Anti-transthyretin (TTR) binding proteins and uses thereof

Abstract

Transthyretin amyloidosis is characterized by progressive deposition of plasma protein transthyretin in myocardium, peripheral nerves and / or other tissues, which can ultimately lead to congestive heart failure, polyneuropathy and death. The present invention provides binding proteins that can specifically bind to isoaspartic acid residues in transthyretin and therapeutic, diagnostic and prognostic uses thereof.

Description

Technical Field

[0001] The present invention can be classified into the field of medicine. Specifically, the present invention provides binding proteins that can specifically bind to isoaspartic acid (isoD) residues in transthyretin (TTR), and their use in the prevention and therapeutic treatment of transthyretin amyloidosis (ATTR). In addition, the present invention also provides the use of binding proteins in prognosis and / or diagnosis methods. Background Art

[0002] Systemic amyloidosis represents a group of rare diseases that are often underdiagnosed due to their diverse symptom profile. In these multisystem diseases, misfolded fibrillar proteins are deposited extracellularly in various organs, leading to progressive loss of function and integrity (Nevone et al., 2020. Front Pharmacol. 11: 1024).

[0003] Transthyretin amyloidosis (ATTR) is characterized by progressive deposition of the plasma protein transthyretin (TTR) in the myocardium, peripheral nerves, and / or other tissues, which can ultimately lead to congestive heart failure, polyneuropathy, and death. Typically, patients die within 4 to 12 years of diagnosis (Grogan et al., 2016. J Am Coll Cardiol. 68(10): 1014-20; Ando et al., 2013. Orphanet J Rare Dis. 8: 31).

[0004] There are two forms of ATTR, wild-type ATTR (ATTRwt) and hereditary ATTR (hATTR). ATTRwt is an often underestimated cause of heart failure that primarily affects older individuals. In contrast, hATTR is an autosomal dominant genetic disease caused by mutations in the TTR gene. To date, more than 130 different mutations are known, with varying ages of onset and clinical manifestations (Thomas et al., 2019. Neurodegener Dis Manag. 9(6): 289-299). Some mutations primarily cause polyneuropathy, while others cause cardiomyopathy or mixed phenotypes. The most common TTR mutation worldwide is the Val30Met mutation, which typically causes peripheral neuropathy. The second most common hereditary mutation is Val122lle. In this case, organ involvement can be limited to the heart (Adams et al., 2021. J Neurol. 268(6): 2109-2122).

[0005] TTR is a 55 kDa homotetrameric protein that binds and transports thyroxine and holo-retinal binding protein in cerebrospinal fluid and plasma (Buxbaum & Reixach, 2009. Cell Mol Life Sci. 66 (19): 3095-101). TTR is mainly produced in the liver. About 5% of the total protein is derived from the choroid plexus and retinal pigment epithelium (Gertz et al., 2015. J Am Coll Cardiol. 66 (21): 2451-2466). Factors such as low pH, high temperature or the presence of mutations in the TTR gene lead to increased instability of the homotetrameric complex. They are conducive to faster dissociation and release of monomers, which in turn facilitates the aggregation process and the formation of amyloid fibrils (Hurshman et al., 2004. Biochemistry. 43 (23): 7365-81). Recently, Schmidt et al. solved the first cryo-EM structure of human ATTR-V30M fibrils (Schmidt et al., 2019. Nat Commun. 10(1):5008). They showed that fibril formation involves almost complete unfolding of the native conformation, allowing the polypeptide chains to rearrange into significantly different conformations. In addition, these structures provide valuable insights into protein regions in the fibrils that may be prone to protein-protein interactions.

[0006] Treatment approaches for ATTR include liver transplantation (Holmgren et al., 1993. Lancet. 341(8853):1113-6), siRNA gene silencing (patisiran, see Adams et al., 2018. N Engl J Med. 379(1):11-21) or antisense oligonucleotides (inotersen, Benson et al., 2018. N Engl J Med. 379(1):22-31), and stabilization of the naturally folded physiological form of the TTR complex (tafamidis, see Maurer et al., 2018. N Engl J Med. 379(11):1007-1016; diflunisal, see Lohrmann et al., 2020. J Card Fail. 26(9):753-759; epigallocatechin-3-gallate, see Ferreira et al., 2012. PLoS One. 7(1):e29933). These therapies are designed to prevent misfolding / unfolding, aggregation, and deposition in tissues.

[0007] Although these advances represent effective approaches to slow the progression of amyloid deposition, alternative measures that can clear pre-existing TTR protein deposits in tissues of diseased patients are still needed. Passive immunotherapy may be a viable approach that involves the administration of antibodies directed against non-natively folded, dissociated monomers and non-native oligomers or amyloid deposits to target them for degradation via Fc-mediated phagocytosis.

[0008] To our knowledge, all mAbs developed to date target a hidden epitope on the surface of amyloid TTR that is exposed during the pathogenic formation of β-pleated fibrils and is therefore unique to non-native TTR. Among them, mAb PRX004 (Higaki et al., 2016. Amyloid. 23(2): 86-97) has passed Phase I clinical trials. However, due to the impact of the COVID-19 pandemic, the trial was terminated early. Michalon et al. successfully developed NI006, another mAb for ATTR cardiomyopathy. A clinical trial involving the administration of NI006 is being planned (Michalon et al. 2021. Nat Commun. 12(1): 3142).

[0009] There remains a need for effective therapies that can treat and / or prevent ATTR. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 : Specificity of TTR mAbs 2F2 and 4D4 characterized by dot blot analysis. Two mAbs specific for isoD-modified TTR (i.e., 2F2 and 4D4) and a polyclonal whole TTR antibody were compared. The mAbs bound to aged amyloid aggregates and extracted fibrils of recombinant TTR protein. No binding was detected to native recombinant TTR monomers and aggregated Aβ1-42 (isoD content verified). Detection was based on alkaline phosphatase activity. The data show that the isoD-specific mAbs are highly specific for aged, post-translationally modified TTR.

[0011] Figure 2: 2F2 / 4D4 mAb-dependent phagocytosis. Recombinant TTRwt (wild-type TTR) protein or extracted human ATTR-V30M fibrils were covalently labeled with the pH-sensitive fluorescent dye pHrodo Red, which shows enhanced fluorescence when exposed to low pH in endocytic vesicles. (A) Overlay of brightfield and fluorescence images of THP-1 cells showing enhanced uptake of aggregated pHrodo-labeled ATTR-V30M fibrils in the presence of isoD38-TTR-specific antibodies 2F2 and 4D4. Scale bar = 50 μm. (B) Phagocytosis of pHrodo-labeled fibrils measured by relative mean fluorescence intensity (MFI) of THP-1 cells. Antibody-dependent phagocytosis was specific for pHrodo-ATTR-V30M fibrils treated with 2F2 or 4D4 antibodies, but both isotype control antibodies failed to induce phagocytosis. 2F2 and 4D4 had no effect on the uptake of native recombinant TTRwt monomers. Data are presented as box plots with 1.5 IQR (interquartile range). Statistical analysis was performed using one-way ANOVA with Tukey post hoc test (***=p<0.001).

[0012] Figure 3 : Kabat numbering scheme applied to the VH and VL fragments of 2F2 and 4D4. This figure shows SEQ ID NOs: 17, 18, 27 and 28. Summary of the invention

[0013] Recent advances in other amyloidosis treatments may provide guidance for the development of effective therapies for ATTR. Donanemab is a mAb currently in Phase III clinical trials for the treatment of Alzheimer's disease (AD), which highlights the potential for customized therapeutic approaches by targeting the pyroglutamate 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 the PTMs of TTR that can affect the development of ATTR.

[0014] Based on a comprehensive analysis of Aβ post-translational modifications and their functions, it can be inferred that some of them may also apply to TTR in ATTR. One of these modifications is the formation of isoaspartate (isoD), which is generated by hydrolysis of L-asparagyl or deamidation of L-asparagine residues (Shimizu et al., 2000. Arch Biochem Biophys. 381(2):225-34), and isomerization occurs spontaneously through an L-succinimide intermediate to form L-isoaspartate or L-aspartate in a ratio of 3:1. This modification determines the half-life of the protein and significantly changes its structure by introducing additional methylene groups into the protein backbone (Robinson & Robinson, 2001. Proc Natl Acad Sci US 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 has potential effects on the solubility, conformation and function of the protein.

[0015] Here, we provide evidence for the formation of isoaspartate at position 38 of the TTR protein and for the presence of isoD38-TTR in amyloid deposits. We developed two polyclonal antibodies, 2F2 and 4D4, that specifically detect this modification in amyloid deposits in human pathological tissues. Native TTR protein does not bind to 2F2 and 4D4. Both antibodies promote the entry of human isoD38-modified TTR fibrils into THP-1 cells via phagocytosis. Therefore, we believe that these antibodies and other binding proteins targeting isoD-modified TTR have diagnostic and therapeutic potential.

[0016] In a first aspect, the present invention provides a binding protein that specifically binds to isoD-modified TTR. The present invention also provides 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.

[0017] The present invention also provides a pharmaceutical composition comprising a 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 can be used as a drug. In particular, the binding protein, nucleic acid, host cell or pharmaceutical composition of the present invention can be used for the treatment and / or prevention of ATTR.

[0018] 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 involve contacting the sample with a binding protein of the present invention.

[0019] In vitro methods involve isolated samples. 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 a binding protein of the present invention, and determining the presence of isoD-modified TTR in the isolated sample.

[0020] The present invention also provides a binding protein of the present invention for use in an in vivo diagnostic and / or prognostic method for ATTR. DETAILED DESCRIPTION

[0021] definition

[0022] The term "affibody" refers to a protein derived from the Z region of protein A and engineered to bind to a specific target (see Frejd & Kim, 2017. Exp Mol Med. 49(3): e306).

[0023] The term "animal" as used in this application refers to any non-human multicellular eukaryotic heterotrophic organism. In some embodiments, the animal is selected from the group consisting of cats, dogs, pigs, ferrets, rabbits, gerbils, hamsters, guinea pigs, horses, rats, mice, cows, sheep, goats, alpacas, camels, donkeys, llamas, yaks, giraffes, elephants, meerkats, lemurs, lions, tigers, kangaroos, koalas, bats, monkeys, chimpanzees, gorillas, bears, dugongs, manatees, seals, and rhinos.

[0024] The term "antibody" refers to a molecule comprising at least one immunoglobulin domain that binds to or is specific for a particular target. The term encompasses intact antibodies and any antigen-binding portion (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 antibody derived therefrom.

[0025] A typical class of antibodies includes at least two heavy chains ("HC") and two light chains ("LC") interconnected by disulfide bonds.

[0026] Each "heavy chain" includes a "heavy chain variable region" (abbreviated herein as "VH") and a "heavy chain constant region" (abbreviated herein as "CH"). The heavy chain constant region generally includes three constant regions, namely CH1, CH2 and CH3.

[0027] Each "light chain|" includes a "light chain variable region" (abbreviated herein as "VL") and a "light chain constant region" ("CL"). The light chain constant region (CL) can be of kappa or lambda type. The VH and VL regions can be further subdivided into hypervariable regions, called "complementarity determining regions" ("CDRs"), interspersed with relatively conserved regions, called "framework regions" ("FW").

[0028] Each VH and VL consists of three CDRs and four FWs, arranged in the following order from the amino terminus to the carboxyl terminus: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The present disclosure specifically proposes VH and VL sequences and subsequences corresponding to CDR1, CDR2 and CDR3.

