Novel immunotherapies for musculoskeletal disorders and conditions

Abstract

Immunotherapy for musculoskeletal disorders and conditions associated with transthyretin amyloidosis is provided.

Description

[Technical Field]

[0001] This invention generally relates to novel immunotherapies for musculoskeletal disorders or conditions in subjects. [Background technology]

[0002] Musculoskeletal conditions are the leading cause of physical disability worldwide, affecting approximately 1.71 billion people globally. Musculoskeletal dysfunction encompasses more than 150 distinct diseases / conditions characterized by impairment of the muscles, bones, joints, and adjacent connective tissues, affecting the system and resulting in temporary or lifelong limitations in function and participation. Musculoskeletal conditions typically feature pain (often persistent), as well as limitations in mobility and dexterity, reducing people's ability to work and participate in society. See the World Health Organization publication, "Musculoskeletal health," dated July 14, 2022. Forms of arthropathy constitute the majority of skeletal muscle diseases, with the remainder consisting of injuries, systemic autoimmune diseases, unspecified chronic widespread pain disorders, and lower neck and back pain.

[0003] Generally, there is no cure for most musculoskeletal disorders, but treatments such as lifestyle changes, physiotherapy, and the administration of nonsteroidal anti-inflammatory drugs (NSAIDs), antistimulants, steroids, and disease-modifying antirheumatic drugs (DMARDs) usually focus on symptom relief, disease improvement, and / or delaying disease progression. Therefore, novel therapeutic strategies for treating musculoskeletal conditions are needed.

[0004] This technical problem is characterized in the claims and is further described below and solved by embodiments shown in the examples and drawings. [Overview of the project]

[0005] This invention is based on the remarkable finding that administration of anti-TTR antibodies to patients with amyloid transthyretin cardiomyopathy (ATTR-CM) resulted in clearance of deposits present in the joints of treated patients. In particular, during clinical trials such as those described in Example 1, a reduction in tracer uptake into the joints of antibody-treated patients was observed, for example, in the shoulder, elbow, and wrist joints, compared to placebo-treated patients. As shown in the attached examples, there is a strong tracer signal in the shoulder joint, which is reduced during treatment with the anti-TTR antibody NI006 (also known as ALXN2220), i.e., 4 and 12 months after treatment (see Patient 3 and Case 6 in Figure 1), and after switching from placebo treatment to NI006 / ALXN2220 treatment (Case 1 in Figure 1). Similarly, a strong tracer signal was observed at the elbow joint, which decreased after 12 months of treatment compared to 4 months of treatment (see Patient 1 in Figure 1), suggesting a reduction in TTR deposits. Deposits in the joints are a common disease pattern in musculoskeletal conditions and diseases, with several musculoskeletal conditions and diseases being associated with deposits, respectively. Patients with advanced chronic arthritis suffer from severe joint deterioration, including bone and cartilage destruction, resulting in long-term pain, deformity, loss of joint function, reduced mobility, and a shortened life expectancy.

[0006] Furthermore, Figure 1 shows that subjects in which a reduction in tracer signal was observed were treated with anti-TTR antibodies at doses of 30 mg / kg and 60 mg / kg once every 28 days. Thus, the present invention generally relates to anti-TTR antibodies for use in the treatment and prevention of musculoskeletal conditions or disorders, wherein the disorder or condition is preferably a disorder or condition affecting the joints, more preferably a disorder or condition related to pathological deposits in the joints. According to the present invention, the anti-TTR antibody is preferably administered once every 28 days at a dose of at least 30 mg / kg, and preferably once every 28 days at a dose of about 30 mg / kg or 60 mg / kg.

[0007] According to the present invention, the anti-TTR antibody specifically recognizes misfolded and mutated wild-type TTR and targets disease-associated amyloid conformations with high affinity, but does not target the physiological form of TTR, i.e., the wild-type TTR tetramer. Preferably, the antibody also does not bind to the monomer and dimer of wild-type TTR.

[0008] Anti-TTR antibodies, for example, are described in International Publication No. 2015 / 092077(A1) and have been shown to be useful in the treatment of transthyretin amyloidosis (ATTR), particularly amyloid-transthyretin cardiomyopathy (ATTR-CM). See European Patent Applications Publications No. 22 207 651.5 and No. 23 020 175.8. ATTR includes two subtypes that differ in their pathogenesis: wild-type ATTR (wtATTR) and variant ATTR (vATTR). Their common precursor protein, transthyretin (TTR), physiologically functions as a transport protein for thyroxine and retinol-binding proteins. TTR is primarily synthesized in the liver and arises in its native form as a tetramer (Alshehri et al., J. Neuroendocrinol. 27 (2015), 303-3239). vATTR, formerly known as hereditary / variant ATTR, is an autosomal dominant disorder. For both wild-type TTR (wtTTR) and variant / variant TTR (vTTR) proteins, the pathogenic mechanism of ATTR is induced by partial unfolding of the TTR protein and subsequent aggregation into beta-pleated sheets that form amyloid fibrils (Eisele et al., Nat. Rev. Drug Discov. 14 (2015), 759-780).

[0009] Clinical trials, such as those described in Example 1, were set up to further confirm the therapeutic applicability of the antibody NI006 / ALXN2220 for the treatment of ATTR-CM and to identify the optimal drug regimen. Indeed, a reduction in cardiac amyloid was observed during the study, and the antibody was found to be effective in treating ATTR-CM. However, surprisingly, strong tracer signals were also found in the joints, for example, in the shoulder and elbow joints of some patients enrolled in the study, i.e., patients diagnosed with ATTR-CM, and it was also shown that the signals were reduced during treatment with the anti-TTR antibody. Thus, treatment with the anti-TTR antibody not only improved the patients' cardiac function but also likely resulted in a reduction of joint deposits. Pathological deposits can be seen in many musculoskeletal disorders and conditions such as carpal tunnel syndrome, arthropathy, e.g., osteoarthritis, lumbar spinal stenosis, amyloid arthropathy, biceps tendon rupture, trigger finger, rotator cuff disorders, and joint pain. Removal of these deposits typically leads to improvement in the symptoms and pathological remission of musculoskeletal disorders, and potentially improves symptoms by resolving / reducing local inflammation after removal of deposits and reduction of mechanical stress on adjacent nerves and filaments. This can be further evaluated by physical assessments such as self-assessment and general tests for diagnosing the disease (e.g., Reiman and Manske J Man Manip Ther. 19(2011), 91-99), or via a 6-minute walk distance assay as described in Example 1.

[0010] Therefore, it is reasonable to assume that NI006 / ALXN2220 can improve joint function and, in the long term, improve arthritis in the treatment of musculoskeletal conditions and diseases associated with such deposits and ATTR, such as ATTR-CM, by inducing the clearance of deposits.

[0011] Furthermore, joint pain has been observed as an adverse event in some patients during treatment with the anti-TTR antibody NI006 / ALXN2220. As previously demonstrated using the applicant's proprietary PDAX mouse model disclosed in International Publication No. 2020 / 094883(A1), the anti-TTR antibody mediates ATTR fibrillary clearance through antibody effector function, particularly antibody-dependent cellular phagocytosis (ADCP). See, for example, Example 3 and Figure 3 in International Publication No. 2023 / 099788(A1). Recent literature has identified the presence of amyloid protein in patients' joints, which appears with joint pain and degenerative arthritis. See Birnbrich et al., JSES Reviews, Reports, and Techniques 2(2022), 201-204. Therefore, although not intended to be theoretically bound, the observed arthralgia may be related to the activation of NI006 / ALXN2220 by phagocytic immune cells targeting musculoskeletal ATTR deposits, which results in the observed clearance of deposits in the patients' joints. This is further confirmed by studies showing that signs of musculoskeletal immune activation are most frequently observed in subjects immediately after high-dose treatment with 10–60 mg / kg of NI006 / ALXN2220, and that signs of musculoskeletal immune activation are delayed in subjects treated with 0.3–3 mg / kg of NI006 / ALXN2220 (see Example 1, Table 12). Thus, the observed musculoskeletal events are dose-dependent, with higher doses resulting in a stronger response, i.e., more efficient removal of TTR deposits, possibly due to the aforementioned activation of phagocytic immune cells. Therefore, preliminary data on immune activation appear to be most pronounced at doses of 30 mg / kg and 60 mg / kg.

[0012] Therefore, it is reasonable to expect that anti-TTR antibodies are suitable for the treatment of musculoskeletal conditions and diseases, particularly those associated with amyloidogenic TTR deposits, especially in the joints. Accordingly, in one embodiment, an anti-TTR antibody for use according to the method of the present invention has an active Fc domain and is capable of inducing ADCP, which can be tested by using the PDAX mouse model disclosed in International Publication No. 2020 / 094883(A1) and the efficacy assay disclosed in International Publication No. 2023 / 099788(A1).

[0013] In fact, there are many musculoskeletal disorders associated with amyloid deposits, particularly TTR deposits. For example, amyloid deposits have been observed in glenoid arthritis (Birnbrich et al., JSES Reviews, Reports, and Techniques 2(2022), 201-204), and various amyloid deposits, including those derived from TTR, have been shown to be present in osteoarthritis of the knee (OA). TTR amyloid deposits have been shown to contribute to cellular and extracellular matrix damage in articular cartilage in human OA, and TTR deposits have been shown to accelerate the progression of OA (see Yanagisawa et al., The Journal of Protein Folding Disorders 23(2016), 26-32; Akasaki et al., Arthritis Rheumatol. 67(2015), 2097-2107; Matsuzaki et al., Aging Cell 16(2017), 1313-1322). Furthermore, amyloidosis in TTR has been identified as one of the causes of osteoarthritis, particularly carpal tunnel syndrome (M'Bappe and Grateau, Best Practice & Research Clinical Rheumatology 26(2012), 459-475), and severe hand pain has been identified as an extracardiac manifestation of TTR amyloidosis (Patel et al., BMJ Case Rep 12(2019), e229677). Generally, orthopedic manifestations of ATTR are frequent and characteristic, including idiopathic bilateral carpal tunnel syndrome, idiopathic lumbar stenosis, nontraumatic rupture of the biceps brachii tendon, and, less frequently, finger disorders and rotator cuff disorders (Perfetto et al., Biomedicines 10(2022), 3226). Furthermore, hip arthroplasty (THA) and knee arthroplasty (TKA) have been observed in patients with ATTR-CM (Rubin et al., Amyloid 24(2017), 226-230).In Wieczorek and Ozyhar, Cells 10(2021), 1768, the role of TTR in biomineralization, calcium deposits, and bone and joint and cardiovascular diseases, as well as TTR-related diseases, and the association between vascular and ligament tissue calcium deposits and TTR levels and TTR structure are discussed.

[0014] The musculoskeletal conditions and diseases mentioned are associated with ATTR-CM and are frequently observed to precede ATTR-CM, in particular. See, for example, Birnbrich et al., Perfetto et al., and Rubin et al. supra. Additionally, the observation of increased tracer uptake in ATTR-CM patients is consistent with a recent single-site retrospective case-control study analyzing 830 patients with cardiac ATTR (ATTR-CA) that showed the prevalence of shoulder lesions and orthopedic symptoms prior to the onset or diagnosis of ATTR-CA. See Basdavanos et al., Am. J. Cardiol. 190(2023), 67-74. Therefore, early detection and treatment of these musculoskeletal conditions and diseases using anti-TTR antibodies may also enable prevention of ATTR-CM. Thus, thanks to the findings made during this clinical trial, it is possible to initiate early treatment of ATTR that can even prevent the establishment of ATTR-CM.

[0015] This disclosure is based in part on the effect of anti-TTR antibodies, such as NI006 / ALXN2220, on joint deposits in patients diagnosed with ATTR-CM. In particular, in human clinical trials conducted in accordance with the present invention, it was first observed that anti-TTR antibodies, such as NI006 / ALXN2220, are capable of reducing tracer signals observed in the joints of human subjects when administered to human patients, suggesting the removal of joint deposits in human subjects. Accordingly, the present disclosure provides a method of treating a subject, such as a human patient, who requires treatment of a musculoskeletal condition and disease associated with amyloid deposits, with an anti-TTR antibody, such as NI006 / ALXN2220. In certain embodiments, the musculoskeletal condition and disease are associated with amyloidogenic TTR deposits. Representative examples include, but are not limited to, for example, arthritis, particularly osteoarthritis, rheumatoid arthritis, and juvenile idiopathic arthritis, carpal tunnel syndrome, joint pain, amyloid arthropathy, lumbar spinal stenosis, and ligament and tendon disorders, particularly biceps tendon rupture, trigger finger, and rotator cuff disease.

[0016] Treatment with an anti-TTR antibody can be assisted by additional agents commonly used to treat musculoskeletal diseases, such as disease-modifying anti-rheumatic drugs and / or anti-inflammatory agents. Accordingly, the present invention further relates to a pharmaceutical composition, component kit, or product comprising an anti-TTR antibody capable of binding to mutant, misfolded, misassembled, and / or aggregated transthyretin (TTR) species and capable of removing ATTR by inducing autophagy, a disease-modifying anti-rheumatic drug, and / or an anti-inflammatory agent.

[0017] Further embodiments of the present invention will become apparent from the following description and examples.

Brief Description of the Drawings

[0018] [Figure 1]Representative images (A-C) of changes in scintigraphy tracer uptake after treatment with NI006 / ALXN2220. Serial bisphosphonate scintigraphy is shown at baseline, 4 months (after completion of placebo-controlled SAD / MAD), and 12 months (after completion of OLE) in two patients randomized to NI006 / ALXN2220 (A and C) and one patient randomized to placebo (B). Cardiac tracer uptake is quantified by the cardiac / whole-body ratio (H / WB ratio (%)). Individual cumulative doses (g) of NI006 / ALXN2220 administered and NI006 / ALXN2220 exposure (* mg / mL AUC) are provided for imaging time points after baseline. [Figure 2] Changes from baseline in Kansas City Cardiomyopathy Questionnaire-Comprehensive Summary and 6-Minute Walk Distance at 12 months in patients randomized to NI006 / ALXN2220. Absolute changes from baseline in Kansas City Cardiomyopathy Questionnaire-Comprehensive Symptom Score and 6-Minute Walk Distance after 12 months of treatment with NI006 / ALXN2220 versus individual cumulative NI006 / ALXN2220 exposure. Linear regression lines with unadjusted 95% confidence intervals are presented for patients randomized to NI006 / ALXN2220. Confidence intervals should not be used to reject or not reject a treatment effect. ACFB shows the absolute change from baseline, KCCQ-OS is the Kansas City Cardiomyopathy Questionnaire-Comprehensive Symptom Score, and 6-MWD is the 6-Minute Walk Distance. The score from the KCCQ questionnaire ranges from 0 to 100, with 0-24 indicating very poor to poor quality of life, 25-49 indicating poor to average quality of life, 50-74 indicating average to good quality of life, and 75-100 indicating good to excellent quality of life. [Modes for carrying out the invention]

[0019] The present invention generally relates to immunotherapies for musculoskeletal conditions and disorders caused and / or associated with transthyretin (TTR) amyloidosis (ATTR). More specifically, the present invention relates to embodiments characterized in the claims, disclosed in the description, and further illustrated in the following examples and drawings.

[0020] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Similar or equivalent methods and materials to those described herein may be used in the implementation or testing of this disclosure, but exemplary methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. Materials, methods, and examples are illustrative and not intended to limit the scope. Unless otherwise specified, terms used herein are given the definitions set forth in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, published 1997, revised 2000, enlarged 2003, ISBN 0-19-850673-2; 2nd edition, published 2006, ISBN 0-19-852917-1 978-0-19852917-0.

[0021] To avoid any doubt, expressions such as “in some embodiments,” “in a particular embodiment,” “in a particular example,” “in some examples,” “in further embodiments,” and “in one embodiment” should be read with the understanding that any of the embodiments described herein combines each of the features of those embodiments, and it is emphasized that this disclosure is used and meant in such a way that any combination of the features of those embodiments should be treated in the same manner as if it were written in one embodiment. The same applies to any combination of embodiments and features shown in the appended claims and examples, which are also intended to be combined with features from the corresponding embodiments disclosed in the description, and for the sake of consistency and brevity only, embodiments are characterized by dependencies, but in practice, each combination of embodiments and features that may be interpreted as having (multiple) dependencies should be considered literally disclosed and not as a choice from different options.

[0022] In relation to the present invention, the term "and / or" is understood to mean that all members of the group connected by the term "and / or" are disclosed either or otherwise, and in each case, cumulatively in any combination. The expression "A, B, and / or C" means that the following disclosures should be understood thereunder: a) A or B or C; or b) (A and B); or c) (A and C); or d) (B and C); or e) (A, B and C).

[0023] When the term "ATTR" is used, unless otherwise specified, it usually refers to vATTR and wtATTR. Similarly, unless otherwise specified, "TTR" also refers to wtTTR and vTTR.

[0024] As used herein, the term “antibody” includes the entire antibody and any antigen-binding fragment (i.e., “antigen-binding moiety”) or its single-chain version. In one embodiment, the term “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or its antigen-binding moiety. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region (CH) consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and complement proteins.

