Anti-thyroxine transporter antibodies, compositions comprising the antibodies, and methods for treating or preventing thyroid hormone transporter-mediated amyloidosis

Abstract

This document provides anti-thyroxine transporter (TTR) antibodies, corresponding polynucleotides and expression vectors, and compositions (e.g., pharmaceutical compositions) containing anti-TTR antibodies as drugs, and related articles. This document also provides methods for treating or preventing thyroxine transporter-mediated amyloidosis (ATTR) in subjects of need using pharmaceutical compositions. Additionally, this document provides methods for validating, identifying, and screening amyloid depletion drugs using high-resolution live-cell imaging, methods for generating pharmaceutical compositions of amyloid depletion drugs, and kits suitable for said methods.

Description

Technical Field

[0001] This disclosure relates to anti-thyroxine transporter (TTR) antibodies, corresponding polynucleotides and expression vectors, compositions comprising said antibodies, and methods for treating or preventing thyroxine transporter-mediated amyloidosis (ATTR). This disclosure also relates to methods for validating, identifying, and screening amyloid depletion drugs using high-resolution live-cell imaging, methods for generating pharmaceutical compositions of amyloid depletion drugs, and kits suitable for said methods. Background Technology

[0002] Thyroxine transporter (TTR) is a soluble protein involved in the transport of thyroxine and retinol in the body. TTR is secreted by the liver in the blood and by the choroid plexus in the cerebrospinal fluid, and is also expressed in specific tissues such as pancreatic α cells or retinal epithelium.

[0003] Under specific conditions that are not yet elucidated and may include acidic pH, oxidative stress, and local factors, TTR proteins adopt misfolded, misassembled, and / or aggregated TTR conformations and become toxic, which may lead to thyroxine transporter-mediated amyloidosis (ATTR).

[0004] There is a need for pharmaceutical products containing antibodies against ATTRs suitable for treating or preventing ATTRs in subjects and antibodies against TTRs that target aggregation (e.g., human anti-TTR antibodies). Summary of the Invention

[0005] This article provides, in particular, an anti-thyroxine transporter (TTR) antibody, a corresponding polynucleotide and expression vector, and a composition (e.g., a pharmaceutical composition) containing the anti-TTR antibody as a drug, and related articles. Furthermore, this article particularly provides a method for treating or preventing thyroxine transporter-mediated amyloidosis (ATTR) in subjects of need using the pharmaceutical composition.

[0006] According to this disclosure, a human anti-TTR antibody has been manufactured that is specific for the disease-associated amyloid-forming form of TTR, characterized by ALXN2220, also known as NI006 and designated CAS Registry No. 2965214-50-8, i.e., by recombinantly expressing the heavy chain (HC) and light chain (LC) of the antibody in the Chinese hamster ovary (CHO) K1 cell line. This recombinant antibody has a unique post-translational modification (PTM) pattern and, in particular, a unique glycosylation profile; see Examples 1 to 3.

[0007] The antibody NI006 / ALXN2220 is a fully human IgG1m3 alloantibody and contains the human constant heavy chain (HC) amino acid sequence as shown in SEQ ID NO:7, 9, and 39, respectively, and the corresponding human constant light chain (LC), here the κ light chain, as shown in SEQ ID NO:8. As further explained below, the NI006 / ALXN2220 IgG1 antibody is formulated as a tetramer consisting of HC and two light LC chains linked by disulfide bridges. Furthermore, the closest human gene / alleles to the variable domain are IGKV1-3901 (93.3%) + IGKJ101 (100%) and IGHV4-30-201 (89.5%) + IGHJ302 (100%).

[0008] The antibody CDR of NI006 / ALXN2220 (e.g., variable heavy chain (VH) and / or variable light chain (VL) CDRs comprising VHCDR1-3 and VLCDR1-3, including variable heavy chain and light chain domains) was first described in WO 2015 / 092077 A1 (designated as antibody NI-301.37F1) and Michalon et al., Nat. Commun. 12 (2021), 3142 (designated as antibody NI301A), the relevant portions of the disclosures in these documents are incorporated herein by reference. This document describes the complete (e.g., full-length) variable heavy chain (VH) and light chain (VL) of the NI006 / ALXN2220 antibody, including its heavy chain and / or light chain constant regions. This disclosure also provides a full-length NI006 / ALXN2220 antibody containing PTM in its heavy chain and / or light chain, which is particularly suitable for providing a stable formulation that can be used as a pharmaceutical composition in ATTR treatment.

[0009] Specifically, the antibodies of this disclosure contain a modified glutamine at the N-terminus of the heavy chain sequence, wherein the modification includes pyroglutamic acid (with an abundance of about 99.9% or higher, e.g., 100%, in the sample, e.g., a recombinant formulation of the antibody). In another embodiment, the antibodies of this disclosure contain a cleaved C-terminal lysine residue in the heavy chain (with an abundance of about 95.8% or higher, e.g., 96%, 97%, 98%, 99%, or even 100% in the sample, e.g., a recombinant formulation of the antibody). Analysis of the antibody heavy chain sequence has identified the NI006 / ALXN2220 antibody isotype containing the deletion of both N-terminal pyroglutamic acid and C-terminal lysine residues as the major post-translational modification.

[0010] N-terminal pyroglutamate modification is beneficial in therapeutic antibodies by enhancing stability, consistency, and reducing immunogenicity. Specifically, N-terminal pyroglutamate modification helps protect the N-terminus of the antibody from enzymatic proteolytic degradation, which increases its stability and prolongs the antibody's half-life in circulation. Furthermore, pyroglutamate formation enhances the homogeneity of antibody preparations by ensuring a consistent N-terminal structure. This consistency can be beneficial for quality control and regulatory approval, as it results in a more uniform product with predictable properties. Additionally, it helps reduce the risk of immunogenicity. By cleaving the N-terminal glutamine or glutamate, unwanted immune responses that might otherwise be triggered by unmodified N-terminal residues are prevented, as these residues might otherwise be recognized as foreign. The enzyme responsible for converting N-terminal glutamine (or sometimes glutamate) to pyroglutamate is called glutamine acylcyclase. The activity level of this enzyme can vary across cell types and can significantly affect the degree of pyroglutamate formation.

[0011] C-terminal lysine cleavage is advantageous because it enhances charge homogeneity, reduces immunogenicity, improves consistency, and does not affect antibody functionality. Specifically, the presence of a C-terminal lysine adds a positive charge to the antibody, which can introduce charge heterogeneity into the antibody population. By cleaving the C-terminal lysine, antibody preparations become more charge-homogeneous, simplifying downstream processes such as purification and improving batch-to-batch consistency and homogeneity, which is beneficial for quality control. Furthermore, antibodies naturally lack C-terminal lysine due to in vivo proteolytic removal. Retaining lysine can make the antibody appear foreign to the immune system, potentially triggering an immune response. Removing this lysine reduces the risk of immunogenicity, making therapeutic antibodies more biocompatible. C-terminal lysine residues do not play a role in the binding of the antibody to its target antigen or in its interaction with the Fc receptor; therefore, their removal does not affect the therapeutic activity of the antibody, making their cleavage beneficial and not impairing efficacy. The efficiency of C-terminal lysine cleavage can depend on the cell line used for antibody production, as different cell lines vary in their protease activity and their efficiency in removing C-terminal lysine residues from antibodies.

[0012] Therefore, antibodies with N-terminal pyroglutamic acid modification and C-terminal lysine cleavage are beneficial for therapeutic purposes and are thus a preferred embodiment of the present invention.

[0013] Furthermore, as shown in Table 3, additional modifications, such as methionine oxidation, asparagine deamidation, and asparagine succinimide formation, have been experimentally identified. Therefore, this disclosure relates to a mature anti-TTR antibody comprising HC having the amino acid sequence SEQ ID NO: 7, wherein...

[0014] X1 is not present; it is either glutamine or pyroglutamic acid (pE).

[0015] X2 is methionine or oxidized methionine;

[0016] X3 is asparagine, deamidated asparagine, or asparagine containing succinimide;

[0017] X4 is asparagine or deamidated asparagine;

[0018] X5 is proline or amidated proline;

[0019] X6 is absent or may be glycine; and

[0020] X7 is absent or may be lysine.

[0021] And LC having the amino acid sequence of SEQ ID NO: 8.

[0022] Therefore, in one embodiment, the antibody of this disclosure comprises HC having the amino acid sequence of SEQ ID NO: 7, wherein the N-terminal glutamine (Q1) represented by X1 in SEQ ID NO: 7 is absent or present, preferably absent. In an embodiment, X1 is present as glutamine or pyroglutamic acid (pE); preferably, X1 is pE.

[0023] Alternatively or concurrently, the antibody of this disclosure comprises HC having the amino acid sequence of SEQ ID NO: 7, wherein the C-terminal lysine (K450) represented by X7 in SEQ ID NO: 7 is absent or present. In a preferred embodiment, X7 is absent.

[0024] In one embodiment, the antibody of this disclosure comprises HC having the amino acid sequence SEQ ID NO: 39, wherein the N-terminal residue X1 is absent or present as glutamine or pE. In an embodiment, X2 is methionine or oxidized methionine. In an embodiment, X3 is asparagine, deamidated asparagine, or asparagine containing succinimide. In an embodiment, X4 is asparagine, deamidated asparagine, or asparagine containing succinimide. In an embodiment, X5 is proline or amidated proline. In an embodiment, X6 is absent or glycine. In an embodiment, X7 is absent or lysine. In an embodiment, X... I It is asparagine or deamidated asparagine. In the implementation scheme, X II It is methionine or oxidized methionine. In the implementation plan, X III It is methionine or oxidized methionine. In the implementation plan, X IV It is aspartic acid or isoaspartic acid. In the implementation plan, X V It is asparagine or glycosylated asparagine. In the embodiment, X VIIt is methionine or oxidized methionine. In the implementation plan, X VII It is methionine or oxymethionine. In the embodiments, the antibody comprises an LC having the amino acid sequence of SEQ ID NO: 8.

[0025] In embodiments, the antibodies of this disclosure comprise HC having amino acid sequences having SEQ ID NO: 7 and 39, respectively. In some embodiments, X1 is pE. In some embodiments, X6 is absent. In some embodiments, X7 is absent. In some embodiments, X5 is amidated. In some embodiments, X2 is oxidized methionine. In some embodiments, X3 is deamidated asparagine. In other embodiments, X3 is asparagine containing succinimide. In some embodiments, X4 is deamidated asparagine.

[0026] In some implementation schemes, X I It is deamidated asparagine. In some embodiments, X II It is oxidized methionine. In some implementations, X III It is oxidized methionine. In some implementations, X IV It is isoflavone. In some implementations, X V It is glycosylated asparagine. In some embodiments, X VI It is oxidized methionine. In some implementations, X VII It is oxymethionine.

[0027] In some implementations, X1 is pE, X2 is methionine, X3 is asparagine, X4 is asparagine, X5 is proline, X6 is glycine, and X7 is absent.

[0028] In some preferred embodiments, X1 is pE, X2 is methionine, X3 is asparagine, X4 is asparagine, X5 is proline, X6 is glycine, X7 is absent, and X... I It's asparagine, X II It's methionine, X III It's methionine, X IV It is aspartic acid, X V It is glycosylated asparagine, X VI It is methionine, and X VII It is methionine.

[0029] In some embodiments, X1 is pE, X2 is oxidized methionine, X3 is deamidated asparagine or asparagine containing succinimide, X4 is deamidated asparagine or asparagine containing succinimide, X5 is proline, X6 is glycine, and X7 is absent.

[0030] In some embodiments, X1 is pE, X2 is oxidized methionine, X3 is deamidated asparagine or asparagine containing succinimide, X4 is deamidated asparagine or asparagine containing succinimide, X5 is amidated proline, X6 is absent, and X7 is absent.

[0031] In some embodiments, X1 is pE, X2 is oxidized methionine, X3 is deamidated asparagine or asparagine containing succinimidylamine, X4 is deamidated asparagine or asparagine containing succinimidylamine, X5 is proline, X6 is glycine, X7 is absent, and X... I It's asparagine, X II It's methionine, X III It's methionine, X IV It is aspartic acid, X V It is glycosylated asparagine, X VI It is methionine, and X VII It is methionine.

[0032] In some embodiments, X1 is pE, X2 is oxidized methionine, X3 is deamidated asparagine or asparagine containing succinimidylamine, X4 is deamidated asparagine or asparagine containing succinimidylamine, X5 is amidated proline, X6 is absent, X7 is absent, X... I It's asparagine, X II It's methionine, X III It's methionine, X IV It is aspartic acid, X V It is glycosylated asparagine, X VI It is methionine, and X VII It is methionine.

[0033] In embodiments, the C-terminal lysine (C450, numbered based on the HC polypeptide sequence containing N-terminal glutamine or pyroglutamic acid) of the antibody of this disclosure can be cleaved (e.g., by proteolytic cleavage). Therefore, in embodiments, the antibody of this disclosure comprises HC having the amino acid sequences of SEQ ID NO: 7 and 39, respectively, wherein the C-terminal lysine represented by X7 in SEQ ID NO: 7 and 39, respectively, is absent or present. Preferably, the C-terminal lysine at X7 in SEQ ID NO: 7 and 39, respectively, is absent.

[0034] In embodiments where the C-terminal lysine in the HC of the antibody of this disclosure is absent, the resulting C-terminal glycine (G449, numbered based on the HC polypeptide sequence containing N-terminal glutamine or pyroglutamic acid) may also be modified. Therefore, in embodiments, the antibody of this disclosure comprises HC having amino acid sequences of SEQ ID NO: 7 and 39, respectively, wherein the C-terminal lysine at X7 is absent, and the amino acid represented by glycine at X6 adjacent to the C-terminus of SEQ ID NO: 7 and 39, respectively, is absent or present.

[0035] In some embodiments, where neither the C-terminal lysine nor glycine is present in the HC of the antibody of this disclosure, the resulting C-terminal proline (P448, numbered based on the HC polypeptide sequence containing N-terminal glutamine or pyroglutamic acid) may be amidated. Therefore, in one embodiment, the antibody of this disclosure comprises HC having amino acid sequences of SEQ ID NO: 7 and 39, respectively, wherein neither the C-terminal lysine at X7 nor the glycine at X6 is present, and the amino acids at the C-terminus of SEQ ID NO: 7 and 39, respectively, and their adjacent amino acids represented by proline at X5, are amidated or unmodified, preferably amidated.

[0036] Alternatively or additionally, in an embodiment, the methionine at position 255 (M255, numbered based on the HC polypeptide sequence containing N-terminal glutamine or pyroglutamic acid) in the heavy chain of the antibody of this disclosure may be oxidized. Therefore, in one embodiment, the antibody of this disclosure comprises HC having the amino acid sequences of SEQ ID NO: 7 and 39, respectively, wherein M255, represented by the methionine at X2 in SEQ ID NO: 7 and 39, is oxidized (modified) or unmodified.

[0037] Alternatively or additionally, in embodiments, in the heavy chain of the antibody of this disclosure, the asparagine at position 318 (N318, numbered based on the HC polypeptide sequence containing N-terminal glutamine or pyroglutamic acid) may be unmodified or deamidated, or may contain succinimide. Thus, in one embodiment, the antibody of this disclosure comprises HC having the amino acid sequences of SEQ ID NO: 7 and 39, respectively, wherein N318, represented by asparagine at X3 in SEQ ID NO: 7 and 39, is modified or unmodified, preferably wherein the modified asparagine at X3 is deamidated (modified) or contains succinimide (modified).

[0038] Alternatively or additionally, in embodiments, in the heavy chain of the antibody of this disclosure, the asparagine at position 387 (N387, numbered based on the HC polypeptide sequence containing N-terminal glutamine or pyroglutamic acid) can be unmodified or modified, for example, wherein the modified amino acid is deamidinated. Therefore, in one embodiment, the antibody of this disclosure comprises HC having the amino acid sequences of SEQ ID NO: 7 and 39, respectively, wherein N387, represented by asparagine at X4 in SEQ ID NO: 7 and 39, is deamidinated (modified) or unmodified.

[0039] In embodiments, the antibodies of this disclosure comprise N-glycosylation. In some embodiments, at least one amino acid in the heavy chain of the antibody of this disclosure is N-glycosylated. Data from Example 2 show that N-glycosylation is a substantial PTM constituting the backbone amino acid sequence of the antibody of this disclosure. In this context, the N-glycosylation site was identified at position Asn300 (HC N300, numbered based on the antibody HC polypeptide sequences of SEQ ID NO: 7 and 39, respectively, having N-terminal glutamine or pyroglutamic acid (X1)).

[0040] In some preferred embodiments, this disclosure relates to an antibody comprising the heavy chain polypeptide sequences shown in SEQ ID NO: 7 and 39, respectively, wherein X1 is pyroglutamic acid, and wherein X7 is cleaved, and wherein asparagine (N) at amino acid position 300 is N-glycosylated.

[0041] In some preferred embodiments, this disclosure relates to anti-TTR antibodies comprising HC containing the sequences shown in SEQ ID NO: 7 and 39, respectively, wherein glutamine (X1) is modified to pyroglutamic acid, lysine (X7) is absent, and wherein the amino acids at positions X2, X3, X4, X5, and X6 are modified or unmodified as described above, preferably unmodified, and at least one amino acid is N-glycosylated. Preferably, in such embodiments, the N-glycosylation site is located at position Asn300 (N300, numbered based on the antibody HC polypeptide sequences of SEQ ID NO: 7 and 39, respectively, having N-terminal glutamine or pyroglutamic acid (X1).

[0042] In one specific embodiment, the antibody of this disclosure contains pyroglutamic acid instead of glutamine at the N-terminus of the heavy chain sequence, wherein the abundance in the sample is about 99.9% or higher, for example, 100%. In another specific embodiment, the antibody of this disclosure contains a C-terminus, wherein the C-terminal lysine residue in the heavy chain is cleaved, wherein the abundance in the sample is about 95.8% or higher, for example, 96%, 97%, 98%, 99%, or even 100%. In yet another specific embodiment, the antibody of this disclosure contains deamidated asparagine at position 58 (based on N58 of the antibody HC polypeptide sequences of SEQ ID NO: 7 and 39, respectively having N-terminal glutamine or pyroglutamic acid (X1)), wherein the abundance in the sample is about 5% or lower, for example, 4%, 3%, 2%, 1%, or 0%. In another specific embodiment, the antibody of this disclosure comprises deamidated asparagine at position 318 (based on N318 of antibody HC polypeptide sequences of SEQ ID NO: 7 and 39 having N-terminal glutamine or pyroglutamic acid (X1), respectively), wherein the abundance in the sample is about 9% or less, for example 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0%. In another specific embodiment, the antibody of this disclosure comprises deamidated asparagine at position 387 (based on N387 of antibody HC polypeptide sequences of SEQ ID NO: 7 and 39 having N-terminal glutamine or pyroglutamic acid (X1), respectively), wherein the abundance in the sample is about 5% or less, for example 4%, 3%, 2%, 1% or 0%. In another specific embodiment, the antibody of this disclosure contains oxymethionine at position 71 (based on M71 of antibody HC polypeptide sequences of SEQ ID NO: 7 and 39 having N-terminal glutamine or pyroglutamic acid (X1), respectively), wherein the abundance in the sample is about 5% or less, such as 4%, 3%, 2%, 1% or 0%. In another specific embodiment, the antibody of this disclosure contains oxymethionine at position 115 (based on M15 of antibody HC polypeptide sequences of SEQ ID NO: 7 and 39 having N-terminal glutamine or pyroglutamic acid (X1), respectively), wherein the abundance in the sample is about 5% or less, such as 4%, 3%, 2%, 1% or 0%. In another specific embodiment, the antibody of this disclosure contains oxymethionine at position 255 (based on M255 of the antibody HC polypeptide sequences of SEQ ID NO: 7 and 39 having N-terminal glutamine or pyroglutamic acid (X1), respectively), wherein the abundance in the sample is about 5% or less, for example 4%, 3%, 2%, 1% or 0%.In another specific embodiment, the antibody of this disclosure contains oxymethionine at position 361 (based on M361 of antibody HC polypeptide sequences of SEQ ID NO: 7 and 39, respectively having N-terminal glutamine or pyroglutamic acid (X1)), wherein the abundance in the sample is about 5% or less, for example 4%, 3%, 2%, 1% or 0%. In another specific embodiment, the antibody of this disclosure contains oxymethionine at position 431 (based on M413 of antibody HC polypeptide sequences of SEQ ID NO: 7 and 39, respectively having N-terminal glutamine or pyroglutamic acid (X1)), wherein the abundance in the sample is about 5% or less, for example 4%, 3%, 2%, 1% or 0%. Furthermore, the antibody of this disclosure may contain glycosylated heavy chains and / or glycosylated light chains.

[0043] As mentioned above, antibodies with N-terminal pyroglutamate modification and C-terminal lysine cleavage are beneficial for therapeutic purposes, but it is preferable that the abundance of the other mentioned PTMs is relatively low. For example, methionine oxidation, isofpartic acid formation, or deamidated asparagine in therapeutic antibodies is generally unfavorable because it can negatively affect stability, function, safety, and efficiency, and therefore, the abundance of methionine oxidation, isofpartic acid formation, and asparagine deamidation should preferably be about 5% or less.

[0044] Therefore, in a preferred embodiment, X1 is pE, X2 is methionine, X3 is asparagine, X4 is asparagine, X5 is proline, X6 is glycine, X7 is absent, and X... I It's asparagine, X II It's methionine, X III It's methionine, X IV It is aspartic acid, X V It is glycosylated asparagine, X VI It is methionine, and X VII It is methionine.

[0045] In one embodiment, the antibody of this disclosure comprises two HCs, each having the amino acid sequences shown in SEQ ID NO: 7 and 39 respectively, and two LCs, each having the amino acid sequence shown in SEQ ID NO: 8, wherein the glutamine at the N-terminus (X1) of the HC sequence is independently modified to pyroglutamic acid (pE) in each case. In another embodiment, the antibody of this disclosure comprises two HCs containing the amino acid sequences shown in SEQ ID NO: 7 and 39 respectively, wherein the C-terminal lysine (X7) of the HC sequence is independently absent in each case. In yet another embodiment, the antibody of this disclosure comprises two HCs containing the amino acid sequences shown in SEQ ID NO: 7 and 39 respectively, wherein the heavy chain is independently N-glycosylated in each case, preferably wherein the N-glycosylation site is at position Asn300. In an embodiment, the antibody of this disclosure comprises two HCs containing the amino acid sequence shown in SEQ ID NO: 7, wherein the amino acids at positions X2, X3, X4 and X5 of SEQ ID NO: 7 and 39, respectively, are modified or unmodified, preferably unmodified.

[0046] In the embodiments, the antibodies of this disclosure comprise two HCs, each having the amino acid sequences shown in SEQ ID NO: 7 and 39 respectively, and two LCs, each having the amino acid sequence shown in SEQ ID NO: 8, wherein the glutamine at the N-terminus (X1) of the HC sequence is independently modified to pyroglutamic acid (pE) in each case; the lysine (X7) at the C-terminus of the HC sequence is independently absent in each case; the heavy chain is independently N-glycosylated in each case, preferably wherein the N-glycosylation site is located at position Asn300 in SEQ ID NO: 7 and 39 respectively, the Asn number corresponding to the polypeptide sequences of SEQ ID NO: 7 and 39 containing glutamine or pyroglutamic acid at X1 respectively; and the amino acids at positions X2, X3, X4 and X5 in SEQ ID NO: 7 and 39 are modified or unmodified, preferably unmodified.

[0047] In embodiments of this disclosure, the antibody comprises two HC chains and two LC chains, wherein each heavy chain independently has the amino acid sequence shown in SEQ ID NO: 9, and each light chain independently has the amino acid sequence shown in SEQ ID NO: 8.

[0048] In a preferred embodiment, the heavy chain of the anti-TTR antibody of this disclosure lacks the C-terminal lysine as shown in SEQ ID NO: 9, and the N-terminal glutamine, as shown in SEQ ID NO: 9, is modified to pyroglutamic acid. The sequence, i.e., the heavy chain sequence having the cleaved C-terminal lysine and cyclized N-terminal glutamine (e.g., glutamine cyclized to pyroglutamic acid), is shown in SEQ ID NO: 12.

[0049] In some embodiments, the antibodies of this disclosure may have undergone other post-translational modifications (PTMs), such as partial cleavage, oxidation, deamidation, succinimide formation, pyroglutamate formation, and isomerization. Multiple such PTMs can be present in any combination. In embodiments, the PTM is in the heavy chain polypeptide sequence; however, the light chain polypeptide sequence can also be post-translational modified. The PTMs experimentally determined in NI006 / ALXN2220 are provided in detail in Example 2. Specifically, in addition to the C-terminal lysine cleavage and N-terminal glutamine cyclization to pyroglutamate mentioned above, the heavy chain and / or light chain of the antibody may contain methionine (M) oxidation, for example at HC position 255; asparagine (N) deamidation, for example at HC position 318 and / or HC position 387; asparagine (N) succinimide formation, for example at HC position 318; and / or C-terminal proline (P) amidation following the deletion of C-terminal lysine and glycine.

[0050] 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 (SEQ ID NO: 9).