[0029] Therefore, it will be understood by those skilled in the art that the sequences of FW1, FW2, FW3 and FW4 are also disclosed. For a particular VH, FW1 is a subsequence between the N-terminus of VH and the N-terminus of HCDR1, FW2 is a subsequence between the C-terminus of HCDR1 and the N-terminus of HCDR2, FW3 is a subsequence between the C-terminus of HCDR2 and the N-terminus of HCDR3, and FW4 is a subsequence between the C-terminus of HCDR3 and the C-terminus of VH. Similarly, for a particular VL, FW1 is a subsequence between the N-terminus of VL and the N-terminus of LCDR1, FW2 is a subsequence between the C-terminus of LCDR1 and the N-terminus of LCDR2. FW3 is a subsequence between the C-terminus of LCDR2 and the N-terminus of LCDR3, and FW4 is a subsequence between the C-terminus of LCDR3 and the C-terminus of VL.

[0030] The variable domains of the heavy and light chains contain regions that interact with a binding target, which are also referred to herein as "antigen-binding sites" or "antigen binding sites". The constant domains of antibodies can mediate the binding of antibodies to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Exemplary antibodies of the present disclosure include typical antibodies, but also include bivalent fragments and variants thereof (e.g., F(ab')2).

[0031] As used herein, the term "antibody" includes intact polyclonal antibodies, intact monoclonal antibodies, bivalent antibody fragments (e.g., F(Ab')2), multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, and any other modified immunoglobulins containing two antigen binding sites.

[0032] Antibodies can be any of the five major classes of immunoglobulins (isotypes): IgA, IgD, IgE, IgG, and IgM, or subtypes thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), which are called α, δ, ε, γ, and μ, respectively, based on the properties of their heavy chain constant domains. Different classes of immunoglobulins have different well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules (e.g., therapeutic or diagnostic agents) to form immunoconjugates.

[0033] The term "anticalin" refers to a protein derived from a lipocalin and engineered to bind to a specific target (see 2008. FEBS J. 275(11):2677-83).

[0034] The term "binding protein" refers to a protein that can specifically bind to a target. Non-limiting examples of binding proteins include antibodies, anticalins, repebodies, monobodies, scFv, scFab, affibodies, fynomers, DARPins, nanobodies, chimeric antigen receptors, chimeric engulfment receptors, and peptide aptamers. The binding protein is preferably an antibody.

[0035] The term "Designed ankyrin repeat protein" or "DARPin" refers to a protein derived from ankyrin repeat sequences that has been engineered to bind to a specific target (see Plückthun, 2015. Annu Rev Pharmacol Toxicol. 55:489-511).

[0036] As used herein, the term "effective amount" of an agent (e.g., a therapeutic agent such as an antibody) refers to an amount sufficient to achieve a beneficial or desired result (e.g., a clinical result), and thus, an "effective amount" depends on the context in which it is used. For example, in the case of administering a therapeutic agent to treat ATTR, an effective amount of the agent refers to, for example, a dose sufficient to reduce or decrease TTR deposits compared to the response obtained when the dose is not administered. The term "effective amount" can be used interchangeably with "effective dose," "therapeutically effective dose," or "therapeutically effective dose."

[0037] The term "fynomer" refers to a protein that is derived from the SH3 domain of human Fyn kinase and has been engineered to bind to a specific target (see Bertschinger et al., 2007. Protein Eng Des Sel. 20(2):57-68).

[0038] The term "identity" or "percent identity", in the context of two or more polypeptide or nucleic acid molecule sequences, refers to when comparing and aligning maximum correspondence in a comparison window or specified region using methods known in the art (e.g., sequence comparison algorithms, by manual alignment or by visual inspection), two or more sequences or subsequences are identical in a specific region, or have a specific percentage of amino acid residues or nucleotides that are identical (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 percentage sequence identity and sequence similarity are 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, respectively. In some embodiments, percent identity is determined over the length of the entire reference sequence disclosed herein.

[0039] The terms "individual", "patient" or "subject" are used interchangeably in this application to refer to humans and are not intended to be limiting in any way. An "individual", "patient" or "subject" may be of any age, gender, and physical condition.

[0040] The terms "isoaspartate-modified transthyretin", "isoAsp-modified transthyretin", "isoAsp-modified TTR", "isoD-modified transthyretin", and "isoD-modified TTR" refer to transthyretin in which an aspartate residue has been post-translationally modified to isoaspartate (preferably L-isoaspartate). An exemplary isoD-modified TTR is isoD38 TTR, in which the aspartate at position 38 (with reference to the numbering of SEQ ID NO: 1) is post-translationally modified to L-isoaspartate. Thus, isoD38 TTR refers to a TTR protein comprising SEQ ID NO: 3.

[0041] SEQ ID NO: 1 is an exemplary TTR having the following amino acid sequence:

[0042] GPTGTGESKCPLMVKVLDAVRGSPAINVAVHVFRKAADDTWEPFASGKTSESGELHGLTTEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTTAVVTNPKE

[0043] The term "K D" refers to the dissociation constant. In a preferred embodiment, K D Determined by surface plasmon resonance or isothermal titration calorimetry. Preferably, K D Measured by surface plasmon resonance at 25°C.

[0044] The term "monomer" refers to a protein derived from a type III fibronectin domain that has been engineered to bind to a specific target (see Koide et al., 2013. J Mol Biol. 415(2):393-405).

[0045] The term "nanobody" refers to a protein comprising a soluble single antigen-binding V domain of a heavy chain antibody, preferably a camelid heavy chain antibody (see Bannas et al., 2017. Front Immunol. 8: 1603).

[0046] The term "peptide aptamer" refers to a short sequence of 5 to 20 amino acid residues that can bind to a specific target. Peptide aptamers are usually inserted into the loop region of a stable protein scaffold (see Reverdatto et al., 2015. Curr Top Med Chem. 15(12): 1082-101).

[0047] As used in this application, the term "prevention" refers to a group of hygienic, pharmacological, surgical and / or physical means used to prevent the onset and / or development of disease and / or symptoms. The term "prevention" encompasses preventive methods because these methods are used to maintain the health of an animal or individual.

[0048] The term "repeat" refers to a protein derived from a leucine-rich repeat module and engineered to bind to a specific target (see Lee et al., 2012. PNAS. 109(9): 3299-3304).

[0049] The term "single-chain antigen-binding fragment" or "scFab" refers to a fusion protein comprising a variable region and a constant region of an antibody light chain linked to a variable region and a constant region of an antibody heavy chain, wherein the heavy and light chains are linked together by a short peptide.

[0050] The term "single-chain variable fragment" or "scFv" refers to a fusion protein comprising the heavy chain variable region and the light chain variable region of an antibody connected to each other with a peptide linker.

[0051] In the context of the present invention, the term "specifically binds" is used to describe a binding protein (e.g., an antibody) that has a binding constant (K) of 1:1 for isoD-modified TTR. D ) compared to the K of the binding protein to TTR without isoD residues DAt least 10 times smaller (e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 times smaller). In some embodiments, the epitope to which the binding protein specifically binds comprises an isoD residue (preferably an isoD residue at position 38).

[0052] As used in this application, the terms "treatment" and "therapy" refer to a group of hygienic, pharmacological, surgical and / or physical means intended to cure and / or alleviate diseases and / or symptoms with the goal of repairing health problems. The terms "treatment" and "therapy" include preventive and therapeutic methods, because both are directed to maintaining and / or reestablishing the health of an individual or animal. Regardless of the source of symptoms, diseases and defects, in the context of this application, the administration of appropriate drugs to alleviate and / or cure health problems should be interpreted as treatment or therapy.

[0053] Binding protein

[0054] In a first aspect, the present invention provides a binding protein that can specifically bind to isoD-modified TTR.

[0055] In some embodiments, the binding protein specifically binds to isoD38 TTR. In some embodiments, the K of the interaction between the binding protein and a polypeptide comprising SEQ ID NO: 3 (e.g., TTR) is D The K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 2 (e.g., TTR) D In a preferred embodiment, the K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 3 (e.g., TTR) is at least 10 times smaller (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 times smaller). D The K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 2 (e.g., TTR) D More preferably, the K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 3 (e.g., TTR) is D The K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 2 (e.g., TTR) D At least 150, 160, 170, 180, 190 or 200 times smaller.

[0056] In some embodiments, the K of the interaction between the binding protein and SEQ ID NO: 3 D The K of the interaction between the binding protein and SEQ ID NO: 2 D In a preferred embodiment, the K of the interaction between the binding protein and SEQ ID NO: 3 is at least 10 times smaller (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 times smaller). D The K of the interaction between the binding protein and SEQ ID NO: 2 D More preferably, the K of the interaction between the binding protein and SEQ ID NO: 3 is at least 100 times smaller. D The K of the interaction between the binding protein and SEQ ID NO: 2 D At least 150, 160, 170, 180, 190 or 200 times smaller.

[0057] Preferably, K D Determined by surface plasmon resonance or isothermal titration calorimetry. More preferably, K D Measured by surface plasmon resonance at 25°C. In some embodiments, K is determined using SEQ ID NO:5 versus SEQ ID NO:3. D (In other words, K D (determined using a peptide consisting of SEQ ID NO:3 and a PEG-biotin moiety covalently bound to the N-terminal residue). In some embodiments, K is determined using SEQ ID NO:6 for SEQ ID NO:2. D (In other words, K D was determined using a peptide consisting of SEQ ID NO: 2 and a PEG-biotin moiety covalently attached to the N-terminal residue).

[0058] In some embodiments, the K of the interaction between the binding protein and SEQ ID NO: 5 D The K of the interaction between the binding protein and SEQ ID NO: 6 D In a preferred embodiment, the K of the interaction between the binding protein and SEQ ID NO: 5 is at least 10 times smaller (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 times smaller). DThe K of the interaction between the binding protein and SEQ ID NO: 6 D More preferably, the K of the interaction between the binding protein and SEQ ID NO: 5 is at least 100 times smaller. D The K of the interaction between the binding protein and SEQ ID NO: 6 D At least 150, 160, 170, 180, 190 or 200 times smaller.

[0059] SEQ ID NO: 2 is TTR (35-43) having the following sequence:

[0060] KAADDTWEP

[0061] SEQ ID NO: 3 is isoD38 TTR (35-43) having the following sequence:

[0062] KAAXDTWEP,

[0063] wherein X is L-isoAsp.

[0064] SEQ ID NO:5 is isoD38 TTR(35-43)PEG-biotin having the following sequence:

[0065] KAAXDTWEP,

[0066] wherein X is L-isoAsp, the N-terminal residue is modified with a PEG-biotin moiety, and the C-terminal residue is amidated.

[0067] In some embodiments, SEQ ID NO:5 is a modified peptide of formula (I):

[0068]

[0069] SEQ ID NO:6 is TTR(35-43)PEG-biotin and has the following sequence:

[0070] KAADDTWEP,

[0071] Among them, the N-terminal residue was modified with PEG-biotin moiety and the C-terminal residue was amidated.

[0072] In some embodiments, SEQ ID NO:6 is a modified peptide of formula (II):

[0073]

[0074] In some embodiments, the K of the interaction between the binding protein and the modified peptide of formula (I) is D The K of the interaction between the binding protein and the modified peptide of formula (II)D In a preferred embodiment, the K of the interaction between the binding protein and the modified peptide of formula (I) is at least 10 times smaller (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 times smaller). D The K of the binding protein and the modified peptide of formula (II) D More preferably, the K of the interaction between the binding protein and the modified peptide of formula (I) is D The K of the binding protein and the modified peptide of formula (II) D At least 150, 160, 170, 180, 190 or 200 times smaller.

[0075] In a preferred embodiment, the K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 3 (eg, TTR) at 25°C is D Less than 200 nM. Preferably, K D Less than 150 nM. More preferably, K D Less than 10 nM. Most preferably, K D Less than 5 nM. Preferably, K D Determined by surface plasmon resonance or isothermal titration calorimetry. More preferably, K D Measured by surface plasmon resonance at 25°C.