[0025] The precise boundaries of the CDRs are defined differently according to different methods. In some embodiments, the location of the CDRs or framework regions within the light chain or heavy chain variable domains may be as defined by Kabat et al., NIH Publication 91(1991), 3242. In such cases, the CDRs may be referred to as "Kabat CDRs" (e.g., "Kabat LCDR2" or "Kabat HCDR1"). In other embodiments, the location of the CDRs in the light chain or heavy chain variable regions may be as defined by Chothia et al., Nature 342(1989), 877-883. Thus, these regions may be referred to as "Chothia CDRs" (e.g., "Chothia LCDR2" or "Chothia HCDR3"). In other embodiments, the location of the CDRs in the light chain and heavy chain variable regions may be as defined by the Kabat-Chothia combined definition. In such embodiments, these regions may be referred to as "combined Kabat-Chothia CDRs". Thomas et al., Mol Immunol 33 (1996), 1389-1401 illustrate the identification of CDR boundaries according to the definitions of Kabat and Chothia. In other embodiments, the location of CDRs or framework regions within light chain or heavy chain variable domains may be as defined by the International Immunogenetics Database (IMGT) standard. Marie-Paule Lefranc et al., Developmental & Comparative Immunology 27 (2003), 55-77 illustrate the identification of CDR boundaries according to the IMGT standard. Thus, these regions may be referred to as "IMGT CDRs" (e.g., "IMGT-LCDR2" or "IMGT-HCDR3").

[0026] As used herein, the terms “antigen” or “antigen target” refer to a molecule or part of a molecule that can be bound by an antibody, one or more Ig-binding domains, or other immunological binding sites, such as engineered polypeptides or fusion proteins disclosed herein. An antigen can be used in an animal to produce an antibody capable of binding to the epitope of that antigen. An antigen may have one or more epitopes.

[0027] As used herein, the term “antigen-binding fragment” (or simply “antibody fragment”) refers to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen. Such “fragments” are, for example, about 8 to about 1500 amino acids long, preferably about 8 to about 745 amino acids long, preferably about 8 to about 300, for example about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids long. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included in the term "antigen-binding fragment" of an antibody include (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of a VH domain (Ward et al., Nature 341 (1989), 544-546); and (vi) isolated complementarity-determining regions (CDRs), or (vii) combinations of two or more isolated CDRs that can be optionally joined by a synthetic linker, such as antibody fragments containing variable heavy chain (VH) CDRs and / or variable light chain (VL) CDRs, such as VHCDR1-3 and / or VLCDR1-3. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using a synthetic linker and recombination. This synthetic linker allows them to form a single protein chain (known as single-chain Fv (scFv)) in which the VL and VH regions pair to form a monovalent molecule. See, for example, Bird et al., Science 242 (1988), 423-426, and Huston et al. Proc. Natl. Acad. Sci. USA 85 (1988), 5879-5883. Such single-chain antibodies are also intended to be included within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using prior art known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.The antigen-binding portion can be produced by recombinant DNA technology, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0028] As used herein, the term “binding domain” refers to a portion of a protein or antibody containing amino acid residues that interact with an antigen. Binding domains include, but are not limited to, antibodies (e.g., full-length antibodies) and their antigen-binding moieties, such as those provided above, including, for example, VHCDR1-3 and VLCDR1-3. Binding domains confer their specificity and affinity to the antigen onto the binder. The term also encompasses any protein having a binding domain that is homologous or largely homologous to an immunoglobulin-binding domain.

[0029] The term "anti-TTR antibody" typically refers to an antibody that binds to misfolded / aggregated TTR but not to physiological TTR tetramers.

[0030] "Musculoskeletal conditions and diseases" are characterized by dysfunction of muscles, bones, joints, and adjacent connective tissues.

[0031] The “therapeutic effective dose” or “clinically active concentration” of a substance means that a given substance is administered to a subject suffering from a condition in an amount sufficient to ensure, alleviate, or partially cessate one or more of the condition or its symptoms. Such therapeutic treatment may result in a reduction in the severity of disease symptoms or an increase in the frequency or duration of symptom-free periods. The effective dose of a given agent for a given purpose will depend on the severity of the disease or injury, as well as the subject's weight and overall condition. As used herein, the term “subject” includes any mammal, preferably human. The therapeutic efficacy and toxicity of such compounds are determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED 50 (Therapeutic dose effective in 50% of the population) and LD 50 It can be determined by (the lethal dose for 50% of the population). The dose ratio between therapeutic effect and toxic effect is the therapeutic index, which is the LD50 / ED 50 It can be expressed as a ratio.

[0032] Furthermore, unless otherwise specified, the terms and expressions used herein to characterize the present invention, and in particular anti-TTR antibodies as used in accordance with the present invention, are given by definition as provided in International Publication No. 2015 / 092077(A1), in particular in subsection I. Definitions on pages 16-42, the disclosure of which is expressly incorporated herein by reference. The same applies to general embodiments disclosed in International Publication No. 2015 / 092077(A1) for antibodies, pharmaceutical compositions, etc.

[0033] The present invention relates to an anti-TTR antibody capable of binding to mutated, misfolded, misassembled, and / or aggregated transthyretin (TTR) species, i.e., amyloid-forming TTR, for use in methods of treating or preventing musculoskeletal disorders or conditions in a subject. In one embodiment, the anti-TTR antibody is capable of binding to mutated and misfolded TTR species. In one embodiment, the anti-TTR antibody is capable of binding to mutated and misassembled TTR species. In one embodiment, the anti-TTR antibody is capable of binding to mutated and aggregated TTR species. In one embodiment, the anti-TTR antibody is capable of binding to misfolded and misassembled TTR species. In one embodiment, the anti-TTR antibody is capable of binding to misfolded and aggregated TTR species. In one embodiment, the anti-TTR antibody is capable of binding to misassembled and aggregated TTR species. In one embodiment, the anti-TTR antibody is capable of binding to mutated, misfolded, and misassembled TTR species. In one embodiment, the anti-TTR antibody can bind to mutated, misfolded, and aggregated TTR species. In one embodiment, the anti-TTR antibody can bind to misfolded, misassembled, and aggregated TTR species. In one embodiment, the anti-TTR antibody can bind to misfolded, aggregated, and mutated TTR species. In one embodiment, the anti-TTR antibody can bind to mutated, misfolded, misassembled, and aggregated TTR species. In one embodiment, the anti-TTR antibody can bind to wild-type and mutant TTR aggregates. In a preferred embodiment, the anti-TTR antibody does not bind to physiological TTR tetramers, more preferably not to wild-type TTR monomers, and preferably not to wild-type TTR dimers.

[0034] Based on the observation that the anti-TTR antibody NI006 / ALXN2220 induces clearance of tracer signals observed in the joints of patients with ATTR-CM, and because it is known that this antibody can remove amyloid-forming TTR, it is reasonable to expect that the anti-TTR antibody can remove amyloid-forming TTR in the joints. Therefore, in one embodiment, the disease or condition treated with the anti-TTR antibody is preferably a musculoskeletal disorder or condition associated with amyloid-forming TTR in the joints.

[0035] Since the anti-TTR antibody is used in the treatment of musculoskeletal conditions and diseases in human subjects according to the present invention, in one embodiment, the anti-TTR antibody is humanized, preferably derived from humans, and is non-immunogenic in humans.

[0036] As illustrated in the examples, the present invention is illustrated using the anti-TTR antibody NI006 / ALXN2220, the parent antibody NI-301.37F1, which was first described in International Publication No. 2015 / 092077(A1). This antibody has the amino acid sequence TTR 41~45 (Sequence ID 51 of International Publication No. 2015 / 092077(A1), and Sequence ID 15 of the present invention) comprising or consisting thereof, wherein the variable region or binding domain contains a complementarity-determining region (CDR), and a variable heavy chain (V) having the amino acid sequence shown in Figure 1C and Figure 1M of International Publication No. 2015 / 092077(A1) (Sequence IDs 2 or 6 and 4 of the present invention, respectively). H ) and variable light chain (V LIt is capable of binding to human TTR epitopes, including ). The antibody NI006 / ALXN2220 is a fully human IgG1 m3 allotyped antibody and contains the human constant heavy chain (HC) amino acid sequence present in SEQ ID NO: 18 and the corresponding human constant light chain (LC), in this case the kappa light chain, as exemplified in SEQ ID NO: 19. As further described below, the IgG antibody is composed of a tetramer consisting of an HC and two light LC chains linked by disulfide crosslinks. The theoretical molecular weight of antibody NI006 / ALXN2220 is 144.2 kDa, and the weights determined by mass spectrometry (MS) are 144.2 kDa (deglycosylated) and 147.0 kDa to 147.6 kDa (intact IgG1), respectively. This antibody and its homologous antibodies are derived from the memory B cell repertoire of healthy aged human donors. Please refer to the description of the examples in International Publication No. 2015 / 092077(A1). Characterization of the antibody's binding properties demonstrated that this antibody exhibits a high binding affinity to misfolded TTR in the sub-nanomole range, is highly selective for the amyloid conformation of TTR, and exhibits similar binding to wtTTR and vTTR. Because this human-derived antibody is selective for misfolded TTR and can induce the removal of joint deposits, as illustrated in the examples, it represents a highly preferred therapeutic candidate for use in the treatment of musculoskeletal disorders.

[0037] The antibody NI006 / ALXN2220 is produced in Chinese hamster ovary (CHO)-K1 cells. CHO cells are the most widely used mammalian cells for the production of recombinant monoclonal antibodies due to their ability to perform post-translational modification (PTM) on antibody molecules, which typically also occurs in the human body. Through mutagenesis, different CHO daughter cells of improved quality have been established. These variants include CHO-K1, CHO-S, CHO-DXB11, and CHO-DG44. Therefore, in one embodiment, the antibody for use according to the present invention is produced in CHO cells, preferably in the CHO-K1 cell line, and purified from the cell culture medium for further use.

[0038] As shown in Example 2, the major PTMs identified in antibody NI006 / ALXN2220 are HC modification of N-terminal glutamine to pyroglutamic acid, loss of C-terminal lysine, and N-glycosylation. In this regard, the N-glycosylation site was identified at position 300 (HC N300, SEQ ID NO: 18).

[0039] Thus, in one embodiment, the antibody for use according to the present invention has lost its C-terminal lysine, i.e., the antibody has undergone C-terminal lysine clipping. In particular, the C-terminal lysine shown in SEQ ID NO: 18 is cleaved from the heavy chain of the antibody, preferably from each heavy chain of the antibody. The sequence, i.e., the sequence of the heavy chain with the C-terminal lysine removed, is set forth in SEQ ID NO: 20. Alternatively, the N-terminal glutamine is modified as pyroglutamic acid, i.e., the heavy chain of the antibody shown in SEQ ID NO: 18 has undergone N-terminal glutaminyl cyclization. The sequence, i.e., the sequence of the heavy chain containing cyclic pyroglutamic acid and not containing N-terminal glutamine, is set forth in SEQ ID NO: 21. Alternatively, the heavy chain of the anti-TTR antibody present in the pharmaceutical composition of the present invention has lost its C-terminal lysine and the N-terminal glutamine is modified as pyroglutamic acid. The sequence, i.e., the sequence of the heavy chain with the C-terminal lysine removed, containing cyclic pyroglutamic acid and not containing N-terminal glutamine, is set forth in SEQ ID NO: 22.

[0040] In addition or alternatively, the antibody is glycosylated, particularly N-glycosylated. More specifically, the heavy chain of the antibody is glycosylated, and even more specifically at N300 of the heavy chain.

[0041] In a preferred embodiment, the anti-TTR antibody for use according to the present invention lacks a C-terminal cysteine, has a modified glutamine at the N-terminus as pyroglutamic acid, and contains at least one N-glycosylation site.

[0042] However, WO 2015 / 092077 (A1) discloses further human-derived antibodies that can be shown to bind to the same human TTR epitope as NI-301.37F1, namely antibodies NI-301.28B3 and NI-301.12D3, including the display of CDRs of V H and V LThe chain amino acid sequences are shown in Figure 1E of International Publication No. 2015 / 092077(A1) for NI-301.28B3 (Sequence IDs 8 and 10 of the present invention) and in Figure 1L for NI-301.12D3 (Sequence IDs 12 and 14 of the present invention). Therefore, anti-TTR antibodies for use according to the present invention generally have the amino acid sequence TTR 41~45 The antibodies may be characterized by binding to human TTR epitopes containing or consisting of (Sequence ID 51 of International Publication No. 2015 / 092077(A1) and Sequence ID 15 of the present invention). Therefore, to reach such antibodies, it is not necessary to rely on the means and methods disclosed in International Publication No. 2015 / 092077(A1) for obtaining such antibodies, and it is possible to perform one or more amino acid substitutions that do not essentially affect the binding characteristics of the antibodies. In addition, any of the other human-derived antibodies disclosed in International Publication No. 2015 / 092077(A1) can also be used in the same way.

[0043] Another class of anti-TTR antibodies, possibly suitable for use in the treatment of musculoskeletal disorders or conditions according to the present invention, is described in an international application by Prothena Biosciences Limited (Prothena). In particular, embodiments of this disclosure relate to the use of Novo Nordisk's anti-TTR antibody NN-6019 (formerly known as PRX004 by Prothena Biosciences) in the treatment of human subjects requiring such treatment, as provided herein. NN-6019 (PRX004) corresponds to the humanized version of antibody 14G8 described in Higaki et al., Amyloid 23(2016)86-97, and disclosed in International Publication Nos. 2016 / 120810(A1) and 2018 / 007922(A2), more specifically in International Publication No. 2019 / 108689(A1), and is a humanized version, with the disclosures of these documents incorporated by reference. NN-6019 (PRX-004) is a monoclonal antibody of the test subject designed to specifically target and eliminate the misfolded (toxic) form of TTR amyloid protein found in ATTR. Therefore, antibody PRX004 has the same epitope as PRX004, namely the amino acid TTR. 89~97 , or amino acid TTR 101~109 Among those that recognize epitopes including and are humanized versions of the first cloned mouse monoclonal antibodies 14G8, 9D5, 5A1, and 6C1 disclosed in International Publication Nos. 2016 / 120810(A1), 2018 / 007924(A2), 2018 / 007924(A2), and 2018 / 007923(A1), another preferred anti-TTR antibody for use in the treatment of musculoskeletal disorders according to the present invention would be, in particular, those which recognize epitopes including and are humanized versions of the first cloned mouse monoclonal antibodies 14G8, 9D5, 5A1, and 6C1 disclosed in International Publication Nos. 2016 / 120810(A1), 2018 / 007924(A2), and 2018 / 007923(A1) (the disclosures in these references, including the antibody sequences relating to these antibody clones and / or their deposits, e.g., their full length and / or CDRs), are incorporated herein by reference.

[0044] Further possibly preferred antibodies are humanized versions of antibody 18C5, or isolated monoclonal antibodies that compete with monoclonal antibody 18C5 for binding to human TTR, preferably the same monoclonal antibody 18C5 that binds to the same epitopes on human TTR, where 18C5 is a mouse antibody characterized by a mature heavy chain variable region having an amino acid sequence including SEQ ID NO: 81 and a mature light chain variable region having an amino acid sequence including SEQ ID NO: 87, as disclosed in International Publication No. 2019 / 071205(A1). The disclosures in these references, including the antibody sequences relating to these antibody clones and / or their deposits, e.g., their full length and / or CDR, are incorporated herein by reference.

[0045] Another class of humanized anti-TTR antibodies, which are likely suitable for use in the treatment of musculoskeletal conditions according to the present invention, is described in international applications by The Chemo-Sero-Therapeutic Research Institute and KM Biologics Co., Ltd., respectively, and is disclosed for antibodies 371M and 313M in International Publication No. 2015 / 115332, and for the antibody described in International Publication No. 2015 / 115331(A1) (designated herein as XY for ease of reference) as an amino acid TTR 78~89 or TR 118~122 Recognizing epitopes including these, the disclosures in these references, including the antibody sequences relating to these antibody clones and / or their depositions, e.g., their full length and / or CDR, are incorporated herein by reference.

[0046] ATTR exists in two forms, namely wtATTR and vATTR, caused by pathological aggregation of wtTTR and vTTR, respectively. wtATTR, in particular, is found in many musculoskeletal disorders such as carpal tunnel syndrome and osteoarthritis. See Perfetto et al., Biomedicines 10(2022), 3226. Therefore, in a preferred embodiment, the antibody binds to amyloid-forming wtTTR and amyloid-forming vTTR.

[0047] As described above, the experiments illustrated in the examples use a complete human IgG1m3 allotype, also known as NI-301.37F1, disclosed in International Publication No. 2015 / 092077(A1), and an amino acid sequence TTR as described in Sequence ID No. 51 (Sequence ID No. 15 of the present invention) of the same publication. 41~45 The procedure was carried out using antibody NI006 / ALXN2220, which binds to the TTR epitope containing the amino acid sequence. Therefore, it is reasonable to expect that further antibodies that bind to epitopes containing the amino acid sequence, such as antibodies NI-301.28B3 and NI-301.12D3, as disclosed in International Publication No. 2015 / 092077(A1), would also be suitable for use in the therapy according to the present invention, and the disclosures in these references, including the antibody sequences relating to these antibody clones and / or their depositions, such as their full length and / or CDR, are incorporated herein by reference.

