sequence having at least 50% identity thereto and VH of SEQ ID NO: 18 or a sequence having at least 50% identity thereto.

9. The binding protein according to any one of claims 1 to 7, which is an antibody.

10. A nucleic acid comprising a sequence encoding the binding protein described in claim 1.

11. A host cell containing the nucleic acid described in claim 10.

12. A binding protein according to any one of claims 1 to 7, a nucleic acid according to claim 10, or a host cell according to claim 11, for use as a pharmaceutical.

13. A binding protein according to any one of claims 1 to 7, a nucleic acid according to claim 10, or a host cell according to claim 11, for use in the treatment and / or prevention of transthyretin amyloidosis.

14. An in vitro diagnostic and / or prognostic method for detecting the presence of isoaspartate-modified transthyretin in an isolated sample, the following: (i) Contacting the isolated sample with the binding protein described in any one of claims 1 to 7, and (ii) Determine whether isoaspartate-modified transthyretin is present in the isolated sample. A method comprising, wherein the isoaspartic acid-modified transthyretin is post-translationally modified, in which the 38th aspartic acid of SEQ ID NO: 1 is L-isoaspartic acid.

15. A binding protein according to any one of claims 1 to 7, for use in a method for in vivo diagnosis and / or prognosis diagnosis of transthyretin amyloidosis.

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