[0051] In a preferred embodiment, the HC chain of the anti-TTR antibody of this disclosure lacks a C-terminal lysine, the N-terminal glutamine is modified to pyroglutamic acid, and it contains at least one N-glycosylation site. Therefore, in a preferred embodiment, the antibody of this disclosure consists of two heavy chains having SEQ ID NO: 9 and two light chains having SEQ ID: 8, wherein in the heavy chains, the N-terminal glutamine is modified to pyroglutamic acid, the C-terminal lysine is cleaved or absent, and the heavy chains are N-glycosylated. In other words, the antibody consists of two heavy chains having SEQ ID NO: 12 (preferably wherein the N-terminus of each antibody HC is cyclized) and two light chains having SEQ ID NO: 8, wherein each heavy chain is N-glycosylated, for example at Asn300. In another preferred embodiment, the antibody has a low abundance of other PTMs (preferably about 5% or less, as further mentioned above).

[0052] Such antibodies are particularly advantageous for therapeutic purposes because they are stable, safe, and effective, possessing a long half-life, reduced immunogenicity, and exhibiting charge homogeneity. These advantageous characteristics are demonstrated in Examples 3 and 4. Specifically, the stability study in Example 4 showed that the antibody formulation was long-term stable, and the batch analysis in Example 3 also confirmed the stability of the antibody. For example, SEC analysis confirmed the near absence of high or low molecular weight substances (no aggregation or degradation) in the sample.

[0053] In one embodiment of this disclosure, the antibody comprises HC having the amino acid sequence of SEQ ID NO: 9 and LC having the amino acid sequence of SEQ ID NO: 8, wherein glutamine (Q) at position 1 of SEQ ID NO: 9 is modified to pE, lysine (K) at position 450 of SEQ ID NO: 9 is cleaved (e.g., by proteolytic digestion), asparagine (N) at position 300 of SEQ ID NO: 9 is glycosylated, and the antibody comprises the following disulfide bridges: 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; and HC:C370-HC:C428. The aforementioned cysteine ​​residues are numbered to their positions in SEQ ID NO: 9 and 8, respectively. Additionally, the antibody comprises a glycan, wherein the glycan is of the Man3+1F, G0-GN, G0F-GN, G0, G0F, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1, and G2FS2 type glycans. In a preferred embodiment, the glycans are predominantly of the G0F and G1F type. In some embodiments, the HC shown in SEQ ID NO: 9 further comprises one or more modifications:

[0054] The asparagine (N) at position 58 is deamidinated;

[0055] The methionine (M) at position 71 is oxidized;

[0056] The methionine (M) at position 115 is oxidized;

[0057] The methionine (M) at position 225 is oxidized;

[0058] The aspartic acid (D) at position 283 is isomerized;

[0059] The asparagine (N) at position 318 is deamidinated or contains succinimide;

[0060] The methionine (M) at position 361 is oxidized;

[0061] The asparagine (N) at position 387 is deamidinated or contains succinimide;

[0062] The methionine (M) at position 431 is oxidized;

[0063] Glycine (G) at position 449 is absent;

[0064] The proline (P) at position 448 is amidated after losing the C-terminal lysine and glycine.

[0065] Furthermore, as mentioned above, each heavy chain of antibody NI006 / ALXN2220 may contain a single N-linked glycosylation site at Asn300. The N-linked glycosylation structure is primarily a complex biantennary glycan fucosylated with 0 galactose residues (GOF) (approximately 49%) or 1 galactose residue (G1F) (approximately 25%). Detailed glycosylation profiles are shown in Examples 2 and 3. 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 and provides valuable information regarding the safety and efficacy of biotherapeutic agents.

[0066] Therefore, in a preferred embodiment, the antibody disclosed herein is an IgG antibody, particularly the IgG1m3 subtype, and has an N-glycosylated heavy chain, preferably wherein the N-linked glycosylation site is Asn300.

[0067] More detailed analysis of the glycan structure revealed that over 85% of the glycans were fucosylated, specifically approximately 89% to 94%. It has also been shown that approximately 80% to 90% of the glycans are part of a predominantly fucosylated glycan type, meaning that the majority of the glycan structure attached to the antibody contains fucose residues. Fucosylation profiles depend on the cell line in which the antibody is generated, and therefore, fucosylation profiles are specific to the antibodies of this invention. Furthermore, such fucosylation profiles are advantageous because fucosylation improves the structural stability of the glycan structure on the antibody, resulting in a longer in vivo half-life, thereby reducing dosing frequency and improving patient compliance. Additionally, highly fucosylated antibodies are generally less likely to elicit an immune response in patients because fucosylated glycans are common in human antibodies and are less likely to be recognized as foreign. High fucosylation levels are more readily and consistently achieved using certain cell lines (such as CHO cells) and are generally more reliable for large-scale manufacturing, yielding consistent products, which is crucial for regulatory approval and therapeutic efficacy.

[0068] Therefore, the antibodies of this disclosure preferably comprise the following glycan types: Man3+1F, G0-GN, G0F-GN, G0, G0F, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1, and G2FS2, preferably wherein the main glycan types are G0F and G1F. More preferably, the antibodies of this disclosure comprise glycans wherein more than 85% of the glycans are fucoidylated, preferably wherein about 85% to 95% of the glycans are fucoidylated, and more preferably wherein about 89% to 94% of the glycans are fucoidylated.

[0069] Furthermore, it has been shown that approximately 27% to 38% of the glycans are galactosylated, preferably approximately 27% to 38%. Therefore, the antibody exhibits a controlled glycosylation profile, which is important for quality control in antibody production, as a consistent glycosylation pattern contributes to predictable efficacy, safety, and stability. Additionally, galactosylation contributes to structural stability (which is crucial for maintaining consistent therapeutic efficacy over time), which is advantageous for long-term antibody administration. Therefore, in one embodiment, the antibody of this disclosure comprises a glycan wherein approximately 20% to 40% of the glycans are galactosylated, preferably approximately 27% to 38%.

[0070] Analysis of the glycan structure revealed that approximately 1% to 4% of the glycans were mannose-containing glycans of the Man5 and Man31F types. Generally, high levels of mannose structures, such as Man5, can indicate incomplete processing in the glycosylation pathway; therefore, a low percentage (as in the case of this invention) is often advantageous in therapeutic antibodies because it suggests that the antibody production process is well-controlled, with most of the glycans being processed into more complex structures. Furthermore, since mannose-rich glycans are less common in human antibodies and can sometimes increase immunogenicity, as the immune system may recognize them as foreign, a low mannose percentage is beneficial for the therapeutic application of the antibody.

[0071] Therefore, in one embodiment, the antibody of this disclosure comprises a glycan, wherein about 1% to 4% of the glycan is a mannose-containing glycan, preferably of the Man5 and Man31F type, and more preferably of the Man5 type. In most batches analyzed, mannose-containing glycans are present in even lower amounts, particularly between 1% and 2%. Therefore, more preferably, the antibody of this disclosure comprises a glycan, wherein about 1% to 2% of the glycan is a mannose-containing glycan, preferably of the Man5 and Man31F type, and more preferably of the Man5 type.

[0072] Finally, glycan analysis showed that about 0.5% to 2% of the glycan was sialylated, and therefore, in one embodiment, the antibody of this disclosure comprises glycans wherein less than 2% of the glycans are sialylated, preferably wherein about 0.5% to 2% of the glycans are sialylated.

[0073] Regarding mannose-containing polysaccharides, it was surprisingly shown that reducing the pH dead zone from 0.20 to 0.05 (where pH was set to 6.9) resulted in a reduction of Man 5 (see Example 3), which was advantageous, as explained above. Specifically, the amount of Man 5-type polysaccharides was almost halved. Therefore, in a particularly preferred embodiment, less than 3% of the polysaccharide is of the Man5 type, preferably less than 2.9%, preferably less than 2.8%, preferably less than 2.7%, preferably less than 2.6%, preferably less than 2.5%, preferably less than 2.4%, preferably less than 2.3%, preferably less than 2.2%, preferably less than 2.1%, preferably less than 2.0%, preferably less than 1.9%, preferably less than 1.8%, preferably less than 1.7%, preferably less than 1.6%, preferably less than 1.5%, preferably less than 1.4%, preferably less than 1.3%, preferably less than 1.2%, preferably less than 1.1%, preferably less than 1.0%, preferably less than 0.9%, preferably less than 0.8%, preferably less than 0.7%, preferably less than 0.6%, preferably less than 0.5%, preferably less than 0.4%, preferably less than 0.3%, preferably less than 0.2%, preferably less than 0.1%, preferably less than 0%. In another preferred embodiment, about 2%, preferably between 2.5% and 1%, more preferably between about 2% and 1%, of the polysaccharide is of the Man5 type (see Table 9).

[0074] Therefore, embodiments of this disclosure also relate to methods and workflows for obtaining ALXN2220 with a desired product profile. Specifically, as described in detail in Example 3, reducing the pH dead zone from 0.20 to 0.05 results in a significant reduction in acidic substances in ALXN2220 process A2, for example, from about 37% to about 29%, and an overall change in the charged variant spectrum. Quite surprisingly, reducing the pH dead zone from 0.20 to 0.05 also results in a significant reduction in Man 5 levels, for example, a reduction of about 50% (from about 3.8% to about 1.9%). Therefore, the implementation of the methods and workflows described herein provides robust control over high-mannose content in batches of antibody pharmaceutical substances without any accompanying adverse effects on other product quality attributes, such as the relative levels (in %) of the major antibody substance relative to the HMW antibody substance relative to the LMW antibody substance (as determined by SEC); the level (in %) of the major antibody substance, as determined by capillary electrophoresis of sodium dodecyl sulfate (CE-SDS-NR) under non-reducing conditions; and the levels (in %) of the antibody heavy and light chains, as determined by capillary electrophoresis of sodium dodecyl sulfate (CE-SDS-R) under reducing conditions.

[0075] Further analysis revealed that, depending on their post-translational modifications, antibody samples comprise both basic and acidic antibody variants. For example, antibodies containing deamidated asparagine residues at positions N58, N328, and / or N387 in their HC, as further mentioned above, containing sialylated N-glycosylation, galactosylated N-glycosylation, HC truncation between N58 and T59, and lysine glycosylation uniformly distributed in all lysines in the HC and LC of the antibody, are acidic. Antibodies containing aspartic isomerization at D283 in their HC, lysine deletion at K450 in their HC, and proline amidation (P448) with glycine and lysine deletions in their HC, as further mentioned above, are basic. Therefore, in one embodiment, the antibody of this disclosure is an acidic antibody variant. In another embodiment, the antibody of this disclosure is a basic antibody variant. In yet another embodiment, the antibody of this disclosure is a neutral antibody variant. In a preferred embodiment, the antibody of this disclosure is an acidic antibody variant.

[0076] In some embodiments, this disclosure relates to recombinant antibodies, for example, produced by expression in suitable host cells such as bacterial (e.g., E. coli) cells or other microbial cells such as yeast cells; or mammalian cells such as CHO cells. In embodiments of this disclosure, the recombinant antibody comprises an N-linked glycosylation structure that is primarily a glycan having 0 galactose residues (GOF) (about 49%) or 1 galactose residue (G1F) (about 25%). Most preferably, the antibody comprises or is substantially composed of the glycosylation profile shown in Example 2 and mentioned above.

[0077] In the embodiments, the theoretical molecular weight (MW) of the antibody of this disclosure is about 144.2 kDa, and the molecular weight determined by mass spectrometry (MS) is about 144.2 kDa (deglycosylated form). In the embodiments, the molecular weight of the antibody of this disclosure, as determined by mass spectrometry (MS), is between 147.0 kDa and 147.6 kDa (intact IgG1).

[0078] Embodiments of this disclosure also relate to pharmaceutical compositions comprising the antibody of this disclosure, as characterized above, having a molecular weight of about 150 kDa, preferably about 147 kDa.

[0079] In the embodiments, the antibody of this disclosure has an experimentally determined pI of about 9.3 and a theoretical pI of about 8.4. In the embodiments, the antibody of the present invention has an experimentally determined extinction coefficient of 1.390 and a theoretically determined extinction coefficient of 1.438.

[0080] In some embodiments, the antibody of this disclosure comprises a human IgG antibody containing two identical heavy chains (HC) and two identical light chains (LC), wherein the identity may be based on sequence identity, i.e., wherein the primary amino acid sequence of the heavy chain polypeptide sequence constituting the antibody tetramer is optionally or together with the primary amino acid sequence of the light chain polypeptide sequence constituting the antibody tetramer is identical. The four chains are stabilized by intra- and inter-chain disulfide bonds, wherein the positions of the disulfide bridges, identified according to Lys-C and trypsin digestion and subsequent LC-MS (see Example 2), are as follows:

[0081] LC:C23-LC:C88

[0082] LC:C134-LC:C194

[0083] LC:C214-HC:C223

[0084] HC:C22-HC:C97

[0085] HC:C147-HC:C203

[0086] HC1:229-HC2:229 and HC1:232-HC2:232

[0087] HC:C264-HC:C324; and

[0088] HC:C370-HC:C428

[0089] The cysteine ​​residues (C) are numbered to correspond to their positions in the antibody HC sequence of SEQ ID NO: 7 and the antibody LC sequence of SEQ ID NO: 8, wherein SEQ ID NO: 7 has an N-terminal glutamine or pyroglutamic acid (X1).

[0090] Therefore, in one embodiment, the antibody of this disclosure comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight disulfide bridges, preferably at the locations identified above. In an embodiment, the antibody of this disclosure comprises a plurality of (e.g., all eight) disulfide (DS) bridges selected from bridges between light chains (LC); bridges between LC and heavy chains (HC); and bridges between HC. In the embodiments, the antibodies of this disclosure comprise DS bridges selected from the following: (1) LC:C23-LC:C88; (2) LC:C134-LC:C194; (3) LC:C214-HC:C223; (4) HC:C22-HC:C97; (5) HC:C147-HC:C203; (6) HC1:229-HC2:229 and HC1:232-HC2:232; (7) HC:C264-HC:C324; and (8) HC:C370-HC:C428, wherein LC represents the antibody light chain; HC represents the antibody heavy chain; C represents a cysteine ​​amino acid; and the number represents the position of the cysteine ​​in the antibody HC sequence of SEQ ID NO: 7 and / or the antibody LC sequence of SEQ ID NO: 8, which contains glutamine or pyroglutamic acid at X1 (i.e., X1 is in SEQ ID NO: 8). NO: 7 does not exist).

[0091] To prevent subjects administering the antibodies described herein or their antigen-binding fragments from developing "anti-drug antibodies" (ADAs), the antibodies are preferably human or humanized antibodies, typically human IgG, and most preferably human IgG1. In a preferred embodiment, the antibody is a human IgG1m3 allotype and preferably contains a κ LC constant region. Most preferably, the closest human gene / alleles to the variable domain are IGKV1-3901 (93.3%) + IGKJ101 (100%) and IGHV4-30-201 (89.5%) + IGHJ302 (100%).

[0092] Antibodies, like many other proteins, are secreted from cells via a co-translational translocation pathway. In eukaryotes, a signal peptide containing 5 to 30 amino acids at the N-terminus of a nascent protein is recognized by a signal recognition particle (SRP) in the cytosol while the protein is still being synthesized on the ribosome. The SRP then delivers the SRP-ribosome-nascent chain (SRP-RNC) complex to the SRP-receptor (SR) in the endoplasmic reticulum (ER) membrane. A GTP-dependent mechanism then delivers the RNC complex to a membrane-bound translocase, which allows the growing polypeptide chain to translocate into the lumen of the ER. After crossing the ER membrane, the signal peptide is cleaved by a signal peptidase (SPP), and human IgG antibodies consist of two identical heavy chains (HC) and two identical light chains (LC). Efficient expression of HC and LC requires the signal peptide to transport the HC and LC polypeptides into the ER for proper folding, assembly, and post-translational modifications.

[0093] Therefore, in one embodiment, the HC of the antibody of this disclosure further comprises a signal peptide derived from the human immunoglobulin heavy chain, and the LC of the antibody of this disclosure further comprises a signal peptide derived from the human immunoglobulin κ light chain. Such signal peptides are well known in the art, and their sequences are available from relevant databases, such as those from the National Institutes of Health (NIH) database, the European Molecular Biology Laboratory (EMBL) database, and the European Bioinformatics Institute (EBI) database of EMBL. Furthermore, WO 2014 / 058389 A1 discloses various signal peptides that can be cloned in principle. In the case of this invention, the signal peptides shown in SEQ ID NO: 17 and 18 have been used to express the HC and LC of the antibody of this disclosure, resulting in efficient expression of the HC and LC, which are then transported to the ER for proper folding, assembly, and post-translational modification, thereby producing antibodies particularly suitable for formulation in pharmaceutical compositions.

[0094] Therefore, in a preferred embodiment, the HC of the antibody of this disclosure further comprises a signal peptide having the amino acid sequence of SEQ ID NO: 17, the sequence of which includes HC and the signal peptide is shown in SEQ ID NO: 19. Alternatively, the LC of the antibody of this disclosure further comprises a signal peptide having the amino acid sequence of SEQ ID NO: 18, the sequence of which includes HC and the signal peptide is shown in SEQ ID NO: 20.

[0095] As understood in the art, some antibodies are tolerant to different signal peptides, while others are more restricted. The aforementioned signal peptides of the heavy chain and light chain antibody sequences represent the optimal signal peptide pair for, for example, recombinant expression of NI006 / ALXN2220 in CHO cells.

[0096] This disclosure also relates to polynucleotides encoding antibodies of this disclosure. Specifically, this disclosure relates to one or more polynucleotides, preferably to two polynucleotides encoding antibodies of this disclosure, i.e., a first polynucleotide comprising a nucleotide sequence encoding the antibody HC, and a second polynucleotide comprising a nucleotide sequence encoding the antibody LC. Preferably, this disclosure relates to polynucleotides encoding antibodies HC and LC, wherein HC has the amino acid sequence of SEQ ID NO: 7 (unmodified, i.e., glutamine or pyroglutamic acid (X1), and wherein lysine (X7) and glycine (X6) are present, and X2, X3, X4, and X5 are unmodified), and wherein LC has the amino acid sequence of SEQ ID NO: 8. This disclosure also relates to polynucleotides encoding antibodies HC and LC, wherein HC has the amino acid sequence of SEQ ID NO: 9 (modified in the sequence of SEQ ID NO: 7, including the deletion of a C-terminal lysine and the N-terminal glutamine cyclization to pyroglutamic acid), and wherein LC has the amino acid sequence of SEQ ID NO: 8.

[0097] Therefore, this disclosure also relates to polynucleotides encoding the aforementioned antibodies, wherein the first polynucleotide comprises the nucleotide sequence encoding antibody HC shown in SEQ ID NO: 13, and the second polynucleotide comprises the nucleotide sequence encoding antibody LC shown in SEQ ID NO: 14.

[0098] This disclosure also relates to codon-optimized nucleic acids, for example, for efficient expression in CHO cell lines. Therefore, in one embodiment, the first polynucleotide of this disclosure comprises the nucleotide sequence encoding antibody HC shown in SEQ ID NO: 15, and the second polynucleotide comprises the nucleotide sequence encoding antibody LC shown in SEQ ID NO: 16.

[0099] In one embodiment, the polynucleotide of this disclosure further comprises a nucleotide sequence encoding a signal peptide. Specifically, in one embodiment, a first nucleotide sequence comprises a nucleotide sequence encoding a signal peptide derived from the human immunoglobulin heavy chain, and a second nucleotide sequence comprises a nucleotide sequence encoding a signal peptide derived from the human immunoglobulin κ light chain. In a preferred embodiment, the nucleotide sequence of the first signal peptide is shown in SEQ ID NO: 21, and the first nucleotide sequence comprising the nucleotide sequence of the signal peptide has the sequence shown in SEQ ID NO: 23. Furthermore, the nucleotide sequence of the second signal peptide is shown in SEQ ID NO: 22, and the first nucleotide sequence comprising the nucleotide sequence of the signal peptide has the sequence shown in SEQ ID NO: 24.

[0100] According to this disclosure, the aforementioned nucleic acid of the signal peptide encoding the heavy chain and light chain antibody sequence represents, for example, the optimal signal peptide encoding nucleic acid pair for recombinant expression of the corresponding heavy chain and light chain polypeptide sequence of NI006 / ALXN2220 in CHO cells.

[0101] This disclosure also relates to a nucleic acid molecule, which is

[0102] (a) A polynucleotide comprising the first nucleotide sequence shown in SEQ ID NO: 13 and the second nucleotide sequence shown in SEQ ID NO: 14;

[0103] (b)(a) messenger RNA (mRNA) equivalents of the first and second nucleotide sequences;

[0104] (c) A polynucleotide containing a sequence complementary to the first and second nucleotide sequences of (a) or its mRNA equivalent in (b); or

[0105] (d) A polynucleotide containing the first and second nucleotide sequences of (a) or the degenerate sequence of its mRNA equivalent (b).

[0106] In a preferred embodiment, the polynucleotide (d) comprises the first nucleotide sequence shown in SEQ ID NO: 15 and the second nucleotide sequence shown in SEQ ID NO: 16. The degenerate sequences disclosed herein represent codon-optimized nucleic acid sequences that enable the corresponding heavy and light chains of the antibody to be optimally recombinantly expressed in cells suitable for commercial manufacturing (e.g., CHO cells).

[0107] In one embodiment, the polynucleotide of this disclosure further comprises a nucleotide sequence encoding a signal peptide. Specifically, in one embodiment, a first nucleotide sequence comprises a nucleotide sequence encoding a signal peptide derived from the human immunoglobulin heavy chain, and a second nucleotide sequence comprises a nucleotide sequence encoding a signal peptide derived from the human immunoglobulin κ light chain. In a preferred embodiment, the nucleotide sequence of the first signal peptide is shown in SEQ ID NO: 21, and the first nucleotide sequence comprising the nucleotide sequence of the signal peptide has the sequence shown in SEQ ID NO: 23. Furthermore, the nucleotide sequence of the second signal peptide is shown in SEQ ID NO: 22, and the first nucleotide sequence comprising the nucleotide sequence of the signal peptide has the sequence shown in SEQ ID NO: 24.

[0108] This disclosure also relates to pre-processed (e.g., precursor protein) and processed (e.g., mature) forms of each of the antibody heavy chain and light chain polypeptide, which are encoded by the aforementioned nucleic acids, such as the nucleic acid sequences shown in SEQ ID NO: 13 or 15 (encoding precursor protein antibody heavy chain) and the nucleic acid sequences shown in SEQ ID NO: 14 or 16 (encoding precursor protein antibody light chain).

[0109] This disclosure also relates to one or more expression vectors comprising the polynucleotide / nucleic acid molecules of this disclosure, and host cells comprising the vectors and / or polynucleotide / nucleic acid molecules of this disclosure.

[0110] As described in Example 1, the antibodies of this disclosure are produced in CHO cells, particularly in the CHO cell line K1. Therefore, in one embodiment, the host cell is a non-human cell, preferably a CHO cell, and most preferably CHO-K1. For recombinant antibody production, microorganisms, such as E. coli cells or insect cells, may also be used.

[0111] This disclosure also relates to a method for manufacturing the antibody of this disclosure, the method comprising at least culturing a host cell containing the polynucleotide of this disclosure (preferably in the form of a vector of this disclosure), preferably CHO cells, most preferably CHO K1 cells, and isolating the antibody from the culture.

[0112] For example, in some embodiments, the antibody manufacturing method described herein produces antibodies comprising HC having the amino acid sequences of SEQ ID NO: 7 and 39, respectively, and LC having the amino acid sequence of SEQ ID NO: 8. In an embodiment, the antibody comprises HC having the amino acid sequences of SEQ ID NO: 7 and 39, respectively, wherein the N-terminal residue X1 is absent or present as glutamine or pE. In an embodiment, X2 is methionine or oxidized methionine. In an embodiment, X3 is asparagine, deamidated asparagine, or asparagine containing succinimide. In an embodiment, X4 is asparagine, deamidated asparagine, or asparagine containing succinimide. In an embodiment, X5 is proline or amidated proline. In an embodiment, X6 is absent or glycine. In an embodiment, X7 is absent or lysine. In an embodiment, X... I It is asparagine or deamidated asparagine. In the implementation scheme, X II It is methionine or oxidized methionine. In the implementation plan, X III It is methionine or oxidized methionine. In the implementation plan, X IV It is aspartic acid or isoaspartic acid. In the implementation plan, X V It is asparagine or glycosylated asparagine. In the embodiment, X VI It is methionine or oxidized methionine. In the implementation plan, X VII It is methionine or oxymethionine. In another embodiment, X1 is pE and / or X7 is absent.