[0048] Therefore, in a particularly preferred embodiment of the present invention, the anti-TTR antibody is derived from a human antibody NI-301.37F1, NI-301.28B3, or NI-301.12D3, and in its variable region, i.e., the binding domain, has a variable heavy chain (V) having the amino acid sequence shown in Figure 1C [NI-301.37F1] (SEQ ID NOs. 2 and 4 of the present invention, respectively) of International Publication No. 2015 / 092077(A1), Figure 1E [NI-301.28B3] (SEQ ID NOs. 8 and 10 of the present invention, respectively), or Figure 1L [NI-301.12D3] (SEQ ID NOs. 12 and 14 of the present invention, respectively) of International Publication No. 2015 / 092077(A1). H ) and variable light chain (V LThe antibody is characterized by containing a complementarity-determining region (CDR) of ), or, in the case of CDR2 and CDR3, having an amino acid sequence that differs by only one, two, three or more amino acids from that shown in Figure 1C, Figure 1E, or Figure 1L of International Publication No. 2015 / 092077(A1), and the antibody exhibits substantially the same or identical characteristics as the anti-TTR antibodies NI-301.37F1, NI-301.28B3, or NI-301.12D3 exemplified in the examples of International Publication No. 2015 / 092077(A1). The location of the CDRs is shown in Figure 1C, Figure 1E, or Figure 1L and is described in the caption for Figure 1 of International Publication No. 2015 / 092077(A1). The corresponding nucleotide sequences are listed in Table II on page 70 of International Publication No. 2015 / 092077(A1) for NI-301.37F1, pages 70-71 for NI-301.28B3, and page 73 for NI-301.12D3. In addition, or alternatively, the framework region or the complete V H and / or V L The chain is 80% identical to the framework region shown in Figure 1C or 1M [NI-301.37F1], Figure 1E [NI-301.28B3] or Figure 1L [NI-301.12D3] of International Publication No. 2015 / 092077(A1), preferably the framework region shown in Figure 1C or 1M, Figure 1E or Figure 1L of International Publication No. 2015 / 092077(A1) and V H and / or V L The chains are 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, respectively. Furthermore, the cloning and expression of antibodies NI-301.37F1, NI-301.28B3, and NI-301.12D3 were carried out as described on pages 110-112 of Examples 1 and 2 of International Publication No. 2015 / 092077(A1), and these methods are incorporated herein by reference.

[0049] Therefore, in one embodiment, the anti-TTR antibody is as shown in Figures 1C and 1M of International Publication No. 2015 / 092077(A1), V H and V LThe antibody is characterized by the CDR of the chain and the entire VH and VL chains. Therefore, the antibody is preferably, (i) Variable heavy (VH) chains containing the following VH complementarity Determining Regions (CDRs) 1, 2, and 3, and / or Variable light (VL) chains containing the following VL CDRs 1, 2, and 3, (a) VH-CDR1: A variant thereof comprising one or two amino acid substitutions at positions 31-35 of Sequence ID No. 10 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 2 of the present invention), (b) VH-CDR2: A variant thereof comprising one or two amino acid substitutions in position 52-67 of Sequence ID No. 10 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 2 of the present invention), (c)VH-CDR3:VH-CDR3:VH-CDR3, International Publication No. 2015 / 092077(A1), containing positions 100-109 of SEQ ID NO: 10 (SEQ ID NO: 2 of the present invention) or a variant thereof comprising one or two amino acid substitutions, (d) VL-CDR1: A variant thereof comprising one or two amino acid substitutions at positions 24-34 of Sequence ID No. 12 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 4 of the present invention), (e) VL-CDR2: A variant thereof comprising the 50-56 position of Sequence ID No. 12 (Sequence ID No. 4 of the present invention) of International Publication No. 2015 / 092077(A1), or one or two amino acid substitutions, and (f) VL-CDR3: A variant thereof comprising positions 89-97 of Sequence ID No. 12 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 4 of the present invention) or one or two amino acid substitutions, and / or (ii) VH chain and / or VL chain, (a) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 10 of International Publication No. 2015 / 092077(A1) (SEQ ID NO: 2 of the present invention) or SEQ ID NO: 53 of the same publication (SEQ ID NO: 6 of the present invention), or a variant thereof comprising one or more amino acid substitutions, (b) The VL chain comprises the amino acid sequence shown in SEQ ID NO: 12 of International Publication No. 2015 / 092077(A1) (SEQ ID NO: 4 of the present invention), or a variant thereof comprising one or more amino acid substitutions. Preferably, the VH chain and / or VL chain include such that the VH chain amino acid sequence and VL chain amino acid sequence are at least 90% identical to Sequence ID No. 10 or 53 and 12 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 2 or 6 and 4 of the present invention, respectively).

[0050] In one embodiment, the antibody comprises a shortened VH chain, i.e., the antibody is a VH and / or VL chain. (a) The VH chain comprises the amino acid sequence shown in Sequence ID No. 16 of the present invention, or a variant thereof comprising one or more amino acid substitutions, and (b) The VL chain comprises the amino acid sequence shown in Sequence ID No. 4 of the present invention, or a variant thereof comprising one or more amino acid substitutions, Preferably, the VH chain and / or VL chain contain amino acid sequences that are at least 90% identical to those of SEQ ID NOs: 16 and 4, respectively.

[0051] In one embodiment, the antibody comprises shortened VH and VL chains, i.e., the antibody is a VH chain and / or a VL chain, (a) The VH chain comprises the amino acid sequence shown in Sequence ID No. 16 of the present invention, or a variant thereof comprising one or more amino acid substitutions, and (b) The VL chain comprises the amino acid sequence shown in Sequence ID No. 17 of the present invention, or a variant thereof comprising one or more amino acid substitutions, Preferably, the VH chain and / or VL chain include such that the VH chain amino acid sequence and the VL chain amino acid sequence are at least 90% identical to those of SEQ ID NOs. 16 and 17, respectively.

[0052] In one embodiment, the anti-TTR antibody is shown in Figure 1E of International Publication No. 2015 / 092077(A1), V H Chain and / or V LThe antibody is characterized by the CDR of the chain, as well as the entire VH and VL chains. Therefore, the antibody is preferably, (i) Variable heavy (VH) chains containing the following VH complementarity Determining Regions (CDRs) 1, 2, and 3, and / or Variable light (VL) chains containing the following VL CDRs 1, 2, and 3, (a) VH-CDR1: A variant thereof comprising positions 31-37 of Sequence ID No. 18 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 8 of the present invention) or one or two amino acid substitutions, (b) VH-CDR2: A variant thereof comprising one or two amino acid substitutions in position 52-67 of Sequence ID No. 18 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 8 of the present invention), (c) VH-CDR3: Positions 100-116 of Sequence ID No. 18 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 8 of the present invention), or a variant thereof comprising one or two amino acid substitutions, (d) VL-CDR1: Positions 24-34 of Sequence ID No. 20 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 10 of the present invention), or a variant thereof comprising one or two amino acid substitutions, (e) VL-CDR2: Positions 50-56 of Sequence ID No. 20 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 10 of the present invention), or variants thereof comprising one or two amino acid substitutions, and (f) VL-CDR3: Positions 89-98 of Sequence ID No. 20 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 10 of the present invention), or variants thereof comprising one or two amino acid substitutions, and / or (ii) VH chain and / or VL chain, (a) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 18 of International Publication No. 2015 / 092077(A1) (SEQ ID NO: 8 of the present invention), or a variant thereof comprising one or more amino acid substitutions, (b) The VL chain comprises the amino acid sequence shown in SEQ ID NO: 20 of International Publication No. 2015 / 092077(A1) (SEQ ID NO: 10 of the present invention), or a variant thereof comprising one or more amino acid substitutions. Preferably, the present invention includes a VH chain and / or VL chain whose VH chain amino acid sequences and VL chain amino acid sequences are at least 90% identical to Sequence ID No. 18 and 20 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 8 and 10 of the present invention, respectively).

[0053] In one embodiment, the anti-TTR antibody is shown in Figure 1E of International Publication No. 2015 / 092077(A1), V H Chain and / or V L The antibody is characterized by the CDR of the chain, as well as the entire VH and VL chains. Therefore, the antibody is preferably, (i) Variable heavy (VH) chains containing the following VH complementarity Determining Regions (CDRs) 1, 2, and 3, and / or Variable light (VL) chains containing the following VL CDRs 1, 2, and 3, (a) VH-CDR1: A variant thereof comprising one or two amino acid substitutions at positions 31-35 of Sequence ID No. 46 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 12 of the present invention), (b) VH-CDR2: A variant thereof comprising one or two amino acid substitutions in positions 50-66 of Sequence ID No. 46 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 12 of the present invention), (c)VH-CDR3:Vase Code 46 of International Publication No. 2015 / 092077(A1) containing positions 99-108 (Sequence Code 12 of the present invention) or a variant thereof comprising one or two amino acid substitutions, (d) VL-CDR1: A variant thereof comprising one or two amino acid substitutions at positions 23-36 of Sequence ID No. 48 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 14 of the present invention), (e) VL-CDR2: A variant thereof comprising positions 52-58 of Sequence ID No. 48 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 14 of the present invention) or one or two amino acid substitutions, and (f) VL-CDR3: A variant thereof comprising positions 91-100 of Sequence ID No. 48 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 14 of the present invention) or one or two amino acid substitutions, and / or (ii) VH chain and / or VL chain, (a) The VH chain comprises the amino acid sequence shown in Sequence ID No. 46 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 12 of the present invention), or a variant thereof comprising one or more amino acid substitutions, (b) The VL chain comprises the amino acid sequence shown in SEQ ID NO: 48 (SEQ ID NO: 14 of the present invention) of International Publication No. 2015 / 092077(A1), or a variant thereof comprising one or more amino acid substitutions. Preferably, the VH chain and / or VL chain include such that the VH chain amino acid sequence and VL chain amino acid sequence are at least 90% identical to Sequence ID No. 46 and 48 of International Publication No. 2015 / 092077(A1) (Sequence ID No. 12 and 14 of the present invention, respectively).

[0054] However, preferably, the antibody NI-301.37F1 / NI006 / ALXN2220 is used in the method according to the present invention.

[0055] The five main classes of immunoglobulins are IgG, IgM, IgA, IgD, and IgE. These are distinguished by the type of heavy chain found in the molecule. IgG molecules have a heavy chain known as the gamma chain; IgM has a mu chain; IgA has an alpha chain; IgE has an epsilon chain; and IgD has a delta chain. For a review, see, for example, Schroeder et al., J. Allergy Clin. Immunol. 125 (2010), s41-s52. Furthermore, different subclasses exist; IgA is further divided into subclasses IgA1 and IgA2, and IgG is further divided into subclasses IgG1, IgG2, IgG3, and IgG4. Additionally, there are two types of light chains: kappa (κ) and lambda (λ).

[0056] In principle, the antibodies used in accordance with the present invention may be of any class and subclass and may contain any type of light chain, as long as the antibody binds to misfolded and preferably aggregated forms of TTR, and preferably the binding specificity to TTR, as shown in the examples of International Publication No. 2015 / 092077(A1) for antibody NI-301.37F1, remains essentially unaffected, and no adverse effects occur when the antibody is administered to a patient, as long as adverse effects can be determined as described in Example 1. However, preferably, a complete IgG antibody containing a constant domain is used. Thus, in one embodiment, the immunoglobulin heavy chain and / or light chain constant domain present in the antibody used in accordance with the present invention is of the IgG, IgM, IgA, IgD, or IgE type, preferably the IgG type. In one embodiment, the constant immunoglobulin heavy chain and / or light chain domains present in the antibody used according to the present invention are IgA1, IgA1, IgG1, IgG2, IgG3, or IgG4 subclasses, preferably IgG1, IgG2, IgG3, or IgG4 subclasses, most preferably IgG1 subclasses.

[0057] Recombinant expression of a fully human IgG1 antibody having a human constant domain or a mouse constant domain can be carried out substantially as described in the examples of International Publication No. 2015 / 092077(A1). Preferably, the antibody is a monoclonal antibody or derived from a monoclonal antibody.

[0058] In addition to the four subclasses of IgG mentioned above, human heavy and light chain genes also exhibit extensive structural polymorphisms, which are closely related and inherited as haplotypes. Allotype variants can be immunogenic and may induce an antibody response as a result of alloimmunity. Therefore, switching allotypes can be of particular interest in providing non-immunogenic antibody therapeutics. To date, a wide range of allotypes (polymorphisms) are known, but the focus has been on serologically defined allotypes. An allotype of the IgG protein is defined by the expression of a unique epitope recognized by a unique serum reagent. Allotypes expressed on the constant region of the IgG heavy chain are called Gm (genetic markers), along with a subclass, e.g., G1m, and an allotype number (or letter), e.g., G1m1 [or G1m(a)], G3m5 [or G3m(b1)]. Human immunoglobulin allotypes are listed in Table 1 of Jefferis and Lefrance, mAbs 1 (2009), 1-7 and in Figure 1A of Irani et al., Molecular Immunology 67 (2015), 171-182, and their contents are incorporated herein by reference. Therefore, in one embodiment, the antibody used in accordance with the present invention is one of the following allotypes, but is not limited to: G1m1, G1m2, G1m3, G1m17, G2m23, G3m21, G3m28, G3m11, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1, A2m2, A2m3, Em1, Km1, Km2 and Km3, preferably G1m2, G1m3 or G1m17, most preferably G1m3.

[0059] As described above, antibody NI006 / ALXN2220 is a fully human IgG1m3 allotyped antibody and consists of two identical heavy chains of the IgG1 subclass and IgG1m3 allotype. In addition, as stated above, the original human antibody NI-301.37F1 is kappa-type, and therefore NI006 / ALXN2220 consists of two identical light chains of the kappa subclass. The sequences of the variable heavy chain (VH) and variable light chain (VL) of NI006 / ALXN2220 are described in SEQ ID NOs: 2, 6, and 16, respectively, and each isotype, such as the IgG1m3 isotype, has a unique amino acid sequence in the constant region of its heavy chain. See Jefferis and Lefrance (2009) above. Therefore, in one embodiment, an antibody used according to the present invention is characterized by two heavy chains, each heavy chain (HC) containing the amino acid sequence described in SEQ ID NO: 18, and two light chains, each light chain (LC) containing the amino acid sequence described in SEQ ID NO: 19. Each heavy chain consists of 450 amino acid residues, and each light chain consists of 214 amino acid residues. The four chains are stabilized by intra- and inter-chain disulfide bonds, and the locations of the disulfide bridges identified for each Lys-C and trypsin digestion and subsequent LC-MS (see Example 2) are as follows: LC:C23-LC:C88 LC:C134-LC:C194 LC:C214-HC:C223 HC:C22-HC:C97 HC:C147-HC:C203 HC1:229-HC2:229 and HC1:232-HC2:232 HC:C264-HC:C324 HC:C370-HC:C428. (The amino acid numbering corresponds to the heavy and light chain sequences described in SEQ ID NOs. 18 and 19.)

[0060] Therefore, in one embodiment, the antibody for use according to the present invention preferably contains at least eight disulfide crosslinks at the positions specified above.

[0061] Furthermore, each heavy chain of the antibody NI006 / ALXN2220 contains a single N-linked glycosylation site in Asn300. The N-linked glycosylation structure is primarily a fucosylated complex branched glycan having either 0 galactose residues (G0F) (approximately 49%) or 1 galactose residue (G1F) (approximately 25%). A detailed glycosylation profile is shown in Example 2. Glycosylation plays a crucial role in the stability, in vivo activity, solubility, serum half-life, and immunogenicity of many therapeutic proteins. N-glycan analysis determines the relative distribution of N-glycans released from glycoproteins, providing insightful information regarding the safety and efficacy of biological therapies.

[0062] Therefore, in one embodiment, the antibody for use according to the present invention has an N-glycosylated heavy chain, preferably the N-linked glycosylation site is Asn300, and preferably the antibody comprises an N-linked glycosylation structure which is a glycan mainly having 0 galactose residues (G0F) (about 49%) or 1 galactose residue (G1F) (about 25%). Most preferably, the antibody has a glycosylation profile as shown in Example 2.

[0063] Furthermore, one or more amino acids at the amino or carboxyl termini of the light chain and / or the heavy chain, such as the C-terminal lysine of the heavy chain, may be missing or derivatized in part or all of the molecule.

[0064] Therefore, in one embodiment, the antibody for use according to the present invention has a heavy chain that does not contain C-terminal lysine. For example, in such an embodiment, the C-terminal lysine contained in SEQ ID NO: 18 is missing. The sequence of such a heavy chain is described in SEQ ID NO: 20.

[0065] In addition, or alternatively, antibodies for use according to the present invention have a heavy chain in which the N-terminal glutamine is derivatized, preferably substituted with pyroglutamic acid. This pyroglutamic acid formation is also called N-terminal cyclization. Sequences of such heavy chains are described in SEQ ID NO: 21 or SEQ ID NO: 22, and are in the absence of N-terminal glutamine.