[0076] In a preferred embodiment, the K of the interaction between the binding protein and SEQ ID NO: 3 at 25°C is D Less than 200 nM. Preferably, K D Less than 150 nM. More preferably, K D Less than 10 nM. Most preferably, K D Less than 5 nM. Preferably, K D Determined by surface plasmon resonance or isothermal titration calorimetry. More preferably, K D Measured by surface plasmon resonance at 25°C. In some embodiments, K is determined using SEQ ID NO:5 versus SEQ ID NO:3. D (In other words, K D was determined using a peptide consisting of SEQ ID NO: 3 and a PEG-biotin moiety covalently attached to the N-terminal residue).

[0077] In a preferred embodiment, the K of the interaction between the binding protein and SEQ ID NO: 5 at 25°C is D Less than 200 nM. Preferably, KD Less than 150 nM. More preferably, K D Less than 10 nM. Most preferably, K D Less than 5 nM. Preferably, K D Determined by surface plasmon resonance or isothermal titration calorimetry. More preferably, K D Measured by surface plasmon resonance at 25°C.

[0078] In a preferred embodiment, the K of the interaction between the binding protein and the modified peptide of formula (I) at 25°C is D Less than 200 nM. Preferably, K D Less than 150 nM. More preferably, K D Less than 10 nM. Most preferably, K D Less than 5 nM. Preferably, K D Determined by surface plasmon resonance or isothermal titration calorimetry. More preferably, K D Measured by surface plasmon resonance at 25°C.

[0079] In some embodiments, the binding protein comprises an Fc domain. An exemplary Fc domain may be composed of the second and third constant regions (CH2-CH3) of the human IgG1 heavy chain. The Fc domain allows 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 phagocytic receptor (see, e.g., WO 2018 / 064076 A1).

[0080] In some embodiments, the binding protein is capable of inducing phagocytosis of isoD-modified TTR.

[0081] In some embodiments, the binding protein is an antibody, scFv, scFab, chimeric antigen receptor, or chimeric phagocytic receptor.

[0082] In a preferred embodiment, the binding protein comprises:

[0083] (a) a heavy chain variable region (VH), wherein the VH comprises a HCDR3 polypeptide selected from the group consisting of NSYYGMDY (SEQ ID NO: 16) and EDY (SEQ ID NO: 26); and / or

[0084] (b) a light chain variable region (VL), wherein the VL comprises a LCDR3 polypeptide selected from the group consisting of HQYLSSRT (SEQ ID NO: 13) and QHFWNIPFT (SEQ ID NO: 23);

[0085] In a preferred embodiment, the binding protein comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH comprises HCDR1, HCDR2 and HCDR3 polypeptides, and the polypeptides are selected from the group consisting of:

[0086] (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

[0087] (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).

[0088] The present invention also contemplates variants of the CDRs set forth in SEQ ID NOs: 11 to 26. Methods for identifying variant CDRs and thus variant binding proteins are known in the art (see, for example, WO 2006 / 050491 A2).

[0089] The amino acids of the binding protein can be replaced by amino acids with similar properties (based on size, polarity, hydrophobicity, etc.). In other words, the amino acids of the binding protein can be replaced by different amino acids of the same class, where amino acids can be classified as:

[0090] -Aromatic: Phe, Tyr, Trp;

[0091] - Non-polar: Leu, Val, Ile, Ala, Met;

[0092] -Aliphatic: Ala, Val, Leu, Ile;

[0093] -Acidic: Asp, Glu;

[0094] - Basic: His, Lys, Arg;

[0095] -Polarity: Gln, Asn, Ser, Thr, Tyr.

[0096] 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 one, two, three, four, five or six (preferably six) CDRs may be replaced according to the following table:

[0097] Amino acids to be replaced Replace amino acids Ala Ser Arg Lys Asn Gln or His Asp Glu Cys Ser or Ala Gln Asn Glu Asp Gly Pro His Asn or Gln Ile Leu or Val Leu Ile or Val Lys Arg, Gln or Glu Met Leu or Ile Phe Met, Leu or Tyr Pro Gly Ser Thr Thr Ser Trp Tyr Tyr Trp or Phe Val Ile or Leu

[0098] The amino acids of the binding protein may be replaced by different amino acids at the same position in related binding proteins (ie, binding proteins capable of binding to the same target). In the present application, 2F2 and 4D4 are related binding proteins.

[0099] In some embodiments, the invention encompasses binding protein variants in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CDR residues differ from the CDRs of 2F2 (ie, SEQ ID NOs: 11-16) or 4D4 (ie, SEQ ID NOs: 21-26).

[0100] In a preferred embodiment, the binding protein comprises:

[0101] (a) a heavy chain variable region (VH), wherein the VH comprises a 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

[0102] (b) a light chain variable region (VL), wherein the VL comprises a 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.

[0103] In a preferred embodiment, the binding protein comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH comprises HCDR1, HCDR2 and HCDR3 polypeptides, and the polypeptides are selected from the group consisting of:

[0104] (a) LCDR1 is KSSQSVLYSSNQKNYLA (SEQ ID NO: 11) or a sequence having at least 70% (preferably at least 90%) identity thereof, LCDR2 is WASTRES (SEQ ID NO: 12) or a sequence having at least 70% (preferably at least 80%) identity thereof, LCDR3 is HQYLSSRT (SEQ ID NO: 13) or a sequence having at least 70% (preferably at least 80%) identity thereof, HCDR1 is GFTFSSFAMH (SEQ ID NO: 14) or a sequence having at least 70% (preferably at least 90%) identity thereof, HCDR2 is FISSGSNTIYYADTVKG (SEQ ID NO: 15) or a sequence having at least 70% (preferably at least 90%) identity thereof, and HCDR3 is NSYYGMDY (SEQ ID NO: 16) or a sequence having at least 70% (preferably at least 80%) identity thereof; and

[0105] (b) LCDR1 is RTSGNIRNSLA (SEQ ID NO:21) or a sequence having at least 70% (preferably at least 90%) identity thereof, LCDR2 is NGKTLAD (SEQ ID NO:22) or a sequence having at least 70% (preferably at least 80%) identity thereof, LCDR3 is QHFWNIPFT (SEQ ID NO:23) or a sequence having at least 70% (preferably at least 80%) identity thereof, HCDR1 is GNTFSSRWIE (SEQ ID NO:24) or a sequence having at least 70% (preferably at least 90%) identity thereof, HCDR2 is EIFPGNGNTNYNEKFKG (SEQ ID NO:25) or a sequence having at least 70% (preferably at least 90%) identity thereof, and HCDR3 is EDY (SEQ ID NO:26) or a sequence having at least 65% identity thereof.

[0106] In a preferred embodiment, the binding protein comprises VL and VH, wherein 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.

[0107] In a preferred embodiment, the binding protein comprises a VL and a VH, wherein the VL and VH are polypeptides selected from the group consisting of: (a) a 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 a 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) a 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 a 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; (c) a VL 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; NO:17 or a VL having at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80% or 85%) identity thereto, and SEQ ID NO:28 or a VH having at least 50% identity thereto; and (d) SEQ ID NO:27 or a VL having at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80% or 85%) identity thereto, and SEQ ID NO:18 or a VH having at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80% or 85%) identity thereto;

[0108] In a preferred embodiment, the binding protein comprises a VL and a VH, wherein the VL and the VH are polypeptides selected from the group consisting of: (a) a VL of SEQ ID NO: 17 or a sequence having 55% identity thereto, and a VH of SEQ ID NO: 18 or a sequence having at least 55% identity thereto; (b) a VL of SEQ ID NO: 27 or a sequence having 55% identity thereto, and a VH of SEQ ID NO: 28 or a sequence having at least 55% identity thereto; (c) a VL of SEQ ID NO: 17 or a sequence having 55% identity thereto, and a VH of SEQ ID NO: 28 or a sequence having at least 55% identity thereto; and (d) a VL of SEQ ID NO: 27 or a sequence having 55% identity thereto, and a VH of SEQ ID NO: 18 or a sequence having at least 55% identity thereto.

[0109] In a preferred embodiment, the binding protein comprises a VL and a VH, wherein the VL and the VH are polypeptides selected from the group consisting of: (a) a VL of SEQ ID NO: 17 or a sequence having 60% identity thereto, and a VH of SEQ ID NO: 18 or a sequence having at least 60% identity thereto; (b) a VL of SEQ ID NO: 27 or a sequence having 60% identity thereto, and a VH of SEQ ID NO: 28 or a sequence having at least 60% identity thereto; (c) a VL of SEQ ID NO: 17 or a sequence having 60% identity thereto, and a VH of SEQ ID NO: 28 or a sequence having at least 60% identity thereto; and (d) a VL of SEQ ID NO: 27 or a sequence having 60% identity thereto, and a VH of SEQ ID NO: 18 or a sequence having at least 60% identity thereto.

[0110] In a preferred embodiment, the binding protein comprises a VL and a VH, wherein the VL and the VH are polypeptides selected from the group consisting of: (a) a VL of SEQ ID NO: 17 or a sequence having 65% identity thereto, and a VH of SEQ ID NO: 18 or a sequence having at least 65% identity thereto; (b) a VL of SEQ ID NO: 27 or a sequence having 65% identity thereto, and a VH of SEQ ID NO: 28 or a sequence having at least 65% identity thereto; (c) a VL of SEQ ID NO: 17 or a sequence having 65% identity thereto, and a VH of SEQ ID NO: 28 or a sequence having at least 65% identity thereto; and (d) a VL of SEQ ID NO: 27 or a sequence having 65% identity thereto, and a VH of SEQ ID NO: 18 or a sequence having at least 65% identity thereto.

[0111] In a preferred embodiment, the binding protein comprises a VL and a VH, wherein the VL and the VH are polypeptides selected from the group consisting of: (a) a VL of SEQ ID NO: 17 or a sequence having 70% identity thereto, and a VH of SEQ ID NO: 18 or a sequence having at least 70% identity thereto; (b) a VL of SEQ ID NO: 27 or a sequence having 70% identity thereto, and a VH of SEQ ID NO: 28 or a sequence having at least 70% identity thereto; (c) a VL of SEQ ID NO: 17 or a sequence having 70% identity thereto, and a VH of SEQ ID NO: 28 or a sequence having at least 70% identity thereto; and (d) a VL of SEQ ID NO: 27 or a sequence having 70% identity thereto, and a VH of SEQ ID NO: 18 or a sequence having at least 70% identity thereto.

[0112] In a preferred embodiment, the binding protein comprises a VL and a VH, wherein the VL and the VH are polypeptides selected from the group consisting of: (a) a VL of SEQ ID NO: 17 or a sequence having 75% identity thereto, and a VH of SEQ ID NO: 18 or a sequence having at least 75% identity thereto; (b) a VL of SEQ ID NO: 27 or a sequence having 75% identity thereto, and a VH of SEQ ID NO: 28 or a sequence having at least 75% identity thereto; (c) a VL of SEQ ID NO: 17 or a sequence having 75% identity thereto, and a VH of SEQ ID NO: 28 or a sequence having at least 75% identity thereto; and (d) a VL of SEQ ID NO: 27 or a sequence having 75% identity thereto, and a VH of SEQ ID NO: 18 or a sequence having at least 75% identity thereto.