[0113] This disclosure also relates to a composition comprising an antibody of this disclosure. Thus, in one embodiment, the composition comprises an antibody having or not having a PTM as defined above. In a preferred embodiment, the composition comprises a mixture of antibodies as defined above. As shown in Examples 2 and 3, approximately 99% to 100% of the antibodies present in samples of typical antibody compositions have an N-terminal pyroglutamic acid residue in their heavy chain, and approximately 96% of the antibodies have a C-terminal lysine residue. Therefore, in one embodiment, approximately 99% (99% to 100%) of the antibodies in the formulation of this disclosure have a heavy chain in which the N-terminal pyroglutamic acid residue is modified with N-terminal glutamine, and / or approximately 96% (95% to 96%) of the antibodies do not have (or lack) a C-terminal lysine residue. The absence of a C-terminal lysine residue may be the result of proteolytic cleavage of the antibody heavy chain containing said lysine residue, or due to antibody engineering, for example, through mutation or deletion of the triplet codon encoding the C-terminal lysine residue in the nucleic acid encoding said HC. Preferably, the antibody present in the composition further comprises N-glycosylated HC as defined above.

[0114] For example, in one embodiment, the composition comprises an antibody, wherein the antibody comprises an HC having the amino acid sequences of SEQ ID NO: 7 and 39, respectively, and a light chain having the amino acid sequence of SEQ ID NO: 8. In another embodiment, the antibody comprises an HC having the amino acid sequences of SEQ ID NO: 7 and 39, respectively, wherein the N-terminal residue X1 is absent or present as glutamine or pE. In another embodiment, for 99% to 100% of the antibody in the composition, X1 is PE, preferably wherein for 99.9% of the antibody in the composition, X1 is PE. In another embodiment, X2 is methionine or oxidized methionine. In another embodiment, X3 is asparagine, deamidated asparagine, or asparagine containing succinimide. In another embodiment, X4 is asparagine, deamidated asparagine, or asparagine containing succinimide. In another embodiment, X5 is proline or amidated proline. In another embodiment, X6 is absent or glycine. In another embodiment, X7 is absent or lysine. In another embodiment, X... I It is asparagine or deamidated asparagine. In the implementation scheme, X II It is methionine or oxidized methionine. In the implementation plan, X III It is methionine or oxidized methionine. In the implementation plan, X IV It is aspartic acid or isoaspartic acid. In the implementation plan, X V It is asparagine or glycosylated asparagine. In the embodiment, X VI It is methionine or oxidized methionine. In the implementation plan, X VII It is methionine or oxymethionine.

[0115] In an embodiment, the composition comprises an antibody, wherein the antibody comprises an HC having the amino acid sequences of SEQ ID NO: 7 and 39, respectively, and a light chain having the amino acid sequence of SEQ ID NO: 8. In an embodiment, the antibody comprises an HC having the amino acid sequences of SEQ ID NO: 7 and 39, respectively, wherein the N-terminal residue X1 is absent or present as glutamine or pE. In an embodiment, X2 is methionine or oxidized methionine. In an embodiment, X3 is asparagine, deamidated asparagine, or asparagine containing succinimide. In an embodiment, X4 is asparagine, deamidated asparagine, or asparagine containing succinimide. In an embodiment, X5 is proline or amidated proline. In an embodiment, X6 is absent or glycine. In an embodiment, X7 is absent or lysine. In an embodiment, X7 is absent in about 95% to about 96% of the antibody in the composition, preferably wherein X7 is absent for 95.8% of the composition. In an embodiment, X... IIt is asparagine or deamidated asparagine. In the implementation scheme, X II It is methionine or oxidized methionine. In the implementation plan, X III It is methionine or oxidized methionine. In the implementation plan, X IV It is aspartic acid or isoaspartic acid. In the implementation plan, X V It is asparagine or glycosylated asparagine. In the embodiment, X VI It is methionine or oxidized methionine. In the implementation plan, X VII It is methionine or oxymethionine.

[0116] In one embodiment, a portion of the antibody in the composition is fragmented in HC via cleavage between asparagine at position 58 and threonine at position 59, as shown in SEQ ID NO: 7 and 39, respectively. In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the antibody in the composition is fragmented.

[0117] Furthermore, but to a lesser extent and preferably in negligible amounts, some antibody material may be present in the composition, which may have undergone other post-translational modifications (PTMs), such as partial cleavage, oxidation, deamidation, succinimide or pyroglutamate formation and isomerization. PTMs identified as present in NI006 / ALXN2220 are mentioned in Example 2. Specifically, following the aforementioned C-terminal lysine cleavage and N-terminal cyclization, the antibody may exhibit methionine (M) oxidation, for example at HC position 255; asparagine (N) deamidation, for example at HC position 318 and / or HC position 387; asparagine (N) succinimide formation, for example at HC position 318; and / or C-terminal proline (P) amidation following the deletion of C-terminal lysine and glycine.

[0118] In other words, in one embodiment, the composition of this disclosure comprises one or more antibodies comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO: 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO: 8, wherein, in one or more of these antibodies, the HC is preferably modified as follows:

[0119] Glutamine (Q) at position 1 was modified to pyroglutamic acid (pE).

[0120] The asparagine (N) at position 300 is glycosylated; and / or

[0121] The lysine (K) at position 450 is absent; preferably, the glutamine (Q) at position 1 is modified to pyroglutamic acid (pE), the asparagine (N) at position 300 is glycosylated, and the lysine (K) at position 450 is absent.

[0122] In one embodiment, the compositions disclosed herein comprise one or more antibodies having the following additional modifications in their HC:

[0123] The methionine (M) at position 255 is oxidized.

[0124] The asparagine (N) at position 318 is deamidinated or contains succinimide;

[0125] The asparagine (N) at position 387 is deamidinated;

[0126] The proline (P) at position 448 is amidated; and / or

[0127] Glycine (G) at position 449 is absent.

[0128] In embodiments, the compositions disclosed herein comprise one or more antibodies having the following additional modifications in their HC:

[0129] Asparagine (N) at position 58 is deamidinated.

[0130] The methionine (M) at position 71 is oxidized.

[0131] The methionine (M) at position 115 is oxidized.

[0132] The methionine (M) at position 255 is oxidized.

[0133] Aspartic acid (D) at position 283 is isoaspartic acid.

[0134] The methionine (M) at position 361 is oxidized, and / or

[0135] The methionine (M) at position 431 is oxidized.

[0136] In a preferred embodiment, the antibody contained in the composition of this disclosure is generated by recombinant expression in CHO-K1 cells.

[0137] In one specific embodiment, the composition of this disclosure comprises one or more antibodies containing pyroglutamic acid instead of glutamine at the N-terminus of the heavy chain sequence, preferably wherein in more than 90%, preferably in about 99% to 100% of the antibodies, glutamine (Q) at position 1 of SEQ ID NO: 9 is modified with pyroglutamic acid. In another specific embodiment, the composition of this disclosure comprises one or more antibodies wherein lysine (K) at position 450 of SEQ ID NO: 9 is absent, preferably wherein lysine (K) at position 450 of SEQ ID NO: 9 is absent in more than 90%, preferably in about 95% to 100%, preferably in about 95% to 96% of the antibodies.

[0138] In another specific embodiment, the composition of this disclosure comprises one or more antibodies wherein lysine (K) at position 450 of SEQ ID NO: 9 is absent, preferably wherein in more than 90%, preferably in about 95% to 100%, preferably in about 95% to 96% of the antibodies, lysine (K) at position 450 of SEQ ID NO: 9 is absent, and the one or more antibodies contain pyroglutamic acid instead of glutamine at the N-terminus of the heavy chain sequence, preferably wherein in more than 90%, preferably in about 99% to 100% of the antibodies, glutamine (Q) at position 1 of SEQ ID NO: 9 is modified to pyroglutamic acid.

[0139] In one preferred embodiment, the asparagine residues (N) at positions 58, 318, and 387 of one or more antibodies are undeamidinated and do not contain succinimidyl ether, and the composition contains no antibody or only a small amount of antibody, such as less than about 5% antibody, wherein the asparagine residues (N) at positions 58, 318, and / or 387 are deamidinated. In another preferred embodiment, the methionine residues at positions 71, 115, 255, 361, and 431 of one or more antibodies are unoxidized, and the composition contains no antibody or only a small amount of antibody, such as less than about 5% antibody, wherein the methionine residues at positions 71, 115, 255, 361, and / or 431 are oxidized. In another preferred embodiment, the aspartic acid at position 283 of one or more antibodies is unisomerized, and the composition contains no antibody or only a small amount of antibody, such as less than about 5% antibody, wherein the aspartic acid at position 283 is isomerized. In another preferred embodiment, the glycine at position 449 in one or more antibodies is not absent; that is, glycine is present, and the composition contains no antibody or only a small amount of antibody, for example, less than about 5% antibody, wherein the glycine at position 449 is absent, i.e., the composition mainly comprises antibodies in which glycine is present. Therefore, preferably, the proline at position 448 in one or more antibodies is unamidinated, and the composition contains no antibody or only a small amount of antibody, for example, less than about 5% antibody, wherein the proline at position 448 is amidated. More specifically, in embodiments, the compositions of this disclosure comprise one or more antibodies, preferably less than about 5%, preferably less than 2%, preferably less than about 1%, preferably from about 0% to 1%, wherein the asparagine (N) at position 58 of SEQ ID NO: 9 is deamidinated. Additionally or alternatively, the compositions of this disclosure comprise one or more antibodies, preferably less than about 8%, preferably from about 6% to 9%, wherein the asparagine (N) at position 318 of SEQ ID NO: 9 is deamidinated. Alternatively or alternatively, the compositions of this disclosure comprise one or more antibodies, preferably less than about 3%, preferably from about 1% to 3%, wherein the asparagine (N) at position 387 of SEQ ID NO: 9 is deamidinated. Alternatively or alternatively, the compositions of this disclosure comprise one or more antibodies, preferably less than about 5%, preferably less than about 2%, preferably less than about 1%, preferably from about 0% to 1%, wherein the methionine (M) at position 71 of SEQ ID NO: 9 is oxidized. Alternatively or alternatively, the compositions of this disclosure comprise one or more antibodies, preferably less than about 5%, preferably less than about 3%, preferably less than about 2%, preferably from about 1% to 3%, wherein the methionine (M) at position 115 of SEQ ID NO: 9 is oxidized.Alternatively or alternatively, the compositions of this disclosure comprise one or more antibodies, preferably less than about 5%, preferably less than about 3%, preferably from about 1% to 30%, wherein the methionine (M) at position 255 of SEQ ID NO: 9 is oxidized. Alternatively or alternatively, the compositions of this disclosure comprise one or more antibodies, preferably less than about 5%, preferably less than about 2%, preferably less than about 1%, preferably from about 0% to 1%, wherein the methionine (M) at position 361 of SEQ ID NO: 9 is oxidized. Alternatively or alternatively, the compositions of this disclosure comprise one or more antibodies, preferably less than about 5%, preferably less than about 3%, preferably less than about 2%, preferably from about 1% to 2%, wherein the methionine (M) at position 431 of SEQ ID NO: 9 is oxidized. Alternatively or alternatively, the compositions of this disclosure comprise one or more antibodies, wherein the aspartic acid (D) at position 283 of SEQ ID NO: 9 is modified to isoaspartic acid. Alternatively or alternatively, the compositions of this disclosure comprise one or more antibodies, wherein the proline (P) at position 448 of SEQ ID NO: 9 is amidated. Alternatively or alternatively, the compositions of this disclosure comprise one or more antibodies, wherein the glycine (G) at position 449 of SEQ ID NO: 9 is absent. Alternatively or alternatively, the compositions of this disclosure comprise one or more antibodies fragmented by cleavage between asparagine at position 58 and threonine at position 59 of SEQ ID NO: 9. Alternatively or alternatively, the compositions of this disclosure comprise one or more antibodies, preferably less than about 6%, preferably less than about 5%, preferably from about 2% to 5%, wherein the heavy chain is glycosylated. Alternatively or alternatively, the compositions of this disclosure comprise one or more antibodies, preferably less than about 4%, preferably less than about 3%, preferably from about 1% to 3%, wherein the light chain is glycosylated.

[0140] In addition, the composition contains one or more antibodies having a glycan spectrum as defined in detail above.

[0141] As described above, compositions of this disclosure containing antibodies having the indicated PTM are advantageous for therapeutic purposes because such compositions are stable, safe, effective, less immunogenic, functional, and reliably reproducible (see Example 4).

[0142] The same characterization can also be used to characterize the antibodies of the present invention.

[0143] The antibody disclosed herein has been shown to be particularly stable in an aqueous formulation at pH 5.8 containing approximately 50 mg / mL or 100 mg / mL of antibody, 20 mM histidine, sucrose at a concentration of 65 mg / mL (6.5% (w / v)) or 80 mg / mL (8% (w / v)), and polysorbate 80 (PS) at a concentration of 0.03% (w / v) (0.3 mg / mL). As shown in Example 4, the shelf life of the pharmaceutical product formulated in the aforementioned aqueous formulation is currently set at 24 months when stored protected from light at 5 ± 3°C.

[0144] Therefore, this disclosure relates to a composition comprising about 25 mg / mL to about 150 mg / mL of the antibody of this disclosure, preferably about 50 mg / mL or 100 mg / mL of the antibody of this disclosure, and further comprising histidine at a concentration of about 20 mM (e.g., L-histidine 1.06 mg / mL and L-histidine monohydrochloride 2.78 mg / mL), sucrose at a concentration of about 50 mg / mL to about 80 mg / mL, preferably 65 mg / mL (6.5% (w / v)) or 80 mg / mL (8% (w / v)), polysorbate 80 (PS) at a concentration of about 0.01% (w / v) to about 0.5% (w / v), preferably about 0.03% (w / v) (0.3 mg / mL), and having a pH of about 5.3 to 6.3, preferably about 5.8. In a preferred embodiment, the antibody comprises two HCs having SEQ ID NO: 7 or 9 and two LCs having SEQ ID: 8, wherein preferably in each HC, the N-terminal glutamine (Q at position X1 of SEQ ID NO: 7 and position 1 of SEQ ID NO: 9, respectively) is modified to pyroglutamic acid, the C-terminal lysine (K at position X7 of SEQ ID NO: 7 and position 450 of SEQ ID NO: 9) is absent, and the heavy chain is N-glycosylated, preferably wherein the N-glycosylation site is at position Asn300 of SEQ ID NO: 9, wherein the amino acids at positions X2, X3, X4, and X5 of SEQ ID NO: 7 are modified or unmodified, preferably unmodified, or wherein the methionine at position M255 of SEQ ID NO: 9 is unoxidized, the asparagine at positions N318 and N387 of SEQ ID NO: 9 is undeamidinated and does not contain succinimide, and SEQ ID NO: The proline at position P448 of SEQ ID NO: 9 is unamidated, and glycine (X6 of SEQ ID NO: 7 and G at position 449 of SEQ ID NO: 9) is present and not cleaved. In a preferred embodiment, the antibody consists of two HCs having SEQ ID NO: 39 or 9 and two LCs having SEQ ID: 8, wherein preferably in each HC, the N-terminal glutamine (X1 of SEQ ID NO: 39 and Q at position 1 of SEQ ID NO: 9, respectively) is modified to pyroglutamic acid, the C-terminal lysine (X7 of SEQ ID NO: 39 and K at position 450 of SEQ ID NO: 9) is absent, and the heavy chain is N-glycosylated, preferably wherein the N-glycosylation site is at position Asn300 (X of SEQ ID NO: 39). VAnd at position 300 (N) of SEQ ID NO: 9, wherein the amino acids at positions X2, X3, X4 and X5 are modified or unmodified, preferably unmodified, or wherein the methionine at position M255 of SEQ ID NO: 9 is unoxidized, the asparagine at positions N318 and N387 of SEQ ID NO: 9 is undeamidinated and does not contain succinimide, and the proline at position P448 of SEQ ID NO: 9 is unamidinated, wherein glycine (X6 of SEQ ID NO: 39 and G at position 449 of SEQ ID NO: 9) is present and not cleaved, and wherein the amino acids at positions X of SEQ ID NO: 39 are modified or unmodified, preferably unmodified, or wherein the methionine at position M255 of SEQ ID NO: 9 is unoxidized, the asparagine at positions N318 and N387 of SEQ ID NO: 9 is undeamidinated and does not contain succinimide, and wherein the proline at position P448 of SEQ ID NO: 9 is unamidinated, wherein glycine (X6 of SEQ ID NO: 39 and G at position 449 of SEQ ID NO: 9) is present and not cleaved, and wherein the amino acids at positions X of SEQ ID NO: 39 are unmodified. I X II X III X IV X VI X VI The amino acids at the specified positions are modified or unmodified, preferably unmodified, or wherein the methionine at positions M71, M115, M361, and M431 of SEQ ID NO: 9 is unoxidized, the asparagine at position N58 of SEQ ID NO: 9 is undeamidinated, and the aspartic acid at position D283 of SEQ ID NO: 9 is unisomerized. In an alternative embodiment, the antibody comprises two heavy chains having SEQ ID NO: 9 and two light chains having SEQ ID: 8, and wherein preferably in the heavy chain, the N-terminal glutamine is modified to pyroglutamic acid, the C-terminal lysine is cleaved or absent, and the heavy chain is N-glycosylated, preferably wherein the N-glycosylation site is at position Asn300.

[0145] In a preferred embodiment, the composition comprises one or more antibodies as defined above, i.e., the composition comprises one or more antibodies wherein lysine (K) at position 450 of SEQ ID NO: 9 is absent, preferably wherein in more than 90%, preferably in about 95% to 100%, preferably in about 95% to 96% of the antibodies, lysine (K) at position 450 of SEQ ID NO: 9 is absent, and the antibody contains pyroglutamic acid instead of glutamine at the N-terminus of the heavy chain sequence, preferably wherein in more than 90%, preferably in about 99% to 100% of the antibodies, glutamine (Q) at position 1 of SEQ ID NO: 9 is modified to pyroglutamic acid.

[0146] In one preferred embodiment, the asparagine residues (N) at positions 58, 318, and 387 of one or more antibodies are undeamidinated and do not contain succinimidyl ether, and the composition contains no antibody or only a small amount of antibody, for example, less than about 10%, preferably 5%, of antibody, wherein the asparagine residues (N) at positions 58, 318, and / or 387 are deamidinated. In another preferred embodiment, the methionine residues at positions 71, 115, 255, 361, and 431 of one or more antibodies are unoxidized, and the composition contains no antibody or only a small amount of antibody, for example, less than about 5%, of antibody, wherein the methionine residues at positions 71, 115, 255, 361, and / or 431 are oxidized. In another preferred embodiment, the aspartic acid at position 283 of one or more antibodies is unisomerized, and the composition contains no antibody or only a small amount of antibody, for example, less than about 5%, of antibody, wherein the aspartic acid at position 283 is isomerized. In another preferred embodiment, the glycine at position 449 in one or more antibodies is not absent; that is, glycine is present, and the composition contains no antibody or only a small amount of antibody, for example, less than about 5% antibody, wherein the glycine at position 449 is absent, i.e., the composition mainly comprises antibodies in which glycine is present. Therefore, preferably, the proline at position 448 in one or more antibodies is unamidinated, and the composition contains no antibody or only a small amount of antibody, for example, less than about 5% antibody, wherein the proline at position 448 is amidinated.

[0147] In a preferred embodiment, the composition is an aqueous composition, also known as a liquid formulation, and therefore also contains water for injection.

[0148] As illustrated in Example 4, several stability tests conducted with the formulation / pharmaceutical composition of this disclosure have demonstrated that the antibody remains stable for at least 1 month under various conditions, such as at 40 ± 2 °C and 75 ± 5% relative humidity (RH) (stress stability study); stable for at least 6 months at 25 ± 2 °C / 60 ± 5% RH (accelerated stability study); and stable for at least 12 to 18 months at 5 ± 3 °C (long-term stability study). Furthermore, the shelf life of the antibody formulation in the dark at 2 °C–8 °C has been determined to be 24 months.

[0149] Therefore, in one embodiment, the composition is characterized by the antibody remaining stable at 40 ± 2 °C and 75 ± 5% RH for at least one week, preferably at least one month; stable at 25 ± 2 °C / 60 ± 5% RH for at least one month, and preferably at least six months; and / or stable at 5 ± 3 °C for at least one month, preferably at least 12 months, more preferably at least 18 months. In one embodiment, the pharmaceutical composition has a shelf life of 24 months when protected from light at 2 °C–8 °C.

[0150] The stability of the formulation can be attributed at least in part to the constant region of the antibody, and more specifically, to the presence of PTM in the heavy chain of the antibody as defined above in this disclosure.

[0151] According to this disclosure, a pharmaceutical formulation of a recombinant human monoclonal antibody specific for TTR, a disease-associated form of amyloid protein, has been developed. The pharmaceutical formulation is characterized by a pH of 5.3 to 6.3, preferably 5.8 ± 0.1, and typically contains a histidine buffer and sucrose, polysorbate, preferably polysorbate 80, as excipients, and water for infusion / injection. The antibody is characterized by ALXN2220, also known as NI006, and has a concentration of about 25 mg / ml to about 150 mg / ml, typically about 50 mg / ml.

[0152] As mentioned, an antibody formulation at 50 mg / ml (pH 5.8) in 20 mM histidine, 6.5% or 8% (w / v) sucrose, and 0.03% (w / v) PS80 was identified in the examples as a lead candidate for further development, and indeed, an antibody formulation at 50 mg / ml (pH 5.8) in 20 mM histidine, 8% (w / v) sucrose, and 0.03% (w / v) PS80 has been successfully used in clinical trials and therefore represents the most preferred embodiment of this disclosure and an equivalent formulation for maintaining the physicochemical properties essential to the stability and function of the antibody as tested and validated in the appended Example 4. When changing the concentration of components and / or alternative excipients in the preferred formulation, the following factors should be considered to ensure the stability and efficacy of the antibody:

[0153] Histidine: Used as a buffer. Buffering capacity is related to histidine's ability to maintain pH close to its pKa value. Since histidine's pKa is approximately 6.0 at pH 5.8, its efficiency is slightly lower, but it can still be used as a buffer. Buffering capacity is also affected by the concentration of the buffer and the closeness of pH to the pKa.

[0154] Sucrose: Stabilizes proteins and increases osmolality during freeze / thaw cycles. Sucrose significantly contributes to the osmolality of the solution, which is important for maintaining the structure of IgG antibodies.

[0155] High concentrations of target protein: Like all proteins, antibodies have buffering capacity due to the presence of ionizable groups in their amino acid side chains. These groups include carboxyl groups of aspartic acid and glutamic acid, amino groups of lysine and arginine, imidazole groups of histidine, hydroxyl groups of tyrosine, thiol groups of cysteine, and terminal amino and carboxyl groups.

[0156] The compositions of this disclosure comprising the above-described formulations shall at least meet the acceptance criteria mentioned in Tables 25 and 26 (these criteria may also be used to determine whether a composition / formulation with altered component concentrations and / or substituted excipients remains long-term stable). Preferably, the compositions of this disclosure have, after storage at 5 ± 3 °C for 6 months, an antibody acid content of ≤40.0% and an antibody basic content of ≤15.0%, as determined by iCIEF; and / or, after storage at 5 ± 3 °C for 6 months, an antibody basic content of ≤5.0%, as determined by SEC-HPLC.

[0157] During the manufacturing process of ALXN2220, as detailed in Example 3, it has been shown that reducing the pH dead zone from 0.20 to 0.05 results in a significant reduction in acidic substances, for example, from about 37% to about 29%. Therefore, in one embodiment, the amount of acidic substances in the compositions of this disclosure is about 30%, preferably between 25% and 33%, more preferably between 25% and 32%. It has further been shown that the charged variant spectrum has been altered overall. Therefore, in another preferred embodiment, the amount of acidic substances in the compositions of this disclosure is equal to or less than about 32%, and preferably, the amount of basic substances is equal to or less than about 5%, and the amount of neutral substances is equal to or greater than about 63%.

[0158] Embodiments of this disclosure relate to a method for generating antibodies comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO: 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO: 8, the method comprising (a) culturing host cells, such as Chinese hamster ovary K1 (CHO-K1) cells, under conditions sufficient to allow expression of the antibodies in a culture, the host cells comprising one or more expression vectors or vector systems containing polynucleotides encoding antibodies HC and LC; (b) setting a pH set point and a pH dead zone of the culture to regulate the levels of acidic, basic, and / or neutral antibody material in the culture; and (c) isolating antibodies HC and LC from the cell culture; and (d) optionally formulating the isolated antibodies into a pharmaceutical formulation comprising the isolated antibodies and a pharmaceutically acceptable carrier.

[0159] In a particular embodiment, this disclosure relates to a method for generating antibodies comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO: 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO: 8, the method comprising (a) culturing host cells, such as Chinese hamster ovary K1 (CHO-K1) cells, under conditions sufficient to allow expression of the antibodies in a culture, the host cells comprising one or more expression vectors or vector systems containing polynucleotides encoding antibodies HC and LC; (b) setting a pH set point and a pH dead zone of the culture to regulate the levels of acidic, basic, and / or neutral antibody material in the culture; and (c) isolating antibodies HC and LC from the cell culture; and (d) optionally formulating the isolated antibodies into a pharmaceutical formulation comprising the isolated antibodies and a pharmaceutically acceptable carrier, wherein step (b) comprises setting the pH set point to about 6.90 and setting the pH dead zone between 0.05 and 0.10 to reduce the levels of acidic antibody material; preferably, the pH dead zone is set to about 0.05.

[0160] In some implementations, the implementation of step (b), which includes setting the pH and pH dead zone, reduces the level of acidic substances in the cultured antibody preparation, while also reducing the level of mannose 5.