[0066] Most preferably, the antibody for use according to the present invention has a heavy chain that does not contain C-terminal lysine, i.e., the C-terminal lysine has undergone C-terminal lysine clipping, and the N-terminal glutamine has been replaced with pyroglutamic acid, i.e., it has undergone N-terminal glutaminyl cyclization (see SEQ ID NO: 22) and is N-glycosylated.

[0067] The amino acid sequences of the heavy and light chains are shown below:

[0068] [Table 1] (SEQ ID NO: 18, NI006 / ALXN2220, heavy chain amino acid sequence, where amino acids in the constant region are underlined, the C-terminal lysine (K) is optional, and / or the N-terminal glutamine (Q) undergoes intramolecular cyclization, resulting in the formation of pyroglutamic acid.)

[0069] [Table 2] (Sequence ID 19, NI006 / ALXN2220, light chain amino acid sequence, where amino acids in the constant region are underlined).

[0070] In addition, however to a small degree, preferably negligible, some antibody species may have undergone other post-translational modifications (PTMs), such as partial cleavage, oxidation, deamidation, succinimide or pyroglutamic acid formation and isomerization. The PTMs identified in NI006 / ALXN2220 are mentioned in Example 2. In particular, antibodies used according to the present invention, following the C-terminal lysine clipping and N-terminal cyclization described above, may preferably exhibit methionine (M) oxidation at the HC255 position, preferably asparagine (N) deamidation at the HC318 and / or HC387 positions, preferably asparagine (N) succinimide formation at the HC318 position, and / or amidation of C-terminal proline (P) after the loss of C-terminal lysine and glycine.

[0071] The anti-TTR having the heavy and light chain variable regions described above is also described in International Publication No. 2015 / 092077(A1) (designated as antibody NI-301.37F1) and Michalon et al., Nat Commun. 12 (2021), 3142 (designated as antibody NI301A), and is capable of binding to human TTR epitopes containing or consisting of amino acid sequences TTR41-45 (sequence number 51 in International Publication No. 2015 / 092077(A1), and sequence number 15 of the present invention). Characterization of the antibody's binding properties has demonstrated that it exhibits high binding affinity to misfolded TTR in the sub-nanomolecal range, is highly selective for the amyloid conformation of TTR, i.e., binds selectively with high affinity to disease-associated ATTR aggregates, exerts similar binding to wild-type TTR and variant TTR associated with sporadic or genetic diseases, and does not bind to physiological TTR monomers. Furthermore, the anti-TTR antibody binds to ATTR deposits in cardiac tissue obtained from autopsies of ATTR-CM patients. Therefore, in one embodiment, an antibody used according to the present invention is an antibody equivalent to the antibody characterized above having heavy and light chain variable regions containing the amino acid sequences of SEQ ID NOs. 10 or 53 and 12 (SEQ ID NOs. 2 or 6 and 4 of the present invention, respectively) of International Publication No. 2015 / 092077(A1), which means that the equivalent antibody has substantially the same binding characteristics as the antibody characterized above having heavy and light chain variable regions containing the amino acid sequences of SEQ ID NOs. 10 or 53 and 12 (SEQ ID NOs. 2 or 6 and 4 of the present invention, respectively) of the same publication. In particular, the equivalent antibody is (i) A human TTR epitope containing or consisting of the amino acid sequence TTR41-45 is bound to it. (ii) It shows high binding affinity to misfolded TTR in the sub-nanomole concentration range, (iii) It is highly selective of the amyloid conformation of TTR, that is, it selectively binds to disease-related ATTR aggregates with high affinity. (iv) Exhibits similar binding to wild-type and variant TTRs associated with sporadic or genetic disorders, (v) Does not bind to physiological TTR monomers, and / or (vi) It binds to ATTR deposits in cardiac tissue obtained from autopsy of ATTR-CM patients.

[0072] In one embodiment, the equivalent antibody exhibits one of the binding properties (i) to (vi). In one embodiment, the equivalent antibody exhibits at least two of the enumerated binding properties. In one embodiment, the equivalent antibody exhibits at least three of the enumerated binding properties. In one embodiment, the equivalent antibody exhibits at least four of the enumerated binding properties. In one embodiment, the equivalent antibody exhibits at least five of the enumerated binding properties. In a preferred embodiment, the equivalent antibody exhibits all of the binding properties (i) to (vi).

[0073] As described above, the antibody NI006 / ALXN2220 reduces ATTR deposits in the joints by phagocytosis, and therefore, in one embodiment, it is prudent to assume that the antibody used according to the present invention has an active Fc domain. Accordingly, the antibody used according to the present invention preferably comprises a constant domain, preferably a human constant domain having an active Fc domain, and has an IgG format, i.e., a complete IgG antibody, preferably an IgG1 antibody or isotype. Recombinant expression of a complete human IgG1 antibody having a human constant domain can be carried out substantially as described in the examples in, for example, Harlow and Lane, "Antibodies, A Laboratory Manual," CSH Press, Cold Spring Harbor (1988), 1st edition, 2nd edition by Edward A. Greenfield, Dana-Farber Cancer Institute (copyright) 2014, ISBN 978-1-936113-81-1 and International Publication No. 2012 / 080518(A1).

[0074] In one embodiment, the antibody used in accordance with the present invention is in the form of a pharmaceutical composition comprising a physiologically acceptable diluent or carrier and optionally an excipient or adjuvant. The antibody can be formulated according to methods well known in the art. See, for example, Remington: The Science and Practice of Pharmacy (2000) by the University of Sciences in Philadelphia, ISBN 0-683-306472. Examples of suitable pharmaceutical carriers, well known in the art, include buffers, water, emulsions such as oil / water emulsions, various types of wetting agents, and sterile solutions. Compositions containing such carriers can be formulated by well known conventional methods. Administration of the suitable compositions can be achieved by different methods known in the art. Exemplary routes of administration include topical, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, intranasal, or intramuscular. In preferred embodiments, the anti-TTR antibody is formulated in a liquid formulation and designed to be administered intravenously (iv), particularly by intravenous infusion as performed in clinical trials outlined in the examples. Furthermore, the antibody can be injected directly into specific tissues where deposits have accumulated, for example, in the joints.

[0075] Antibodies and formulations can be administered to subjects at a suitable dose, i.e., a therapeutically effective dose, to reduce the risk, reduce the severity, or delay the onset of at least one sign or symptom of musculoskeletal disorder in subjects susceptible to the disease or otherwise at risk of the disease (preventive approach), or to improve or at least inhibit the further exacerbation of at least one sign or symptom of the disease in subjects suffering from musculoskeletal disorder (therapeutic approach). A regimen is considered therapeutically or preventively effective if, for example, individual treated subjects achieve a more favorable outcome than the mean outcome in a control population of comparators not treated by the methods disclosed herein. For example, a regimen is therapeutically effective if the deposition observed in a patient is reduced during treatment.

[0076] The administration plan will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors, including the patient's size, body surface area, age, the specific compound being administered, sex, time and route of administration, general health status, and other drugs being administered concurrently. Generally, subjects may be administered according to any other schedule determined by empirical analysis.

[0077] As seen in the examples, administration of NI006 / ALXN2220 at doses of 10 mg / kg, 30 mg / kg, and 60 mg / kg once every 28 days for at least 4 months to a maximum of 12 months resulted in a reduction of tracer signals observed in the heart and joints. Therefore, in one embodiment, the antibody used according to the present invention is administered in doses of about 10 mg / ko, about 100 mg / kg, preferably at least 10 mg / kg, 30 mg / kg, 60 mg / kg, or 100 mg / kg, i.e., a method for treating or preventing musculoskeletal disorders or conditions includes administering the antibody in doses of at least about 10 mg / kg, 30 mg / kg, 60 mg / kg, or 100 mg / kg. In one embodiment, the antibody used according to the present invention is administered once every 28 days, i.e., a method for treating or preventing musculoskeletal disorders or conditions includes administering the antibody once every 28 days. In one embodiment, the antibody used in accordance with the present invention is administered for at least 4 to 12 months, i.e., a method for treating or preventing a musculoskeletal disorder or condition includes administering the antibody for at least 4 to 12 months. Preferably, the antibody used in accordance with the present invention is administered at a dose of at least about 10 mg / kg, 30 mg / kg, or 60 mg / kg, once every 28 days for at least 4 to 12 months.

[0078] Considering the results of clinical trials illustrated in the examples, the antibody dose in the therapeutic method according to the present invention is such that the dose results in a reduction of the median amyloid level when the dose corresponds to a dose of 30–60 mg / kg in the patient. Therefore, dosing regimens that result in amyloid removal to substantially the same extent as the 30 mg / kg and 60 mg / kg treatment groups in clinical trials can be applied according to the present invention. In this context, fixed doses, lower / higher doses with shorter or longer dosing intervals can generally be selected and can be considered equivalent to the preferred dosing amounts of 30 mg / kg once every 28 days and 60 mg / kg once every 28 days. Since the dose refers to the molecular weight of antibody NI006 / ALXN2220 having approximately 147 kDa for intact IgG1 antibody, the dose may be adjusted accordingly for the use of antibodies or conjugated fragments with significantly different MWs. Similarly, if the antibody is used with a lower or longer serum half-life due to modifications such as altered glycosylation and / or PEGylation, the dose and dosing interval can be recalculated, respectively. When considering alternative drug regimens, exposure (Cmax and AUC) should be within the range observed at 30 and 60 mg / kg over 28 days for SAD / MAD doses.

[0079] As shown in the clinical trial studies outlined in the Examples, reduced tracer uptake was observed in the shoulder and elbow joints 4–12 months after therapy with anti-TTR antibodies. Therefore, the progression of musculoskeletal disorders or conditions, or the progress of treatment, can be monitored by determining the relative changes in scintigraphy tracer uptake in the joints or bones. Increased tracer uptake means that more deposits are detected, and these deposits are associated with musculoskeletal disorders or conditions. Therefore, increased tracer uptake in the joints or bones indicates the progression of the disorder or condition, while decreased tracer uptake suggests the effectiveness of the treatment. In principle, any tracer capable of detecting deposits and amyloid deposits in the subject can be used. In a preferred embodiment, the tracer is a bisphosphonate, preferably, 99mTc-hydroxyl-methylene-diphosphonate (HMDP) or 99m Contains Tc-3,3-diphosphono-1,2-propanodicarboxylic acid (DPD).

[0080] This finding can also be used in screening methods. Therefore, subjects treated according to the present invention can be screened for the presence of musculoskeletal disorders or conditions associated with deposits in the joints and / or bones, preferably by determining the uptake of scintigraphy tracers in the joints and / or bones, and increased uptake compared to healthy controls indicates the onset or presence of the disorder or condition.

[0081] As described above, musculoskeletal events, primarily arthralgia and arthritis, increased with increasing doses of NI006 / ALXN2220 in the placebo-controlled dose-escalating phase, likely due to the phagocytic activity of the antibody, and also increased when patients who received placebo were switched to NI006 / ALXN2220 in the open-label continuation phase. The majority of these events were mild and were treated with nonsteroidal anti-inflammatory drugs or low-dose glucocorticoids. Therefore, in one embodiment, the antibody used according to the present invention is administered in combination with a disease-modifying antirheumatic drug or an anti-inflammatory agent. In a preferred embodiment, such agents are corticosteroids (disease-modifying antirheumatic drugs) and / or nonsteroidal anti-inflammatory drugs (NSAIDs). In one embodiment, the disease-modifying antirheumatic drug and the anti-inflammatory agent are administered in combination with an anti-TTR antibody.

[0082] The administration of disease-modifying antirheumatic drugs and / or anti-inflammatory agents is preferably carried out after the administration of anti-TTR antibodies and after the onset of arthralgia and arthritis, as is done in clinical trials outlined in the examples.

[0083] Drugs acting as TTR tetramer stabilizers, silencers, and gene-editing therapies are designed to prevent ATTR accumulation, but these drugs do not directly target amyloid already deposited in the heart, for example. Based on surrogate markers in scintigraphy and cardiac MRI, there is some evidence that patients who have received therapies to stabilize or silence TTR may have a lower amyloid load than untreated patients, although significant reductions are rare. However, thanks to the present invention, these alternative strategies may function better in combination with anti-TTR antibodies for use, for example, in methods of treating ATTR-related joint and bone disorders.

[0084] Accordingly, the present invention further relates to implementing the therapies described herein using, for example, combination therapies comprising an ATTR antibody, such as those provided in the preceding paragraph, and a TTR tetramer stabilizer, a drug acting as a silencer, as well as gene editing therapy. Preferably, combination therapy is used with an anti-TTR antibody, as exemplified based on the antibody NI006 / ALXN2220, which was also designed as NI-301.37F1 and is used with a TTR stabilizer disclosed in International Publication No. 2021 / 228987(A1), and combination therapy with a TTR tetramer stabilizer such as tafamidis, diflunisal, and AG10 has been shown to result in a significant clearance rate of TTR fibrils in mice. See, for example, Example 2 of International Publication No. 2021 / 228987(A1). Diflunisal is an anti-amyloid agent and acts as a TTR tetramer stabilizer, and is marketed by Merck Sharp & Dohme for the treatment of rheumatoid arthritis (RA) and for the treatment of rheumatic pain and osteoarthritis (Dolobid). Accordingly, certain embodiments of this disclosure provide combinations of an anti-TTR antibody and a TTR tetramer stabilizer, particularly diflunisal, for the treatment of musculoskeletal disorders in human patients requiring treatment of musculoskeletal disorders, such as human patients with arthritis, rheumatic conditions, and osteoarthritis.

[0085] Accordingly, in additional or alternative embodiments, anti-TTR antibodies, such as those used according to the present invention, are administered in combination with another agent effective in treating or preventing musculoskeletal disorders or conditions, preferably arthritis, rheumatic conditions, and osteoarthritis. Such agents may include TTR tetramer stabilizers, such as diflunisal or tafamidis. In this regard, the disclosures of International Publication No. 2021 / 228987(A1) relating to this combination are incorporated herein by reference.

[0086] The present invention further relates to pharmaceutical compositions, component kits, or products. (i) an antibody capable of binding to mutated, misfolded, misassembled, and / or aggregated transthyretin (TTR) species and removing ATTR by inducing phagocytosis, and (ii) Includes disease-modifying antirheumatic drugs.

[0087] The present invention further relates to pharmaceutical compositions, component kits, or products. (i) an antibody capable of binding to mutated, misfolded, misassembled, and / or aggregated transthyretin (TTR) species and removing ATTR by inducing phagocytosis, and (ii) Contains anti-inflammatory agents

[0088] The present invention further relates to pharmaceutical compositions, component kits, or products. (i) an antibody capable of binding to mutated, misfolded, misassembled, and / or aggregated transthyretin (TTR) species and removing ATTR by inducing phagocytosis, and (ii) Includes disease-modifying antirheumatic drugs and anti-inflammatory agents.

[0089] The present invention further relates to pharmaceutical compositions, component kits, or products. (i) an antibody capable of binding to mutated, misfolded, misassembled, and / or aggregated transthyretin (TTR) species and removing ATTR by inducing phagocytosis, and (ii) TTR tetramer stabilizers, such as diflunisal.

[0090] In one embodiment, the pharmaceutical composition, component kit, or product further comprises a pharmaceutically acceptable carrier.

[0091] The present invention further relates to pharmaceutical compositions, component kits, or products. (i) an antibody capable of binding to mutated, misfolded, misassembled, and / or aggregated transthyretin (TTR) species and removing ATTR by inducing phagocytosis, (ii) Disease-modifying antirheumatic drugs, and (iii) Contains a TTR tetramer stabilizer.

[0092] The present invention further relates to pharmaceutical compositions, component kits, or products. (i) an antibody capable of binding to mutated, misfolded, misassembled, and / or aggregated transthyretin (TTR) species and removing ATTR by inducing phagocytosis, (ii) Anti-inflammatory agents, (iii) TTR tetramer stabilizers, such as diflunisal.

[0093] The present invention further relates to pharmaceutical compositions, component kits, or products. (i) an antibody capable of binding to mutated, misfolded, misassembled, and / or aggregated transthyretin (TTR) species and removing ATTR by inducing phagocytosis, (ii) Disease-modifying antirheumatic drugs and anti-inflammatory agents, (iii) TTR tetramer stabilizers, such as diflunisal.

[0094] In one embodiment, the pharmaceutical composition, component kit, or product further comprises a pharmaceutically acceptable carrier.

[0095] In one embodiment, the antibody contained in the pharmaceutical composition, component kit, or product of the present invention is a human-derived anti-TTR antibody.

[0096] In addition, or alternatively, the antibodies contained in the pharmaceutical composition, component kit, or product of the present invention have a constant region of an IgG isotype, preferably an IgG1 isotype. Most preferably, the antibody is of the IgG1m3 allotype.

[0097] In addition, or alternatively, the antibodies contained in the pharmaceutical composition, component kit, or product of the present invention are characterized by the VH and VL regions, as well as the CDR, as described in detail above.