[0113] In a preferred embodiment, the binding protein comprises a VL and a VH, wherein the VL and the VH are polypeptides selected from the group consisting of: (a) a VL of SEQ ID NO: 17 or a sequence having 80% identity thereto, and a VH of SEQ ID NO: 18 or a sequence having at least 80% identity thereto; (b) a VL of SEQ ID NO: 27 or a sequence having 80% identity thereto, and a VH of SEQ ID NO: 28 or a sequence having at least 80% identity thereto; (c) a VL of SEQ ID NO: 17 or a sequence having 80% identity thereto, and a VH of SEQ ID NO: 28 or a sequence having at least 80% identity thereto; and (d) a VL of SEQ ID NO: 27 or a sequence having 80% identity thereto, and a VH of SEQ ID NO: 18 or a sequence having at least 80% identity thereto.

[0114] In a preferred embodiment, the binding protein comprises a VL and a VH, wherein the VL and the VH are polypeptides selected from the group consisting of: (a) a VL of SEQ ID NO: 17 or a sequence having 85% identity thereto, and a VH of SEQ ID NO: 18 or a sequence having at least 85% identity thereto; (b) a VL of SEQ ID NO: 27 or a sequence having 85% identity thereto, and a VH of SEQ ID NO: 28 or a sequence having at least 85% identity thereto; (c) a VL of SEQ ID NO: 17 or a sequence having 85% identity thereto, and a VH of SEQ ID NO: 28 or a sequence having at least 85% identity thereto; and (d) a VL of SEQ ID NO: 27 or a sequence having 85% identity thereto, and a VH of SEQ ID NO: 18 or a sequence having at least 85% identity thereto.

[0115] SEQ ID NO: 17 is the VL fragment present in the 2F2 fragment, having the following sequence:

[0116] NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQYLSSRTFGAGTKLELK

[0117] SEQ ID NO: 18 is the VH fragment present in the 2F2 fragment, having the following sequence:

[0118] DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFAMHWVRQAPDKGLEWVAFISSGSNTIYYADTVKGRFTVSRDNPKNTLFLQMTSLRSEDTAMYYCASNSYYGMDYWGQGTSVTVSS

[0119] SEQ ID NO:27 is the VL fragment present in the 4D4 fragment, having the following sequence:

[0120] DIQMTQSPASSLSASVGETVTITCRTSGNIRNSLAWYRQKQGKSPQLLVFNGKT LADGVPSRFSGSGSGTQYSLKINSLQPEDLGTYYCQHFWNIPFTFGSGTKLEIK

[0121] SEQ ID NO:28 is the VH fragment present in the 4D4 fragment, having the following sequence:

[0122] QVQLQQSGAELMKPGASVKISCKAPGNTFSSRWIEWVKERPGHGLEWIGEIFPGNGNTNYNEKFKGKATFTADTSSNTAYIQFSSLASEDSGVYYCVSEDYWGQGTLVTVSS

[0123] Preferably, the binding protein is an antibody.

[0124] Phage display and combinatorial methods for generating antibodies are known in the art (described in, e.g., Ladner et al., U.S. Pat. No. 5,223,409; Kang et al., International Publication No. WO 92 / 18619; Dower et al., International Publication No. WO 91 / 17271; Winter et al., International Publication No. WO 92 / 20791; Markland et al., International Publication No. WO 92 / 15679; Breitling et al., International Publication No. WO 93 / 01288; McCafferty et al., International Publication No. WO 92 / 01047; Garrard et al., International Publication No. WO 92 / 09690; Ladner et al., International Publication No. WO 90 / 02809; Fuchs et al., (1991) Bio / Technology 9:1370-1372; Hay et al. (1992) Human Antibody Hybridoma 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths 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).

[0125] In one embodiment, the antibody is a fully human antibody (e.g., an antibody made in a mouse that is genetically engineered to produce antibodies derived from human immunoglobulin sequences), or a non-human antibody, such as 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.

[0126] Rather than using the mouse system, human monoclonal antibodies can be generated using transgenic mice carrying human immunoglobulin genes. Splenocytes from transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinity for epitopes from human proteins. (See, e.g., Wood et al. International Application WO 91 / 00906, Kucherlapati et al. PCT Application WO 91 / 10741; Lonberg et al. International Application WO 92 / 03918; Kay et al. International Application 92 / 03917; Lonberg, N et al. 1994 Nature 368:856-859; Green, LL et al. 1994 Nature Genet. 7:13-21; Morrison, SL 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).

[0127] The antibody may be an antibody in which the variable region or a portion thereof (e.g., CDR) is generated in a non-human organism (e.g., rat or mouse). Chimeric antibodies, CDR-grafted antibodies, and humanized antibodies are all within the scope of the present invention. Antibodies generated in a non-human organism (e.g., rat or mouse) and subsequently modified (e.g., in the variable framework region or constant region) to reduce antigenicity in humans are also within the scope of the present invention.

[0128] Chimeric antibodies can be produced by recombinant DNA techniques known in the art (see Robinson et al., International Patent Publication No. PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, M., European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., International Application No. WO 86 / 01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application No. 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).

[0129] The humanized or CDR-grafted antibody will have at least one or two, but usually all three acceptor CDRs (acceptor CDRs of the immunoglobulin heavy chain and / or immunoglobulin light chain) replaced by donor CDRs. The antibody may be replaced by at least a portion of the non-human CDRs or only some of the CDRs may be replaced by non-human CDRs. It is only necessary to replace the number of CDRs required for the humanized antibody to bind to the isoD-modified TTR. Preferably, the donor will be a rodent antibody, such as a rat or mouse antibody, and the acceptor will be a human framework region or a human consensus framework region. Typically, the immunoglobulin providing the CDR is called a "donor" and the immunoglobulin providing the framework region is called an "acceptor." In one embodiment, the donor immunoglobulin is a non-human (e.g., rodent) immunoglobulin. The acceptor framework region is a naturally occurring (e.g., human) framework region or a consensus framework region or a sequence having more than 85%, preferably 90%, 95%, or 99% identity therewith.

[0130] As used herein, the term "consensus sequence" refers to a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a protein family, each position in the consensus sequence is occupied by the most frequently occurring amino acid at that position in the family. If two amino acids occur with the same frequency, either amino acid may be included in the consensus sequence. "Consensus framework region" refers to a framework region in a consensus immunoglobulin sequence.

[0131] Antibodies can be humanized by methods known in the art (see, eg, Morrison, SL, 1985, Science 229: 1202-1207, Oi et al., 1986, BioTechniques 4: 214, Queen et al. US 5,585,089, US 5,693,761 and US 5,693,762).

[0132] Humanized or CDR grafted antibodies are produced by CDR grafting or CDR replacement, wherein one, two or all CDRs of an immunoglobulin chain can be replaced. See, e.g., U.S. Pat. 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. Pat. No. 5,225,539. Winter describes a CDR grafting method that can be used to prepare humanized antibodies of the present invention (British patent application GB 2188638A filed March 26, 1987; Winter U.S. Pat. No. 5,225,539).

[0133] The scope of the present invention also includes humanized antibodies in which specific amino acids are replaced, deleted or added. The criteria for selecting amino acids from donors are described in U.S. Patent No. 5,585,089, for example, columns 12 to 16 of US 5,585,089. Other techniques for humanizing antibodies are described in EP 519596A1, et al., published on December 23, 1992, by Padlan et al.

[0134] In other embodiments, the antibody has, for example, a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; in particular, for example, a heavy chain constant region selected from (e.g., human) heavy chain constant regions of IgG1, IgG2, IgG3 and IgG4. In another embodiment, the antibody has a light chain constant region selected from (e.g., human) light chain constant regions of κ or λ. The constant region can be changed, such as mutated, to change the properties of the antibody (e.g., increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, 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 or fix complement. In another embodiment, the ability of the antibody to bind to Fc receptors is weakened or there is no ability to bind to Fc receptors. For example, it is an isotype or subtype, fragment or other mutant that does not support binding to Fc receptors, for example, it has a mutagenic or missing Fc receptor binding region.

[0135] Methods for changing constant regions are known in the art. Antibodies with altered functions, such as altered affinity for effector ligands (e.g., FcR on cells) or the C1 component of complement, can be produced by replacing at least one amino acid residue in the constant portion of an antibody with a different residue (see, e.g., EP 388,151A1, U.S. Patent No. 5,624,821, and U.S. Patent No. 5,648,260). Similar types of changes can be described, where if applied to mouse or other species immunoglobulins, these functions will be reduced or eliminated.

[0136] Antibodies can be derivatized or connected with another functional molecule (e.g., another peptide or protein). As used herein, "derivatized" antibody molecules are modified antibody molecules. Derivatization methods include, but are not limited to, adding fluorescent moieties, radionucleotides, toxins, enzymes, or affinity ligands such as biotin. Therefore, antibody molecules of the present invention are intended to include antibodies described herein in derivatized and other modified forms, including immunoadhesion molecules. For example, antibody molecules can be functionally connected (by chemical conjugation, genetic fusion, non-covalent binding or other means) to one or more other molecular entities, such as another antibody (e.g., bispecific antibodies or diabodies), detectable substances, cytotoxic agents, pharmaceutical agents, and / or proteins or peptides (e.g., streptavidin core regions or polyhistidine tags) that can mediate the binding of the antibody or antibody portion to another molecule.

[0137] One type of derivatized antibody molecule is produced by cross-linking two or more antibodies (cross-linking antibodies of the same type or different types, for example, to produce bispecific antibodies). Suitable cross-linking agents include those that are heterobifunctional agents (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional agents (e.g., disuccinimidyl suberate) having two differently reactive groups separated by a suitable spacer. Such linkers are available from Pierce Chemical Company, Rockford, Ill.

[0138] The antibody may be conjugated to another molecular entity, typically a label or therapeutic agent (e.g., a cytotoxic or cytostatic agent) or moiety. Radioisotopes may be used in diagnostic or therapeutic applications. Such radioisotopes include, but are not limited to, iodine ( 131 I or 125 I), yttrium ( 90 Y), Lutetium ( 177 Lu), Actinium ( 225 Ac), praseodymium, astatine ( 211 At), rhenium ( 186 Re、Bi( 212 Bior 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 (75Se), or gallium (67Ga). Radioactive isotopes used as labels (e.g., for diagnostics) 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 therapeutic isotopes listed above.

[0139] The present invention provides radiolabeled antibody molecules and methods for labeling antibody molecules. In one embodiment, a method for labeling antibody molecules is disclosed. The method includes contacting the antibody molecule with a chelating agent, thereby producing a conjugated antibody. The conjugated antibody is then radiolabeled with a radioisotope (e.g., 111 indium, 90 yttrium, and 177 lutetium), thereby producing a labeled antibody molecule.

[0140] The antibody molecule can be conjugated to a therapeutic agent. The antibody can be labeled. For example, the antibody can be labeled with a biotin molecule, an enzyme, or a fluorophore.

[0141] The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (numbering scheme "Kabat"), Al-Lazikani et al., (1997) JMB 273, 927-948 (numbering scheme "Chothia"). As used herein, CDRs defined by the "Chothia" numbering scheme are sometimes also referred to as "hypervariable loops." In some embodiments, the amino acid sequence boundaries of a CDR are determined using the Kabat numbering scheme. Figure 3 Exemplary applications of the Kabat numbering scheme are provided.

[0142] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises two LCs and two HCs, wherein each LC and each HC is a polypeptide selected from the following groups: (a) LC of SEQ ID NO:31 and HC of SEQ ID NO:32; (b) LC of SEQ ID NO:33 and HC of SEQ ID NO:34; (c) LC of SEQ ID NO:31 and HC of SEQ ID NO:34; (c) LC of SEQ ID NO:33 and HC of SEQ ID NO:32.