[0161] In some embodiments, compared to a production method in which step (b) is performed at pH 6.90 and a pH dead zone of 0.20, the method includes setting the pH to 6.90 and the pH dead zone between 0.05 and 0.10; preferably, the implementation of step (b) with the pH set to 6.90 and the pH dead zone set to about 0.05 reduces the level of N-linked mannose-5-glycan (Man5) in the antibody preparation. In such embodiments, the reduction in Man5 level is greater than >20% compared to the Man5 level in antibody preparations produced using the same method except that step (b) is performed at pH 6.90 and a pH dead zone of 0.20; more preferably, the reduction in Man5 level is about 40% to 50%.

[0162] In some implementations, the step (b) includes setting the pH to 6.90 and the pH dead zone between 0.05 and 0.10; preferably, the step (b) of setting the pH to 6.90 and the pH dead zone to about 0.05 is performed during the production phase, which includes days 5 to 14 of the culture process, for example, between days 5 and 14 after CHO-K1 cell seeding.

[0163] In some embodiments, the implementation of step (b) of setting the pH to 6.90 and the pH dead zone between 0.05 and 0.10, preferably setting the pH to 6.90 and the pH dead zone to about 0.05, controls the high mannose content in the antibody preparation without adversely affecting the secondary quality properties of the antibody preparation, for example, wherein the secondary quality properties are selected from (1) the relative levels (in %) of the major antibody substance in the preparation relative to the high molecular weight (HMW) antibody substance relative to the low molecular weight (LMW) antibody substance, for example, as determined by size exclusion chromatography (SEC); (2) the relative levels (in %) of the major antibody substance in the preparation, for example, as determined by capillary electrophoresis of sodium dodecyl sulfate (CE-SDS-NR) under non-reducing conditions; and / or (3) the relative levels (in %) of the antibody heavy chain and light chain in the preparation, for example, as determined by capillary electrophoresis of sodium dodecyl sulfate (CE-SDS-NR) under reducing conditions.

[0164] Embodiments of this disclosure relate to antibodies generated according to the above method, for example, antibodies comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO: 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO: 8, the antibodies being generated using a method comprising the steps of: (a) culturing host cells, such as Chinese hamster ovary K1 (CHO-K1) cells, under conditions sufficient to allow the antibodies to be expressed in a culture, the host cells comprising one or more expression vectors or vector systems containing polynucleotides encoding antibodies HC and LC; (b) setting a pH set point and a pH dead zone of the culture to regulate the levels of acidic, basic, and / or neutral antibody material in the culture, preferably wherein regulation comprises reducing the acidic material of the antibody culture by setting the pH to 6.90 and setting the pH dead zone between 0.05 and 0.10, preferably setting the pH dead zone to about 0.05; and (c) isolating antibodies HC and LC from the cell culture; and (d) optionally formulating the isolated antibodies into a pharmaceutical preparation comprising the isolated antibodies and a pharmaceutically acceptable carrier.

[0165] In the implementation scheme, the pH dead zone is set to less than 0.2, preferably ≤0.19, preferably ≤0.18, preferably ≤0.175, preferably ≤0.17, preferably ≤0.16, preferably ≤0.15, preferably ≤0.14, preferably ≤0.13, preferably ≤0.125, preferably ≤0.12, preferably ≤0.11, preferably ≤0.1, preferably ≤0.09, preferably ≤0.08, preferably ≤0.075, preferably ≤0.07, preferably ≤0.06, and most preferably ≤0.05. In a preferred implementation scheme, the pH dead zone is set to about 0.1 to 0.05, preferably 0.05 or 0.1, and most preferably 0.05.

[0166] In the implementation, the antibody of this disclosure is formulated as a high-concentration pharmaceutical composition, for example, for administration to a patient at pH 5.8. The pH is slightly below the pKa of histidine, which will still provide a buffering effect. For example, if the initial buffer system has a concentration of 20 mM histidine and a pH of 5.8, the ratio of conjugate base to acid can be calculated using the Henderson-Hasselbalch equation. To maintain the pH, the concentrations of the protonated form (HA) and deprotonated form (A-) can be doubled to 40 mM. Since changing the total concentration of the buffer will affect the ionic strength and osmolality of the solution, which may influence the stability and solubility of the protein, the concentration and / or properties of the osmolality-concentrating agent (here, sucrose) can be adjusted, and the corresponding formulation can be tested according to the examples.

[0167] As mentioned above, the heavy and light chain complementarity-determining regions (VHCDR and VLCDR), including the heavy and light chain antigen-binding domains of NI006 / ALXN2220, have been described in WO 2015 / 092077 A1 (named antibody NI-301.37F1) and Michalon et al., Nat. Commun. 12 (2021), 3142 (named antibody NI301A). As disclosed in WO 2015 / 092077 A1, NI006 (NI-301.37F1) is characterized in particular by binding to aggregated human wild-type thyroxine transporter (wtATTR), which in Figures 2 to 4 and Figure 7 The following are shown and described in Examples 3 through 6, and further described in the last paragraph on page 46. Furthermore, WO 2015 / 092077 A1 discloses monomers and dimers of NI006 (NI-301.37F1) that do not bind to the human natural thyroxine transporter (TTR), as in Example 5 and... Figure 4As shown. This binding spectrum is advantageous because the antibody selectively binds to aggregated wtTTR, thus allowing for consideration not only of hereditary thyroxine transporter amyloidosis (hATTR) with polyneuropathy (formerly known as familial amyloid polyneuropathy, FAP), caused by mutations in the gene encoding TTR, but also of wild-type thyroxine transporter amyloidosis (wtATTR), known as senile systemic amyloidosis (SSA). Furthermore, the antibody does not pose a risk of interfering with the assembly of natural monomers into physiological tetramers. Additionally, the antibody has been shown to remove ATTR deposits ex vivo from patient-derived myocardium via macrophages, and in vivo from mice transplanted with patient-derived ATTR fibrils in a dose- and time-dependent manner, where the bioactivity of ATTR removal involves antibody-mediated activation of phagocytic immune cells, including macrophages; see Michalon et al., Nat. Commun. 12 (2021), 3142.

[0168] Therefore, this disclosure relates to a composition, preferably a pharmaceutical composition, for use in a method of treating a subject with TTR amyloidosis (ATTR), comprising, in an aqueous formulation further comprising L-histidine (L-histidine and L-histidine monohydrochloride), sucrose, polysorbate 80, and water for injection, an antibody of the present disclosure as defined above. Preferably, the composition is a composition of the present disclosure for use in a method of treating a subject with TTR amyloidosis (ATTR), i.e., the composition comprises, in an aqueous formulation further comprising L-histidine (L-histidine and L-histidine monohydrochloride), sucrose, polysorbate 80, and water for injection, an antibody of the present disclosure at a concentration of about 50 mg / mL to about 100 mg / mL, preferably about 50 mg / mL, or 100 mg / mL, preferably about 50 mg / mL.

[0169] In a preferred embodiment, the corresponding composition of this disclosure and the formulation of the composition used according to this disclosure have a pH of 5.8.

[0170] Furthermore, preferably, the corresponding compositions of this disclosure and the formulations of the compositions used according to this disclosure have an osmolar concentration of ≥240 mOsm / Kg.

[0171] Furthermore, as mentioned above, the formulation of the composition is stable; therefore, in one embodiment, the pharmaceutical composition used according to this disclosure has a shelf life of 24 months at 2°C-8°C and protected from light, and preferably exhibits the stability criteria mentioned above.

[0172] In one embodiment, the compositions of this disclosure and the compositions used according to this disclosure comprise a formulation, which, when aggregated to about 2 ml, consists essentially of the following:

[0173] 50mg or 100mg of antibodies,

[0174] 2.12 mg of L-histidine,

[0175] 5.56 mg L-histidine hydrochloride

[0176] 160mg sucrose,

[0177] 0.6mg PS80, and

[0178] Water for injection.

[0179] In cases where the formulation is intended to provide another volume (e.g., 2.25 ml, 20 ml, or 22.5 ml), the corresponding amounts of antibody and excipient can be calculated.

[0180] As mentioned in Example 1, antibody NI006 / ALXN2220 can be produced in Chinese hamster ovary (CHO) cells. CHO cells are the most widely used mammalian cells for producing recombinant monoclonal antibodies because they are capable of post-translational modification (PTM) of antibody molecules, which also typically occurs in humans. Different CHO daughter cells with improved quality have been established through mutagenesis-induced genetic manipulation. Among these variants are CHO-K1, CHO-S, CHO-DXB11, and CHO-DG44. Therefore, in one embodiment, antibodies used in the compositions of this disclosure and in the compositions used according to this disclosure are produced in CHO cells, preferably in CHO-K1 cells, and purified from cell culture medium for further use.

[0181] In addition, the compatibility of the antibody formulation with materials used in clinical use was evaluated. Specifically, compatibility with PVC IV bags, IV lines and filters, as well as with PVC syringes and corresponding non-PVC materials, was assessed when glucose or saline was used as a diluent. Three concentrations of 1 mg / mL, 20 mg / mL, and 50 mg / mL were evaluated, and the data provided in Example 4 indicate that the formulation is generally compatible with the evaluated materials used in clinical use. Specifically, in the saline group, visible particles were observed, indicating that NI006 / ALXN2220 is less stable in saline, with data showing no significant changes in appearance, protein concentration, subvisible particles, SEC-HPLC, and ELISA binding assays when diluted in glucose solution. Therefore, NI006 / ALXN2220 at concentrations of 1.0 mg / mL, 20.0 mg / mL, and 50.0 mg / mL were demonstrated to be stable for 24 hours at 2°C–8°C, followed by 6 hours at 25°C (a total of 30 hours), and compatible with the evaluated materials used in clinical use.

[0182] The most common route of administration of monoclonal antibodies in therapy is intravenous (IV) infusion. This method is preferred because it allows the antibody to be delivered directly into the bloodstream, ensuring immediate distribution throughout the body and enabling precise dosing.

[0183] For monoclonal antibodies, intravenous administration is particularly important because their large molecular size typically prevents them from being effectively absorbed through the intestines or skin. This means that oral or transdermal delivery methods are not suitable for these types of drugs. Furthermore, IV administration bypasses first-pass metabolism in the liver, which can significantly alter the drug's efficacy and safety.

[0184] Therefore, the corresponding compositions of this disclosure and the compositions used according to this disclosure have been specifically developed for IV administration, and are therefore preferably formulations suitable for intravenous administration.

[0185] In some embodiments, the corresponding compositions of this disclosure and the compositions used according to this disclosure have not been reconstituted from lyophilized anti-TTR antibodies or their antigen-binding fragments and / or have not been further lyophilized.

[0186] Since sucrose is used as a tension modulator and additional stabilizer for antibodies in the formulations of the corresponding compositions of this disclosure and the compositions used according to this disclosure, NaCl is not required, especially since the primary purpose of NaCl is to stabilize proteins, which sucrose can achieve without increasing the ionic strength of the solution. Therefore, the pharmaceutical compositions of this disclosure are preferably substantially free of sodium chloride.

[0187] Alternatively or concurrently, the corresponding compositions of this disclosure and the compositions used according to this disclosure are substantially free of (or, for example, completely lacking) poloxamer.

[0188] The corresponding compositions of this disclosure and formulations of the compositions used according to this disclosure are preferably provided in vials. Although in a preferred embodiment, the formulation is provided in a 2 ml glass vial, preferably a type I clear glass vial, preferably with a 12.5% ​​overfill (2.25 ml), this disclosure also covers other pharmaceutical containers with different fill volumes, such as 20 ml vials, for example 20 ml glass vials, preferably with a 12.5% ​​overfill (22.5 ml), or designed such that the amount of the corresponding vial / container and the amount of antibody provided totals a fixed dose, optionally plus, for example, about 12.5% ​​of other containers / vias provided by the overfill.

[0189] In a preferred embodiment, the formulation is provided in a 2 mL glass vial with an aluminum flip-top seal above a rubber stopper, preferably with a volume of 2.25 mL added to the vial.

[0190] In a preferred embodiment, the corresponding composition of this disclosure and the formulation of the composition used according to this disclosure are preservative-free concentrates for infusion solutions, which are sterile, colorless to pale yellow, clear to pale milky white liquids substantially free of visible particles, wherein preferably, the concentrate for the solution is diluted in sterile glucose prior to administration as an intravenous infusion.

[0191] This disclosure also relates to corresponding pharmaceutical containers that contain the compositions / formulations of this disclosure.

[0192] This disclosure also relates to a stable aqueous formulation for use in a method for treating TTR amyloid cardiomyopathy (ATTR-CM), wherein the formulation

[0193] (i) It is basically composed of the following items:

[0194] (a) The antibody disclosed herein, preferably

[0195] The antibody comprises two HCs having SEQ ID NO: 7 or 9 and two LCs having SEQ ID: 8, wherein preferably in each HC, the N-terminal glutamine (Q at position X1 of SEQ ID NO: 7 and position 1 of SEQ ID NO: 9, respectively) is modified to pyroglutamic acid, the C-terminal lysine (K at position X7 of SEQ ID NO: 7 and position 450 of SEQ ID NO: 9) is absent, and the heavy chain is N-glycosylated, preferably wherein the N-glycosylation site is at position Asn300 of SEQ ID NO: 9, wherein the amino acids at positions X2, X3, X4, and X5 of SEQ ID NO: 7 are modified or unmodified, preferably unmodified, or wherein the methionine at position M255 of SEQ ID NO: 9 is unoxidized, the asparagine at positions N318 and N387 of SEQ ID NO: 9 is undeamidinated and does not contain succinimide, and SEQ ID NO: 8 is also present in the LC. The proline at position P448 of NO:9 is unamidated, and the glycine (X6 of SEQ ID NO: 7 and G at position 449 of SEQ ID NO: 9) is preferably present and not cleaved. In a preferred embodiment, the antibody consists of two HCs having SEQ ID NO: 39 or 9 and two LCs having SEQ ID: 8, wherein preferably in each HC, the N-terminal glutamine (X1 of SEQ ID NO: 39 and Q at position 1 of SEQ ID NO: 9, respectively) is modified to pyroglutamic acid, the C-terminal lysine (X7 of SEQ ID NO: 39 and K at position 450 of SEQ ID NO: 9) is absent, and the heavy chain is N-glycosylated, preferably wherein the N-glycosylation site is at position Asn300 (X of SEQ ID NO: 39). V And at position 300 (N) of SEQ ID NO: 9, wherein the amino acids at positions X2, X3, X4 and X5 are modified or unmodified, preferably unmodified, or wherein the methionine at position M255 of SEQ ID NO: 9 is unoxidized, the asparagine at positions N318 and N387 of SEQ ID NO: 9 is undeamidinated and does not contain succinimide, and the proline at position P448 of SEQ ID NO: 9 is unamidinated, wherein glycine (X6 of SEQ ID NO: 39 and G at position 449 of SEQ ID NO: 9) is preferably present and not cleaved, and wherein the amino acids at positions X of SEQ ID NO: 39 are modified or unmodified, preferably unmodified, or wherein the amino acids at positions X of SEQ ID NO: 39 are unmodified, and the amino acids at positions X of SEQ ID NO: ...9 are unmodified, and the amino acids at positions X of SEQ ID NO: 9 are unmodified, and the amino acids at positions X of SEQ ID NO: 9 are unmodified, and the amino acids at positions X of SEQ ID NO: 9 are unmodified, and the amino acids at positions X of SEQ ID NO: 9 are unmodified, and the amino acids at positions X of SEQ ID NO: 9 are unmodified, and the amino acids at positions X of SEQ ID NO: I X II X III X IV X VIX VI The amino acids at the specified positions are modified or unmodified, preferably unmodified, or wherein the methionine at positions M71, M115, M361, and M431 of SEQ ID NO: 9 is unoxidized, the asparagine at position N58 of SEQ ID NO: 9 is undeamidinated, and the asparagine at position D283 of SEQ ID NO: 9 is unisomerized; alternatively, the antibody consists of two heavy chains having SEQ ID NO: 9 and two light chains having SEQ ID: 8, and wherein preferably in the heavy chain, the N-terminal glutamine is modified to pyroglutamic acid, the C-terminal lysine is cleaved or absent, and the heavy chain is N-glycosylated, preferably wherein the N-glycosylation site is at position Asn300.

[0196] (b) Histidine at a concentration of approximately 20 mM

[0197] (c) Sucrose at a concentration of approximately 8% (w / v),

[0198] (d) PS80 at a concentration of approximately 0.03% w / v

[0199] (e) Water for injection,

[0200] (ii) It has a pH of approximately 5.8;

[0201] (iii) Contain in a 2 mL disposable vial containing approximately 100 mg of antibody or in a 20 mL disposable vial containing 1000 mg of antibody:

[0202] (iv) Dilute with sterile glucose solution before administration, preferably to a concentration of not less than 1 mg / mL of antibody; and

[0203] (v) Administered via intravenous infusion.

[0204] This disclosure also relates to a kit comprising one or more containers of this disclosure and a device for delivering the formulation / composition to a human subject, optionally wherein the device comprises an infusion bag or syringe. In a preferred embodiment, the formulation is a preservative-free, clear to milky white and colorless to pale yellow solution provided in a vial at 100 mg / 2 mL.

[0205] This disclosure also relates to an article comprising one or more containers and labels of this disclosure, wherein the label specifies that the antibody is indicated for the treatment of ATTR, particularly ATTR cardiomyopathy (ATTR-CM), such as amyloid cardiomyopathy mediated by wild-type or hereditary thyroxine transporters (wtATTR-CM or hATTR-CM). In a preferred embodiment, the kit includes instructions specifying that the antibody will be administered intravenously, and may contain instructions for, for example, IV dosing. In a preferred embodiment, the container is a glass vial as defined above.

[0206] In another preferred embodiment, the kit may also include a diluent. In this embodiment, the diluent comprises a 5% glucose solution, which may be provided in a separate container. In a preferred embodiment, the antibody is prepared as a preservative-free, clear to milky white and colorless to pale yellow solution provided in vials at 100 mg / 2 mL.

[0207] This disclosure also relates to a method for treating ATTR, preferably ATTR cardiomyopathy (ATTR-CM) and / or ATTR polyneuropathy (ATTR-PN), wherein the method comprises administering the corresponding antibody and composition of this disclosure to a subject in need, preferably wherein the administration is intravenous. Preferably, the antibody / composition is diluted and administered as an intravenous infusion over approximately one to two hours, wherein the dilution is preferably performed in a 5% glucose solution.

[0208] In some embodiments, patients to be treated with the antibody of this disclosure or a pharmaceutical composition containing the antibody have been pre-screened, for example, to identify that they have wtATTR-CM or hATTR-CM, and treatment is selectively provided to such patients who have been positively identified.

[0209] In some implementations, the subject has previously been treated with and / or is concurrently receiving a disease modifier, such as a TTR tetramer stabilizer, like tafamidis (VYNDAQEL). ® Or VYNDAMAX ® ) or acoramidis (AG10). Currently, chlorpromazine, which provides relief for oral diseases, is the only approved drug that specifically targets wild-type and hereditary forms of ATTR-CM.

[0210] This disclosure also provides a method for manufacturing the corresponding antibody and the corresponding pharmaceutical product of this disclosure.

[0211] Methods for generating antibodies or their antigen-binding fragments include

[0212] a) Cloning the corresponding nucleic acid molecules and polynucleotides of the present disclosure, comprising a first nucleotide sequence and preferably a first signal peptide, into an expression vector, and cloning the corresponding nucleic acid molecules and polynucleotides of the present disclosure, comprising a second nucleotide sequence and preferably a second signal peptide, into an expression vector, wherein the first nucleotide sequence and the second nucleotide sequence can be provided in the same or different expression vectors, preferably different expression vectors;

[0213] b) Transform the expression vector into non-human host cells, preferably into CHO cells, and more preferably into CHO-K1 cells;

[0214] c) Culture host cells under conditions that allow the expression of immunoglobulin chains containing both heavy and light chains; and

[0215] d) Separate the immunoglobulin chains and the resulting IgG antibodies from the culture, and optionally digest the IgG antibodies to generate antigen-binding fragments of the antibodies, for example using enzymatic digestion including papain cleavage to generate Fab fragments.

[0216] In embodiments, the method of this disclosure includes culturing host cells, particularly CHO-K1 cells, with a pH dead zone of less than 0.2, preferably ≤0.19, preferably ≤0.18, preferably ≤0.175, preferably ≤0.17, preferably ≤0.16, preferably ≤0.15, preferably ≤0.14, preferably ≤0.13, preferably ≤0.125, preferably ≤0.12, preferably ≤0.11, preferably ≤0.1, preferably ≤0.09, preferably ≤0.08, preferably ≤0.075, preferably ≤0.07, preferably ≤0.06, and most preferably ≤0.05. In a preferred embodiment, the pH dead zone is set to about 0.1 to 0.05, preferably 0.05 or 0.1, and most preferably 0.05. Preferably, the method of this disclosure includes culturing host cells, particularly CHO-K1 cells, at pH 6.9 with a pH dead zone less than 0.2, preferably ≤0.19, preferably ≤0.18, preferably ≤0.175, preferably ≤0.17, preferably ≤0.16, preferably ≤0.15, preferably ≤0.14, preferably ≤0.13, preferably ≤0.125, preferably ≤0.12, preferably ≤0.11, preferably ≤0.1, preferably ≤0.09, preferably ≤0.08, preferably ≤0.075, preferably ≤0.07, preferably ≤0.06, and most preferably ≤0.05. In a preferred embodiment, the pH dead zone is set to about 0.1 to 0.05, preferably 0.05 or 0.1, and most preferably 0.05. In an embodiment, the pH is set to pH 6.9 and has the pH dead zone indicated above.

[0217] This method can also be used to generate the pharmaceutical compositions and corresponding pharmaceutical products of this disclosure. Specifically, after generating the antibody, the method further includes...

[0218] e) The antibody is prepared in an aqueous solution containing an antibody at a concentration of about 50 mg / ml or about 100 mg / ml and histidine at a concentration of about 20 mM, sucrose at a concentration of about 6.5% by weight / volume (w / v) or about 8% (w / v) sucrose, and PS80 at a concentration of about 0.03% w / v, wherein the preparation has a pH of about 5.8 to generate a pharmaceutical composition containing the antibody; and optionally

[0219] f) Fill the composition into vials, and further optionally...

[0220] g) The pharmaceutical composition and vial are packaged together with instructions for use in human patients, such as intravenous administration of the antibody, in the kit.

[0221] As mentioned above, the antibodies of the present invention have been shown to remove ATTR deposits ex vivo from patient-derived myocardium via macrophages and in vivo from mice transplanted with patient-derived ATTR fibrils in a dose- and time-dependent manner, wherein the bioactivity of ATTR removal involves antibody-mediated activation of phagocytic immune cells, including macrophages; see Michalon et al., Nat. Commun. 12 (2021), 3142.

[0222] To further investigate the mechanisms of macrophage-mediated amyloid depletion, a high-resolution live-cell imaging assay has been developed. This assay involves incubating cardiac tissue sections containing ATTR with THP-1-derived macrophages in the presence of an anti-TTR antibody, followed by live-cell imaging.

[0223] In one method, the method includes staining amyloid deposits in tissue sections with the amyloid-specific red fluorescent dye Amytracker 680 and labeling ALXN2220 with the green fluorescent dye Vivotag-680. After incubating the stained tissue sections with macrophages in the presence of labeled ALXN2220, high-resolution live-cell imaging is performed as described in Example 5. Overlapping displays of fluorescent images visualized using either the red fluorescent dye for staining amyloid deposits or the green fluorescent dye for labeling antibodies show the same portion of tissue stained with the red fluorescent dye for directly staining amyloid deposits and stained with the green fluorescent dye for staining amyloid deposits by the binding of labeled antibodies to the amyloid deposits; see also Figure 7 Therefore, the binding of the antibody to ATTR in the tissue section was confirmed.

[0224] In another method, the procedure involves staining tissue sections with the amyloid-specific red fluorescent dye Amytracker 680. ALXN2220 was unlabeled. The stained tissue sections were then incubated with macrophages in the presence of an antibody and subjected to high-resolution live-cell imaging as described in Example 5. Figure 8 As shown, ALXN2220 triggers macrophage phagocytosis of amyloid protein. Dotted and intracellular red fluorescence patterns reveal the presence of ATTR amyloid protein in phagocytic vesicles and thus demonstrate macrophage-mediated ATTR internalization.

[0225] In another method, ALXN2220 is labeled with the green fluorescent dye Vivotag-680. Unstained tissue sections are incubated with macrophages in the presence of the labeled antibody and then subjected to high-resolution live-cell imaging as described in Example 5. Figure 9 and Figure 10 The visible fluorescent signal represents amyloid staining caused by antibody binding to ATTR. For example... Figure 9 and Figure 10 As shown, the punctate fluorescence signal separated from the fluorescence signal of amyloid over time, or the thin and elongated fluorescence signal separated from adjacent cardiomyocytes, and the punctate fluorescence signal indicate that macrophages mediate amyloid fragmentation and the separation of ATTR deposits from adjacent cardiomyocytes, followed by amyloid fragmentation.