[0098] In addition, or alternatively, the antibodies contained in the pharmaceutical composition, component kit, or product of the present invention are characterized by two heavy chains and two light chains as described in detail above.

[0099] In addition, or alternatively, the antibodies contained in the pharmaceutical composition, component kit, or product of the present invention are characterized by having the PTMs listed above.

[0100] Therefore, in preferred embodiments, the antibody is an antibody or antigen-binding fragment thereof, as used in accordance with the present invention and characterized in each of the above.

[0101] In addition, or as an alternative, the disease-modifying antirheumatic drug contained in the pharmaceutical composition, component kit, or product of the present invention is a corticosteroid.

[0102] In addition, or as an alternative, the anti-inflammatory agent contained in the pharmaceutical composition, component kit, or product of the present invention is an NSAID.

[0103] The present invention further relates to a method for treating a musculoskeletal condition or disorder as defined herein, the treatment comprising administering to a subject in need an anti-TTR antibody as defined herein, and optionally one or more supporting agents such as an anti-inflammatory agent, a disease-modifying antirheumatic agent, and / or a TTR tetramer stabilizer as defined herein.

[0104] The therapeutic benefits of the therapy according to the present invention can be evaluated, for example, by physical assessments such as self-assessment and general tests for diagnosing disease (see, e.g., Reiman and Manske J Man Manip Ther. 19 (2011), 91-99), or by a 6-minute walk distance assay as described in Example 1. Furthermore, the therapeutic benefits of the therapy according to the present invention can be evaluated, for example, by reduction of local inflammation accompanied by simultaneous reduction of pain, and improvement of functional capacity and quality of life, using EULAR or ACR response criteria (see, for example, Van Gestel et al., Arthritis Rheum 39 (1996), 34-40 and Felson et al., Arthritis Rheum 38 (1995), 727-35).

[0105] An anti-TTR antibody as defined herein earlier, i.e., an anti-TTR antibody NI006 / ALXN2220 capable of binding to mutated, misfolded, misassembled, and / or aggregated transthyretin (TTR) species, and in a preferred embodiment comprising the indicated sequence, most preferably its CDR and its VH and VL regions, most preferably its two heavy chains and two light chains, can be used in methods for diagnosing subjects, e.g., musculoskeletal disorders or conditions in human patients, particularly those related to amyloid-forming TTR deposits in joints / bones. Musculoskeletal disorders are muscle disorders as defined above. Compared to a control, increased antibody binding in the patient's joints and / or bones, and in each tissue, respectively, indicates the disease.

[0106] The present invention further relates to a kit for treating or preventing a musculoskeletal disorder or condition as defined herein earlier in human subjects, wherein the kit comprises a dose of an anti-transthyretin (TTR) antibody capable of binding to a mutated, misfolded, misassembled, or aggregated transthyretin (TTR) species, preferably the antibody NI006 / ALXN2220, characterized by the antibody or its antigen-binding fragment as defined herein earlier, most preferably its CDR and its VH and VL regions, most preferably its two heavy chains and two light chains, and instructions for using the anti-TTR species in a method of treatment as defined herein earlier in human subjects, wherein the kit comprises a dose of an anti-transthyretin (TTR) antibody capable of binding to a mutated, misfolded, misassembled, or aggregated transthyretin (TTR) species, most preferably the antibody NI006 / ALXN2220, characterized by its two heavy chains and two light chains, respectively, and instructions for using the anti-TTR species in a method of treatment as defined herein earlier in human subjects.

[0107] Throughout this specification, several documents are cited. The contents of all cited references (including documents cited throughout this application, including published patents and patent applications, including the background art section and manufacturer specifications and instructions) are expressly incorporated herein by reference; however, this does not constitute an admission that the cited documents are actually prior art relating to the present invention.

[0108] A more complete understanding can be obtained by referring to the following specific examples, which are provided herein for illustrative purposes only and are not intended to limit the scope of the invention. [Examples]

[0109] Example 1: Phase 1 (1a / 1b) first-in-human, double-blind, placebo-controlled, international, multicenter, single-dose and repeated-escalation randomized clinical trial in patients with ATTR-CM and chronic heart failure, followed by an open-label continuation phase. This study is the first-in-human investigation of NI006, also known as ALXN2220, aimed at evaluating the safety and tolerability of intravenous infusion of NI006 in patients with ATTR-CM. NI006 is a recombinant human anti-ATTR monoclonal IgG1 antibody generated from a comprehensive immunorepertory analysis of memory B cell complement in healthy aged human subjects, as described in International Publication No. 2015 / 092077(A1), where the antibody was named NI-301.37F1. Amyloid-transthyretin cardiomyopathy (ATTR-CM) is a progressive and fatal disease caused by misfolded transthyretin. Despite progress in slowing disease progression, there are no available treatments to deplete ATTR from the heart and improve cardiac dysfunction. NI006 / ALXN2220 is a recombinant human anti-ATTR antibody developed to remove ATTR amyloid by phagocytic immune cells.

[0110] method Test design This study was a phase 1 (1a / 1b) double-blind, placebo-controlled, international, multicenter, randomized clinical trial of single-ascending dose (SAD) and multiple-ascending dose (MAD) in patients with ATTR-CM and chronic heart failure, followed by an open-label extension (OLE) phase. Patients were randomized in a 4:2 ratio to receive either NI006 / ALXN2220 or placebo. Patients randomized to NI006 / ALXN2220 were sequentially enrolled into six escalating dose cohorts ranging from 0.3 mg / kg to 60 mg / kg. The study included a 4-month placebo-controlled single-ascending and multiple-ascending dose phase, followed by an 8-month open-label extension phase, during which all patients (placebo and NI006 / ALXN2220 treatments) received NI006 / ALXN2220. More specifically, the first two patients in each dose cohort (sentinel) were randomized in a 1:1 ratio to receive either NI006 / ALXN2220 or placebo. If no relevant safety signals occurred in the sentinel, four subsequent patients in each cohort were randomized in a 3:1 ratio to receive either NI006 / ALXN2220 or placebo. After a good review of sufficient single-dose escalation safety data, the next high-dose cohort was initiated. Patients received a total of four doses (q4w) of either NI006 / ALXN2220 or placebo during the single-dose / multiple-dose escalation combination phase. If a patient discontinued treatment during the single-dose / multiple-dose escalation phase for reasons other than suspected drug toxicity, replacement patients were recruited (four replacement patients were recruited, one each for the 1 mg / kg, 10 mg / kg, 30 mg / kg, and 60 mg / kg dose cohorts). Patients who entered the open-label continuation phase were entitled to eight doses of NI006 / ALXN2220 every four weeks, regardless of their treatment allocation in the single-dose / repeated-dose escalation phase, which remained blinded at that time. As new safety information became available from the single-dose / repeated-dose escalation phase of the higher-dose cohort, patients who started at lower dose levels were escalated to the maximum safe dose level at each dose.

[0111] NI006 / ALXN2220 was administered intravenously via IV infusion over approximately 2 hours (±10 minutes, excluding a maximum of 3 hours at 60 mg / kg) without premedication. Subsequent infusions were administered over approximately 50–70 minutes without premedication. Patients were hospitalized for 4 nights after the initial single dose escalation and after the first open-label continuation (i.e., the first NI006 / ALXN2220 administration in patients randomized to placebo), and for 1–2 nights after each of the three MAD administrations. All further administrations in the open-label continuation phase were performed on an outpatient basis.

[0112] Patient group The study population included individuals with a confirmed diagnosis of ATTR-CM, and at the time of screening, they had a left ventricular wall thickness of at least 14 mm, a left ventricular ejection fraction of at least 40%, NYHA class I-III, and a heart rate of 30 mL / min / 1.73 m². 2 The study consisted of patients with an eGFR exceeding 5000 pg / mL and NT-proBNP levels of 600–6000 pg / mL. See Figure 1 below. Combination therapy with tafamidis was approved, but treatment with other ATTR-specific drugs was not. Patients were recruited from six specialized amyloidosis centers in four European countries between February 2020 and April 2022.

[0113] [Table 3-1]

[0114] [Table 3-2]

[0115] Safety evaluation The primary objective was to determine the safety and tolerability profile of NI006 / ALXN2220. Dose escalation was induced by evaluating clinically relevant adverse events (AEs) occurring during treatment, as well as safety markers including clinical laboratory tests, echocardiography, and ECG. Blinded safety data were continuously monitored by both the principal investigator and the sponsor and reviewed by the Data Evaluation Committee.

[0116] Pharmacokinetic profile and immunogenicity assessment Sequential serum NI006 / ALXN2220 concentrations were measured throughout the study in all patients using a validated assay. Individual total serum NI006 / ALXN2220 exposure was calculated as the area under the curve from simulated pharmacokinetic profiles. More specifically, serum NI006 / ALXN2220 concentrations were measured using a validated sandwich ELISA assay constructed based on two anti-idiotype Fab fragments that selectively bind to NI006 / ALXN2220. The assay lower limit of quantification (LLOQ) was 0.17 ug / mL. Non-compartmental analysis (NCA) was performed on serum NI006 / ALXN2220 concentration-vs-time data using Nuventra (Durham, NC, USA) with a validated installation of Phoenix WinNonlin version 8.2.2 utilizing an intravenous (IV) infusion model, using actual blood collection time and dosage level, for the calculation of standard PK parameters. Population pharmacokinetic modeling was performed using LYO-X (Switzerland) with a two-compartment model involving linear antibody clearance from the central compartment, antibody binding to its target ATTR, and elimination of the NI006 / ALXN2220:ATTR complex in the peripheral compartment. Population pharmacokinetic parameters were estimated using a probabilistic approximation algorithm for expectation maximization performed in Monolix. Individual total serum NI006 / ALXN2220 exposures for each patient were calculated at 4 and 12 months from simulated pharmacokinetic profiles using individual parameter estimates and a linear trapezoidal rule.

[0117] Furthermore, we monitored for anti-drug antibodies. The presence of potential anti-drug antibodies was evaluated using an electrochemiluminescence immunoassay validated by QPS (Netherlands), with biotinylated NI006 / ALXN2220 and sulfotag-labeled NI006 / ALXN2220 used as detection reagents.

[0118] Cardiac amyloid imaging research All enrolled patients underwent either serial bisphosphonate scintigraphy or cardiac MRI (cMRI) to assess changes at 4 and 12 months. Imaging methods were selected according to standard practice guidelines and analyzed uniformly in a blinded manner. The effect of NI006 / ALXN2220 was evaluated using changes in cardiac tracer retention of the cardiac-to-whole-body ratio in planar scintigraphy. The effect of NI006 / ALXN2220 was assessed by changes in left ventricular extracellular volume using cMRI. Both imaging methods were performed according to standardized protocols and read at a central laboratory by readers blinded to treatment assignments.

[0119] Scintigraphy During test preparation, follow the standardized acquisition protocol established in the laboratory manual. 99m Tc-hydroxyl-methylene-diphosphonate (HMDP) or 99m Bisphosphonate scintigraphy was performed at the clinical trial site using Tc-3,3-diphosphono-1,2-propanodicarboxylic acid (DPD) as a tracer. Adherence to laboratory manuals and image quality were continuously monitored. Planar whole-body images were acquired 3 hours after tracer injection. Image interpretation was performed centrally in the imaging core lab by two independent nuclear medicine physicians using Syngovia software (Siemens), blinded to pseudonymized patient ID, treatment assignment, dose cohort, and acquisition time. To calculate the heart-to-whole body retention ratio (H / WB ratio), areas with potentially high tracer retention (e.g., bladder, kidney, injection site) were considered rejection areas in the following formula:

[0120]

number

[0121] A consensus reading was performed when the discrepancy between the two interpreters exceeded a predetermined range of ±10% of the important parameters.

[0122] Cardiac MRI Cardiac MRI scans were recorded at the research facility using local scanners. The acquisition protocol followed the latest guidelines, was standardized across all facilities during the study preparation phase, and adherence to laboratory manuals was continuously monitored. MRI scans were interpreted by two independent leaders (specialized radiologists or cardiologists) in the imaging core lab using Medis software (Medis, Leiden, The Netherlands). All MRI scans were analyzed individually. The central leader was blinded to pseudonymized patient IDs, treatment assignments, dose cohorts, and acquisition time. MRI acquisition and analysis procedures were similar to those described in Martinez-Naharro et al., J Am Coll Cardiol 70 (2017), 466-477. Measurements were performed before and after contrast agent administration, defining the entire left ventricular myocardium at the basal and central ventricular levels as the region of interest. T1 measurements were performed in short-axis view for both ventricular myocardium and blood. Hematocrit information was available. ECV was calculated according to the following formula:

[0123]

number

[0124] Cardiac biomarkers and other parameters Changes in NT-proBNP and troponin T levels at 4 and 12 months were assessed in a unified manner. In addition, overall quality of life, functional capacity, and cardiac structure and function were investigated using the Kansas City Cardiomyopathy Questionnaire-Overall Summary (KCCQ-OS), 6-minute walk distance (6-MWD), and echocardiography. Scores from the KCCQ-OS questionnaire range from 0 to 100, with 0-24 indicating very poor to poor, 25-49 indicating poor to average, 50-74 indicating average to good, and 75-100 indicating good to excellent quality of life.

[0125] Echocardiography Standardized echocardiograms were acquired at three time points throughout the trial, and complete echocardiograms for efficacy analysis were recorded at baseline before initial treatment, after completion of MAD (4 months), and after completion of open-label continued administration (12 months). A unified acquisition protocol was applied to all facilities, and the analysis was centrally performed by experienced cardiologists at the Imaging Core Lab (Biotrial, Rennes, France), demonstrating low intra-leader variability with pseudonymized patient IDs, treatment assignments, dose cohorts, and acquisition time blinded. Measurements were taken using EchoPAC CE Medical software (GE Healthcare, Milwaukee, MI, USA) in 3-loop (sinus rhythm) or 5-loop (atrial fibrillation) sequences, and the mean of the 3-loop / 5-loop sequences was used for further analysis. If the recording did not allow for measurement of individual parameters (e.g., low echogenicity, insufficient number of recorded loops, suboptimal sections), the measurement was reported as missing.

[0126] In addition, to rule out sudden deterioration of systolic left ventricular function or the occurrence of pericardial effusion, non-standardized oriented echocardiography was performed by the principal investigator or co-investigator at the participating center before each of the first five treatment sessions, both before infusion and before discharge.

[0127] statistical analysis The primary results presented here were generated after all patients in the highest-dose cohort completed placebo-controlled single-dose and repeated escalation-dose phases. Results are presented for all patients (safety population) who received at least one dose of NI006 / ALXN2220 or placebo. All available data from open-label extension phases are included (data cutoff November 3, 2022). No formal statistical hypotheses were tested, and missing data were not supplemented. Patients randomized to NI006 / ALXN2220 were grouped according to their nominal dose cohort at enrollment, and patients assigned to placebo were pooled from all dose cohorts. Open-label extension data were aggregated using nominal group assignments. The effect of NI006 / ALXN2220 was investigated by pre-identified grouping of patients receiving higher (at least 10 mg / kg) or lower (up to 3 mg / kg) doses. Absolute or relative changes from baseline at 4 and 12 months were calculated. Baseline assessments prior to open-label continuation were used to calculate changes during open-label continuation for patients randomized to placebo. Data handling and analysis were performed using Statistical Analysis System® (SAS®) version 9.4, and the GGplot2 package for R was used for data visualization.

[0128] result Patient characteristics A total of 40 participants (median age 72 years, range 28–87 years, 39 males) were enrolled in the study. 27 were assigned to NI006 / ALXN2220, sequentially enrolled in dose levels from 0.3–60 mg / kg, and 13 were assigned to placebo (Table 2). The majority of patients (33 out of 40) had ATTRwt, and 36 of the 40 patients received combination therapy with tafamidis for a median treatment duration of 7 months (interquartile range [IQR], 4–16 months). Patients assigned to NI006 / ALXN2220 appeared to have more advanced disease than those randomized to placebo (Tables 2 and 3). Adherence to the clinical trial protocol was high, with 34 out of 40 patients receiving all four planned placebo-controlled doses in the study's single-dose and repeated-escalation dose phases, and 34 out of 35 eligible patients enrolled in the study's open-label extension phase.

[0129] [Table 4] * The collection of ethnic materials was not permitted at the French facility. $ Local values ​​during screening. NT-proBNP: N-terminal pro-B-type natriuretic peptide, GFR: glomerular filtration rate, which is estimated using the formula from the Chronic Renal Disease Epidemiology Collaborative Study, NAC National Amyloidosis Center, and NYHA: New York Heart Association.