[0143] SEQ ID NO:31 is the LC present in 2F2, having the following sequence:

[0144] NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQYLSSRTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0145] SEQ ID NO:32 is the HC present in 2F2 and has the following sequence:

[0146] DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFAMHWVRQAPDKGLEWVAFISSGSNTIYYADTVKGRFTVSRDNPKNTLFLQMTSLRSEDTAMYYCASNSYYGMDYWGQGTSVTVSSATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPGK

[0147] SEQ ID NO:33 is the LC present in 4D4 and has the following sequence:

[0148] DIQMTQSPASSLSASVGETVTITCRTSGNIRNSLAWYRQKQGKSPQLLVFNGKTLADGVPSRFSGSGSGTQYSLKINSLQPEDLGTYYCQHFWNIPFTFGSGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0149] SEQ ID NO:34 is the HC present in 4D4, having the following sequence:

[0150] QVQLQQSGAELMKPGASVKISCKAPGNTFSSRWIEWVKERPGHGLEWIGEIFPGNGNTNYNEKFKGKATFTADTSSNTAYIQFSSLASEDSGVYYCVSEDYWGQGTLVTV SSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPP CKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTI SKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0151] In a preferred embodiment, the antibody is a monoclonal antibody.

[0152] Nucleic Acids

[0153] In a second aspect, the present invention provides a nucleic acid comprising a sequence encoding a 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 genetic construct codons are known in the art (Quax et al., 2015. Mol Cell. 59 (2): 149-61).

[0154] In a preferred embodiment, the nucleic acid is contained in a vector. The vector can be a cloning vector, an expression vector, a transport vector or a viral vector.

[0155] In some embodiments, the nucleic acid is a modified mRNA molecule suitable for expressing a binding protein in vivo. The mRNA can be delivered using lipid nanoparticles (see Hou et al., 2021. Nat Rev Mater. 6(12): 1078-1094).

[0156] In some embodiments, the nucleic acid comprises:

[0157] (a) SEQ ID NO: 19 and SEQ ID NO: 20;

[0158] (b) SEQ ID NO:29 and SEQ ID NO:30;

[0159] (c) SEQ ID NO:29 and SEQ ID NO:20; or

[0160] (d) SEQ ID NO:19 and SEQ ID NO:30.

[0161] SEQ ID NO: 19 is the nucleic acid sequence encoding the VL fragment of 2F2:

[0162] AACATTATGATGACGCAGTCGCCATCTCTGGCTGTGTCTGCAGGAGAAAAGGTCACTATGAGCTGTAAGTCCAGTCAGAGTGTTTTACAGTTCAAATCAGAAGAATTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGG GCATCCACTAGGGAATCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGTGTACAAGCTGAAGACCTGGCAGTTTTATTACTGTCATCAATACCTCTCCTCGCGCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAG

[0163] SEQ ID NO:20 is the nucleic acid sequence encoding the VH fragment of 2F2:

[0164] GATGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGCAATGCACTGGGTTCGTCAGGCTCCAGACAAGGGGCTGGAGTGGGTCGCATTCATTAGTAGTGGCAGTAATACCATCTACTATGCAGACACAGTGAAGGGCCGATTCACCGTCTCCAGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGTAATTCGTACTATGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0165] SEQ ID NO:29 is the nucleic acid sequence encoding the VL fragment of 4D4:

[0166] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAACAAGTGGGAATATTCGCAATTCATTAGCATGGTATCGACAGAAACAGGGAAAATCCCCTCAGCTCCTGGTCTTTAATGGCAAAACTTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCTGAAGATTTAGGGACTTATTACTGTCAACATTTTTGGAATATTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA

[0167] SEQ ID NO:30 is the nucleic acid sequence encoding the VH fragment of 4D4:

[0168] CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTCCTGGCAACACATTCAGTAGCAGGTGGATAGAGTGGGTAAAGGAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTTCCTGGAAATGGT AATACTAACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTATATACAATTCAGCAGCCTGGCATCTGAGGACTCTGGCGTCTATTACTGTGTATCTGAAGACTATTGGGGCCAAGGTACCCTGGTCACCGTCTCCTCA

[0169] In some embodiments, the nucleic acid comprises:

[0170] (a) SEQ ID NO:35 and SEQ ID NO:36;

[0171] (b) SEQ ID NO:37 and SEQ ID NO:38;

[0172] (c) SEQ ID NO:35 and SEQ ID NO:38; or

[0173] (d) SEQ ID NO:37 and SEQ ID NO:36.

[0174] SEQ ID NO:35 is the nucleic acid sequence encoding the LC of 2F2:

[0175] AACATTATGATGACGCAGTCGCCATCATCTCTGGCTGTGTCTGCAGGAGAAAAGGTCACTATGAGCTGTAAGTCCAGTCAGAGTGTTTTATACAGTTCAAATCAGAAGAATTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGTGTACAAGCTGAAGACCTGGCAGTTTATTACTGTCATCAATACCTCTCCTCGCGCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAGCGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG

[0176] SEQ ID NO:36 is the nucleic acid sequence encoding the HC of 2F2:

[0177]

[0178] SEQ ID NO:37 is the nucleic acid sequence encoding the LC of 4D4:

[0179] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAACAAGTGGGAATATTCGCAATTCATTAGCATGGTATCGACAGAAACAGGGAAAATCCCCTCAGCTCCTGGTCTTTAATGGCAAAACTTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCTGAAGATTTAGGGACTTATTACTGTCAACATTTTTGGAATATTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG

[0180] SEQ ID NO:38 is the nucleic acid sequence encoding the HC of 4D4:

[0181]

[0182] In some embodiments, the heavy and light chain sequences of an antibody of the invention are encoded on one or two nucleic acid molecules.

[0183] Host cells

[0184] In a third aspect, the invention provides a host cell comprising a nucleic acid of the invention.

[0185] In some embodiments, the host cells are used in a method for producing a binding protein of the invention, which comprises culturing the host cells under conditions suitable for protein expression, and then isolating the binding protein.

[0186] In some embodiments, the host cell is an immune cell (eg, a T cell or a natural killer cell) and the binding protein (eg, a chimeric antigen receptor or a chimeric engulfment receptor) is expressed on the cell surface.

[0187] Pharmaceutical composition

[0188] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the binding protein, nucleic acid or host cell of the present invention, and a pharmaceutically acceptable carrier or diluent.

[0189] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable diluent" refers to 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 pharmaceutically active substances is known in the art. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, without limiting the scope of the invention, additional buffers; preservatives; cosolvents; antioxidants (including ascorbic acid and methionine); chelating agents (e.g., EDTA); metal complexes (e.g., Zn-protein complexes); biodegradable polymers (e.g., polyesters); salt-forming counterions (e.g., sodium, polysaccharide alcohols); amino acids (e.g., alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid and threonine); organic sugars. or sugar alcohols (e.g., lactitol, stachyose, mannose, sorbitol, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol); sulfur-containing reducing agents (e.g., urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, [α]-monothioglycerol, and sodium thiosulfate); low molecular weight proteins (e.g., human serum albumin, bovine serum albumin, gelatin or other immunoglobulins); and hydrophilic polymers (e.g., polyvinyl pyrrolidone).

[0190] The pharmaceutical compositions described herein may also include other substances. These substances include, but are not limited to, cryoprotectants, lyoprotectants, surfactants, fillers, antioxidants, and stabilizers. In some embodiments, the pharmaceutical composition may be lyophilized.

[0191] The term "cryoprotectant" as used herein includes agents that provide stability to active ingredients against stress caused by freezing by preferentially excluding them from the surface of proteins. Cryoprotectants can also provide protection during primary and secondary drying and long-term product storage. Non-limiting examples of cryoprotectants include sugars (e.g., sucrose, glucose, trehalose, mannitol, mannose, and lactose); polymers (e.g., dextran, hydroxyethyl starch, and polyethylene glycol); surfactants (e.g., polysorbates (e.g., PS-20 or PS-80)); and amino acids (e.g., glycine, arginine, leucine, and serine). Cryoprotectants that exhibit low toxicity in biological systems are generally used.

[0192] In one embodiment, a lyoprotectant is added to the pharmaceutical composition described herein. The term "lyoprotectant" as used herein includes agents that provide stability to the active ingredient during the freeze-drying or dehydration process (primary and secondary freeze-drying cycles): by providing an amorphous glassy matrix and binding to the surface of the active ingredient through hydrogen bonds, replacing the water molecules removed during the drying process. This helps to minimize product degradation during the freeze-drying cycle and improves product long-term stability. Non-limiting examples of lyoprotectants include: sugars, such as sucrose or trehalose; amino acids, such as monosodium glutamate, amorphous glycine or histidine; methylamines, such as betaine; lyotropic salts, such as magnesium sulfate; polyols, such as trivalent or higher sugar alcohols, such as glycerin, erythritol, glycerol, arabinol, xylitol, sorbitol and mannitol; propylene glycol; polyethylene glycol; pluronics; and combinations thereof. The amount of lyoprotectant added to the pharmaceutical composition is generally an amount that does not result in unacceptable bacterial degradation when the pharmaceutical composition is lyophilized.

[0193] In some embodiments, fillers are included in the pharmaceutical composition. The term "filler" as used herein includes agents that provide the structure of the freeze-dried product without directly interacting with the drug product. In addition to providing a pharmaceutically sophisticated block, fillers can also impart useful qualities in improving collapse temperature, providing freeze-thaw protection, and enhancing long-term storage strain stability. Non-limiting examples of fillers include mannitol, glycine, lactose, and sucrose. Fillers can be crystalline (e.g., glycine, mannitol, or sodium chloride) or amorphous (e.g., dextran, hydroxyethyl starch), and are typically used in formulations in an amount of 0.5% to 10%.

[0194] Other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed., (1980) may also be included in the pharmaceutical compositions described herein, provided that they do not adversely affect the desired properties of the pharmaceutical compositions. As used herein, "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents that are compatible with drug administration. The use of such media and agents for pharmaceutically active substances is known in the art. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include: additional buffers; preservatives; cosolvents; antioxidants, including ascorbic acid and methionine; chelating agents, such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters; salt-forming counterions, such as sodium, polyols; amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, Acid, glutamic acid and threonine; organic sugars or sugar alcohols, such as lactitol, stachyose, mannose, sorbitol, xylose, ribose, ribitol, inositol, galactose, galactitol, glycerol, cyclic alcohols (e.g., inositol), polyethylene glycol; sulfur-containing reducing agents, such as urea, glutathione, lipoic 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 polyvinyl pyrrolidone.

[0195] Pharmaceutical compositions can be prepared for oral administration, sublingual administration, buccal administration, intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, conjunctival administration, rectal administration, transdermal administration, intrathecal administration, topical administration and / or inhalation-mediated administration. In a preferred embodiment, the pharmaceutical composition can be a solution suitable for intravenous administration, intramuscular administration, conjunctival administration, transdermal administration, intraperitoneal administration and / or subcutaneous administration. In another embodiment, the pharmaceutical composition can be a gel or solution suitable for intrathecal administration.

[0196] The pharmaceutical composition may further include common excipients and carriers known in the prior art. For solid pharmaceutical compositions, conventional non-toxic solid carriers may be used, including for example pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, etc. For injection solutions, the pharmaceutical composition may further include cryoprotectants, lyophilization protectants, surfactants, fillers, antioxidants, stabilizers, and pharmaceutically acceptable carriers. For aerosol administration, the pharmaceutical composition is usually provided in a subdivided form with a surfactant and a propellant. The surfactant must certainly be non-toxic and is usually dissolved in the propellant. Representatives of such agents are esters or partial esters of fatty acids (e.g., caproic acid, caprylic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric acid, and oleic acid) containing 6 to 22 carbon atoms and aliphatic polyols or their cyclic anhydrides. Mixed esters may be used, such as mixed glycerides or natural glycerides. If desired, a carrier may also be included, such as the lecithin for intranasal delivery. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides.