[0226] Therefore, this study demonstrates for the first time that live-cell imaging is a suitable method for detecting antibody binding to amyloid deposits and for visualizing the removal of amyloid deposits through activation of phagocytic macrophages. These findings open the possibility of developing methods for screening and validating anti-amyloid antibodies and other amyloid-depleting compounds using live-cell imaging.

[0227] Therefore, in one aspect, the present invention relates to a method for verifying an amyloid depletion drug, also known as an amyloid depletion compound, comprising incubating a tissue section containing amyloid deposits with macrophages in the presence of the amyloid depletion drug, wherein the tissue section is stained with an amyloid-specific fluorescent dye and / or the amyloid depletion drug is labeled with a fluorescent dye, and performing high-resolution live-cell imaging, wherein (a) the overlap of antibody fluorescence signals and amyloid fluorescence signals indicates the binding of the amyloid depletion drug to amyloid; (b) the presence of punctate and intracellular fluorescence signals in phagocytic vesicles indicates macrophage-mediated amyloid internalization; and (c) the sequential separation of punctate fluorescence signals from the fluorescence signal of amyloid indicates macrophage-mediated amyloid fragmentation. Thus, live-cell imaging can be used to visualize the binding of the amyloid depletion drug to amyloid deposits, macrophage-mediated amyloid internalization, and macrophage-mediated amyloid fragmentation, wherein these mechanisms lead to intracellular amyloid degradation. Therefore, if any of (a) to (c), preferably all of (a) to (c), more preferably at least one of (b) and (c), or (b) and (c) are observed during live-cell imaging, then the amyloid depletion drug is confirmed to have amyloid depletion activity.

[0228] To confirm the specificity of the results, i.e., attributable to the activity of the amyloid depletion drug, the observed mechanisms (binding of the amyloid depletion drug to amyloid deposits, macrophage-mediated amyloid internalization, and macrophage-mediated amyloid fragmentation) should not be observed when tissue sections and macrophages with amyloid deposits are incubated not in the presence of the amyloid depletion drug, but, for example, in the presence of a control compound. The control compound preferably belongs to the same class of substances as the amyloid depletion drug, and more preferably to the same class of substances as the antibody, but exhibits nonspecific binding. The same applies to the methods further described below.

[0229] In another aspect, the present invention relates to a screening method for identifying and optionally obtaining amyloid depletion drugs from a variety of test compounds, the method comprising incubating tissue sections with amyloid deposits with macrophages in the presence of the test compound, wherein the tissue sections are stained with an amyloid-specific fluorescent dye and / or the test compound is labeled with a fluorescent dye, and performing high-resolution live-cell imaging, wherein (a) the overlap of the fluorescence signal of the test compound and the fluorescence signal of amyloid indicates the binding of the amyloid depletion drug to amyloid; (b) the presence of punctate and intracellular fluorescence signals in phagocytic vesicles indicates macrophage-mediated amyloid internalization; and (c) the sequential separation of the punctate fluorescence signal from the fluorescence signal of amyloid indicates macrophage-mediated amyloid fragmentation, wherein the presence of any one of items (a) to (c), preferably all of items (a) to (c), or at least one of items (b) and (c), preferably items (b) and (c), indicates the suitability of the test compound as an amyloid depletion drug.

[0230] The present invention also relates to a method for screening an amyloid depletion drug for its ability to bind to amyloid and mediate macrophage recruitment to amyloid deposits, followed by amyloid fragmentation and internalization, i.e., for its ability to activate macrophage-mediated amyloid fragmentation and internalization, thereby leading to intracellular degradation of amyloid. The method includes incubating tissue sections containing amyloid deposits with macrophages in the presence of the amyloid depletion drug, wherein the tissue sections are stained with an amyloid-specific fluorescent dye and / or the amyloid depletion drug is labeled with a fluorescent dye, and performing high-resolution live-cell imaging, wherein (a) overlap of antibody fluorescence signals and amyloid fluorescence signals indicates binding of the amyloid depletion drug to amyloid; (b) the presence of punctate and intracellular fluorescence signals in phagocytic vesicles indicates macrophage-mediated amyloid internalization; and (c) the sequential separation of punctate fluorescence signals from the amyloid fluorescence signal indicates macrophage-mediated amyloid fragmentation.

[0231] In one embodiment, the method of the present invention includes testing a control sample, for example, incubating tissue sections with macrophages in the presence of a control. To qualify as an amyloid depletion drug, the test substance used in the screening method should exhibit enhanced binding of the amyloid depletion drug to amyloid, enhanced amyloid internalization, and / or enhanced amyloid fragmentation compared to the control, and / or, for suitability for treating amyloidosis or amyloid-related diseases, the amyloid depletion drug should exhibit enhanced binding of the amyloid depletion drug to amyloid, enhanced amyloid internalization, and / or enhanced amyloid fragmentation compared to the control. The control sample can be any control exhibiting nonspecific binding to amyloid and / or macrophages.

[0232] The present invention also relates to a method for producing a pharmaceutical composition of an amyloid depletion drug, the method comprising: (i) providing, optionally producing, an amyloid depletion drug; (ii) subjecting the amyloid depletion drug to a method as defined above; (iii) using the information obtained in step (ii) as part of an evaluation of whether the amyloid depletion drug is suitable for use as a pharmaceutical composition; and optionally formulating the amyloid depletion drug, which is found to be suitable for use as a pharmaceutical composition in step (iii), together with a pharmaceutically acceptable carrier such as a buffer, a tensioning agent, and / or a surfactant, most preferably all three components.

[0233] In another aspect, the present invention relates to a method for characterizing, validating, developing, and / or quality controlling (e.g., batch control of amyloid depletion drugs), the method comprising: (i) providing, optionally producing, an amyloid depletion drug; (ii) subjecting the amyloid depletion drug to the method as described above; (iii) communicating the information obtained in (i) to a customer, contracting party, or partner and / or selecting a drug that has been identified as a suitable amyloid depletion drug; and optionally (iv) treating amyloidosis or amyloid-related diseases with the amyloid depletion drug or a pharmaceutical composition comprising the amyloid depletion drug.

[0234] Combination therapy has also attracted considerable attention in the treatment of amyloidosis. For example, as disclosed in WO 2021 / 228987 A1, the combination of anti-TTR antibodies, which are amyloid depletion agents, with TTR tetramer stabilizers (such as cloxacillin and diflunisal) or with TTR gene silencing agents is a promising approach for treating TTR amyloidosis. During the development of combination therapies, the effects of the drugs on each other must be carefully tested to avoid adverse effects such as reduced treatment efficacy. Therefore, the present invention also relates to a method for analyzing the effect of an agent on the amyloid depletion activity of an amyloid depletion drug, the agent being, for example, a second drug, a tracer, radiation, labeling, preferably wherein the agent is a second drug, and particularly a second drug or analgesic for treating amyloidosis or amyloid-related diseases, such as a nonsteroidal anti-inflammatory drug such as ibuprofen or acetaminophen, the method comprising incubating a tissue section having amyloid deposits with macrophages, preferably THP-1-derived macrophages, in the presence of the amyloid depletion drug and the agent; and performing high-resolution live-cell imaging, wherein (a) the overlap of antibody fluorescence signals and amyloid fluorescence signals indicates the binding of the amyloid depletion drug to amyloid; ( (b) The presence of punctate and intracellular fluorescent signals in phagocytic vesicles indicates macrophage-mediated amyloid internalization; and (c) the sequential separation of punctate fluorescent signals from the fluorescent signals of amyloid indicates macrophage-mediated amyloid fragmentation, wherein, compared to a control, the control is preferably a tissue section with amyloid deposits incubated with macrophages and an amyloid depletion drug but not with the drug, the substantially unchanged fluorescence patterns of items (a) to (c) indicate the suitability of the combination of the amyloid depletion drug and the drug in the treatment of amyloidosis or amyloid-related diseases, and the substantially changed fluorescence patterns of any of items (a) to (c) indicate the effect of the drug on the amyloid depletion activity of the amyloid depletion drug.

[0235] Enhanced fluorescence signals emitted from amyloid deposits indicate more efficient binding of the amyloid-depleting drug in the presence of the agent, and therefore indicate a synergistic effect on the amyloid-depleting activity of the drug. Conversely, decreased fluorescence signals emitted from amyloid deposits indicate less efficient binding of the drug in the presence of the agent, and therefore indicate a detrimental effect on the amyloid-depleting activity of the drug.

[0236] Alternatively, enhanced intracellular fluorescence in phagocytic vesicles indicates enhanced macrophage-mediated amyloid internalization, and therefore, a synergistic effect on the amyloid depletion activity of amyloid-depleting drugs. Decreased intracellular fluorescence in phagocytic vesicles indicates decreased macrophage-mediated amyloid internalization, and therefore, an impaired effect on the amyloid depletion activity of amyloid-depleting drugs.

[0237] Alternatively, enhanced separation of the punctate fluorescence signal from the fluorescence signal of amyloid indicates enhanced macrophage-mediated amyloid fragmentation, and therefore, a synergistic effect on the amyloid depletion activity of amyloid depletion drugs. Decreased separation of the punctate fluorescence signal from the fluorescence signal of amyloid indicates decreased macrophage-mediated amyloid fragmentation, and therefore, an impairing effect on the amyloid depletion activity of amyloid depletion drugs.

[0238] In a preferred embodiment of the method of the present invention, the amyloid depletion agent is an anti-amyloid antibody, and most preferably an anti-TTR antibody. Therefore, the amyloid deposits are preferably TTR amyloid deposits, and the amyloidosis and amyloid-related diseases are preferably TTR amyloidosis or TTR amyloid-related diseases, most preferably cardiac TTR amyloidosis. Therefore, the tissue section is preferably a cardiac tissue section. In this context, the agent is preferably a second drug, which is preferably a drug that can be used to treat TTR amyloidosis, such as a TTR tetramer stabilizer, such as chlorpromazine or diflunisal, or a TTR gene silencer.

[0239] The amyloid depletion drug to be verified by the methods of the present invention, characterized by the methods of the present invention, used for quality control by the methods of the present invention, or used to analyze the effect of a drug (e.g., a second drug) on ​​its amyloid depletion activity is preferably the anti-TTR antibody of the present invention, and most preferably the antibody ALXN2220 as characterized above. The antibody can also be used as a positive control in the methods of the present invention, particularly in screening and identification methods. Therefore, the methods of the present invention include the use of a positive control, which is preferably the antibody of the present invention as defined above, and most preferably the antibody ALXN2220.

[0240] In a preferred embodiment, high-resolution live-cell imaging uses refractive index imaging for cell visualization and fluorescence microscopy for amyloid imaging. If the amyloid in the tissue section is stained with an amyloid-specific fluorescent dye, and the antibody is also labeled with a fluorescent dye, it is preferable to use two different dyes, preferably dyes of different colors, such as red and green fluorescent dyes.

[0241] Tissue sections containing amyloid protein are preferably derived from subjects suffering from amyloidosis or amyloid-related diseases, more preferably from TTR amyloidosis, and most preferably from subjects suffering from cardiac TTR amyloidosis.

[0242] The present invention also relates to a kit for carrying out the methods of the present invention, the kit comprising at least a first fluorescent dye for staining amyloid and / or a second fluorescent dye for labeling amyloid depletion drugs, and optionally instructions for use or consumables for carrying out the method, such as microculture dishes. Preferably, the kit may also comprise negative and / or positive controls, wherein the positive control may be, for example, an antibody as defined above, preferably antibody ALXN2220.

[0243] In the past, additional assays have been developed to determine the elimination or reduction of amyloid fibrils in non-human animal models containing amyloid fibril implants (PDAX) following administration of amyloid depletion drugs (particularly anti-TTR antibodies) to animals (see WO 2020 / 094883 A1), or to determine the efficacy of target antigen-binding molecules (particularly anti-TTR antibodies) in mediating the phagocytosis of amyloid-producing TTRs (see WO 2023 / 099788A1). These assays can be combined with the methods of the present invention to improve the accuracy of the results. For example, the method of the present invention can be performed in conjunction with a corresponding method using a patient-derived amyloid xenograft (PDAX) nonhuman animal model, wherein the animal is characterized by an implant of amyloid fibrils derived from tissues or organs of a patient suffering from amyloidosis or amyloid-related diseases, wherein the corresponding amyloid protein and amyloid fibrils contain amyloid-thyroxine transporter (ATTR), and wherein the amyloid fibrils are implanted subcutaneously or subcapsularly, or implanted in the kidney, peritoneum, muscle, brain, ventricle, nerve, eye, tongue, or heart, wherein the corresponding method includes administering an amyloid depletion drug or test substance to the PDAX nonhuman animal model and determining the amyloid fibrils in the model, wherein, compared to a control, the accelerated elimination or reduction of amyloid fibrils after administration of the drug or test substance respectively indicates the suitability of the amyloid depletion drug for the treatment of amyloidosis or amyloid-related diseases and the amyloid depletion activity of the test substance.

[0244] definition

[0245] To avoid any doubt, it should be emphasized that the use of expressions such as "in some embodiments," "in certain embodiments," "in some cases," "in some situations," "in another embodiment," "in one embodiment," "in another aspect," "in the first aspect," "in the second aspect," etc., and is intended to mean that any embodiment described herein should be read in conjunction with each feature of those embodiments, and must be treated in the same manner as the combination of features of those embodiments and aspects will be detailed in one embodiment. The same applies to any combination of embodiments and features exemplified in the appended claims and examples, which are also intended to be combined with features of the corresponding embodiments disclosed in the specification, wherein, for the sake of consistency and brevity only, the features of the embodiments are characterized by dependence, and in practice, each combination of embodiments and features that may be interpreted due to dependence must be considered as literally disclosed, and not as a choice between different options. In this context, those skilled in the art will understand that the embodiments and features disclosed in the examples are intended to be generalized to any anti-TTR antibody and its equivalents having substantially the same properties.

[0246] As used herein, the term "about" means a value within ±10%, preferably ±5%, of the stated value. For example, "about 8%" can mean any percentage between 7.2% and 8.8%, preferably between 7.6% and 8.4%. Similarly, "about 2 mL" can mean any volume between 1.8 mL and 1.2 mL (e.g., 1.8 mL, 1.9 mL, 1.95 mL, 2 mL, 2.05 mL, 2.10 mL, 2.15 mL, and 2.2 mL). In the context of antibody amounts as referred to herein, such as 50 mg / mL or 100 mg / mL, the term "about" means a concentration in the range of 45 mg / mL to 50 mg / mL, and preferably 48 mg / mL to 52 mg / mL, and a concentration in the range of 90 mg / mL to 113 mg / mL, preferably 96 mg / mL to 113 mg / mL. Therefore, even if the term "about" is not explicitly used, the above concentration ranges apply. For example, when 50 mg / mL is mentioned, it includes concentrations in the range of 45 mg / mL to 55 mg / mL, and when 100 mg / mL is mentioned, it includes concentrations in the range of 90 mg / mL to 113 mg / mL, because these ranges are within experimental deviations.

[0247] In connection with this disclosure, the term “and / or” should be understood to mean that all members of the group connected by the term “and / or” are disclosed cumulatively in any combination (alternating with each other and in each case). For the expression “A, B and / or C”, this 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 and B and C).

[0248] As used herein, “antibody” refers to a population of immunoglobulin molecules that specifically bind to a target antigen. The term “antibody” should be understood to encompass not only a single molecular entity but also antibody variants that can be produced by post-translational modifications such as glycosylation, deamidation, lysine cleavage, and pyroglutamate formation. These modifications can result in a heterogeneous population, where individual molecules within a batch vary in the number or type of modifications present. Therefore, the term “antibody” refers to this population as a whole, including both modified and unmodified forms.

[0249] In the context of the formulations / pharmaceutical compositions disclosed herein, the phrases "substantially NaCl-free" and "largely NaCl-free" mean that the pharmaceutical composition / formulation contains no NaCl or contains only trace amounts that are considered negligible for the intended use of the product; that is, NaCl is present at low levels that do not affect the performance, stability, safety, or efficacy of the formulation. Furthermore, this phrase can also refer to a formulation / pharmaceutical composition in which NaCl was not intentionally added, but which may contain NaCl due to the presence of other excipient salts, such as histidine-HCl. 2+ or Cl - .

[0250] In the context of the formulations / pharmaceutical compositions disclosed herein, the phrases “substantially free” and “largely free” mean that the pharmaceutical composition / formulation does not contain the mentioned substance or contains only trace amounts that are considered negligible for the intended use of the product, i.e., the substance is present at low levels that do not affect the performance, stability, safety, or efficacy of the formulation.

[0251] As used herein, the term "binding potency" refers to a characteristic corresponding to a quantitative measurement of biological activity (e.g., TTR binding). Binding potency assays (e.g., ELISA assays) can be used to measure the ability of the anti-TTR antibody of this disclosure or its antigen-binding fragment to elicit a specific response in a disease-associated system (e.g., a subject with ATTR-CM such as WT-ATTR-CM). The activity measured in the assay is a surrogate for the expected biological effect and can be used to assess the maintenance of that effect over time (e.g., after storage).

[0252] As used herein, the expressions “capable of binding” and “bind to” refer to the ability of an antibody or its antigen-binding fragment to bind to, for example, aggregated TTRs under experimental conditions (e.g., in an ELISA assay).

[0253] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent (e.g., an anti-TTR antibody as described herein), optionally in combination with one or more pharmaceutically acceptable excipients, diluents, and / or carriers. The pharmaceutical composition is, for example, formulated for administration to a subject, such as a mammal, like a human, to prevent, treat, or control a specific disease or condition that affects or may affect the subject (e.g., ATTR-CM, such as WT-ATTR-CM).

[0254] As used herein, the term “pharmaceutically acceptable” means those compounds, materials, compositions, and / or dosage forms that are suitable for contact with the tissues of a subject (such as, in mammals, e.g., humans) without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the bounds of reasonable medical judgment, and in proportion to a reasonable benefit / risk ratio.

[0255] As used in this article, the term "between" includes endpoints.

[0256] As used in this article, according to the European Pharmacopoeia, room temperature (RT) is defined as between 15°C and 25°C.

[0257] The "sequence identity percentage (%)" relative to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference polynucleotide or polypeptide sequence after sequence alignment and, where necessary, nicking to achieve the maximum sequence identity percentage. Alignment used to determine the nucleic acid or amino acid sequence identity percentage can be performed in various ways within the capabilities of those skilled in the art, such as using publicly available computer software, such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. For example, the sequence identity percentage value can be generated using the sequence comparison computer program BLAST. As an example, the sequence identity percentage of a given nucleic acid or amino acid sequence A relative to a given nucleic acid or amino acid sequence B, A to B or A to B (which may alternatively be expressed as a given nucleic acid or amino acid sequence A relative to a given nucleic acid or amino acid sequence B, A to B or A to B having a specific sequence identity percentage) is calculated as follows:

[0258] 100 multiplied by (fraction X / Y)

[0259] Where X is the number of nucleotides or amino acids that receive the same match score in the alignment of A and B by a sequence alignment program (e.g., BLAST), and Y is the total number of nucleic acids in B. It should be understood that when the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percentage of sequence identity between A and B will not be equal to the percentage of sequence identity between B and A.

[0260] As used herein, the term "treat" refers to therapeutic treatment and preventative or preventive measures aimed at preventing or mitigating (alleviating) undesirable physiological changes or conditions, such as the development of a heart defect. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and detectable or undetectable remission (whether partial or complete). "Treatment" can also mean extended survival compared to expected survival without treatment (e.g., extending the survival of a subject with ATTR by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 years or more, e.g., the subject's lifetime). Those who need treatment include those who already have symptoms or conditions, as well as those who are susceptible to symptoms or conditions or those who need to prevent the manifestation of symptoms or conditions.

[0261] "pH dead zone" refers to a small range around a set point within which pH fluctuations are permissible before correction occurs. "pH dead zone of 0.05" means that pH fluctuations of ±0.05 units around the target pH are permissible before correction actions (such as adjustment with acid or base) are applied.

[0262] In the context of peptides or antibodies, the term "isolated" refers to a molecule that has been removed from its natural environment (e.g., cell culture). An "isolated" antibody is substantially free of material from the cell from which it originates. In some embodiments, isolated molecules, such as peptides, soluble proteins, antibodies, polynucleotides, carriers, and cells, are those that have been purified to the point that they no longer exist in forms found in nature. In some embodiments, the isolated molecules are substantially pure. As used herein, the term "substantially pure" means material that is at least 50% pure (e.g., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure. In some embodiments, the purified molecules are pharmaceutical grade.

[0263] The term "polynucleotide" is intended to include both single and multiple nucleic acids, and refers to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA) or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide. "Isolated" nucleic acids or polynucleotides are intended to be nucleic acid molecules, DNA or RNA, that have been removed from their natural environment. For example, for the purposes of this disclosure, recombinant polynucleotides encoding antibodies contained in a vector are considered isolated. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides purified (partially or substantially) in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of this disclosure. Isolated polynucleotides or nucleic acids according to this disclosure also include synthetically produced such molecules. Furthermore, polynucleotides or nucleic acids may be or may include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators. Attached Figure Description

[0264] Figure 1 : cIEF acidity of ALXN2220 GMP pharmaceutical substance batch.

[0265] Figure 2 The amount of mannosaccharide (Man5) in the ALXN2220 GMP pharmaceutical substance batch.

[0266] Figure 3 : Small-scale study of iCIEF acidity.

[0267] Figure 4 Overview of Man 5 in small-scale studies.

[0268] Figure 5 : Online pH of small-scale bioreactors.

[0269] Figure 6 Glycosylation spectrum of ALXN2220 determined by LCMS.

[0270] Figure 7ATTR deposits in myocardial tissue sections were identified using Amytracker 680, a red fluorescent dye specific for amyloid. ALXN2220, labeled with the green fluorescent dye A488, selectively binds to ATTR, as indicated by the overlap between red and green fluorescence. (A) Refractive index imaging of myocardial tissue sections; (B) Identification of ATTR deposits in myocardial tissue sections using the red fluorescent dye Amytracker 680; (C) Visualization of ALXN2220 labeled with the green fluorescent dye A488; (D) The overlap between red and green fluorescence indicates ALXN2220 binding to ATTR.

[0271] Figure 8 ALXN2220 triggers macrophage phagocytosis of amyloid protein. Dotted and intracellular red fluorescence patterns reveal the presence of ATTR amyloid protein in phagocytic vesicles. Macrophages exhibit a broad range of phagocytic activity indicated by the number of red fluorescent vesicles. Figures (A), (B), (C), and (D) each represent exemplary fluorescence images, with the right image being a magnified portion of the left image.

[0272] Figure 9 Multinucleated macrophages continuously dissociate amyloid protein fragments from large deposits. Fluorescence images show the fragment dissociation process over time (after 2.5 h, 3 h, 4 h, 12 h, 13 h, and 13.5 h).

[0273] Figure 10 Macrophages were observed to separate thin, elongated ATTR deposits from adjacent cardiomyocytes, allowing the deposits to migrate more than 20 µm. Macrophages also cleaved large, protruding amyloid deposits, thereby dissociating small and large amyloid fragments. Fluorescence images show the migration of the deposits over time (after 1 h, 4 h, 5.5 h, 6 h, 11 h, 13.5 h, and 16 h). Detailed Implementation

[0274] This article provides, in particular, an anti-thyroxine transporter (TTR) antibody, the corresponding polynucleotide and expression vector, and compositions (e.g., pharmaceutical compositions) containing the anti-TTR antibody as a drug, and related articles. Furthermore, this article provides, in particular, a method for treating or preventing thyroxine transporter-mediated amyloidosis (ATTR) in subjects of need using the pharmaceutical composition. In addition, this article provides methods for validating, identifying, and screening amyloid depletion drugs using high-resolution live-cell imaging, methods for generating pharmaceutical compositions of amyloid depletion drugs, and methods for quality control, including kits suitable for said methods.

[0275] The antibodies and antibody compositions disclosed herein

[0276] This disclosure relates to a monoclonal human anti-TTR antibody comprising an immunoglobulin light chain (LC) and an immunoglobulin heavy chain (HC) having the amino acid sequences shown in Table 1 below.

[0277] The antibody may comprise HC having the amino acid sequence of SEQ ID NO: 7 and LC having the amino acid sequence of SEQ ID NO: 8, wherein

[0278] X1 is not present; it is either glutamine or pyroglutamic acid (pE).

[0279] X2 is methionine or oxidized methionine;

[0280] X3 is asparagine, deamidated asparagine, or asparagine containing succinimide;

[0281] X4 is asparagine or deamidated asparagine;

[0282] X5 is proline or amidated proline;

[0283] X6 is absent or may be glycine; and

[0284] X7 is absent or may be lysine.

[0285] And LC having the amino acid sequence of SEQ ID NO: 8.

[0286] In one embodiment, the antibody of this disclosure comprises HC as shown in SEQ ID NO: 7, wherein glutamine (X1), glycine (X6) and lysine (X7) are present, and wherein the amino acids at positions X2, X3, X4 and X5 are modified (e.g., oxidized, deamidinated, containing succinimide and / or amidated) or unmodified as shown in Table 1.