[0130] [Table 5] Data is presented as mean ± standard deviation or median (interquartile range). * The Mayo clinical staging system predicts outcomes for patients with amyloidosis based on troponin and NT-proBNP levels. Stage I indicates no risk factors, while Stage III indicates two risk factors. The score from the KCCQ questionnaire ranges from 0 to 100, with 0-24 indicating very poor to poor quality of life, 25-49 indicating poor to average quality of life, 50-74 indicating average to good quality of life, and 75-100 indicating good to excellent quality of life. 6MWD indicates 6-minute walk distance, ACE indicates angiotensin-converting enzyme, ARB indicates angiotensin receptor blocker, ECV indicates extracellular volume, E / e' indicates the ratio of initial mitral valve inflow velocity to initial mitral annular diastolic velocity, ED-IVS indicates end-diastolic interventricular septum, H / WB indicates the heart-to-whole-body ratio, LAV indicates left atrial volume, LVEF indicates left ventricular ejection fraction, LVESV / LVEDV indicates left ventricular end-systolic / end-diastolic volume, LAV indicates left atrial volume, NAC indicates National Amyloidosis Center, KCCQ-OS indicates Kansas City Cardiomyopathy Questionnaire-Overall Summary Score, and SGLT2 indicates sodium-glucose cotransporter-2.

[0131] Safety and tolerability NI006 / ALXN2220 appeared to have a favorable safety profile up to the highest dose level, with no dose-limiting toxicity experienced by patients and no serious adverse events (SAEs) considered to be associated with NI006 / ALXN2220 occurring (Table 4). Of the 40 patients, a total of 38 experienced at least one adverse event (AE) during the single-dose and repeated dose-escalation phases, the majority of which were considered to be mild or moderate in severity (124 and 60 of a total of 191 events were reported as Grade 1 and 2 according to the Common Terminology Criteria for Adverse Events, respectively), and were not dose-dependent (Tables 5 and 6). During the study, there were two deaths attributed to the progression of amyloidosis that occurred during the open-label continuation phase. See the detailed explanation below.

[0132] The most frequently observed adverse events (AEs) were heart failure and arrhythmias, which is expected in this patient population. The frequency and type of AEs appeared similar across the NI006 / ALXN2220 dose cohort. Three patients assigned to the 10 mg / kg or 30 mg / kg cohort experienced cytokine release syndrome with associated increases in cardiac biomarkers, or non-severe grade 1 or 2 AEs, during the single-dose and repeated escalation phases. All three patients completed treatment without recurrence throughout the study and open-label continuation phases. See the explanation below. Numerical increases in musculoskeletal events, primarily arthralgia and arthropathy, were observed in placebo patients during escalation phases in the single-dose and repeated escalation phases, and when switched to NI006 / ALXN2220 in the open-label continuation phase. Most of these events were of mild intensity and were managed with nonsteroidal anti-inflammatory drugs or low-dose corticosteroids, which were tapered during continued treatment. However, one patient in the 10 mg / kg dose group discontinued the study, and one patient in the 30 mg / kg dose group withdrew consent to participate in the open-label continuation phase. Two patients experienced transient, asymptomatic decreases in platelet count, and one patient (60 mg / kg group) discontinued the study due to a moderate-severity, non-critical event of thrombocytopenia following two drug infusions. The thrombocytopenia in this individual was considered related to NI006 / ALXN2220 infusion due to the temporal relationship between the decrease in platelet count and its recurrence after resumption. No bleeding or other related AEs occurred in this patient, and complete recovery of platelet count occurred within two weeks. Two patients discontinued the study due to COVID-19 infection at the recommendation of the Data Evaluation Committee during the single-dose and repeated dose-escalation phases. There was no response to the infusion, and no laboratory safety parameters, including plasma levels of TTR, or other apparent clinically significant changes from baseline in vital signs were observed throughout the cohort. Cardiac safety monitoring at participating centers, including echocardiography and outpatient ECG monitoring, did not identify any apparent evidence of new cardiac dysfunction, pericardial effusion, or increased arrhythmias.During the open-label, continued administration phase, three patients prematurely dropped out of the study; one was due to COVID-19 infection, and two were documented fatal cases.

[0133] The following is a description of the case. Fatal serious adverse events Patient 1 (a 68-year-old patient with ATTRv, NYHA stage II, NAC stage II, MAYO stage II, and a baseline NT-proBNP level of 4088 pg / mL) was initially assigned to placebo and received only four doses of NI006 / ALXN2220 (10 mg / kg dose at each dose) during the open-label continuation phase. He was hospitalized once for heart failure during the single-dose escalation / repeated-dose escalation phase while receiving placebo, once at the end of the open-label continuation phase, and once during the follow-up period after the open-label continuation phase. He died 99 days after the last dose of NI006 / ALXN2220. The patient's cause of death was disease progression.

[0134] The second patient was 85 years old and had ATTRwt and NAC stage III (NYHA stage II, MAYO stage III, baseline NT-proBNP level of 3776 pg / mL) at baseline. This patient experienced progressive exacerbation with multiple hospitalizations for heart failure during the open-label continuation phase. This patient received a total of eight infusions (six 3 mg / kg dose infusions and two 10 mg / kg dose infusions) when the patient's condition no longer permitted participation in the study. The patient died at home 77 days after the last dose of NI006 / ALXN2220. The cause of death was disease progression.

[0135] Non-serious adverse events of cytokine release syndrome Three participants treated with NI006 / ALXN2220 at the same institution—one in Cohort 4 (Case 1) and two in Cohort 5 (Cases 2 and 3)—experienced infusion-related reactions reported as adverse events occurring during treatment for cytokine release syndrome. No events met the dose-limiting toxicity criteria, and all three participants continued to receive the study drug, including in an open-label extension phase, without further evidence of cytokine release syndrome. All adverse events occurring under treatment, as described below, were assessed by the principal investigator as being related to the investigational product.

[0136] Case 1 (Cohort 4, 10 mg / kg dose group) was a 74-year-old male with NYHA class I heart failure who experienced moderate-intensity cytokine release syndrome approximately 30 hours after completion of the first administration of NI006 / ALXN2220, as evidenced by a transient increase in body temperature (38°C) and heart rate (95 bpm), accompanied by a simultaneous decrease in blood pressure to 93 / 51 mmHg and oxygen saturation (88% room air). Treatment for cytokine release syndrome included IV corticosteroids, IV solution, oxygen via nasal cannula, and antipyretics. No infectious etiology was identified for the changes in vital signs. Laboratory evaluation on day 3 post-infusion showed significant increases in NTproBNP (9269 ng / l, pre-infusion: 5366 ng / l), CRP (69 mg / l, pre-infusion: 5 mg / l), and troponin T (68 pg / ml, pre-infusion: 55 mg / dl). Echocardiography and ECG on day 3 post-infusion showed no relevant changes, and cMRI showed individual signs of septal myocarditis, the interpretation of which was complicated by pre-existing inflammatory changes in the septum on cMRI prior to the study. Furthermore, an adverse event (12 non-sustained ventricular tachycardias) occurring during treatment for grade 1 ventricular arrhythmia was reported on day 4. This participant was otherwise asymptomatic throughout hospitalization and was discharged on day 5 according to protocol, and did not experience any further events of cytokine release syndrome or myocarditis during continued participation, including after escalation to 30 mg / kg.

[0137] Case 2 (Cohort 5, 30 mg / kg dose group), a 69-year-old male with NYHA class II heart failure and bilateral carpal tunnel syndrome, experienced mild-intensity cytokine release syndrome approximately 9 hours after completion of the second dose of NI006 / ALXN2220, characterized by progressive swelling, warmth, and pain in both wrists, muscle pain in both thighs, and elevated C-reactive protein (23 mg / l, within the normal range of <5 mg / l) and interleukin-6 (108 pg / ml). For this reason, he was treated with a cool pack and antipyretics. There were no clinically significant changes in blood pressure, heart rate, body temperature, or oxygen saturation at the same time. Following resolution of symptoms and improvement of inflammatory markers, this participant was discharged according to protocol on day 3 post-infusion. This participant continued treatment as planned and experienced recurrent pain in different small joints of both the upper and lower extremities, including a moderate recurrence of reactive arthritis of the wrist with a minimal elevation of C-reactive protein (11.4 mg / l) occurring a few days after the third dose, for which he was treated with glucocorticoids. This patient also had two reported cardiac-related events: myocarditis (day 53) associated with a peak troponin of 45.3 pg / ml (pre-medication level: 31.8 pg / ml) without any other cardiac abnormalities (cMRI was not performed), and myocarditis associated with a peak troponin of 45.3 pg / ml (pre-medication level: 31.8 pg / ml) following the second dose of the study drug, without associated symptoms. The troponin subsequently returned to baseline by day 65, and a further event of troponin elevation (days 99–120, peak level: 45 pg / ml) occurred after the third dose of the study drug. This participant continued treatment as planned during the open-label continuation, including a total of 10 doses of NI006 / ALXN2220 at 30 mg / kg.

[0138] Case 3 (Cohort 5, 30 mg / kg dose group), a 60-year-old male with NYHA class II heart failure, experienced mild-intensity cytokine release syndrome approximately 30 minutes after completion of the first dose of NI006 / ALXN2220, characterized by facial and arm flushing, headache, and elevated body temperature (39.2°C). For this reason, he was treated with glucocorticoids and antipyretics. Laboratory findings demonstrated a moderate increase in interleukin-6 (45.3 pg / ml, <12.9 pg / ml baseline) on day 3 and a moderate increase in C-reactive protein (65.7 mg / l) on day 4. This participant was asymptomatic with resolved inflammatory markers upon discharge according to protocol on day 5 and did not experience any further events of cytokine release syndrome during continued participation. However, non-serious events (mild, related), an increase in troponin T (troponin T level was 57.8 pg / ml on day 29 but resolved by day 37 [pre-infusion level: 40.8 pg / ml]), and right bundle branch block (currently day 29–422) were reported at the 8th visit (reported to resolve 29 days after the initial infusion and 2 weeks after cytokine release syndrome). This patient completed a single dose escalation / repeated dose escalation phase and was escalated to 60 mg / kg during open-label continuation without further events.

[0139] [Table 6] The severity of adverse events (AEs) is classified according to the Common Terminology Criteria for Adverse Events (CTCAE) grading scale, with Grade 0 being asymptomatic and Grade 5 being death related to the adverse event. The data is presented as n (percentage in the cohort) of patients and [number of events]. IMP indicates the investigational drug (NI006 / ALXN2220 or placebo), and SAE indicates a serious adverse event.

[0140] [Table 7]

[0141] [Table 8] The numbers represent n patients (percentage within the group) and [n events]. * Patients are counted only once at the highest grade of the Common Terminology Criteria for Adverse Events (CTCAE). Terminology is provided for adverse events that lead to temporary discontinuation (d / c) of the investigational drug (IMP) (NI006 or placebo) or discontinuation of the study.

[0142] [Table 9]

[0143] [Table 10-1]

[0144] [Table 10-2] The numbers represent n patients (percentage within the group) and [n events]. * Patients are counted only once at the highest grade of the Common Terminology Criteria for Adverse Events (CTCAE). Terminology is provided for fatal adverse events and adverse events leading to temporary discontinuation (d / c) of the investigational drug (IMP) (NI006 / ALXN2220 or placebo) or discontinuation of the study.

[0145] Pharmacokinetic and immunogenicity profiles The pharmacokinetic profile of NI006 / ALXN2220 was consistent with human IgG characteristics, exhibiting low to moderate inter-subject variability. After a single intravenous administration, serum NI006 / ALXN2220 concentrations decreased biphasically, with elimination half-lives ranging from 15.5 to 19.2 days. Exposure to NI006 / ALXN2220, measured as maximum concentration and area under the curve, increased proportionally with higher doses. No patients developed anti-drug antibodies throughout the study, including during open-label continuation phases.

[0146] Amyloid depletion After 4 months of therapy, higher total NI006 / ALXN2220 exposure appeared to be associated with reduced cardiac tracer uptake on scintigraphy and reduced extracellular volume on cMRI compared to baseline. After up to 12 months of continued treatment with NI006 / ALXN2220, cardiac tracer uptake and extracellular volume appeared to decrease further. In contrast, among patients randomized to placebo, there was a clear increase in cardiac tracer uptake on scintigraphy and increased extracellular volume on cMRIs at 4 months, and after these patients entered open-label continued administration and received NI006 / ALXN2220 for 8 months, these parameters appeared to decrease. Representative scintigraphy and cMRI images from NI006 / ALXN2220-treated and placebo-treated patients are shown in Figure 1. At a dose of at least 10 mg / kg, there was a significant reduction in cMRI extracellular volume from 63.4±13% to 54.1±9.9% (median 59.4% (IQR 56.2~68.7) to 49.0% (IQR 48.4~57.3)) at 4 months, and from 45.0±10.4% (median 41.6% (IQR 39.1~49.2)) at 12 months (n=3) (Table 7), as well as from 5.9±2.2% to 5.0±2.7% (n=11) (median 5.7% (IQR 4.3~6.9) to 3.8% (IQR 3.3~6.2)) at 4 months, and A significant reduction in the scintigraphy cardiac-to-whole-body ratio was observed at 12 months (n=6) (Table 8), with a median of 2.7±0.6% (IQR 2.3-3.2). Furthermore, a reduction in tracer uptake at the shoulder joint was observed after 4 months of treatment, and after up to 12 months of continuous treatment with NI006 / ALXN2220, tracer uptake at the shoulder appeared to be further reduced. See Patient 3 in Figure 1. In Patient 1, a reduction in tracer uptake at the elbow joint was observed after 12 months of treatment compared to 4 months of treatment.

[0147] [Table 11] The numbers represent the mean ± standard deviation and the median (interquartile range). aFor patients switched from placebo to NI006 / ALXN2220, absolute and relative changes from baseline at 12 months were calculated using baseline data from before open-label continued administration.

[0148] [Table 12] The numbers represent the mean ± standard deviation and the median (interquartile range). a For patients switched from placebo to NI006 / ALXN2220, absolute and relative changes from baseline at 12 months were calculated using baseline data from before open-label continued administration.

[0149] Cardiac biomarkers and other parameters Changes in NT-proBNP and troponin T appeared to be associated with higher doses of NI006 / ALXN2220. At doses of at least 10 mg / kg, the significant reduction from baseline in NT-proBNP at 12 months was from 2798±1577 (n=15) to 1398±1292 (n=9) (median 2460 pg / mL (IQR1443~4188, n=15) to 778 pg / mL (IQR234~2416, n=9)), and the significant reduction in troponin was from 51±22 (n=15) to 35±15 (n=8) (median 43 pg / mL (IQR35~72, n=15) to 35 pg / mL (IQR21~48, n=8)) (Table 9). In patients in the 30 mg / kg and 60 mg / kg cohorts, baseline NT-proBNP levels were 2691±1499 (n=5) and 2358±1906 (n=5), respectively (median 2658 pg / mL (IQR1403~4018, n=5) and 1482 pg / mL (IQR1193~2760, n=5), respectively), and decreased to 953±1267 (n=3) and 477±276 (n=3), respectively (median 223 (IQR221~1320, n=3) and 420 pg / mL (327~599, n=3)).

[0150] In the low-dose and high-dose cohorts, the median increase in KCCQ-OS scores from baseline to 12 months was 6.8 points among 11 out of 12 patients in the low-dose group and 6.0 points among 9 out of 15 patients in the high-dose group (Table 10). Changes in echocardiographic parameters and 6-minute walk distance at 12 months are shown in Figure 2 and Table 11.

[0151] [Table 13-1]

[0152] [Table 13-2]

[0153] [Table 14] The numbers represent the mean ± standard deviation and the median (interquartile range). a For patients switched from placebo to NI006 / ALXN2220, absolute and relative changes from baseline at 12 months were calculated using baseline data from before open-label continued administration.

[0154] [Table 15] The numbers represent the mean ± standard deviation and the median (interquartile range). aFor patients switched from placebo to NI006 / ALXN2220, absolute and relative changes from baseline at 12 months were calculated using baseline data prior to open-label extension. The Kansas City Cardiomyopathy Questionnaire-Comprehensive Summary Questionnaire (KCCQ-OS) was administered by investigators blinded to treatment allocation. Both the KCCQ-OS and the 6-minute walk test were administered by investigators blinded to treatment allocation. Patients switched from placebo received 4 months of placebo and, depending on the starting dose level, 8 months of either low-dose or high-dose NI006 / ALXN2220 during the open-label extension phase. Scores from the KCCQ-OS questionnaire ranged from 0 to 100, with 0-24 indicating very poor to poor, 25-49 indicating poor to average, 50-74 indicating average to good, and 75-100 indicating good to excellent quality of life.

[0155] [Table 16] The numbers represent the mean ± standard deviation and the median (interquartile range). a For patients switched from placebo to NI006 / ALXN2220, absolute and relative changes from baseline at 12 months were calculated using baseline levels prior to open-label continuation. Patients switched from placebo received 4 months of placebo and, depending on the starting dose level, 8 months of either low-dose or high-dose NI006 / ALXN2220 during open-label continuation. Echocardiography was analyzed blindly at the Central Imaging Core Laboratory. ED-IVS indicates the end-diastolic interventricular septum, LVEF indicates the left ventricular ejection fraction, LVESV indicates the left ventricular end-systolic volume, LVEDV indicates the left ventricular end-diastolic volume, LAV indicates the left atrial volume, and E / e' indicates the ratio of the initial mitral valve inflow velocity to the initial diastolic velocity of the mitral annulus.