[0197] Medical Uses

[0198] In a fifth aspect, the present invention provides the use of the binding protein, nucleic acid, host cell or pharmaceutical composition of the present invention as a drug. In a sixth aspect, the present invention provides the use of the binding protein, nucleic acid, host cell or pharmaceutical composition of the present invention for treating and / or preventing transthyretin amyloidosis (ATTR).

[0199] In a specific embodiment, the binding protein of the invention is capable of binding to and clearing or removing isoD-modified TTR in biological fluids and tissues for preventing and / or treating diseases associated with TTR aggregates and / or amyloid formation.

[0200] It is believed that the binding proteins of the invention induce clearance of aggregated TTR, oligomeric TTR or monomeric TTR. For example, clearance is achieved by targeting aggregated, oligomeric or monomeric TTR for phagocytosis.

[0201] The binding proteins, nucleic acids, host cells or pharmaceutical compositions of the invention can be used in methods for treating and / or preventing ATTR, wherein a therapeutically effective amount of the binding protein is administered to a subject in need thereof. Patients susceptible to treatment include individuals at risk for ATTR but not showing symptoms, as well as patients currently showing symptoms. Some patients can be treated in the prodromal stage of ATTR.

[0202] In some 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).

[0203] In some embodiments, ATTR is Portuguese amyloidosis. Portuguese amyloidosis (transthyretin amyloidosis, ATTR V30M) is the most common form of systemic hereditary amyloidosis, which is autosomal dominant. The disease is also called familial amyloid polyneuropathy type I (FAP-I) and is caused by mutated transthyretin (TTR), which is synthesized by the liver. In Portuguese patients, it was found that the valine at position 30 of the TTR molecule was replaced by methionine (TTR V30M) (see Lobato, 2003, J Nephrol. 16 (3): 438-42).

[0204] An exemplary dosage range of the binding protein can be about 0.1 mg / kg to 20 mg / kg, or 0.5 mg / kg to 5 mg / kg body weight (e.g., 0.5, 1, 2, 3, 4 or 5 mg / kg body weight) or a fixed dose of 10 mg to 1500 mg. The dosage depends on the patient's condition, the response to previous treatment (if any), whether the treatment is preventive or therapeutic, and whether the disease is acute or chronic.

[0205] The compositions of the present invention can be administered at such dosages daily, every other day, weekly, every two weeks, monthly, quarterly, or any other schedule determined by empirical analysis. Exemplary treatments involve administration of multiple doses over a longer period of time (e.g., at least six months). Other exemplary treatment regimens involve administration once every two weeks, once a month, or once every 3 to 6 months.

[0206] The composition can be administered by a peripheral route. The route of administration includes topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, intranasal or intramuscular. The route of administration of the antibody can be intravenous or subcutaneous. Intravenous administration can be, for example, infusion within 30 minutes to 90 minutes. This injection is most often performed in the arm or leg muscle. In some embodiments, the reagent (e.g., a binding protein, nucleic acid, host cell or pharmaceutical composition) is injected directly into a specific tissue where deposits accumulate.

[0207] The binding protein, nucleic acid, host cell or pharmaceutical composition of the present invention can be used in combination with another agent that effectively treats or prevents the disease being treated. Such agents may include siRNA or Vyndaqel (a stabilizer that stabilizes the formation of TTR tetramers) for inhibiting TTR expression.

[0208] The efficacy of treatment can be monitored by determining the level of TTR deposition in a subject using positron emission tomography (PET) before and after administration of a binding protein, nucleic acid, host cell, or pharmaceutical composition disclosed herein.

[0209] diagnosis

[0210] IsoD-modified TTR has been detected in extracted human ATTR-V30M fibrils. Without being bound by a particular theory, this suggests that isoD in TTR is a suitable biomarker for diagnosis and prognosis of ATTR.

[0211] In a seventh aspect, the present invention provides an in vitro diagnostic and / or prognostic method for detecting the presence of isoD-modified TTR in an isolated sample. In some embodiments, the isolated sample is derived from a patient suspected of having ATTR. In some embodiments, the method comprises:

[0212] (i) contacting the separated sample with a binding protein of the present invention; and

[0213] (ii) Determine whether isoD-modified TTR is present in the isolated sample.

[0214] In some 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 specifically binds to TTR but does not distinguish between different forms of TTR. An exemplary antibody that can detect total TTR is "TTR, rabbit, polyclonal" sold by BIOZOL (Biozol, product number: CSB-PA041819).

[0215] In an eighth aspect, the present invention also provides a binding protein of the present invention for use in an in vivo diagnostic and / or prognostic method for ATTR. In some embodiments, the diagnostic and / or prognostic method comprises administering the binding protein to an individual. In some embodiments, the binding protein is labeled with a detectable moiety (e.g., a radioactive label), which allows the localization of TTR fibrils.

[0216] In a preferred embodiment, the binding protein is derivatized in any of the ways previously discussed. For example, the antibody can be fused to catalase and used in an ELISA for diagnosis and / or prognosis of ATTR.

[0217] Sandwich immunoassays are common in the art. There are numerous articles for reference. For example: Cox KL, Devanarayan V, Kriauciunas A et al. Immunoassay Methods. May 1, 2012. [Updated December 24, 2014]. In: Sittampalam GS, Coussens NP, Brimacombe K et al., eds. Assay Guide [Internet]. Bethesda (MD): Eli Lilly and Company and 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.

[0218] Compositions and methods for producing binding proteins

[0219] In a ninth aspect, the present invention provides a method for producing a binding protein (eg, a binding protein of the present invention), comprising immunizing an animal (eg, a mouse) with a polypeptide comprising SEQ ID NO:3.

[0220] In a tenth aspect, the present invention provides a method for screening proteins (eg, binding proteins of the present invention), comprising contacting a group of binding proteins with a polypeptide comprising SEQ ID NO:3.

[0221] In an eleventh aspect, the present invention provides a polypeptide (e.g., an isolated polypeptide) comprising SEQ ID NO: 3. In some embodiments, the isolated polypeptide comprising SEQ ID NO: 3 is used to produce and / or screen for binding proteins (e.g., binding proteins of the present invention). In some embodiments, the present invention provides a container comprising a polypeptide (e.g., an isolated polypeptide) comprising SEQ ID NO: 3.

[0222] Invention Project

[0223] The present invention also includes the following items, which can be combined with any embodiment or aspect disclosed in this application:

[0224] [1] A binding protein that specifically binds to isoaspartate-modified transthyretin.

[0225] [2] The binding protein according to item [1], wherein the isoaspartic acid-modified transthyretin comprises SEQ ID NO: 3 and / or the binding protein specifically binds to an epitope comprising an isoaspartic acid (preferably L-isoaspartic acid) residue.

[0226] [3] The binding protein according to item [1] or [2], wherein the K of the interaction between the binding protein and SEQ ID NO: 5 is D The K of the interaction between the binding protein and SEQ ID NO: 6 D At least 10 times smaller, wherein optionally, the K of the interaction between the binding protein and SEQ ID NO: 5 D The K of the interaction between the binding protein and SEQ ID NO: 6 D At least 200 times smaller.

[0227] [4] The binding protein according to any one of items [1] to [3], wherein the K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 3 (eg, TTR) is D The K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 2 (e.g., TTR) is D At least 10 times smaller, wherein, optionally, the K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 3 (e.g., TTR) is D The K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 2 (e.g., TTR) is D At least 200 times smaller.

[0228] [5] The binding protein according to item [4], wherein the K of the interaction between the binding protein and SEQ ID NO: 3 D The K of the interaction between the binding protein and SEQ ID NO: 2 D At least 10 times smaller, wherein optionally, the K of the interaction between the binding protein and SEQ ID NO: 3 D The K of the interaction between the binding protein and SEQ ID NO: 2 D At least 200 times smaller.

[0229] [6] The binding protein according to any one of items [1] to [5], wherein the K of the interaction between the binding protein and the polypeptide comprising SEQ ID NO: 3 (e.g., TTR) at 25°C is D Less than 200nM.

[0230] [7] The binding protein according to item [6], wherein the K of the binding protein interacting with SEQ ID NO: 3 at 25°C D Less than 200nM.

[0231] [8] The binding protein according to any one of items [1] to [7], wherein the K of the binding protein interacting with SEQ ID NO: 5 at 25°C D Less than 200nM.

[0232] [9] The binding protein according to any one of items [1] to [8], wherein the binding protein comprises:

[0233] (a) a heavy chain variable region (VH), wherein the VH comprises a HCDR3 polypeptide selected from the group consisting of:

[0234] (i) NSYYGMDY (SEQ ID NO: 16), or a sequence at least 70% (preferably at least 80%) identical thereto; and

[0235] (ii) EDY (SEQ ID NO: 26) or a sequence at least 65% identical thereto; and / or

[0236] (b) a light chain variable region (VL), wherein the VL comprises a LCDR3 polypeptide selected from the group consisting of:

[0237] (i) HQYLSSRT (SEQ ID NO: 13) or a sequence having at least 70% (preferably at least 80%) identity thereto; and

[0238] (ii) QHFWNIPFT (SEQ ID NO: 23), or a sequence at least 70% (preferably at least 80%) identical thereto.

[0239]

[10] The binding protein according to any one of items [1] to [9], wherein the binding protein comprises VL and VH, wherein the VL comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH comprises HCDR1, HCDR2 and HCDR3 polypeptides, which are selected from the group consisting of:

[0240] (a) LCDR1 is KSSQSVLYSSNQKNYLA (SEQ ID NO: 11) or a sequence having at least 70% (preferably at least 90%) identity thereof, LCDR2 is WASTRES (SEQ ID NO: 12) or a sequence having at least 70% (preferably at least 80%) identity thereof, LCDR3 is HQYLSSRT (SEQ ID NO: 13) or a sequence having at least 70% (preferably at least 80%) identity thereof, HCDR1 is GFTFSSFAMH (SEQ ID NO: 14) or a sequence having at least 70% (preferably at least 90%) identity thereof, HCDR2 is FISSGSNTIYYADTVKG (SEQ ID NO: 15) or a sequence having at least 70% (preferably at least 90%) identity thereof, and HCDR3 is NSYYGMDY (SEQ ID NO: 16) or a sequence having at least 70% (preferably at least 80%) identity thereof; and

[0241] (b) LCDR1 is RTSGNIRNSLA (SEQ ID NO:21) or a sequence having at least 70% (preferably at least 90%) identity thereof, LCDR2 is NGKTLAD (SEQ ID NO:22) or a sequence having at least 70% (preferably at least 80%) identity thereof, LCDR3 is QHFWNIPFT (SEQ ID NO:23) or a sequence having at least 70% (preferably at least 80%) identity thereof, HCDR1 is GNTFSSRWIE (SEQ ID NO:24) or a sequence having at least 70% (preferably at least 90%) identity thereof, HCDR2 is EIFPGNGNTNYNEKFKG (SEQ ID NO:25) or a sequence having at least 70% (preferably at least 90%) identity thereof, and HCDR3 is EDY (SEQ ID NO:26) or a sequence having at least 65% identity thereof.

[0242]

[11] The binding protein according to any one of items 1 to 10, wherein the binding protein comprises VL and VH, wherein the VL comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH comprises HCDR1, HCDR2 and HCDR3 polypeptides, and the polypeptides are selected from the group consisting of:

[0243] (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

[0244] (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).

[0245]

[12] The binding protein according to any one of items 1 to 11, wherein the binding protein comprises 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 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 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 50% identity thereto, and VH of SEQ ID NO: 28 or a sequence having at least 50% identity thereto; and (d) VL of SEQ ID NO: 27 or a sequence having 50% identity thereto, and VH of SEQ ID NO: 18 or a sequence having at least 50% identity thereto.