[0287] In one embodiment, the antibody of this disclosure comprises HC as shown in SEQ ID NO: 7, wherein glutamine (X1) is absent or modified to pyroglutamic acid, preferably modified to pyroglutamic acid, and wherein glycine (X6) and lysine (X7) are present, and wherein the amino acids at positions X2, X3, X4 and X5 are modified or unmodified as shown above.

[0288] In one embodiment, the antibody of this disclosure comprises HC as shown in SEQ ID NO: 7, wherein lysine (X7) is absent, glycine (X6) and glutamine (X1) are present, and wherein the amino acids at positions X2, X3, X4 and X5 are modified or unmodified as shown above.

[0289] In one embodiment, the antibody of this disclosure comprises HC as shown in SEQ ID NO: 7, wherein lysine (X7) and glycine (X6) are absent, glutamine (X1) is present, and the amino acids at positions X2, X3, X4 and X5 are modified or unmodified as shown above.

[0290] In one embodiment, the antibody of this disclosure comprises HC as shown in SEQ ID NO: 7, wherein glutamine (X1) is absent or modified to pyroglutamic acid, preferably modified to pyroglutamic acid, wherein lysine (X7) is absent, wherein glycine (X6) is present, and wherein the amino acids at positions X2, X3, X4 and X5 are modified or unmodified as shown above.

[0291] In one embodiment, the antibody of this disclosure comprises HC as shown in SEQ ID NO: 7, wherein glutamine (X1) is absent or modified to pyroglutamic acid, preferably modified to pyroglutamic acid, wherein lysine (X7) and glycine (X6) are absent, and wherein the amino acids at positions X2, X3, X4 and X5 are modified or unmodified as shown above.

[0292] In a preferred embodiment, the antibody of this disclosure comprises HC as shown in SEQ ID NO: 7, wherein glutamine (X1) is modified to pyroglutamic acid, wherein lysine (X7) is absent, and wherein the amino acids at positions X2, X3, X4 and X5 are modified or unmodified as shown above.

[0293] In a preferred embodiment, X2, X3, X4, and X5 are unmodified. Therefore, preferably, the antibody of this disclosure comprises HC as shown in SEQ ID NO: 7, wherein glutamine (X1) is modified to pyroglutamic acid, wherein lysine (X7) is absent, and wherein the amino acids at positions X2, X3, X4, and X5 are unmodified.

[0294] The antibody disclosed herein preferably comprises two HCs, each having the amino acid sequence shown in SEQ ID NO: 7, and two LCs, each having the amino acid sequence shown in SEQ ID NO: 8, wherein in each HC, the N-terminal glutamine (X1) is modified to pyroglutamic acid, the C-terminal lysine (X7) is absent, and the heavy chain is N-glycosylated (e.g., at least one amino acid in the HC is N-glycosylated), preferably wherein the N-glycosylation site is at position Asn300, and wherein the amino acids at positions X2, X3, X4, and X5 are modified or unmodified, preferably unmodified.

[0295] In one embodiment, the antibody comprises an HC having the amino acid sequence of SEQ ID NO: 39 and PTM as detailed in the Summary of the Invention section, and an LC having the amino acid sequence of SEQ ID NO: 8. Specifically, the antibody may comprise an HC having the amino acid sequence of SEQ ID NO: 39 and an LC having the amino acid sequence of SEQ ID NO: 8, wherein...

[0296] X1 is absent; it is either glutamine or pyroglutamic acid (pE), with pE being preferred.

[0297] X2 is methionine or oxidized methionine, preferably methionine;

[0298] X3 is asparagine, deamidated asparagine, or asparagine containing succinimide, preferably asparagine;

[0299] X4 is asparagine or deamidated asparagine, preferably asparagine;

[0300] X5 is proline or amidated proline, preferably proline;

[0301] X6 is either absent or glycine, with glycine being preferred;

[0302] X7 is absent or contains lysine, preferably absent;

[0303] X I It is asparagine or deamidated asparagine, with asparagine being preferred;

[0304] X II It is methionine or oxidized methionine, with methionine being preferred;

[0305] X III It is methionine or oxidized methionine, with methionine being preferred;

[0306] X IV It is either aspartic acid or isoaspartic acid, with aspartic acid being preferred;

[0307] X V It is asparagine or glycosylated asparagine, preferably glycosylated asparagine;

[0308] X VI It is methionine or oxidized methionine, preferably methionine; and

[0309] X VII It is methionine or oxidized methionine, preferably methionine.

[0310] And LC having the amino acid sequence of SEQ ID NO: 8.

[0311] The antibody may comprise HC having the amino acid sequence of SEQ ID NO: 9 and LC having the amino acid sequence of SEQ ID NO: 8.

[0312] In one embodiment, the heavy chain of the antibody of this disclosure has been deleting a C-terminal lysine, i.e., the antibody has undergone C-terminal lysine cleavage, and the C-terminal lysine is either cleaved or absent. Specifically, the C-terminal lysine is absent as shown in SEQ ID NO: 9, preferably from each heavy chain of the antibody. The sequence (i.e., the heavy chain sequence with the absent C-terminal lysine) is shown in SEQ ID NO: 10.

[0313] Alternatively, the N-terminal glutamine is modified with pyroglutamic acid, i.e., the heavy chain of the antibody as shown in SEQ ID NO: 9 has undergone N-terminal glutamine acyl cyclization. This sequence (i.e., the heavy chain sequence without N-terminal glutamine) is shown in SEQ ID NO: 11. Conversely, the N-terminus is cyclized and contains pyroglutamic acid, respectively.

[0314] In a preferred embodiment, the heavy chain of the anti-TTR antibody of this disclosure has been deleting the C-terminal lysine as shown in SEQ ID NO: 9, i.e., the C-terminal lysine is either cleaved or absent, and the N-terminal glutamine as shown in SEQ ID NO: 9 is modified to pyroglutamic acid. The sequence (i.e., the heavy chain sequence without C-terminal lysine and without N-terminal glutamine) is shown in SEQ ID NO: 12. Conversely, the N-terminus is cyclized and contains pyroglutamic acid.

[0315] 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 (SEQ ID NO: 9).

[0316] Therefore, this disclosure provides different antibody substances, namely, antibodies encoded by the same gene and containing the same primary amino acid sequence but modified post-translationally to varying degrees. As mentioned above, the most prominent PTM is achieved through C-terminal modification by cleaving lysine residues, N-terminal pyroglutamate formation, and N-glycosylation.

[0317] Furthermore, the theoretical molecular weight of the antibody disclosed herein is 144.2 kDa, and its weight, determined by mass spectrometry (MS), is 144.2 kDa (deglycosylated) and between 147.0 kDa and 147.6 kDa (intact IgG1). Therefore, in one embodiment, the antibody included in the pharmaceutical composition of this disclosure has a molecular weight of about 150 kDa, preferably about 147 kDa.

[0318] In one embodiment, the antibody disclosed herein comprises at least eight disulfide bridges, preferably located at the following positions:

[0319] LC:C23-LC:C88

[0320] LC:C134-LC:C194

[0321] LC:C214-HC:C223

[0322] HC:C22-HC:C97

[0323] HC:C147-HC:C203

[0324] HC1:229-HC2:229 and HC1:232-HC2:232

[0325] HC:C264-HC:C324; and

[0326] HC:C370-HC:C428

[0327] The cysteine ​​residues (C) are numbered to their positions in SEQ ID NO: 7 and 8, where SEQ ID NO: 7 has N-terminal glutamine (X1), and to their positions in SEQ ID NO: 9 and 8, where SEQ ID NO: 9 has N-terminal glutamine (Q).

[0328] In one embodiment, the HC of the antibody of this disclosure may further comprise a signal peptide having the amino acid sequence of SEQ ID NO: 17, wherein the sequence comprising HC and the signal peptide is shown in SEQ ID NO: 19. Alternatively or additionally, the LC of the antibody of this disclosure may further comprise a signal peptide having the amino acid sequence of SEQ ID NO: 18, wherein the sequence comprising HC and the signal peptide is shown in SEQ ID NO: 20.

[0329] This disclosure also relates to compositions comprising any of the above-described antibodies, preferably antibodies comprising LC as shown in SEQ ID NO: 8 and HC as shown in SEQ ID NO: 7, wherein glutamine (X1) is modified to pyroglutamic acid and wherein lysine (X7) is absent, and wherein the amino acids at positions X2, X3, X4, and X5 are unmodified, and wherein X6 is present; or antibodies comprising LC as shown in SEQ ID NO: 8 and HC as shown in SEQ ID NO: 9, wherein the C-terminal lysine (K) is cleaved and wherein the N-terminal glutamine is modified to pyroglutamic acid; or antibodies comprising HC as shown in SEQ ID NO: 39, wherein glutamine (X1) is modified to pyroglutamic acid and wherein lysine (X7) is absent, and wherein the amino acids at positions X2, X3, X4, and X5 are unmodified, wherein the amino acid at X6 is present, and preferably wherein asparagine (X) is present. V ) is glycosylated, and position X is therein. I X II X III X IV X VI X VI The amino acids at that location are unmodified.

[0330] More preferably, the composition comprises a mixture of the above-described antibody substances, wherein, preferably, in about 90% to 100% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, preferably about 100% (99.9%)) of the antibody present in the composition, the N-terminus is modified with pyroglutamic acid, and in about 90% to 100% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, preferably about 96% (95.8%)) of the antibody present in the composition, the C-terminal lysine is cleaved off.

[0331] Table 1. Anti-TTR antibody sequences

[0332]

[0333]

[0334]

[0335]

[0336] CDR = Complementary Determinant Region; VH = Heavy Chain Variable Region; VL = Light Chain Variable Region; HC = Heavy Chain; LC = Light Chain

[0337] N-terminal glutamine is absent and / or has been modified to pyroglutamic acid, also known as N-terminal cyclization. Preferably, N-terminal glutamine has been modified to pyroglutamic acid.

[0338] In some embodiments, the CDR may contain one or more amino acid residues, particularly in CDRs containing multiple tryptophan (W) residues. These minor differences in CDR length can be attributed to different applications of Martin's rule, according to which VHCDR1 ends just before tryptophan (W), typically before the motif W-valine (WV), W-isoleucine (WI), or W-alanine (WA). Since the heavy chain CDR1 of NI006 / ALXN2220 contains sequence 31-SRSSYWGWI-39 (SEQ ID NO: 37), the boundary of this CDR can end with either Y or G, both preceding W, depending on how the rule is applied. For the avoidance of doubt, the most widely used definition of VHCDR1 containing SRSSY is used herein. In embodiments, VHCDR1 contains SRSSYWG (SEQ ID NO: 38).

[0339] The polynucleotides and nucleic acid molecules disclosed herein

[0340] The polynucleotide encoding the antibodies disclosed herein can be composed of any polynucleotide or polydeoxynucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, the polynucleotide encoding the antibody can be composed of single-stranded and double-stranded DNA, DNA consisting of a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA and RNA consisting of a mixture of single-stranded and double-stranded regions, or a hybrid molecule containing DNA and RNA that can be single-stranded or more commonly double-stranded or a mixture of single-stranded and double-stranded regions. Additionally, the polynucleotide encoding the antibody can be composed of a triple-stranded region containing RNA or DNA or both RNA and DNA. The polynucleotide encoding the antibody may also contain one or more modified bases or a DNA or RNA backbone modified for stability or other reasons. "Modified" bases include, for example, triphenylmethylated bases and rare bases such as inosine. DNA and RNA can be modified in various ways; therefore, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms. Isolated polynucleotides encoding non-natural variants of polypeptides derived from immunoglobulins (e.g., the heavy or light chain portion of an immunoglobulin) can be generated by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence of an immunoglobulin, such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conserved amino acid substitutions are made at one or more non-essential amino acid residues.

[0341] RNA can be isolated from primitive B cells, hybridoma cells, or other transformed cells using standard techniques such as guanidine isothiocyanate extraction and precipitation, followed by centrifugation or chromatography. When desired, mRNA can be isolated from total RNA using standard techniques such as oligo dT cellulose chromatography. Suitable techniques are well known in the art. In one embodiment, cDNA encoding the light and heavy chains of an antibody can be prepared simultaneously or separately using reverse transcriptase and DNA polymerase according to well-known methods. PCR can be initiated using common constant region primers or more specific primers based on published heavy and light chain DNA and amino acid sequences. As discussed above, PCR can also be used to isolate DNA clones encoding the light and heavy chains of antibodies. In this case, the library can be screened using common primers or larger homologous probes, such as human constant region probes.

[0342] DNA, typically plasmid DNA, can be isolated from cells using techniques known in the art, and subjected to restriction mapping and sequencing according to standard, well-known techniques, such as those detailed in the foregoing references relating to recombinant DNA technology. Of course, the DNA can be synthesized according to this disclosure at any point during the isolation process or subsequent analysis.

[0343] In this context, this disclosure also relates to a polynucleotide encoding an antibody of this disclosure. Specifically, this disclosure relates to a polynucleotide encoding at least one chain of an immunoglobulin chain of an antibody of this disclosure.

[0344] In a preferred embodiment of this disclosure, the polynucleotide comprises a nucleic acid having a polynucleotide sequence of HC or LC as depicted in Table 1, and is substantially composed of or constitutes thereof.

[0345] Polynucleotides can be produced or manufactured by any method known in the art. For example, if the nucleotide sequence of an antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides, as described, for example, in Kutmeier et al., BioTechniques 17 (1994), 242, which briefly includes the synthesis of overlapping oligonucleotides containing a portion of the sequence encoding the antibody, the annealing and ligation of those oligonucleotides, and the subsequent amplification of the ligated oligonucleotides by PCR.

[0346] Alternatively, the polynucleotide encoding the antibody or its antigen-binding fragment, variant, or derivative may be produced from a nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a specific antibody is unavailable, but the sequence of the antibody molecule is known, the nucleic acid encoding the antibody may be chemically synthesized or derived from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing a TTR-specific antibody, such as hybridoma cells selected to express the antibody, or nucleic acids isolated from that tissue or cell, preferably polynucleotides). + RNA is obtained by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific to a particular gene sequence, to identify, for example, a cDNA clone from a cDNA library encoding the antibody. The amplified nucleic acid produced by PCR can then be cloned into a reproducible cloning vector using any method well known in the art. Thus, in one embodiment of this disclosure, cDNA encoding an antibody, an immunoglobulin chain, or a fragment thereof is used to generate an anti-TTR antibody.

[0347] In other embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, the polynucleotide comprising the nucleic acid encoding a polypeptide may typically include a promoter and / or other transcriptional or translational control elements operatively associated with one or more coding regions. An operative association is a situation where the coding region of a gene product (e.g., a polypeptide) is associated with one or more regulatory sequences in such a way that the expression of the gene product is under the influence or control of the regulatory sequences. Two DNA segments (such as a polypeptide coding region and its associated promoter) are "operatively associated" or "operatively linked" if the induction of promoter function leads to transcription of mRNA encoding the desired gene product, and if the nature of the connection between the two DNA segments does not interfere with the ability of the expression regulatory sequence to direct the expression of the gene product or the ability of the DNA template to be transcribed. Thus, if a promoter is capable of influencing the transcription of a nucleic acid encoding a polypeptide, the promoter region will be operatively associated with that nucleic acid. The promoter may be a cell-specific promoter that directs large-scale transcription of DNA only in a predetermined cell. Other transcriptional control elements besides promoters, such as enhancers, operons, repressors, and transcription termination signals, may be operatively associated with polynucleotides to direct cell-specific transcription. This article discloses suitable promoters and other transcriptional control regions.

[0348] Various transcriptional control regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (immediate early promoter, binding to intron A), simian virus 40 (early promoter), and retroviruses (such as Raoul's sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit β-globulin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters induced by interferon or interleukin).

[0349] Similarly, a variety of translation control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation start and stop codons, and elements derived from microRNAs (particularly internal ribosome entry sites or IRES, also known as CITE sequences).

[0350] In other embodiments, the polynucleotides disclosed herein are RNA, for example, in the form of messenger RNA (mRNA).

[0351] The polynucleotide and nucleic acid coding regions of this disclosure can be associated with additional coding regions that encode secretory or signal peptides that guide the secretion of polypeptides encoded by the polynucleotides of this disclosure. According to the signal hypothesis, proteins secreted by mammalian cells have signal peptides or secretory leader sequences that are cleaved from mature proteins once the growing protein chain begins to exit through the rough endoplasmic reticulum. Those skilled in the art will know that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the whole or “full-length” polypeptide to produce the secretory or “mature” form of the polypeptide. In some embodiments, a natural signal peptide, such as an immunoglobulin heavy or light chain signal peptide, or a functional derivative of the ability of the retained sequence to guide the secretion of the polypeptide operatively associated with it, is used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. In a preferred embodiment, signal peptides as shown in Table 1 are used in the context of this disclosure.

[0352] expression of antibody peptides

[0353] Typically, a polynucleotide encoding an antibody is inserted into an expression vector to be introduced into a host cell, which can then be used to produce a desired amount of antibody. Once the polynucleotide encoding the heavy or light chain of an antibody molecule or an antibody of this disclosure is obtained, a vector for producing the antibody molecule can be generated using recombinant DNA techniques, as well as those well known in the art as described in the appended Example 1. Therefore, methods for preparing proteins by expressing polynucleotides containing antibody-encoding nucleotide sequences are described herein. Expression vectors containing antibody-encoding sequences and appropriate transcription and translation control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Therefore, this disclosure provides reproducible vectors comprising a nucleotide sequence encoding an antibody molecule of this disclosure or its heavy or light chain, operatively linked to a promoter. Such vectors may include nucleotide sequences encoding constant regions of an antibody molecule, and variable domains of an antibody may be cloned into such vectors for expression of the entire heavy or light chain.

[0354] The terms “vector” or “expression vector” are used herein to refer to a vector used according to this disclosure for introducing a desired gene into a host cell and expressing that gene in the host cell. Such vectors may be selected from the group consisting of free plasmids, bacteriophages, viruses, and retroviruses, as known to those skilled in the art. Generally, vectors compatible with this disclosure will contain selection markers, appropriate restriction sites, and the ability to facilitate the cloning and entry into and / or replication of the desired gene into eukaryotic or prokaryotic cells. Many expression vector systems may be employed for the purposes of this disclosure. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells that have integrated DNA into their chromosomes can be selected by introducing one or more selection markers of host cells that allow transfection. These markers may provide protrophic status in auxotrophic hosts, resistance to biocides (e.g., antibiotics), or resistance to heavy metals such as copper. Selective marker genes can be directly ligated to the DNA sequence to be expressed or introduced into the same cell via co-transformation. Optimal mRNA synthesis may also require additional elements. These elements may include signal sequences, splicing signals, and transcription promoters, enhancers, and termination signals, as also described in Example 1.

[0355] In a particularly preferred embodiment, the cloned light and heavy chain genes are inserted into an expression vector along with the signal peptide sequences explained above, preferably into two different expression vectors (one for the heavy chain gene and one for the light chain gene). Examples of suitable vectors include, but are not limited to, plasmids pcDNA3, pHCMV / Zeo, pCR3.1, pEF1 / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAX1, and pZeoSV2 (available from Invitrogen, San Diego, CA) and plasmid pCI (available from Promega, Madison, WI). Typically, screening for cells expressing appropriately high levels of immunoglobulin heavy and light chains in a large number of transformed cells is a routine experiment that can be performed, for example, by a robotic system. Vector systems are also taught in U.S. Patent Nos. 5,736,137 and 5,658,570, each of which is incorporated herein by reference in its entirety. This system provides high expression levels, such as >30 pg / cell / day. Other exemplary vector systems are disclosed, for example, in U.S. Patent No. 6,413,777.

[0356] In other preferred embodiments, the antibodies of this disclosure or their antigen-binding fragments, variants, or derivatives may be expressed using polycistronic constructs such as those disclosed in U.S. Patent Application Publication No. 2003-0157641 A1, the entire contents of which are incorporated herein by reference. In these expression systems, a variety of gene products of interest, such as the heavy and light chains of antibodies, can be generated from a single polycistronic construct. These systems advantageously utilize internal ribosome entry sites (IRES) to provide relatively high levels of antibody. Compatible IRES sequences are disclosed in U.S. Patent No. 6,193,980, which is also incorporated herein by reference. Those skilled in the art will recognize that such expression systems can be used to efficiently generate the full range of antibodies disclosed in this application. Therefore, in one embodiment, this disclosure provides a vector comprising a polynucleotide encoding at least a binding domain or variable region of an immunoglobulin chain of an antibody, optionally combined with a polynucleotide encoding a variable region of another immunoglobulin chain of the binding molecule.

[0357] More typically, once the vector or DNA sequence encoding the monomeric subunit of the antibody has been prepared, the expression vector can be introduced into a suitable host cell. The introduction of plasmids into host cells can be achieved using a variety of techniques well known to those skilled in the art. These techniques include, but are not limited to, transfection (including the use of, for example, Fugene) ®Lipid transfection with lipofectamine, protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus are all possible methods. Typically, plasmid introduction into the host is achieved via standard calcium phosphate co-precipitation. Host cells carrying the expression construct are grown under conditions suitable for the production of light and heavy chains, and heavy and / or light chain protein synthesis is measured. Exemplary assays include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting assay (FACS), immunohistochemistry, etc.

[0358] Expression vectors are transferred into host cells using conventional techniques, and the transfected cells are then cultured using conventional techniques to produce antibodies used in the methods described herein. Therefore, this disclosure includes host cells containing polynucleotides encoding an antibody of this disclosure or its heavy or light chain, preferably operably linked to a heterologous promoter. Alternatively or additionally, this disclosure also includes host cells containing a vector as defined above, the vector containing a polynucleotide encoding at least a binding domain or variable region of an immunoglobulin chain encoding an antibody, optionally combined with a polynucleotide encoding a variable region of another immunoglobulin chain encoding the binding molecule. In one embodiment expressing a double-stranded antibody, a single or multiple vectors encoding both the heavy and light chains may be co-expressed in a host cell for the expression of the entire immunoglobulin molecule, as detailed below.

[0359] Host cells can be co-transfected using the two expression vectors disclosed herein: a first vector encoding a heavy-chain-derived polypeptide and a second vector encoding a light-chain-derived polypeptide. Both vectors may contain the same selectivity markers that enable equal expression of the heavy-chain and light-chain polypeptides. Alternatively, a single vector encoding both the heavy-chain and light-chain polypeptides may be used. In such cases, the light chain is advantageously placed before the heavy chain to avoid excessive amounts of toxic free heavy chain; see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad. Sci. USA 77 (1980), 2197. The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

[0360] As used herein, “host cell” means a cell carrying a vector constructed using recombinant DNA technology that encodes at least one heterologous gene. In the description of methods for isolating antibodies from a recombinant host, the terms “cell” and “cell culture” are used interchangeably to indicate the source of the antibody, unless otherwise explicitly stated. In other words, recovering polypeptides from “cells” can mean from centrifuged whole cells or from cell cultures containing culture medium and suspension cells.

[0361] Various host-expression vector systems can be used to express antibody molecules used in the methods described herein. These host-expression systems represent media by which the coding sequences of interest can be generated and subsequently purified, but also represent cells that can express the antibody molecules disclosed herein in situ when transformed or transfected with appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms such as bacteria (e.g., *Escherichia coli*) transformed with recombinant phage DNA, plasmid DNA, or copious DNA expression vectors containing antibody coding sequences. *Bacillus coli*, *Bacillus subtilis*; yeast transformed with a recombinant yeast expression vector containing an antibody-encoding sequence (e.g., *Saccharomyces*, *Pichia*); insect cell systems infected with a recombinant viral expression vector containing an antibody-encoding sequence (e.g., baculovirus); plant cell systems infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with a recombinant plasmid expression vector containing an antibody-encoding sequence (e.g., Ti plasmid); or mammalian cell systems carrying a recombinant expression construct (e.g., COS, CHO, NSO, BLK, 293, 3T3 cells) containing a promoter derived from a mammalian cell genome (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). Preferably, bacterial cells such as *Escherichia coli* are used, and more preferably, eukaryotic cells, especially eukaryotic cells for expressing complete recombinant antibody molecules, are used to express recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, together with vectors such as major intermediate early gene promoter elements from human cytomegalovirus, are efficient antibody expression systems; see, for example, Foecking et al., Gene 45 (1986), 101; Cockett et al., Bio / Technology 8 (1990), 2.

[0362] Host cell lines used for protein expression are typically of mammalian origin; those skilled in the art are deemed capable of preferentially identifying specific host cell lines in which the desired gene product is most well expressed. Exemplary host cell lines include, but are not limited to, CHO (Chinese hamster ovary), DG44 and DUXB11 (Chinese hamster ovary lines, DHFR negative), HELA (human cervical cancer), CVI (monkey kidney line), COS (a derivative of CVI with the SV40 T antigen), VERY, BHK (juvenile hamster kidney), MDCK, WI38, R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney line), SP2 / O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney). CHO and 293 cells are particularly preferred. Host cell lines are typically available from commercial services, the American Tissue Culture Collection, or published literature.

[0363] Furthermore, host cell lines can be selectively regulated to modulate the expression of inserted sequences or to modify and process gene products in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be crucial for protein function. Different host cells possess characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure proper modification and processing of expressed exogenous proteins. For this purpose, eukaryotic host cells with cellular machinery for the proper processing, glycosylation, and phosphorylation of primary transcripts of gene products can be used.