[0156] Dose-response relationship for signs reported by the principal investigator of musculoskeletal immune activation To identify signs of musculoskeletal immune activation, clinically reported (adverse) events encoded in the System Organ Class (SOC) musculoskeletal and connective tissue disorders were used. As shown in Table 12, in the high-dose cohort (≥10 mg / kg) (Cohorts 4–7 of the NI006 / ALXN2220 group), signs of musculoskeletal immune activation were most frequent immediately after the initiation of treatment (SAD / MAD phase). Over time, the signs were reported less frequently. In placebo patients switched to high-dose NI006 / ALXN2220 during OLE (Cohorts 4–7 of the placebo group), signs of musculoskeletal immune activation were most frequently observed early after the initiation of treatment (OLE phase). In the low-dose cohorts (Cohorts 1-3 of the NI006 / ALXN2220 or placebo-NI006 / ALXN2220 and placebo groups, respectively), signs of musculoskeletal immune activation appeared with a delay when patients were escalated to doses of 10 mg / kg or higher (OLE2).

[0157] [Table 17]

[0158] In summary, the safety profile of NI006 / ALXN2220 appeared favorable up to the highest dose tested. There were no apparent dose-limiting toxicities, and no serious drug-related adverse reactions occurred. The pharmacokinetic profile was consistent with IgG antibodies, and no anti-drug antibodies were detected. At doses of at least 10 mg / kg, scintigraphy tracer uptake and extracellular volume assessed by cardiac MRI (both imaging surrogates for amyloid deposits) appeared to be reduced. In addition, scintigraphy tracer uptake in the shoulder and elbow also appeared to be reduced. Median NT-proBNP levels and troponin T levels also appeared to decrease over 12 months.

[0159] Consideration This Phase I study found that the safety, tolerability, pharmacokinetics, and pharmacodynamic profiles of the human anti-ATTR antibody NI006 / ALXN2220 appeared favorable in patients with ATTR-CM and chronic heart failure. As demonstrated by scintigraphy and cMRI, two imaging techniques considered surrogates for cardiac amyloid load, NI006 / ALXN2220 administration was associated with changes in extracellular volume on cMRI and cardiac-to-whole-body ratio on scintigraphy, particularly at doses exceeding 10 mg / kg every four weeks. These observations were supported by clear changes in cardiac biomarkers and functional measures.

[0160] The favorable safety profile of NI006 / ALXN2220, as well as the absence of anti-drug antibodies and dose-limiting toxicities, may be related to the human source of the amino acid sequence of NI006 / ALXN2220 and its selectivity for misfolded ATTRs that do not bind to physiological TTRs. See Michalon et al., Nat Commun 21(2021), 3142. In fact, no variation in TTR plasma levels was observed between dose cohorts (Table 13). The types, proportions, and severity of cardiac adverse events appeared to be comparable to those reported in larger Phase 3 trials of tafamidis (Maurer et al., N Engl J Med 379 (2018), 1007-1016) and patisiran (Maurer et al., Primary Results from APOLLO-B, A Phase 3 Study of Patisiran in Patients with Transthyretin-Mediated Amyloidosis with Cardiomyopathy, Heart Failure Society of America (HFSA) Annual Scientific Meeting (2022), Gaylord National Harbor, Washington, DC), which recruited similar patient samples. A notable exception between the AEs in these studies and those in our study is the presence of arthralgia in our study (see Table 12), which is more common with NI006 / ALXN2220 and may be related to the activation of NI006 / ALXN2220 by phagocytic immune cells targeting musculoskeletal ATTR deposits (see Basdavanos et al., Am J Cardiol 190 (2023), 67-74; Rubin et al., Amyloid 24 (2017), 226-230), which may explain the simultaneous reduction in tracer uptake in the shoulder and elbow joints observed during this study. Currently, the primary mechanism for cardiac dysfunction in ATTR-CM is hypothesized to be mechanical dysfunction caused by amyloid deposits, leading to increased ventricular rigidity and diastolic dysfunction.See Griffin et al., Circ Res 128(2021), 1554-1575. Therefore, reducing the amyloid load is a rational therapeutic target in ATTR-CM. Available data from ATTR-CM patients show that extracellular volume on cMRI correlates with amyloid load determined by histological analysis, is associated with other markers of ATTR disease status, and predicts mortality. See Martinez-Naharro et al., J Am Coll Cardiol 70(2017), 466-477, Pucci et al., J Am Heart Assoc 10(2021), e020358, Morioka et al., J Am Heart Assoc 11(2022), e024717. Bisphosphonate scintigraphy uptake is also associated with histological cardiac amyloid load and outcomes in ATTR-CM patients. See Morioka et al., J Am Heart Assoc 11 (2022), e024717; Castano et al., JAMA Cardiol 1 (2016), 880-889; Rapezzi et al., JACC Cardiovasc Imaging 4 (2011), 659-70; Hutt et al., Eur Heart J Cardiovasc Imaging 18 (2017), 1344-1350. TTR tetramer stabilizers, drugs acting as silencers, and TTR gene editing therapies are designed to prevent ATTR accumulation, but they do not directly target amyloid already deposited in the heart. See Ioannou et al., Circulation 146(2022), 1657-1670, Lopez-Sainz et al., Rev Esp Cardiol (Engl Ed) 74(2021), 149-158, Gillmore et al., N Engl J Med 385(2021), 493-502.While there is some evidence that therapies stabilizing or silencing TTR can reduce amyloid load compared to untreated populations, as demonstrated by scintigraphy and cardiac MRI imaging proxies (Chamling et al., Clin Res Cardiol 11(2023), 353-362; Fontana et al., JACC Cardiovasc Imaging 14(2021), 189-199; Odouard et al., JACC Cardiovasc Imaging 15(2022), 2149-2151; Rettl et al., Eur Heart J Cardiovasc Imaging 2023, doi:10.1093 / ehjci / jead030), substantial improvement is rare (Wu et al., ESC Heart Fail 9(2022), 4335-4339). In contrast, NI006 / ALXN2220 at doses of at least 10 mg / kg appeared to reduce cardiac-to-whole-body ratio on scintigraphy and extracellular volume on cMRI at 4 and 12 months after treatment. These findings support the proof of concept for using NI006 / ALXN2220 to treat ATTR-CM patients and appear consistent with preclinical data demonstrating the activity of NI006 / ALXN2220 in inducing ATTR removal. See Michalon et al., Nat Commun 21(2021), 3142.

[0161] In ATTR-CM patients, NT-pro-BNP and troponin levels are frequently elevated and associated with a worse prognosis. See Grogan et al., J Am Coll Cardiol 68 (2016), 1014-20; Gillmore et al., Eur Heart J 39 (2018), 2799-2806; and Law et al., ESC Heart Fail 7 (2020), 3942-3949. Echocardiographic assessments and functional and quality-of-life measures, such as the 6-minute walk distance and KCCQ-OS score, are also frequently used to measure disease progression. See Garcia-Pavia et al., Eur J Heart Fail 23 (2021), 895-905. In a recent study, a clinically significant increase in NT-proBNP (500 pg / ml from baseline to 12 months) was independently associated with a worse prognosis. See Law et al., Heart 108 (2022), 474-478. Biomarkers including NT-proBNP and troponin T, echocardiographic evaluation, and clinical evaluation suggest clinical improvement.

[0162] [Table 18]

[0163] In conclusion, this Phase I proof-of-concept study demonstrates the safety profile of NI006 / ALXN2220 in patients with ATTR-CM and supports the suitability of NI006 / ALXN2220 for the treatment of patients with ATTR-CM.

[0164] Example 2: Characterization of mature NI006 / ALXN2220 The antibody NI006 / ALXN2220 was produced in the CHO-K1 cell line (ATCC number CCL 61), cultured in a large-scale production bioreactor, and then obtained from the cell culture. The amino acid sequences of the mature heavy chain (HC) and light chain (LC) of NI006 / ALXN2220 are shown in SEQ ID NOs. 18 and 19, with the following modifications. The total number of amino acids is 1328, the number of heavy chain amino acids is 450, and the number of light chain amino acids is 214.

[0165] Further characterization of the antibody NI006 / ALXN2022 was performed primarily by standard procedures, such as mass spectrometry. For example, post-translational modifications of NI006 / ALXN2220 were identified using liquid chromatography analysis by tandem mass spectrometry (LC-MS / MS) of fragments of NI006 / ALXN2220 obtained from sequential lys-C and trypsin digestion and free sulfhydryl analysis. Characterization of antibody-based therapeutics by LC-MS analysis is a standard procedure and can be performed by those skilled in the art. See, for example, Robotham and Kelly, Approaches to the Purification, Analysis and Characterization of Antibody-Based Therapeutics (2020), 1–33.

[0166] N-glycans were released using PNGase F, subsequently labeled with 2-AB, separated by HILIC (Hydrophilic Interaction Chromatography), and then detected by fluorescence (FLD) using a UPLC system for N-glycan profiling. Individual N-glycans and unknown peaks were quantified by their peak area percentage relative to the total peak area.

[0167] The results are described below.

[0168] The molecular weight of antibody NI006 / ALXN2220, as determined by standard mass spectrometry, is approximately 147.1 kDa for intact IgG1 and 144.2 kDa for the deglycosylated variant.

[0169] The monoclonal antibody NI006 / ALXN2220 is an IgG1 subclass antibody, consisting of two heavy chains of the IgG1 subclass and two light chains of the kappa subclass. The four chains are stabilized by multiple disulfide bonds. In particular, as determined by standard procedures, namely Lys-C and trypsin digestion and subsequent LC-MS, at least the following disulfide crosslinks are present in NI006 / ALXN2220. LC:C23-LC:C88 LC:C134-LC:C194 LC:C214-HC:C223 HC:C22-HC:C97 HC:C147-HC:C203 HC1:229-HC2:229 and HC1:232-HC2:232 HC:C264-HC:C324 HC:C370-HC:C428

[0170] NI006 / ALXN2220 is a glycoprotein, and the constant region of each heavy chain contains one N-linked glycan site at residue N300. During the determination of the glycosylation profile, the major N-glycan types were shown to be G0F (approximately 49.0%) and G1F (approximately 25.4%). More specifically, the following glycosylation profile (sugar type, location of glycosylation site, etc.) has been determined for NI006 / ALXN2220.

[0171] [Table 19] Note 1. The nomenclature for glycans follows the order HexNac-Hexose-Fucose-NeuAc-NeuGc. For example, 23000 is HexNac(2)-Hexose(3)-Fucose(0)-NeuAc(0)-NeuGc(0). 2. G1Fa and G1Fb are isomers and are classified as G1F. G1F is calculated as the sum of G1Fa and G1Fb using the original unrounded numbers.

[0172] Furthermore, N-terminal glutamine modified with pyroglutamic acid (abundance in sample: 99.9%) and C-terminal lysine clipping of the heavy chain (abundance in sample: 95.8%) have been identified as major post-translational modifications. In addition, minor ratio modifications such as methionine oxidation, asparagine deamide, and asparagine succinimide formation were experimentally determined, as shown in Table 15 below.

[0173] [Table 20] Note 1. HC refers to the heavy chain, and LC refers to the light chain. 2. The peptide sequences in the underlined font were identified as PTM sites. 3. * refers to the N-terminal peptide of the heavy chain, and # refers to the C-terminal peptide of the heavy chain. 4. / indicates that no PTM (Post-Treatment Machine) has been reported. 5. pE(Q) represents an N-terminal glutamine modified with pyroglutamic acid. 6.-K refers to the loss of C-terminal lysine. 7. KG amidation (P) refers to the amidation of C-terminal proline after the loss of C-terminal lysine and glycine.

[0174] In summary, N-linked glycosylation of the heavy chain, N-terminal pyroglutamic acid modified from N-terminal glutamine, and C-terminal lysine clipping of the heavy chain are the major post-translational modifications of NI006 / ALXN2220.

Claims

1. An antibody capable of binding to mutated, misfolded, misassembled, and / or aggregated transthyretin (TTR) species for use in, for example, a method of treating or preventing musculoskeletal disorders or conditions in human patients, wherein the antibody is administered once every 28 days at a dose of at least about 30 mg / kg.

2. The antibody for use according to claim 1, wherein the treatment results in the removal of TTR aggregates associated with the musculoskeletal disorder or condition.

3. The antibody for use according to claim 1 or 2, wherein the method comprises administering the antibody once every 28 days at a dose of about 30 mg / kg or about 60 mg / kg.

4. The antibody for use according to any one of claims 1 to 3, wherein the method comprises administering the antibody for at least 4 to 12 months.

5. The antibody for use according to any one of claims 1 to 4, wherein the treatment results in a reduction of the median amyloid level when the dose of the antibody corresponds to a dose of 30 to 60 mg / kg in the patient.

6. The antibody for use according to any one of claims 1 to 5, wherein the subject is screened for the presence of the musculoskeletal disorder or condition by determining scintigraphy tracer uptake in joints and / or bones, and increased uptake compared to a healthy control indicates the onset or presence of the disorder or condition.

7. The antibody for use according to any one of claims 1 to 6, wherein the progression of the musculoskeletal disorder or condition or the progression of the treatment is monitored by determining a relative change in scintigraphy tracer uptake in the joints and / or bones, with an increase in uptake indicating the progression of the disorder or condition and a decrease in uptake indicating the effectiveness of the treatment.

8. The tracer is a bisphosphonate, preferably, ９９ｍ Tc-hydroxyl-methylene-diphosphonate (HMDP) or ９９ｍ An antibody for use according to claim 6 or 7, comprising Tc-3,3-diphosphono-1,2-propanodicarboxylic acid (DPD).

9. An antibody for use according to any one of claims 1 to 8, which binds to mutated and wild-type agglutinated TTR species and does not recognize physiological TTR species.

10. An antibody for use according to any one of claims 1 to 9, which does not bind to monomers or dimers of human natural TTR.

11. An antibody for use according to any one of claims 1 to 10, capable of binding to a TTR epitope comprising or consisting of the amino acid sequence WEPFA (SEQ ID NO: 15).

12. An antibody for use according to any one of claims 1 to 11, which is a human-derived anti-TTR antibody.

13. The antibody or its antigen-binding fragment has the following complementarity-determining regions (CDRs) of the VH and VL region amino acid sequences in its variable region or binding domain: a) VH-CDR1: Positions 31-35 of Sequence ID No. 2 VH-CDR2: Positions 52-67 of Sequence ID No. 2 VH-CDR3: Sequence ID 2, positions 100-109 VL-CDR1: Positions 24-34 of Sequence ID No. 4 VL-CDR2: Positions 50-56 of Sequence ID No. 4 VL-CDR3: Positions 89-97 of Sequence ID No. 4, or b) An antibody for use according to any one of claims 1 to 12, comprising VH-CDR1, VH-CDR2, and VH-CDR3 and VL-CDR1, VL-CDR2, and VL-CDR3, the amino acid sequences of which are at least 80% identical to the amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 of (a).

14. The antibody or its antigen-binding fragment has a variable region or binding domain. (a) The amino acid sequence of the VH region described in Sequence ID No. 2 or 6, and the amino acid sequence of the VL region described in Sequence ID No. 4, or (b) The aforementioned V of (a) Ｈ and V Ｌ V is at least 80% identical to the amino acid sequence of the region. Ｈ and V Ｌ The amino acid sequence of the region, or (c) The amino acid sequence of the VH region described in SEQ ID NO: 16, and the amino acid sequence of the VL region described in SEQ ID NO: 17, or (d) The V of (c) Ｈ and V Ｌ and a V that is at least 80% identical to the amino acid sequence of the V Ｈ region, or Ｌ the amino acid sequence of the V region, or (e) The amino acid sequence of the VH region described in SEQ ID NO: 16, and the amino acid sequence of the VL region described in SEQ ID NO: 4, or (f)(e) The aforementioned V Ｈ and V Ｌ V is at least 80% identical to the amino acid sequence of the region. Ｈ and V Ｌ An antibody for use according to any one of claims 1 to 13, characterized by comprising the amino acid sequence of a region.

15. The antibody for use according to any one of claims 1 to 11, characterized in that the antibody or its antigen-binding fragment contains in its variable region or binding domain the amino acid sequence of the VH region described in SEQ ID NO: 6 and the amino acid sequence of the VL region described in SEQ ID NO:

4.

16. The antibody for use according to any one of claims 1 to 11, characterized in that the antibody or its antigen-binding fragment contains in its variable region or binding domain the amino acid sequence of the VH region described in SEQ ID NO: 16 and the amino acid sequence of the VL region described in SEQ ID NO:

17.

17. An antibody for use according to any one of claims 1 to 16, comprising a human Ig constant region.

18. An antibody for use according to any one of claims 1 to 17, wherein the antibody is human IgG, preferably human IgG1.

19. An antibody for use according to any one of the above claims 1 to 18, which is a human IgG1m3 allotype.

20. An antibody for use according to any one of claims 17 to 19, comprising a kappa (κ) light chain.

21. An antibody for use according to any one of claims 1 to 20, wherein the antibody is NI006 / ALXN2220 and a human IgG1m3 allotype.