[0246]

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

[12] , wherein the binding protein is an antibody.

[0247]

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

[13] .

[0248]

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

[14] .

[0249]

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

[13] , the nucleic acid described in item

[14] or the host cell described in item

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

[0250]

[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 medicament.

[0251]

[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 treating and / or preventing transthyretin amyloidosis.

[0252]

[19] An in vitro diagnostic and / or prognostic method for detecting the presence of isoaspartate-modified transthyretin in an isolated sample, the method comprising:

[0253] (i) contacting the separated sample with the binding protein of any one of items 1 to 20; and

[0254] (ii) determining the presence or absence of isoaspartate-modified transthyretin in the isolated sample.

[0255]

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

[13] , which is used in a method for in vivo diagnosis and / or prognosis of transthyretin amyloidosis.

[0256]

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

[0257]

[23] The binding protein according to item

[22] , wherein the isoaspartate-modified transthyretin comprises SEQ ID NO: 3 and / or the binding protein specifically binds to an epitope comprising an isoaspartate residue.

[0258]

[24] The binding protein according to item

[22] or

[23] , wherein the K of the interaction between the binding protein and SEQ ID NO: 5 is D The K of the interaction between the binding protein and SEQ ID NO: 6 D At least 10 times smaller, wherein optionally, the K of the interaction between the binding protein and SEQ ID NO: 5 D The K of the interaction between the binding protein and SEQ ID NO: 6 D At least 200 times smaller.

[0259]

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

[22] to

[24] , wherein the K of the binding protein interacting with SEQ ID NO: 5 at 25°C D Less than 200nM.

[0260]

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

[22] to

[25] , wherein the binding protein comprises:

[0261] (a) a heavy chain variable region (VH), wherein the VH comprises a HCDR3 polypeptide selected from the group consisting of:

[0262] (i) NSYYGMDY (SEQ ID NO: 16), or a sequence at least 70% (preferably at least 80%) identical thereto; and

[0263] (ii) EDY (SEQ ID NO: 26) or a sequence at least 65% identical thereto; and / or

[0264] (b) a light chain variable region (VL), wherein the VL comprises a LCDR3 polypeptide selected from the group consisting of:

[0265] (i) HQYLSSRT (SEQ ID NO: 13) or a sequence having at least 70% (preferably at least 80%) identity thereto; and

[0266] (ii) QHFWNIPFT (SEQ ID NO: 23), or a sequence at least 70% (preferably at least 80%) identical thereto.

[0267]

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

[22] to

[26] , wherein the binding protein comprises VL and VH, wherein the VL comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH comprises HCDR1, HCDR2 and HCDR3 polypeptides, and the polypeptides are selected from the group consisting of:

[0268] (a) LCDR1 is KSSQSVLYSSNQKNYLA (SEQ ID NO: 11) or a sequence having at least 70% (preferably at least 90%) identity thereof, LCDR2 is WASTRES (SEQ ID NO: 12) or a sequence having at least 70% (preferably at least 80%) identity thereof, LCDR3 is HQYLSSRT (SEQ ID NO: 13) or a sequence having at least 70% (preferably at least 80%) identity thereof, HCDR1 is GFTFSSFAMH (SEQ ID NO: 14) or a sequence having at least 70% (preferably at least 90%) identity thereof, HCDR2 is FISSGSNTIYYADTVKG (SEQ ID NO: 15) or a sequence having at least 70% (preferably at least 90%) identity thereof, and HCDR3 is NSYYGMDY (SEQ ID NO: 16) or a sequence having at least 70% (preferably at least 80%) identity thereof; and

[0269] (b) LCDR1 is RTSGNIRNSLA (SEQ ID NO:21) or a sequence having at least 70% (preferably at least 90%) identity thereof, LCDR2 is NGKTLAD (SEQ ID NO:22) or a sequence having at least 70% (preferably at least 80%) identity thereof, LCDR3 is QHFWNIPFT (SEQ ID NO:23) or a sequence having at least 70% (preferably at least 80%) identity thereof, HCDR1 is GNTFSSRWIE (SEQ ID NO:24) or a sequence having at least 70% (preferably at least 90%) identity thereof, HCDR2 is EIFPGNGNTNYNEKFKG (SEQ ID NO:25) or a sequence having at least 70% (preferably at least 90%) identity thereof, and HCDR3 is EDY (SEQ ID NO:26) or a sequence having at least 65% identity thereof.

[0270]

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

[22] to

[27] , wherein the binding protein comprises VL and VH, wherein the VL comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH comprises HCDR1, HCDR2 and HCDR3 polypeptides, and the polypeptides are selected from the group consisting of:

[0271] (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

[0272] (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).

[0273]

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

[22] to

[28] , wherein the binding protein comprises 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 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 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 50% identity thereto, and VH of SEQ ID NO: 28 or a sequence having at least 50% identity thereto; and (d) VL of SEQ ID NO: 27 or a sequence having 50% identity thereto, and VH of SEQ ID NO: 18 or a sequence having at least 50% identity thereto.

[0274]

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

[22] to

[29] , wherein the binding protein is an antibody.

[0275]

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

[22] to

[30] .

[0276]

[32] A host cell comprising the nucleic acid of item

[31] .

[0277]

[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.

[0278]

[34] The binding protein of any one of items

[22] to

[30] , the nucleic acid of item

[31] or the host cell of item

[32] , for use in treating and / or preventing transthyretin amyloidosis.

[0279]

[35] An in vitro diagnostic and / or prognostic method for detecting the presence of isoaspartate-modified transthyretin in an isolated sample, the method comprising:

[0280] (i) contacting the separated sample with the binding protein of any one of items 22 to 30; and

[0281] (ii) determining the presence or absence of isoaspartate-modified transthyretin in the isolated sample.

[0282]

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

[22] to

[30] , for use in a method for in vivo diagnosis and / or prognosis of transthyretin amyloidosis.

[0283] Example

[0284] Example 1: Preparation and screening of monoclonal antibodies against IsoAsp38TTR

[0285] 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 (eg SEQ ID NO: 3) but not with the same molecule containing aspartic acid at position 38 (eg SEQ ID NO: 2).

[0286] A peptide comprising amino acids 35 to 43 of human TTR was synthesized by Peptide & Elephants (Berlin, Germany), with an isoaspartic acid residue at position 38 instead of aspartic acid, and an artificial C-terminal amino acid was added to facilitate coupling (i.e., KAAXDTWEPC, where X is L-isoAsp) (SEQ ID NO: 4). The peptide was covalently linked to keyhole limpet hemocyanin via a C-terminal cysteine ​​residue. To generate monoclonal antibodies, 8-week-old female BALB / c mice were used. Mice were immunized subcutaneously with a 1:1 emulsified KLH-conjugated TTR peptide and complete Freund's adjuvant (VWR), and then boosted with an additional 95 μg in incomplete Freund's adjuvant 44 and 73 days later, with a final boost once a day starting 4 days before fusion. Antibody titers were confirmed by enzyme-linked immunosorbent assay (ELISA). Homogenized splenocytes were fused with SP2 / 0 myeloma cells using the PEG method ( & Milstein, 1975. Nature. 256(5517): 495-7). The cells were immediately plated onto prepared feeder cell plates, placed in HAT medium, and screened after 10 days. Hybridomas were selected based on specific binding to isoD38-TTR(35-43)-PEG-biotin (SEQ ID NO: 5) but not to TTR(35-43)-PEG-biotin (SEQ ID NO: 6) using indirect ELISA and surface plasmon resonance (SPR). Hybridomas producing stable antibodies were selected and subsequently cloned by limiting dilution and additional single cell isolation to ensure the monoclonality of the hybridomas.

[0287] Hybridoma cells are expanded and cultured in T175 flasks. Collect supernatant and carry out sterile filtration (0.22 μm, TPP) before purification. For affinity purification mAb, corresponding supernatant is mixed with equal-volume binding buffer (40mM Na2HPO4, 150mM NaCl, pH 7.0), and pass through the Protein G Sepharose Fast Flow column (GEHealthcare Life Sciences) of pre-equilibration. Use 40mM Na2HPO4 and 2M KSCN (pH 7.0) to elute the bound antibody. The fraction collected is dialyzed overnight in PBS (138mM NaCl, 8mM Na2HPO4, 1.5mM KH2PO4, 3mM KCI, pH 7.0) at 4 ° C.

[0288] Example 2: Study of Antibody Specificity by Dot Blot Analysis

[0289] Dot blot analysis was performed to get an overall idea about the specificity and cross-reactivity of the antibodies obtained in Example 1.

[0290] method

[0291] 0.5 μg of recombinantly expressed TTR protein (TTRwt (SEQ ID NO: 1), TTR-V30M (SEQ ID NO: 7), or TTR-V122I (SEQ ID NO: 8)), ATTR fibrils, or 0.5 μg of freshly solubilized Aβ(1-42) (SEQ ID NO: 9) or aggregated Aβ(1-42) (SEQ ID NO: 10) with confirmed isoAsp content were spotted on nitrocellulose membranes. The membranes were blocked in blocking solution (5% (w / v) milk powder + 0.05% (v / v) Tween-20 (Carl Roth) in TBS) for 1 hour. Antibodies 2F2, 4D4, or polyclonal total TTR antibody (Biozol, product number: CSB-PA041819) were diluted to 1 μg / mL in blocking solution and incubated overnight at 4°C. Detection was then performed using alkaline phosphatase-conjugated anti-mouse or rabbit IgG antibodies (Abcam) and the chromogenic substrates BCIP and NBT (Carl Roth).

[0292] SEQ ID NO:7 is TTR-V30M and has the following sequence:

[0293] GPTGTGESKCPLMVKVLDAVRGSPAINVAMHVFRKAADDTWEPFASGKTSESGELHGLTTEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTTAVVTNPKE

[0294] SEQ ID NO:8 is TTR-V122I and has the following sequence:

[0295] GPTGTGESKCPLMVKVLDAVRGSPAINVAVHVFRKAADDTWEPFASGKTSESGELHGLTTEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTTAVITNPKE

[0296] SEQ ID NO: 9 is Aβ(1-42) and has the following sequence:

[0297] DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

[0298] SEQ ID NO: 10 is isoD7-Aβ(1-42) and has the following sequence:

[0299] DAEFRHXSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA,

[0300] wherein X is L-isoAsp.

[0301] result

[0302] like Figure 1 As shown, the 2F2 and 4D4 antibodies failed to recognize the recombinantly expressed monomeric proteins TTRwt, TTR-V30M, and TTR-V122I (lanes 1 to 3), indicating the absence of an isoaspartate residue at position 38. However, 2F2 and 4D4 recognized aggregated, one-year-old recombinant TTR-derived fibril samples (lanes 4 to 6). In addition, isoD38 TTR species were observed in fibrils extracted from human heart tissue of patients with pathological ATTR-V30M as detected by 2F2 and 4D4 antibodies (lane 7). In contrast, the total TTR pAb was unable to distinguish between recombinant monomeric TTR variants and aggregated, aged TTR fibrils generated in vitro or extracted from human tissue (lane 3). Neither TTR mAb showed reactivity to other isoaspartate-containing amyloid fibrils (isoD7-Aβ) or their monomeric peptides (lanes 8 and 9).