[0364] In a preferred embodiment, CHO cells, and most preferably the CHO cell line K1 as shown in Example 1, are used to express the polynucleotides disclosed herein to generate antibodies and immunoglobulin chains, respectively.

[0365] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines stably expressing antibody molecules can be engineered. Instead of using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectivity markers. After introducing exogenous DNA, engineered cells can be grown in enriched medium for 1–2 days, then switched to selective medium. Selectivity markers in the recombinant plasmid confer resistance to selection and enable cells to stably integrate the plasmid into their chromosome and grow to form focal clusters (foci), which can then be cloned and amplified into cell lines. This method is advantageously suited for engineering cell lines stably expressing antibody molecules.

[0366] Many selection systems can be used, including but not limited to herpes simplex virus thymidine kinase (Wigler et al., Cell 11 (1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48 (1992), 202) and adenine phosphoribosyltransferase (Lowy et al., Cell 22 (1980), 817) genes that can be used for tk-, hgprt-, or aprt- cells, respectively. In addition, antimetabolite resistance can be used as a basis for selecting the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl.Acad.Sci.USA 77 (1980), 357; O'Hare et al., Proc.Natl.Acad.Sci.USA 78 (1981), 1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc.Natl.Acad.Sci.USA 78 (1981), 2072); neo, which confers resistance to aminoglycoside G-418, Goldspiel et al., Clinical Pharmacy 12 (1993), 488-505; Wu and Wu, Biotherapy 3 (1991), 87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32 (1993), 573-596; Mulligan, Science 260 (1993), 926-932; and Morgan and Anderson, Ann. Rev. Biochem. 62 (1993), 191-217; TIB TECH 11 (1993), 155-215; and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30 (1984), 147).The well-known methods available in the field of recombinant DNA technology are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated herein by reference in their entirety.

[0367] In vitro production allows for scale-up to produce large quantities of the desired peptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include, for example, homogeneous suspension culture in an airlift reactor or a continuous stirred-tank reactor, or immobilized or entrapped cell culture, for example, in hollow fibers, microcapsules, agarose beads, or ceramic cylinders. If necessary and / or desired, the peptide solution can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, DEAE-cellulose chromatography, or (immuno)affinity chromatography, for example, after the preferential biosynthesis of the synthetic hinge region peptide or before or after the HIC chromatographic steps described herein.

[0368] Genes encoding the antibodies disclosed herein, or their antigen-binding fragments, variants, or derivatives, may also be expressed in non-mammalian cells, such as bacterial, insect, yeast, or plant cells. Bacteria that readily take up nucleic acids include members of the following families: Enterobacteriaceae, such as strains of *Escherichia coli* or *Salmonella*; Bacillaceae, such as *Bacillus subtilis*; *Pneumococcus*; *Streptococcus*; and *Haemophilus influenzae*. It should also be understood that when expressed in bacteria, the heterologous polypeptide typically becomes part of an inclusion body. The heterologous polypeptide must be isolated, purified, and then assembled into a functional molecule. When a tetravalent antibody is desired, the subunit will then self-assemble into a tetravalent antibody; see, for example, International Application WO 02 / 096948.

[0369] In bacterial systems, a variety of expression vectors can be advantageously selected based on the intended use of the expressed antibody molecule. For example, when producing large quantities of such a protein, a vector that directs the expression of a high-level fusion protein product that is easily purified may be needed to generate a pharmaceutical composition of the antibody molecule. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2 (1983), 1791), in which the antibody-coding sequence can be individually linked to a frame containing the lacZ coding region in the vector to produce a fusion protein; pIN vectors (Inouye and Inouye, Nucleic Acids Res. 13 (1985), 3101-3109; Van Heeke and Schuster, J. Biol. Chem. 24 (1989), 5503-5509); and so on. pGEX vectors can also be used to express exogenous peptides as fusion proteins with glutathione S-transferase (GST). Typically, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption and binding to a glutathione-agarose bead matrix, followed by elution in the presence of free glutathione. The pGEX vector is designed to include thrombin or factor Xa protease cleavage sites, allowing the cloned target gene product to be released from the GST moiety.

[0370] Besides prokaryotes, eukaryotic microorganisms can also be used. *Saccharomyces cerevisiae* or *Bacillus bakerella* are the most commonly used eukaryotic microorganisms, but many other strains are also readily available, such as *Pichia pastoris*. For expression in yeast species, plasmids such as YRp7 are commonly used (Stinchcomb et al., *Nature* 282 (1979), 39; Kingsman et al., *Gene* 7 (1979), 141; Tschemper et al., *Gene* 10 (1980), 157). This plasmid already contains the TRP1 gene, which provides a selection marker for yeast mutants lacking the ability to grow in tryptophan (e.g., ATCC number 44076 or PEP4-1) (Jones, *Genetics* 85 (1977), 12). The presence of the trpl-deficient region (lesion), a characteristic of the yeast host cell genome, provides an effective environment for detecting transformations achieved through growth in the absence of tryptophan.

[0371] In insect systems, the alfalfa silver-striped armyworm nucleopolyhedrovirus (AcNPV) is commonly used as a vector for expressing foreign genes. This virus grows in Spodoptera frugiperda cells. Antibody-coding sequences can be cloned separately into non-essential regions of the virus (e.g., polyhedrosis protein genes) and placed under the control of AcNPV promoters (e.g., polyhedrosis protein promoters).

[0372] Once the antibody molecules of this disclosure have been recombinantly expressed, the complete antibody, its dimer, individual light and heavy chains, or other immunoglobulin forms of this disclosure can be purified according to standard procedures in the art, including, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for a specific antigen after protein A, and fractional column chromatography), centrifugation, differential solubility (e.g., ammonium sulfate precipitation), or by any other standard technique for purifying proteins; see, for example, Scopes, “Protein Purification”, Springer Verlag, NY (1982). Alternatively, preferred methods for increasing the affinity of the antibodies of this disclosure are disclosed in U.S. Patent Publication 2002-0123057 A1. Therefore, in one embodiment, this disclosure also provides a method for preparing an anti-TTR antibody or an antibody or immunoglobulin chain thereof that recognizes mutated, misfolded, misassembled, or aggregated TTR material and / or fragments thereof, said method comprising:

[0373] (a) Culturing a host cell as defined above, the cell containing a polynucleotide or carrier as defined above; and

[0374] (b) Isolate the antibody or its immunoglobulin chain from the culture.

[0375] Furthermore, in one embodiment, this disclosure also relates to an antibody or immunoglobulin chain thereof encoded by a polynucleotide as defined above or obtained by the methods described herein for preparing an anti-TTR antibody or an antibody or immunoglobulin chain thereof that recognizes mutated, misfolded, misassembled or aggregated TTR material and / or fragments thereof.

[0376] Pharmaceutical Compositions / Formulations

[0377] This disclosure provides pharmaceutical compositions comprising a mixture of anti-TTR antibodies and anti-TTR antibody substances as defined above. The pharmaceutical compositions of this disclosure can be formulated as described below. For example, a pharmaceutical composition containing an anti-TTR antibody can be formulated to include sucrose, polysorbate 80, and histidine. Furthermore, the pharmaceutical composition containing an anti-TTR antibody can be formulated at a desired pH (e.g., pH 5.8) as described herein. The pharmaceutical composition containing an anti-TTR antibody may also contain pharmaceutically acceptable excipients or diluents as described herein.

[0378] For example, the pharmaceutical composition may contain, in a volume of 2.0 mL, the disclosed anti-TTR antibody at a concentration of about 50 mg / mL, a histidine buffer at a pH of about 5.8, 6.5% w / v sucrose, and 0.03% w / v polysorbate 80.

[0379] In another example, the pharmaceutical composition may contain, in a total volume of 2.0 mL, the disclosed anti-TTR antibody at a concentration of about 50 mg / mL, a histidine buffer at a pH of about 5.8, 8.0% w / v sucrose, and 0.03% w / v polysorbate 80.

[0380] In a preferred embodiment, the antibody comprises an HC chain having the amino acid sequence shown in SEQ ID NO: 7, 39 or 9 and an LC chain having the amino acid sequence shown in SEQ ID NO: 8.

[0381] As shown in Example 2, approximately 99% to 100% of the antibodies present in the pharmaceutical formulation have an N-terminal pyroglutamic acid residue in the heavy chain, and approximately 96% of the antibodies have a C-terminal lysine residue. Therefore, in one embodiment, approximately 99% of the antibodies in the formulation of this disclosure have a heavy chain in which the N-terminal pyroglutamic acid residue is modified from N-terminal glutamine, and / or approximately 96% of the antibodies have a C-terminal lysine residue.

[0382] In a preferred embodiment, the antibody of this disclosure comprises two HCs having the amino acid sequence shown in SEQ ID NO: 7 and two LCs having the amino acid sequence shown in SEQ ID NO: 8, wherein in each HC, the N-terminal glutamine (X1) is modified to pyroglutamic acid, the C-terminal lysine (X7) is absent, and the heavy chain is N-glycosylated, preferably wherein the N-glycosylation site is at position Asn300, and wherein the amino acids at positions X2, X3, X4, and X5 are modified or unmodified, preferably unmodified. In another preferred embodiment, the antibody of this disclosure comprises two HCs having the amino acid sequence shown in SEQ ID NO: 39 and two LCs having the amino acid sequence shown in SEQ ID NO: 8, wherein in each HC, the N-terminal glutamine (X1) is modified to pyroglutamic acid, the C-terminal lysine (X7) is absent, and the heavy chain is N-glycosylated, preferably wherein the N-glycosylation site is at position Asn300 (X1). V At position X1, X2, X3, X4, and X5, the amino acids are either modified or unmodified, preferably unmodified, and at position X... I X II X III X IV X VI X VI The amino acids at the site can be modified or unmodified, with unmodified being preferred.

[0383] In another preferred embodiment, the antibody of this disclosure comprises two HC chains having SEQ ID NO: 9 and two LC chains having SEQ ID: 8, wherein in the HC chains, the N-terminal glutamine is modified with pyroglutamic acid, the C-terminal lysine is deleted (i.e., the C-terminal lysine is cleaved or absent), and the heavy chain is N-glycosylated, preferably wherein the N-glycosylation site is at position Asn300. In other words, the antibody comprises two heavy chains having SEQ ID NO: 12 (preferably wherein the N-terminus is cyclized) and two light chains having SEQ ID NO: 8, wherein the heavy chain is N-glycosylated, preferably wherein the N-glycosylation site is at position Asn300.

[0384] Furthermore, but to a lesser extent and preferably in negligible amounts, some antibody material may be present in the analyzed antibody composition, which may have undergone other post-translational modifications (PTMs), such as partial cleavage, oxidation, deamidation, succinimide or pyroglutamic acid formation and isomerization. PTMs identified as present in NI006 / ALXN2220 are mentioned in Examples 2 and 3. Specifically, following the C-terminal lysine cleavage and N-terminal cyclization mentioned above, the antibody may exhibit methionine (M) oxidation at HC position 255; asparagine (N) deamidation at HC positions 318 and / or HC positions 387; asparagine (N) succinimide formation at HC position 318; and / or C-terminal proline (P) amidation following the deletion of C-terminal lysine and glycine.

[0385] antibody concentration

[0386] The antibodies disclosed herein can be formulated into pharmaceutical compositions having a concentration of about 25 mg / mL to about 150 mg / mL (e.g., about 25 mg / mL, about 50 mg / mL, about 75 mg / mL, about 100 mg / mL or about 125 mg / mL), preferably about 50 mg / mL to about 100 mg / mL (e.g., about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 95 mg / mL or about 100 mg / mL), and most preferably 50 mg / mL or 100 mg / mL of the anti-TTR antibody of this disclosure.

[0387] sucrose

[0388] The pharmaceutical composition further comprises sucrose, for example, in an amount of about 6% to about 9%, about 6% to about 7%, or about 7.5% to about 8.5% by weight per unit volume (w / v) (e.g., about 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, or 9% w / v sucrose). For example, the pharmaceutical composition may comprise about 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.45%, 6.5%, 6.55%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8%, 8.05%, 8.1%, 8.2%, 8.3%, 8.4%, or 8.5% w / v sucrose. Specifically, the pharmaceutical composition comprises about 6.5% w / v sucrose or about 8% w / v sucrose, most preferably 8% w / v sucrose.

[0389] Polysorbate

[0390] The pharmaceutical composition further comprises polysorbate, such as polysorbate 20 or polysorbate 80, preferably polysorbate 80 (PS80), for example in an amount of about 0.001% to about 0.1% w / v (e.g., about 0.001%, 0.005%, 0.01%, 0.05%, or 0.1% w / v PS (80)). For example, the pharmaceutical composition may comprise about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% w / v PS80. Specifically, the pharmaceutical composition contains approximately 0.03% w / v PS(80).

[0391] buffer

[0392] The pharmaceutical composition further comprises a histidine buffer. The pharmaceutical composition may contain a buffer in an amount of about, for example, about 1 mM to about 100 mM (e.g., about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM). For example, the drug may contain about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 19.5 mM, about 20 mM, about 20.5 mM, about 21 mM, about 22 mM, about 23 mM, about... 24mM, approximately 25mM, approximately 26mM, approximately 27mM, approximately 28mM, approximately 29mM, approximately 30mM, approximately 31mM, approximately 32mM, approximately 33mM, approximately 34mM, approximately 35mM, approximately 36mM, approximately 37mM, approximately 38mM, approximately 39mM, approximately 40mM, approximately 41mM, approximately 42mM, approximately 43mM, approximately 44mM, approximately 45mM, approximately 46mM, approximately 47mM, approximately 48mM, approximately 49mM Approximately 50mm, approximately 51mm, approximately 52mm, approximately 53mm, approximately 54mm, approximately 55mm, approximately 56mm, approximately 57mm, approximately 58mm, approximately 59mm, approximately 60mm, approximately 61mm, approximately 62mm, approximately 63mm, approximately 64mm, approximately 65mm, approximately 66mm, approximately 67mm, approximately 68mm, approximately 69mm, approximately 70mm, approximately 71mm, approximately 72mm, approximately 73mm, approximately 74mm, approximately 75mm The amounts are approximately 76 mM, 77 mM, 78 mM, 79 mM, 80 mM, 81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, or 100 mM. Specifically, the pharmaceutical composition comprises approximately 20 mM of histidine, preferably L-histidine and L-histidine hydrochloride.

[0393] pH

[0394] The pharmaceutical composition may have a pH of about 5.0 to about 8.0 (e.g., about 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0). For example, the pharmaceutical composition may have a pH of about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.75, 5.8, 5.85, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. Specifically, the pharmaceutical composition has a pH of about 5.8.

[0395] volume

[0396] The pharmaceutical composition may be about 1 mL to about 100 mL, about 1 mL to about 50 mL, about 5 mL to about 25 mL, about 20 mL to about 25 mL, or about 1 mL to about 10 mL (e.g., about 1 mL to about 80 mL, about 1 mL to about 70 mL, about 1 mL to about 60 mL, about 1 mL to about 50 mL, about 1 mL to about 40 mL, about 1 mL to about 30 mL, about 1 mL to about 20 mL, about 1 mL to about 10 mL, about 5 mL to about 20 mL, about 5 mL to about 15 mL, about 5 mL to about 10 mL). Volumes of approximately 10 mL to approximately 20 mL, approximately 15 mL to approximately 20 mL, approximately 20 mL to approximately 22.5 mL, approximately 20 mL to approximately 25 mL, approximately 1 mL to approximately 9 mL, approximately 1 mL to approximately 8 mL, approximately 1 mL to approximately 7 mL, approximately 1 mL to approximately 6 mL, approximately 1 mL to approximately 5 mL, approximately 1 mL to approximately 4 mL, approximately 1 mL to approximately 3 mL, approximately 1 mL to approximately 2.25 mL, approximately 1 mL to approximately 2 mL, or approximately 2 mL to approximately 2.25 mL are provided (e.g., in vials or other containers, as described herein). For example, the pharmaceutical composition may be from about 1 mL to about 2.25 mL (e.g., about 1 mL to about 2.2 mL, about 1 mL to about 2 mL, about 1 mL to about 1.8 mL, about 1 mL to about 1.6 mL, about 1 mL to about 1.4 mL, about 1 mL to about 1.2 mL, about 1.5 mL to about 1.25 mL, about 1.5 mL to about 2 mL, about 1.9 mL to about 1.2 mL, about 2.1 mL to about 2.25 mL) or from about 1 mL to about 100 mL (e.g., It exists in volumes of approximately 1 mL, 1.8 mL, 1.9 mL, 2 mL, 2.1 mL, 2.2 mL, 2.25 mL, 2.3 mL, 2.4 mL, 2.5 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, or 100 mL.

[0397] In one specific example, the pharmaceutical composition may be present in a volume of about 2.25 mL or about 2 mL. In another example, the pharmaceutical composition may be present in a volume of about 20 mL or about 22.5 mL.

[0398] adjuvants

[0399] Pharmaceutical compositions can be prepared such that they are not reconstituted from lyophilized anti-TTR antibodies and / or are not further lyophilized. Furthermore, pharmaceutical compositions can be prepared such that they are substantially free of sodium chloride and / or substantially free of poloxamer. Pharmaceutical compositions can also be prepared as sterile compositions.

[0400] In the most preferred embodiment of this disclosure, the pharmaceutical composition comprises or consists of the following: 50 mg / mL of the antibody of this disclosure, 20 mM histidine (1.06 mg / mL of L-histidine and 2.78 mg / mL of L-histidine monohydrochloride), 65 mg / mL or 80 mg / mL of sucrose (preferably 80 mg / mL of sucrose), 0.3 mg / mL of polysorbate 80, and water for infusion / injection (pH 5.8).

[0401] The contents of PCT Publication WO2024105092 A1 are incorporated herein by reference in all respects.

[0402] feature

[0403] This disclosure is characterized by the disclosure of pharmaceutical compositions having improved properties (e.g., stability, solubility, storage, etc.) as described herein (e.g., see Example 4). The pharmaceutical compositions may be characterized by a shelf life of 24 months at 2 to 8°C, preferably protected from light.

[0404] The pharmaceutical compositions disclosed herein are characterized by any combination of one, two, three, or all four stability criteria (i) to (iv): (i) a decrease in the main peak (indicating monomer content) of less than 5%, preferably less than 4%, more preferably less than 3%, and even more preferably less than 2%, as measured by SEC-HPLC, under heat stress conditions of about 40°C for 1 month, or about 25°C for 6 months, or during long-term storage of about 5°C for 18 months; (ii) the pharmaceutical composition exhibits a low content of acidic substances of the anti-TTR antibody or its antigen-binding fragment under heat stress conditions of about 40°C for 2 weeks or about 25°C for 3 months. (iii) The pharmaceutical composition exhibits an acidic content of less than 40%, preferably less than 35%, of the anti-TTR antibody or its antigen-binding fragment after long-term storage at about 5°C for 12 or 18 months, as measured by cIEF; and / or (iv) The anti-TTR antibody or its antigen-binding fragment retains at least 80%, preferably at least 90%, of its binding potency to the TTR protein after storage at about 25°C for 6 months or at about 5°C for 12 or 18 months, as measured by ELISA and relative to a control (e.g., not stored for a long time).

[0405] Preferably, the pharmaceutical composition exhibits any or all of the characteristics shown in the table of Example 4.

[0406] Specifically, the pharmaceutical composition has an osmolar concentration of ≥240 mOsm / Kg.

[0407] The pharmaceutical composition is stable upon freezing and thawing. As used herein, and in the context of the pharmaceutical composition described herein, the term "stable" or "stable" refers to the maintenance of the physical and functional characteristics of the composition over time. For example, a stable composition may be described as one that retains its appearance (e.g., color, opalescence, number of visible particles and / or number of subvisible particles), pH, antibody concentration, and / or osmolality after prolonged storage.

[0408] The pharmaceutical compositions disclosed herein have been shown to be stable for at least 1 month at 40±2°C and 75±5% RH (stress stability study); stable for at least 6 months at 25±2°C and 60±5% RH (accelerated stability study); and / or stable for at least 12 to 18 months at 5±3°C (long-term stability study). Furthermore, the extinction coefficient of the pharmaceutical compositions has been determined to be 1.438 (mg / mL). -1 cm -1 Furthermore, the pharmaceutical preparation has been proven to be a sterile, colorless to pale yellow, clear to pale milky white solution, substantially free of visible particles, with a pH of 5.8.

[0409] Treatment

[0410] Antibodies of the present disclosure, polynucleotides of the present disclosure, vectors of the present disclosure, host cells of the present disclosure, or compositions of the present disclosure containing anti-TTR antibodies of the present disclosure may be used for the preventive or therapeutic treatment of diseases associated with TTR amyloidosis (such as ATTR), such as ATTR-CM, ATTR polyneuropathy (ATTR-PN), familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), senile systemic amyloidosis (SSA), systemic familial amyloidosis, pia mater / central nervous system (CNS) amyloidosis (including Alzheimer's disease), TTR-associated ocular amyloidosis, TTR-associated renal amyloidosis, TTR-associated hyperthyroxinemia, TTR-associated ligamentous amyloidosis (including carpal tunnel syndrome, rotator cuff tears, and lumbar spinal stenosis), and preeclampsia. Furthermore, the antibodies disclosed herein can be used for in vivo detection or imaging of TTRs in humans or animals, or for targeting therapeutic agents and / or diagnostic agents to the TTRs. Preferably, the in vivo imaging includes scintillation scanning, positron emission tomography (PET), single-photon emission tomography (SPECT), near-infrared (NIR), optical imaging, or magnetic resonance imaging (MRI).

[0411] Specifically, the pharmaceutical compositions described herein can be used in methods for treating or preventing thyroxine transporter-mediated amyloidosis (ATTR). Additionally, the pharmaceutical compositions described herein can be used in methods for treating or preventing ATTR amyloid cardiomyopathy (ATTR-CM, such as WT-ATTR-CM). For example, a pharmaceutical composition containing about 50 mg / mL of the human anti-TTR antibody of this disclosure, a histidine buffer having a pH of about 5.8, 6.5% or 8.0% w / v sucrose, and 0.03% w / v polysorbate 80 can be used to treat ATTR or ATTR-CM, such as WT-ATTR-CM.

[0412] Pharmaceutical compositions containing anti-TTR antibodies may be administered to human subjects to treat, prevent, or control transthyretin-mediated amyloidosis (ATTR), including ATTR amyloid cardiomyopathy (ATTR-CM, such as WT-ATTR-CM). The pharmaceutical compositions are preferably administered by intravenous injection or infusion.

[0413] The compositions and methods described herein may be used to treat subjects with: ATTR, ATTR-CM, ATTR polyneuropathy (ATTR-PN), familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), senile systemic amyloidosis (SSA), systemic familial amyloidosis, pia mater / central nervous system (CNS) amyloidosis, Alzheimer's disease, TTR-associated ocular amyloidosis, TTR-associated renal amyloidosis, TTR-associated hyperthyroxinemia, TTR-associated ligamentous amyloidosis, carpal tunnel syndrome, rotator cuff tear, lumbar spinal stenosis, preeclampsia, or a known pathogenic TTR mutation (e.g., a mutation that causes amyloidosis). Subjects may have sporadic WT-ATTR-CM and have negative genetic tests for TTR mutations.

[0414] The pharmaceutical composition may be ready-to-use for administration to a subject in need, preferably via intravenous infusion. The pharmaceutical composition may be diluted in glucose or a polymer thereof prior to infusion, preferably wherein the polymer is dextran. The concentration of glucose or its polymer may be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% w / v, preferably in 5% glucose.

[0415] container

[0416] This document also provides containers comprising the pharmaceutical compositions of this disclosure. Suitable containers include, for example, bottles (e.g., infusion bottles), vials (e.g., type I clear glass vials), syringes, IV solution bags, etc. Containers can be formed from a variety of materials, such as glass or plastic. Containers may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle).

[0417] The containers, such as type I clear glass vials or infusion bottles, may be 1 mL, 2 mL, 5 mL, 10 mL, 15 mL, 20 mL, or 25 mL in size and are capable of holding about 10% to about 15% (e.g., 10% or 12.5%) of the volume of the pharmaceutical composition overfilled. The container may include a volume of the pharmaceutical composition described herein, such as 0.5 mL to 10 mL, 2 mL to 2.25 mL, 10 mL to 20 mL, 15 mL to 25 mL, 20 mL to 22.5 mL, for example 0.5 mL, 1 mL, 2 mL, 2.25 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 20.5 mL, 21 mL, 21.5 mL, 22 mL, 22.5 mL, 23 mL, 24 mL, or 25 mL. In a preferred embodiment, the pharmaceutical composition (i.e., the pharmaceutical product described herein) is provided as a concentrate of a solution for infusion, presenting as a sterile, colorless to pale yellow, clear to pale milky liquid, substantially free of visible particles, and in a 2 mL (2R) glass vial with an aluminum flip-top on a 13 mm rubber stopper. The product is preferably diluted prior to administration with sterile glucose, which is a commercially available product and, in one embodiment, is not accompanied by the pharmaceutical product. Preferably, the container is a 2 ml or 20 ml vial, and most preferably, a glass vial with an aluminum flip-top on a 13 mm rubber stopper, which preferably contains approximately 12.5% ​​volume overfill, or a total volume of 2.25 ml or 22.5 ml of the pharmaceutical formulation.