22. a) Each heavy chain consists of 450 amino acid residues having SEQ ID NO: 18, and each light chain consists of 214 amino acid residues having SEQ ID NO: 19, or b) Each heavy chain consists of 449 amino acid residues having SEQ ID NO: 20, and each light chain consists of 214 amino acid residues having SEQ ID NO: 19, or c) Each heavy chain consists of 449 amino acid residues having SEQ ID NO: 21, and each light chain consists of 214 amino acid residues having SEQ ID NO: 10, or d) The antibody for use according to claim 21, wherein each heavy chain is composed of 448 amino acid residues having SEQ ID NO: 22, and each light chain is composed of 214 amino acid residues having SEQ ID NO:

10.

23. An antibody for use according to any one of claims 1 to 22, which is produced in CHO-K1 host cells and purified from a cell culture.

24. The antibody for use according to any one of claims 15 to 23, wherein the heavy chain of the antibody has undergone N-terminal glutaminyl cyclization and / or C-terminal lysine clipping.

25. An antibody for use according to any one of claims 15 to 24, wherein it is N-glycosylated, and preferably, the N-linked glycan is located on N300 of the heavy chain.

26. The antibody for use according to any one of claims 1 to 25, wherein the musculoskeletal disorder or condition is accompanied by or related to amyloid-transthyretin cardiomyopathy (ATTR-CM).

27. The antibody for use according to any one of claims 1 to 26, wherein the antibody is in the form of a pharmaceutical composition comprising a physiologically acceptable diluent or carrier.

28. The antibody for use according to any one of claims 1 to 27, wherein the musculoskeletal disorder or condition is related to ATTR and is preferably selected from the group consisting of osteoarthritis, carpal tunnel syndrome, joint pain, shoulder joint pain, amyloid arthropathy, lumbar spinal stenosis, biceps brachii tendon rupture, trigger finger, and rotator cuff disorder, and preferably the musculoskeletal disorder or condition is osteoarthritis or amyloid arthropathy, preferably amyloid arthropathy.

29. The antibody for use according to claim 28, wherein the musculoskeletal disorder or condition is characterized by aggregated TTR, preferably wild-type TTR, deposits in a joint, and most preferably the disorder or condition is shoulder TTR amyloidosis, particularly wild-type TTR amyloidosis.

30. The antibody for use according to any one of claims 1 to 28, wherein the antibody is administered by intravenous injection or by direct injection into / at the joint.

31. The antibody for use according to any one of claims 1 to 29, wherein the method comprises administering the antibody in combination with a disease-modifying antirheumatic drug, preferably the disease-modifying antirheumatic drug being a corticosteroid, and / or the method comprises administering the antibody in combination with an anti-inflammatory agent, preferably the anti-inflammatory agent being a nonsteroidal anti-inflammatory drug (NSAID).

32. The antibody for use according to any one of claims 1 to 31, wherein the method comprises administering the antibody in combination with a TTR tetramer stabilizer.

33. A kit for use in methods for treating or preventing musculoskeletal disorders or conditions in human patients, wherein the kit is (a) A dose of an anti-transthyretin (TTR) antibody capable of binding to a mutated, misfolded, misassembled, or aggregated transthyretin (TTR) species, preferably the antibody being an antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 25, (b) A kit comprising instructions for using the anti-TTR antibody in the method according to any one of claims 1 to 32 or 44 to 76.

34. The kit according to claim 33, wherein the musculoskeletal disorder or condition is related to ATTR and is preferably selected from the group consisting of osteoarthritis, carpal tunnel syndrome, joint pain, shoulder joint pain, amyloid arthropathy, lumbar spinal stenosis, biceps brachii tendon rupture, trigger finger, and rotator cuff disorder, and preferably the musculoskeletal disorder or condition is osteoarthritis or amyloid arthropathy, preferably amyloid arthropathy.

35. The antibody or its antigen-binding fragment has the following complementarity-determining regions (CDRs) of the VH and VL region amino acid sequences in its variable region or binding domain: VH-CDR1: Positions 31-35 of Sequence ID No. 2 VH-CDR2: Positions 52-67 of Sequence ID No. 2 VH-CDR3: Sequence ID 2, positions 100-109 VL-CDR1: Positions 24-34 of Sequence ID No. 4 VL-CDR2: Positions 50-56 of Sequence ID No. 4 VL-CDR3: Includes positions 89-97 of sequence number 4, Preferably, the kit according to claim 32 or 33, wherein the antibody is NI006 / ALXN2220 and human IgG1m3 allotype.

36. A pharmaceutical composition, component kit, or product comprising: (i) an antibody capable of binding to mutated, misfolded, misassembled, or aggregated transthyretin (TTR) species and capable of removing ATTR by inducing phagocytosis, preferably the antibody being an antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 25; (ii) a disease-modifying antirheumatic drug, anti-inflammatory agent, or TTR tetramer stabilizer; and optionally (iii) a pharmaceutically acceptable carrier.

37. The antibody or its antigen-binding fragment has the following complementarity-determining regions (CDRs) of the VH and VL region amino acid sequences in its variable region or binding domain: VH-CDR1: Positions 31-35 of Sequence ID No. 2 VH-CDR2: Positions 52-67 of Sequence ID No. 2 VH-CDR3: Sequence ID 2, positions 100-109 VL-CDR1: Positions 24-34 of Sequence ID No. 4 VL-CDR2: Positions 50-56 of Sequence ID No. 4 VL-CDR3: Includes positions 89-97 of sequence number 4, Preferably, the antibody is NI006 / ALXN2220 and human IgG1m3 allotype, a pharmaceutical composition, component kit, or product.

38. Use of a pharmaceutical composition, component kit, or product comprising an antibody capable of binding to mutated, misfolded, misassembled, or aggregated transthyretin (TTR) species and removing ATTR by inducing phagocytosis, preferably the antibody being an antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 25, and optionally together with (i) a disease-modifying antirheumatic agent, or (ii) an anti-inflammatory agent, or (iii) a TTR tetramer stabilizer, in the prevention, treatment, or diagnosis of musculoskeletal disorders or conditions in a subject.

39. The use according to claim 38, wherein the musculoskeletal disorder or condition is related to ATTR and is preferably selected from the group consisting of osteoarthritis, carpal tunnel syndrome, joint pain, shoulder joint pain, amyloid arthropathy, lumbar spinal stenosis, biceps brachii tendon rupture, trigger finger, and rotator cuff disorder, and preferably the musculoskeletal disorder or condition is osteoarthritis or amyloid arthropathy, preferably amyloid arthropathy.

40. The antibody or its antigen-binding fragment has the following complementarity-determining regions (CDRs) of the VH and VL region amino acid sequences in its variable region or binding domain: VH-CDR1: Positions 31-35 of Sequence ID No. 2 VH-CDR2: Positions 52-67 of Sequence ID No. 2 VH-CDR3: Sequence ID 2, positions 100-109 VL-CDR1: Positions 24-34 of Sequence ID No. 4 VL-CDR2: Positions 50-56 of Sequence ID No. 4 VL-CDR3: Includes positions 89-97 of sequence number 4, Preferably, the use according to claim 38 or 39, wherein the antibody is NI006 / ALXN2220 and human IgG1m3 allotype.

41. An antibody capable of binding to mutated, misfolded, misassembled, or aggregated transthyretin (TTR) species for use in a method for diagnosing musculoskeletal disorders or conditions in human patients, preferably the antibody being an antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 25.

42. An antibody for use in the method of claim 41, wherein the musculoskeletal disorder or condition is related to ATTR and is preferably selected from the group consisting of osteoarthritis, carpal tunnel syndrome, joint pain, shoulder joint pain, amyloid arthropathy, lumbar spinal stenosis, biceps brachii tendon rupture, trigger finger, and rotator cuff disorder, and preferably the musculoskeletal disorder or condition is osteoarthritis or amyloid arthropathy, preferably amyloid arthropathy.

43. The antibody or its antigen-binding fragment has the following complementarity-determining regions (CDRs) of the VH and VL region amino acid sequences in its variable region or binding domain: VH-CDR1: Positions 31-35 of Sequence ID No. 2 VH-CDR2: Positions 52-67 of Sequence ID No. 2 VH-CDR3: Sequence ID 2, positions 100-109 VL-CDR1: Positions 24-34 of Sequence ID No. 4 VL-CDR2: Positions 50-56 of Sequence ID No. 4 VL-CDR3: Includes positions 89-97 of sequence number 4, Preferably, the antibody for use in the method according to claim 41 or 42 is NI006 / ALXN2220 and human IgG1m3 allotype.

44. A method for treating or preventing a musculoskeletal disorder or condition in a human subject, comprising administering to the subject an anti-TTR antibody capable of binding to mutated, misfolded, misassembled, and / or aggregated transthyretin (TTR) species.

45. The method according to claim 44, wherein the treatment results in the removal of TTR aggregates associated with the musculoskeletal disorder or condition.

46. The method according to claim 44 or 45, wherein the antibody is administered in a dose of at least about 10 mg / kg, 30 mg / kg, or 60 mg / kg.

47. The method according to any one of claims 44 to 46, wherein the antibody is administered approximately once every 28 days for at least about 4 to 12 months.

48. The method according to any one of claims 44 to 46, wherein the antibody is administered once every 28 days at a dose of at least about 30 mg / kg.

49. The method according to any one of claims 44 to 48, wherein the treatment results in a reduction of the median amyloid level when the dose of the antibody corresponds to a dose of 30 to 60 mg / kg in the patient.

50. The method according to any one of claims 44 to 49, wherein the subject is screened for the presence of the musculoskeletal disorder or condition by determining scintigraphy tracer uptake in the joints and / or bones, and increased uptake compared to a healthy control indicates the onset or presence of the disorder or condition.

51. The method according to any one of claims 44 to 50, wherein the progression of the musculoskeletal disorder or condition or the progression of the treatment is monitored by determining relative changes in scintigraphy tracer uptake in the joints and / or bones, wherein an increase in uptake indicates the progression of the disorder or condition, and a decrease in uptake indicates the effectiveness of the treatment.

52. The tracer is a bisphosphonate, preferably ９９ｍ Tc-hydroxyl-methylene-diphosphonate (HMDP) or ９９ｍ The method according to claim 50 or 51, comprising Tc-3,3-diphosphono-1,2-propanodicarboxylic acid (DPD).

53. The method according to any one of claims 44 to 52, wherein the antibody binds to mutated and wild-type agglutinated TTR species and does not recognize physiological TTR species.

54. The method according to any one of claims 44 to 53, wherein the antibody does not bind to monomers and dimers of human natural TTR.

55. The method according to any one of claims 44 to 53, wherein the antibody is capable of binding to a TTR epitope comprising or consisting of the amino acid sequence WEPFA (SEQ ID NO: 15).

56. The method according to any one of claims 44 to 55, wherein the antibody is a human-derived anti-TTR antibody.

57. The antibody or its antigen-binding fragment has the following complementarity-determining regions (CDRs) of the VH and VL region amino acid sequences in its variable region or binding domain: a) VH-CDR1: Positions 31-35 of Sequence ID No. 2 VH-CDR2: Positions 52-67 of Sequence ID No. 2 VH-CDR3: Sequence ID 2, positions 100-109 VL-CDR1: Positions 24-34 of Sequence ID No. 4 VL-CDR2: Positions 50-56 of Sequence ID No. 4 VL-CDR3: Positions 89-97 of Sequence ID No. 4, or b) The method according to any one of claims 44 to 56, comprising VH-CDR1, VH-CDR2, and VH-CDR3, and VL-CDR1, VL-CDR2, and VL-CDR3, the amino acid sequence of which is at least 80% identical to the amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 of (a).

58. The antibody or its antigen-binding fragment has a variable region or binding domain. (a) The amino acid sequence of the VH region described in Sequence ID No. 2 or 6, and the amino acid sequence of the VL region described in Sequence ID No. 4, or (b) The aforementioned V of (a) Ｈ and V Ｌ The V is identical to the amino acid sequence of the region by at least 80%. Ｈ and V Ｌ The amino acid sequence of the region, or (c) The amino acid sequence of the VH region described in SEQ ID NO: 16, and the amino acid sequence of the VL region described in SEQ ID NO: 17, or (d)(c) The aforementioned V Ｈ and V Ｌ The V is identical to the amino acid sequence of the region by at least 80%. Ｈ and V Ｌ The amino acid sequence of the region, or (e) The amino acid sequence of the VH region described in SEQ ID NO: 16, and the amino acid sequence of the VL region described in SEQ ID NO: 4, or (f)(e) The aforementioned V Ｈ and V Ｌ The V is identical to the amino acid sequence of the region by at least 80%. Ｈ and V Ｌ The method according to any one of claims 44 to 57, characterized by comprising the amino acid sequence of a region.

59. The method according to any one of claims 44 to 58, characterized in that the antibody or its antigen-binding fragment contains in its variable region or binding domain the amino acid sequence of the VH region described in SEQ ID NO: 6 and the amino acid sequence of the VL region described in SEQ ID NO: 4, or the amino acid sequence of the VH region described in SEQ ID NO: 16 and the amino acid sequence of the VL region described in SEQ ID NO:

17.

60. The method according to any one of claims 44 to 59, wherein the antibody comprises a human Ig constant region.

61. The method according to any one of claims 44 to 60, wherein the antibody is human IgG.

62. The method according to any one of claims 44 to 61, wherein the antibody is human IgG1.

63. The method according to any one of claims 44 to 62, wherein the antibody is a human IgG1m3 allotype.

64. The method according to any one of claims 60 to 63, wherein the antibody comprises a kappa (κ) light chain.

65. The method according to any one of claims 44 to 64, wherein the antibody is NI006 / ALXN2220 and human IgG1m3 allotype.

66. a) Each heavy chain of the antibody is composed of 450 amino acid residues having SEQ ID NO: 18, and each light chain is composed of 214 amino acid residues having SEQ ID NO: 19, or b) Each heavy chain of the antibody is composed of 449 amino acid residues having SEQ ID NO: 20, and each light chain is composed of 214 amino acid residues having SEQ ID NO: 19, or c) Each heavy chain of the antibody is composed of 449 amino acid residues having SEQ ID NO: 21, and each light chain is composed of 214 amino acid residues having SEQ ID NO: 10, or d) The method according to claim 65, wherein each heavy chain of the antibody is composed of 448 amino acid residues having SEQ ID NO: 22, and each light chain is composed of 214 amino acid residues having SEQ ID NO:

10.

67. The method according to any one of claims 44 to 66, wherein the antibody is produced in CHO-K1 host cells and purified from the cell culture.

68. The method according to any one of claims 58 to 67, wherein the heavy chain of the antibody has undergone N-terminal glutaminyl cyclization and / or C-terminal lysine clipping.

69. The method according to any one of claims 58 to 68, wherein the antibody is N-glycosylated, and preferably, the N-linked glycan is located on N300 of the heavy chain.

70. The method according to any one of claims 44 to 69, wherein the musculoskeletal disorder or condition is accompanied by or related to amyloid-transthyretin cardiomyopathy (ATTR-CM).

71. The method according to any one of claims 44 to 70, wherein the antibody is in the form of a pharmaceutical composition comprising a physiologically acceptable diluent or carrier.

72. The method according to any one of claims 44 to 71, wherein the musculoskeletal disorder or condition is related to ATTR and is preferably selected from the group consisting of osteoarthritis, carpal tunnel syndrome, joint pain, shoulder joint pain, amyloid arthropathy, lumbar spinal stenosis, biceps brachii tendon rupture, trigger finger, and rotator cuff disorder, and preferably the musculoskeletal disorder or condition is osteoarthritis or amyloid arthropathy, preferably amyloid arthropathy.

73. The method according to claim 73, wherein the musculoskeletal disorder or condition is characterized by aggregated TTR, preferably wild-type TTR, deposits in a joint, and most preferably the disorder or condition is shoulder TTR amyloidosis, particularly wild-type TTR amyloidosis.

74. The method according to any one of claims 44 to 73, wherein the antibody is administered by intravenous injection or by direct injection into / at the joint.

75. The method according to any one of claims 44 to 74, wherein the method comprises administering the antibody in combination with a disease-modifying antirheumatic drug, preferably the disease-modifying antirheumatic drug being a corticosteroid, and / or the method comprises administering the antibody in combination with an anti-inflammatory agent, preferably the anti-inflammatory agent being a nonsteroidal anti-inflammatory drug (NSAID).

76. The method according to any one of claims 44 to 75, wherein the method comprises administering the antibody in combination with a TTR tetramer stabilizer.

77. Use of antibodies capable of binding to mutated, misfolded, misassembled, and / or aggregated transthyretin (TTR) species for the manufacture of a pharmacopoeia for the treatment or prevention of musculoskeletal disorders or conditions in human subjects requiring such treatment.

78. The use according to claim 77, wherein the treatment or prevention comprises administering the antibody at a dose of at least about 30 mg / kg once every 28 days.

79. The use according to claim 77 or 78, wherein the antibody is an antibody as defined in any one of claims 1 to 78.

80. The use according to claim 77 or 78, wherein the subject is one of those defined in any one of claims 1 to 79.

81. The use according to any one of claims 77 to 79, wherein the administration of the antibody is carried out as defined in any one of claims 1 to 80.

82. The use according to any one of claims 77 to 80, wherein the treatment is performed as defined in any one of claims 1 to 81.

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