[0303] Example 3: Determination of the binding of immobilized 2F2 or 4D4 to TTR(35-43) by surface plasmon resonance (SPR) analysis or K of isoD38-TTR(35-43) D value

[0304] method

[0305] The binding kinetics of antibodies to TTR(35-43)-PEG-biotin (SEQ ID NO: 5) and isoD38-TTR(35-43)-PEG-biotin (SEQ ID NO: 6) were analyzed by using SPR on a BIAcore 3000 instrument at 25°C. To this end, the surface of a CM5 sensor chip (Cytiva) was modified with goat anti-mouse antibodies using NHS / EDC chemistry. To immobilize anti-mouse IgG, the carboxymethylated dextran surface of the sensor chip was activated by mixing 0.1 M N-hydroxysuccinimide (NHS) and 0.4 M N-ethyl-N'-(dimethylaminopropyl)carbodiimide hydrochloride (EDC) 1:1. 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 in 10 mM sodium acetate (pH 5.5) and injected at a flow rate of 30 μL / min with a contact time of 2x 4 minutes. Inactivation was performed by applying 1M ethanolamine (pH 8.5) at a flow rate of 10 μL / min with a contact time of 2x 7 minutes. Approximately 20,000 RU of goat anti-mouse antibody was immobilized on the chip surface. Antibodies 2F2 and 4D4 were captured using an antibody diluent of 25 μg / mL in HBS-EP buffer at a flow rate of 2 μL / min to achieve a level of about 2000 RU of both antibodies on the chip surface. For the 4D4 antibody, multi-cycle kinetic 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 were performed with 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).

[0306] The binding kinetics of antibodies to isoD38-TTR (28-50) (SEQ ID NO: 39) were analyzed by using SPR on a Bruker SPR-24Pro device at 25°C. For this purpose, approximately 4000 RU of antibodies 2F2 and 4D4 were immobilized to an IgG-Capture Sensor (Bruker) using an antibody dilution of 10 μg / mL in HBS-EP buffer at a flow rate of 5 μL / min. For both antibodies, single injection cycle kinetic analysis was performed by applying 0.5 nM to 50 nM of isoD38-TTR (28-50) (SEQ ID NO: 39).

[0307] SEQ ID NO:39 is isoD38-TTR (28-50) having the following sequence:

[0308] VAVHVFRKAAXDTWEPFASGKTS,

[0309] Where X is L-isoAsp, the N-terminal residue is acetylated, and the C-terminal residue is amidated.

[0310] result

[0311] The dissociation constants for binding of 2F2 and 4D4 to 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 the isoaspartic acid-containing TTR(35-43) peptide (SEQ ID NO:5) and TTR(28-50) peptide (SEQ ID NO:39). In contrast, the dissociation constant of the unmodified peptide TTR (35-43) (SEQ ID NO: 6) was 233-fold (4D4) and 464-fold (2F2) higher than that of isoD38-TTR (35-43)-PEG-biotin (SEQ ID NO: 5), and 1078-fold (4D4) and 317-fold (2F2) higher than that of isoD38-TTR (28-50) peptide (SEQ ID NO: 39). Higher dissociation constant values ​​indicate lower binding affinity.

[0312] Table 1: K of isoD38-TTR specific antibodies and TTR peptides D value

[0313]

[0314] Example 4: Engulfment and degradation by phagocytosis using isoD38-TTR mAbs 2F2 and 4D4 TTR fibrils

[0315] To analyze whether antibodies 2F2 and 4D4 can induce Fc-mediated phagocytosis of TTR fibrils, we established a cell phagocytosis assay using macrophage-like THP-1 cells in vitro. THP-1 cells are commonly used to evaluate the ability of monoclonal antibodies to induce Fc-mediated phagocytosis (see, for example, Gogesch et al., 2021. Int J Mol Sci. 22(16):8947, Section 2.4.2).

[0316] method

[0317] Samples containing heart-extracted human ATTR-V30M fibrils or 1 mg / mL of native recombinant TTRwt (SEQ ID NO: 1) were amine-coupled with pHrodo Red dye (Thermo Fisher, see e.g., Miksa et al., 2009. J Immunol Methods. 342(1-2):71-7) at room temperature for 1 hour, with a protein to dye ratio of 10: 1 applied. In the case of native TTRwt, excess pHrodo Red dye was removed using a 5 kDa molecular weight cutoff filter (Merck). For amyloid fibrils, excess free dye label was removed by three centrifugation steps at 17000 x g for 10 minutes each, followed by resuspension in PBS.

[0318] For stimulation, 2x 10 5 Human monocytic 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 medium containing PMA was replaced with fresh RPMI + 10% low IgG FBS and cultured for another 2 days.

[0319] 20 μg / mL of pHrodo-labeled TTR (monomer TTRwt or extracted human TTR-V30M fibrils) was pre-incubated with 40 μg / mL of antibody in cell culture medium for 1 hour at 37°C. 2x 10 5 After PMA treatment of THP-1 cells, tissue culture was incubated for 3 hours. Antigen-antibody complexes were removed by washing the cells three times with PBS.

[0320] The extent of phagocytosis was monitored by flow cytometry using a FACSCalibur (Becton Dickinson). Forward and side scattered light were used to identify cell populations and measure cell size and granularity. After excitation at 560 nm, red fluorescence emitted by cells at 585 nm was recorded. The remaining cells were transferred to a glass chamber slide and imaged by bright field and fluorescence microscopy using a Keyence BZ-X800.

[0321] result

[0322] Extracted human ATTR-V30M fibrils 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 extremely weak, while in acidic environments (such as in endocytic vesicles), the fluorescence increases significantly. This allows the uptake of pHrodo-labeled proteins by cells to be monitored. THP-1 cells were added after treatment of pHrodo-labeled TTR with two antibodies 2F2 and 4D4 or isotype controls. Cellular uptake was observed by bright field and fluorescence microscopy ( Figure 2 A), measured by flow cytometry and displayed as mean fluorescence intensity (MFI, Figure 2 B) Low fluorescence was observed when TTRwt monomers were treated with either the isoD38-TTR-specific antibody or the isotype control. In contrast, a significant increase in fluorescence was observed after treatment of ATTR-V30M fibrils with the 2F2 or 4D4 antibodies, indicating that these antibodies mediate phagocytosis of TTR fibrils.

[0323] These data indicate that targeting isoD38 allows clearance of TTR fibrils by antibody-dependent cellular phagocytosis. The ability of antibodies to reduce TTR amyloid load is a therapeutic goal that is expected to translate into improved patient survival. (See Michalon et al., 2021. Nat Commun. 12(1):3142). Therefore, the present data confirm that the binding proteins disclosed herein are suitable for treating transthyretin amyloidosis.

Claims

1. A binding protein that specifically binds to isoaspartate-modified transthyretin, wherein: With reference to SEQ ID NO: 1 numbering, the aspartic acid at position 38 has been 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 comprising an isoaspartate residue.

3. The binding protein according to claim 1 or 2, wherein The K of the interaction between the binding protein and SEQ ID NO: 5 determined by surface plasmon resonance at 25°C D The K of the interaction between the binding protein and SEQ ID NO: 6 determined by surface plasmon resonance at 25°C D at least 10 times smaller, wherein, optionally, the K of the interaction between the binding protein and SEQ ID NO: 5 as determined by surface plasmon resonance at 25°C D The K of the interaction between the binding protein and SEQ ID NO: 6 determined by surface plasmon resonance at 25°C D At least 200 times smaller.

4. The binding protein according to any one of claims 1 to 3, wherein The K of the binding protein for interaction with SEQ ID NO: 5 determined by surface plasmon resonance at 25°C D Less than 200nM.

5. The binding protein according to any one of claims 1 to 4, wherein The binding protein comprises: (a) a heavy chain variable region (VH), wherein the VH comprises a HCDR3 polypeptide selected from the group consisting of: (i) NSYYGMDY (SEQ ID NO: 16), or a sequence at least 70% (preferably at least 80%) identical thereto; and (ii) EDY (SEQ ID NO: 26) or a sequence at least 65% identical thereto; and / or (b) a light chain variable region (VL), wherein the VL comprises a LCDR3 polypeptide selected from the group consisting of: (i) HQYLSSRT (SEQ ID NO: 13) or a sequence having at least 70% (preferably at least 80%) identity thereto; and (ii) QHFWNIPFT (SEQ ID NO: 23), or a sequence at least 70% (preferably at least 80%) identical thereto.

6. The binding protein according to any one of claims 1 to 5, wherein The binding protein comprises VL and VH, wherein the VL comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH comprises HCDR1, HCDR2 and HCDR3 polypeptides, and the polypeptides are selected from the group consisting of: (a) LCDR1 is KSSQSVLYSSNQKNYLA (SEQ ID NO: 11) or a sequence having at least 70% (preferably at least 90%) identity thereof, LCDR2 is WASTRES (SEQ ID NO: 12) or a sequence having at least 70% (preferably at least 80%) identity thereof, LCDR3 is HQYLSSRT (SEQ ID NO: 13) or a sequence having at least 70% (preferably at least 80%) identity thereof, HCDR1 is GFTFSSFAMH (SEQ ID NO: 14) or a sequence having at least 70% (preferably at least 90%) identity thereof, HCDR2 is FISSGSNTIYYADTVKG (SEQ ID NO: 15) or a sequence having at least 70% (preferably at least 90%) identity thereof, and HCDR3 is NSYYGMDY (SEQ ID NO: 16) or a sequence having at least 70% (preferably at least 80%) identity thereof; and (b) LCDR1 is RTSGNIRNSLA (SEQ ID NO:21) or a sequence having at least 70% (preferably at least 90%) identity thereof, LCDR2 is NGKTLAD (SEQ ID NO:22) or a sequence having at least 70% (preferably at least 80%) identity thereof, LCDR3 is QHFWNIPFT (SEQ ID NO:23) or a sequence having at least 70% (preferably at least 80%) identity thereof, HCDR1 is GNTFSSRWIE (SEQ ID NO:24) or a sequence having at least 70% (preferably at least 90%) identity thereof, HCDR2 is EIFPGNGNTNYNEKFKG (SEQ ID NO:25) or a sequence having at least 70% (preferably at least 90%) identity thereof, and HCDR3 is EDY (SEQ ID NO:26) or a sequence having at least 65% identity thereof.

7. The binding protein according to any one of claims 1 to 6, wherein The binding protein comprises VL and VH, wherein the VL comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH comprises HCDR1, HCDR2 and HCDR3 polypeptides, and the polypeptides are selected from the group consisting of: (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).

8. The binding protein according to any one of claims 1 to 7, wherein The binding protein comprises a VL and a VH, wherein the VL and the VH are polypeptides selected from the group consisting of: (a) a VL of SEQ ID NO: 17 or a sequence having 50% identity thereto, and a VH of SEQ ID NO: 18 or a sequence having at least 50% identity thereto; (b) a VL of SEQ ID NO: 27 or a sequence having 50% identity thereto, and a VH of SEQ ID NO: 28 or a sequence having at least 50% identity thereto; (c) a VL of SEQ ID NO: 17 or a sequence having 50% identity thereto, and a VH of SEQ ID NO: 28 or a sequence having at least 50% identity thereto; and (d) a VL of SEQ ID NO: 27 or a sequence having 50% identity thereto, and a 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 8, wherein The binding protein is an antibody. 10 . A nucleic acid comprising a sequence encoding the binding protein according to claim 1 .

11. A host cell containing the nucleic acid of claim 10.

12. The binding protein according to any one of claims 1 to 9, the nucleic acid according to claim 10 or the host cell according to claim 11 for use as a medicament.

13. The binding protein according to any one of claims 1 to 9, the nucleic acid according to claim 10 or the 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 method comprising: (i) contacting the separated sample with the binding protein according to any one of claims 1 to 9; as well as (ii) determining the presence or absence of isoaspartate-modified transthyretin in the isolated sample; With reference to the numbering of SEQ ID NO: 1, the 38th aspartic acid of the isoaspartic acid-modified transthyretin has been post-translationally modified to L-isoaspartic acid.

15. The binding protein according to any one of claims 1 to 9, for use in a method for in vivo diagnosis and / or prognosis of transthyretin amyloidosis.

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