[0418] Products / Therapeutic Kits

[0419] This disclosure also provides an article of manufacture (e.g., a kit, particularly a therapeutic kit) containing material that can be used to treat or prevent thyroxine transporter-mediated amyloidosis (ATTR) in human subjects. The article of manufacture comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 7, 48, 49, 50, or 50 or more) containers of this disclosure as described above, and a label or packaging instruction located on or associated with one or more containers, wherein the containers contain the composition of this disclosure.

[0420] The label or package insert indicates that the composition is for the treatment of ATTR. The article may at least include a first container having the pharmaceutical composition. Optionally, the article may also include a second container having a second therapeutic agent (such as a TTR tetramer stabilizer, for example, chlorpromazine). The article in this embodiment of the present disclosure may also include a package insert indicating that the composition is for the treatment of ATTR. Alternatively or additionally, the article may include a second (or third) container containing a pharmaceutically acceptable buffer, such as BWFI, PBS, Ringer's solution, and dextran solution.

[0421] The product may also include other materials desired from a commercial and user perspective, including additional buffers, diluents, filters, needles, and syringes. For example, the product (e.g., a kit) may include a second container with a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and / or glucose or polymers thereof, such as dextran (e.g., at a concentration of about 5% w / v). The product may include other materials, including buffers, diluents, filters, needles, syringes, and packaging instructions with instructions for use.

[0422] In one preferred embodiment, the article comprises a vial, preferably a clear glass vial sealed with a (grey) rubber stopper and a (blue) aluminum-plastic flip-top. Preferably, the antibody is present in the vial at a concentration of 50 mg / mL and is provided as a concentrate of a solution for infusion, which is a sterile, colorless to pale yellow, clear to pale milky liquid, substantially free of visible particles. In another preferred embodiment, the article comprises a dosing syringe or infusion bag in a metering pump containing the diluted antibody formulation.

[0423] The live-cell imaging method disclosed herein

[0424] Based on findings obtained during experiments conducted within the scope of this invention, methods for screening, validating, characterizing, and quality controlling amyloid depletion drugs have been developed.

[0425] Specifically, methods for validating amyloid depletion drugs, screening methods for identifying and optionally obtaining amyloid depletion drugs from a variety of test compounds, methods for analyzing the effect of agents (such as second drugs) on the amyloid depletion activity of amyloid depletion drugs, and methods for screening amyloid depletion drugs for their ability to bind to amyloid and mediate macrophage recruitment to amyloid deposits, followed by amyloid fragmentation and internalization, all include the following steps: incubating tissue sections containing amyloid deposits with macrophages in the presence of the amyloid depletion drug or test compound (in the case of screening methods), and performing high-resolution live-cell imaging. Live-cell imaging uses refractive index imaging for cell visualization and fluorescence microscopy for amyloid imaging. Therefore, tissue sections are stained with amyloid-specific fluorescent dyes, which results in the staining of amyloid deposits in the tissue sections; corresponding amyloid depletion drugs and test compounds are labeled with fluorescent dyes, which results in the labeling of amyloid deposits in the tissue sections when the corresponding amyloid depletion drugs and test compounds bind to amyloid; or tissue sections are stained with amyloid-specific dyes and labeled with corresponding amyloid depletion drugs and test compounds.

[0426] In one embodiment, before adding macrophages and the amyloid depletion drug (or, in the case of a screening method, the test compound), tissue sections are stained with an amyloid-specific fluorescent dye, and the corresponding amyloid depletion drug and test compound are labeled with the fluorescent dye. This method is preferably used to visualize and detect the binding of the corresponding amyloid depletion drug and test compound to amyloid deposits by generating an overlay image of the fluorescence signals of the corresponding amyloid depletion drug and test compound and the fluorescence signals emitted by the amyloid-specific fluorescent dye. In this embodiment, the corresponding amyloid depletion drug and test compound are labeled with a fluorescent dye different from the fluorescent dye used to stain the amyloid deposits.

[0427] In one embodiment, tissue sections are stained with an amyloid-specific fluorescent dye before the addition of macrophages and the corresponding amyloid depletion drug and test compound, and the corresponding amyloid depletion drug and test compound are unlabeled. This method is preferably used to visualize and determine macrophage-mediated amyloid internalization by detecting intracellular fluorescence signals in phagocytic vesicles. Such fluorescence signals are typically punctate fluorescence signals.

[0428] In one embodiment, a corresponding amyloid depletion drug and test compound are labeled with a fluorescent dye, and the tissue sections are not stained prior to the addition of macrophages and the corresponding amyloid depletion drug and test compound. This method is preferably used to visualize and determine macrophage-mediated amyloid fragmentation by detecting sequential separation of fluorescent signals (preferably punctate fluorescent signals) from the fluorescent signals of amyloid deposits, these fluorescent signals being generated by the binding of the corresponding labeled amyloid depletion drug and test compound to the amyloid deposits.

[0429] In principle, any fluorescent dye suitable for staining amyloid, i.e., amyloid-specific fluorescent dyes, or any fluorescent dye suitable for labeling drugs, can be used in the method of the present invention. In a preferred embodiment, the corresponding amyloid-depleted drug and test compound are labeled with the fluorescent dye Vivotag-680. Alternatively or additionally, amyloid deposits are preferably stained with the amyloid-specific dye Amytracker 680 or any other Amytracker variant, such as Amytracker 480, 520, 540, or 630. Alternatively, Thioflavin T staining can be performed.

[0430] In one embodiment, live-cell imaging is performed using a Nanolive CX-A instrument, employing refractive index imaging for cell visualization and fluorescence microscopy (Cy5 channel) for amyloid imaging. The Nanolive CX-A is preferably placed in a temperature-controlled room and mounted on a vibration-damped table. During imaging, cell culture dishes containing tissue sections are maintained at 37°C with controlled humidity and a 5% CO2 supply. Preferably, imaging is performed every 15 minutes, and the experiment duration is preferably approximately 20 hours.

[0431] Therefore, with the aid of high-resolution live-cell imaging, the binding of the corresponding amyloid depletion drugs and test compounds to amyloid deposits can be observed, as well as the recruitment of macrophages to amyloid deposits, leading to the internalization and fragmentation of the corresponding amyloid and amyloid deposits, followed by intracellular degradation of amyloid.

[0432] If any of the following is observed in the method of the present invention: (a) binding of the corresponding amyloid depletion drug and test compound to amyloid, (b) amyloid internalization, and (c) amyloid fragmentation, this respectively confirms that the amyloid depletion compound indeed possesses amyloid depletion activity and that the test compound possesses amyloid depletion activity. Preferably, if (b) amyloid internalization or (c) amyloid fragmentation is observed, this respectively indicates that the amyloid depletion compound indeed possesses amyloid depletion activity and that the test compound possesses amyloid depletion activity. More preferably, if (b) amyloid internalization and (c) amyloid fragmentation are observed, this respectively indicates that the amyloid depletion compound indeed possesses amyloid depletion activity and that the test compound possesses amyloid depletion activity. More preferably, if (a) the binding of the corresponding amyloid depletion drug and test compound to amyloid and (b) amyloid internalization and (c) amyloid fragmentation are observed, this indicates that the amyloid depletion compound does indeed have amyloid depletion activity and the test compound does have amyloid depletion activity, respectively.

[0433] The method of this invention utilizes macrophages. Macrophages are immune cells derived from embryonic development or differentiation from monocytes. They can exhibit a variety of phenotypes depending on their origin, tissue distribution, and response to different stimuli and tissue environments. Therefore, in vivo, macrophages possess a continuous lineage of phenotypes, which are rarely strictly pro-inflammatory or anti-inflammatory, and exhibit a broad expression profile across the entire polarization spectrum. Schematically, three major macrophage subsets coexist in human tissues: immature macrophages, also known as M0; pro-inflammatory macrophages, also known as M1 macrophages; and anti-inflammatory macrophages, also known as M2 macrophages. Immature macrophages exhibit phagocytic function, recognize pathogens, and rapidly undergo polarization towards pro-inflammatory or anti-inflammatory macrophages to acquire their full suite of functions. Pro-inflammatory macrophages are widely involved in the inflammatory response, during which they exert antimicrobial and antitumor functions. Conversely, anti-inflammatory macrophages are involved in the resolution of inflammation, phagocytosis of cellular debris, and tissue repair after injury. Macrophages also play important, albeit harmful, roles in the initiation and progression of various pathophysiological environments, including solid tumors and hematopoietic system cancers. In principle, any type of macrophage can be used, as long as they are capable of phagocytizing amyloid-producing proteins.

[0434] As described in Example 5, macrophages are generated through the differentiation of THP1 cells; therefore, in a preferred embodiment, the macrophages used in the method of the present invention are THP1 cell-derived macrophages. In a preferred embodiment, macrophages are generated by differentiating THP1 cells with phorbol 12-myristate-13-acetate (PMA), preferably at a concentration of 100 ng / mL, wherein incubation is preferably performed for 3 days, preferably followed by resting for 3 days without PMA. In the presence of the amyloid depletion agent, macrophages are preferably distributed in tissue sections at a density of 400,000 cells. If the amyloid depletion agent to be analyzed is an antibody, preferably an anti-TTR antibody and most preferably an anti-TTR antibody as defined above, the antibody is preferably used at a concentration of 10 nM (1.5 μg / mL). This concentration has previously been identified as triggering maximal antibody-dependent phagocytosis of ATTR aggregates by macrophages in vitro.

[0435] Amyloid depletion drugs, also known as amyloid depletion compounds, are any compounds capable of removing or reducing amyloid protein from tissues, preferably by recruiting macrophages that internalize and degrade amyloid protein. In a preferred embodiment, the compound is an anti-amyloid antibody. This mechanism of action is also known as antibody-dependent cell-mediated phagocytosis (ADCP). It is defined as a highly regulated process by which an antibody eliminates a target by attaching its Fc domain to a specific receptor on a phagocyte and initiating phagocytosis. In the context of this invention, ADCP refers to the mechanism by which the Fc receptor of a phagocyte (here, a macrophage) binds to an amyloid depletion drug (e.g., an anti-amyloid antibody), which binds to amyloid protein and stimulates the phagocyte to internalize both the protein and the cyclic compound. Therefore, amyloid depletion drugs are preferably antibodies containing an Fc domain or fragments or derivatives thereof, or antibody fragments fused to a domain capable of inducing phagocytosis, such as the Fc domain.

[0436] The suitability of the live-cell imaging method has been verified using tissue sections containing TTR amyloid protein and anti-TTR antibodies, particularly the anti-TTR antibody ALXN2200 as described above. Therefore, the preferred amyloid depletion agent is an anti-TTR antibody, most preferably an anti-TTR antibody as defined above, which is capable of depleting amyloid protein TTR, wherein the TTR is wild-type or mutant, preferably wild-type TTR.

[0437] However, it is cautiously anticipated that the method of the present invention can be used not only for the verification, screening, quality control, and characterization of anti-TTR antibodies, but also for the verification, screening, quality control, and characterization of any compound capable of depleting TTR amyloid protein through macrophage recruitment and phagocytosis. Furthermore, since macrophage-mediated amyloid protein depletion is not limited to TTR amyloid protein depletion, this method can be applied to any amyloid protein-depleting compound, such as depleting amyloid-producing α-synuclein (α-syn), tau, prion protein (PrP), amyloid β (Aβ), β2-microglobulin (β2-m), immunoglobulin light chain (LC), immunoglobulin heavy chain (HC), serum amyloid A (SAA), amylin (IAPP), chromosome 9 open reading frame 72 (C9orf72), and TAR. Compounds including DNA-binding protein 43 (TDP-43), superoxide dismutase 1 (SOD1), RNA-binding protein fused to sarcoma (FUS), huntingtin (htt), optic nerve protein (OPTN), neuroserpin, ABri, Adan, ubiquitin, optic nerve protein, leukocyte chemotactic factor 2 (LECT2), coagulin, apolipoprotein AI (ApoAI), apolipoprotein AII (ApoAII), apolipoprotein AIV (ApoAIV), apolipoprotein CII (ApoCII), apolipoprotein CIII (ApoCIII), fibrinogen, cystatin C, and lysozyme. Other amyloid fibrillation proteins may be derived from AmyPro, an open-access database that provides a collection of amyloid fibrillation proteins (Varadi et al., Nucleic Acids Research 46 (2018), D387-D392, DOI: 10.1093 / nar / gkx950), and / or may be those listed in Table 1 of Benson et al., Amyloid 25 (2018), 215-219.

[0438] In a preferred embodiment, the amyloid-generating proteins are involved in systemic amyloidosis, and are more preferably selected from the following list: thyroxine transporter (TTR) (particularly wild-type TTR and variant TTR, preferably wild-type TTR), immunoglobulin light chain (LC), immunoglobulin heavy chain (LH), serum amyloid A (SAA), leukocyte chemokine 2 (LECT2), coagulants, apolipoprotein AI (ApoAI), apolipoprotein AII (ApoAII), apolipoprotein AIV (ApoAIV), apolipoprotein CII (ApoCII), apolipoprotein CIII (ApoCIII), fibrinogen, β2-microglobulin (particularly wild-type and variant β2-microglobulin), cystatin C, ABriPP, prions, and lysozyme; see, for example, Benson et al., Amyloid 25 (2018), 215-219 and Muchtar et al., Journal of Internal Medicine 289 (2021), 268-292.

[0439] The tissue sections are preferably sections of heart tissue, kidney tissue, liver tissue, gastrointestinal tissue, skin tissue, muscle tissue, tongue tissue, adipose tissue, salivary gland tissue, lymph node tissue, brain tissue, pancreatic tissue, or any amyloidoma. In a preferred embodiment, the tissue section is a heart tissue section. Most preferably, the tissue section is obtained from a subject suffering from amyloidosis or amyloid protein-related disease, preferably suffering from TTR amyloidosis, and most preferably suffering from cardiac TTR amyloidosis. The tissue section obtained from the patient is a tissue section containing amyloid protein deposits.

[0440] The method of the present invention may further include the analysis of a control compound, which is, for example, an isotype control antibody, particularly when the amyloid depletion compound is an antibody. This ensures the observed effects, namely, the binding of the corresponding amyloid depletion drug and test compound to amyloid, (b) amyloid internalization, and (c) amyloid fragmentation observed in the method of the present invention, which are attributed to the action of the amyloid depletion drug rather than any nonspecific side effects.

[0441] As mentioned above, in a preferred embodiment, an amyloid depletion drug was analyzed, which is suitable for TTR amyloid depletion and therefore suitable for treating thyroxine transporter (TTR) amyloidosis or TTR amyloid-related diseases, preferably cardiac TTR amyloidosis.

[0442] As shown in Example 5, the mechanism of action of the ATTR-specific antibody was analyzed, and it has been shown that the antibody binds to amyloid in tissue sections and induces macrophage-mediated amyloid internalization and fragmentation. Therefore, in one embodiment of the method of the present invention, the drug comprises an anti-amyloid antibody or an amyloid-binding molecule. In another embodiment, the drug is an antibody, and the control is a corresponding isotype control antibody. Suitable antibody candidates are known in the art, for example, anti-thyroxine transporter (TTR) antibodies disclosed in WO 2015 / 092077 A1, WO 2014 / 124334 A2, WO 2018 / 007923 A3, WO 2016 / 120810 A1, US 2017 / 0058023 A1, and US 9,879,080 B2. Of course, it is contemplated within the present invention that novel antibodies or compounds, in particular, may be applied to the methods of the present invention to evaluate their suitability as amyloid depletion drugs. Besides antibodies, all types of drugs can be conveniently tested for suitability as anti-amyloid drugs using the method of the present invention, as long as they can recruit macrophages.

[0443] Therefore, the method of the present invention enables the acquisition and selection of amyloid depletion compounds, particularly antibodies, which can be reasonably expected to be specific in order to selectively bind to target amyloid in the patient to be treated at desired and necessary locations, such as toxic amyloid deposits, and mediate the depletion of amyloid.

[0444] In a particularly preferred embodiment, the candidate antibody is a humanized antibody, a human-like antibody, or a human antibody, preferably a human antibody, most preferably isolated from human memory B cells, and a recombinant variant thereof, which generally comprises essentially the variable heavy and light chains of the original human antibody and a human constant domain, which preferably belongs to the IgG1 or IgG4 subtype, but is not necessarily the same as the constant domain of the original human antibody.

[0445] Another aspect of the invention relates to a method for manufacturing a pharmaceutical composition comprising an amyloid depletion drug and a pharmaceutically acceptable carrier. In this method, an amyloid depletion drug, which has been determined to be suitable by the method of the present invention as described above, is mixed with a pharmaceutically acceptable carrier.

[0446] Pharmaceutically acceptable carriers and routes of administration are available from corresponding literature known to those skilled in the art. The pharmaceutical compositions of the present invention can be formulated according to methods well known in the art; see, for example, Remington: The Science and Practice of Pharmacy (2000), University of Sciences in Philadelphia, ISBN 0-683-306472; Vaccine Protocols, 2nd ed., Robinson et al., Humana Press, Totowa, New Jersey, USA, 2003; Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, 2nd ed., Taylor and Francis. (2006), ISBN: 0-8493-1630-8. Examples of suitable drug carriers are well known in the art and include phosphate-buffered saline solutions, water, emulsions (such as oil / water emulsions), various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be formulated using well-known conventional methods. These pharmaceutical compositions can be administered to subjects at appropriate doses. The administration of suitable compositions can be achieved in various ways. Examples include compositions containing a pharmaceutically acceptable carrier administered via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods. Aerosol formulations, such as nasal spray formulations, comprise purified aqueous or other solutions of an active agent with preservatives and isotonic agents. Preferably, such formulations are adjusted to a pH and isotonic state compatible with the nasal mucosa. Pharmaceutical compositions for oral administration, such as single-domain antibody molecules (e.g., "nanobodies"), are also contemplated in this invention. ™Such oral formulations may be in tablet, capsule, powder, liquid, or semi-solid form. Tablets may contain solid carriers such as gelatin or adjuvants. Formulations for rectal or vaginal administration may be available as suppositories with suitable carriers; see also O'Hagan et al., Nature Reviews, Drug Discovery 2(9) (2003), 727-735. Further guidance on formulations suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17th edition (1985) and corresponding updates. For a brief overview of drug delivery methods, see Langer, Science 249 (1990), 1527-1533. Preferred carriers according to the invention are buffers, tensioning agents, and / or surfactants, with all three components being most preferred.

[0447] In one embodiment of the method of the present invention, the pharmaceutical composition is designed to treat ATTR or ATTR-related diseases. Thus, amyloidosis is characterized by ATTR deposits in the patient, particularly the corresponding precursor protein. Most preferably, the pharmaceutical composition is an aqueous formulation containing an amyloid depletion drug as defined above, particularly an anti-amyloid antibody, and most preferably an anti-TTR antibody.

[0448] On the other hand, the present invention relates to a method for the characterization, validation, development, and / or quality control (including batch control) of an amyloid depletion drug suitable for treating amyloidosis or amyloid-related diseases. For example, the amyloid depletion drug is characterized using a live-cell imaging method, and information regarding the drug having undergone the validation method of the present invention as described above is communicated to customers, contracting parties, or partners. Furthermore, drugs identified as suitable amyloid depletion drugs may be selected, and optionally, the amyloid depletion drug or a pharmaceutical composition comprising the amyloid depletion drug may be used to treat amyloidosis or amyloid-related diseases.

[0449] The method of the present invention can be performed in addition to another method that can be used to verify the therapeutic suitability of a candidate drug and / or to detect the ability of a drug to deplete amyloid-producing proteins and amyloid deposits, respectively. Such a method is, for example, a method using a patient-derived amyloid xenograft (PDAX) nonhuman animal model as disclosed in WO 2020 / 094883 A1, the contents of which are incorporated herein by reference.

[0450] Example

[0451] The following embodiments provided for implementing specific aspects of this disclosure are for illustrative purposes only and are not intended to limit the scope of this disclosure in any way.

[0452] Example 1. Cloning and expression of NI006 / ALX2220

[0453] NI006 / ALXN2220 was produced in the CHO K1 cell line (ATCC number CCL 61).

[0454] Construction and sequence confirmation of expression plasmids

[0455] First, the signal peptide was added to the sequences of the heavy chain gene (shown in SEQ ID NO: 15) and the light chain gene (shown in SEQ ID NO: 16) encoding NI006 / ALXN2220, and each expression cassette (the heavy chain gene containing the signal peptide and the light chain gene containing the signal peptide) was cloned into a separate expression vector.

[0456] Expression vectors contain typical elements such as promoters that control gene expression, ribosome entry sites, two selective markers (one for maintenance in mammalian cells and one for maintenance in E. coli), terminators, and origins of replication for proliferation in E. coli.

[0457] Prior to transfection, the CHO K1 host cell line was thawed and cultured. Linearized light and heavy chain expression vectors were transfected into the CHO-K1 host cell line at a 1:1 ratio using Freestyle Max reagent. Forty-eight hours post-transfection, cells were plated in four 96-well plates in selective medium containing the appropriate antibiotics for maintenance in mammalian cells. The 96-well plates were incubated in a static CO2 incubator (36.5°C, 6% CO2) for 2 to 3 weeks, changing the medium every 3 to 4 days.

[0458] Then, single clones were tested, and master cell banks (MCBs) were prepared.

[0459] Example 2. Characterization of mature NI006 / ALXN220

[0460] Antibody NI006 / ALXN2220 was produced in the CHO-K1 cell line (ATCC number CCL 61) and obtained from cell culture as described in Example 1. The amino acid sequences of the mature heavy chain (HC) and light chain (LC) of NI006 / ALXN2220 are shown in SEQ ID NO: 7 and 8, with the modifications mentioned below. The total number of amino acids, the number of amino acids in the heavy chain, and the number of amino acids in the light chain are 1328, 450, and 214, respectively.

[0461] Further characterization of the antibody NI006 / ALXN2022 was performed primarily through standard procedures, such as mass spectrometry. For example, post-translational modifications of NI006 / ALXN2220 were identified using liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis of the NI006 / ALXN2220 fragment obtained from sequential digestion with Lys-C and trypsin, along with free thiol analysis. Characterization of antibody-based therapeutics via LC-MS analysis is a standard procedure and can be performed by a technician; see, for example, Robotham and Kelly, Approaches to the Purification, Analysis and Characterization of Antibody-Based Therapeutics (2020), 1-33.

[0462] Exemplary method for releasing N-glycans to determine glycosylation profiles :

[0463] The N-glycans of ALXN2220 were analyzed using a release N-glycan method. N-glycans from ALXN2220 were released using PNGase F and labeled with 2-AB, followed by HILIC (hydrophilic interaction chromatography) separation and fluorescence detection (FLD) using an UPLC system. Individual N-glycans were quantified by their percentage of peak area relative to the total peak area.

[0464] Exemplary Peptide Mapping Analysis Using LCMS :

[0465] Primary structure and post-translational modifications were analyzed using peptide mapping and high-resolution mass spectrometry (MS). ALXN2220 was used for reduction, denaturation, and alkylation. The reduced and alkylated protein was sequentially digested with the endonuclease Lys-C / trypsin. The resulting digested peptides were separated by reverse-phase C18 column analysis and subsequently detected by high-resolution mass spectrometry. The peptide identity was confirmed by comparing the theoretical mass of the control peptide with MS / MS. Post-translational modifications were quantified as the percentage of peak area of ​​the modified peptide relative to the sum of the peak areas of the modified and unmodified forms of the peptide.

[0466] An exemplary icIEF method (charge method) for determining charge variants. :

[0467] Imaging capillary isoelectric focusing (iCIEF) is used to separate charged variants of proteins based on their isoelectric point (pI). Samples are prepared by mixing with a master mixture consisting of a carrier ampholyte, a pI marker, urea, and water. ALXN2220 is separated into a main peak, acidic regions, and basic regions based on its isoelectric point by a pH gradient formed by the carrier ampholyte within the capillary. The focused protein regions are detected by full-capillary UV detection. Acidic, main, and basic components are quantified as a percentage of their peak area relative to the sum of all detected protein peaks.

[0468] The molecular weight of antibody NI006 / ALXN2220, determined by standard mass spectrometry, is approximately 147.1 kDa for intact IgG1 and approximately 144.2 kDa for the deglycosylated variant. The pI values ​​of antibody NI006 / ALXN2220 are 8.4 (theoretical) and 9.3 (determined experimentally). The theoretical extinction coefficient (A) of antibody NI006 / ALXN2220 is... 0.1% (mL / (mg)) The values ​​for (cm) are 1.390 (theoretical) and 1.438 (determined experimentally).

[0469] The monoclonal antibody NI006 / ALXN2220 is an IgG1 subclass antibody, composed of two heavy chains from the IgG1 subclass and two light chains from the κ subclass. The four chains are stabilized by multiple disulfide bonds. Specifically, as determined by standard procedures, namely by Lys-C and trypsin digestion followed by LC-MS, at least the following disulfide bonds are present in NI006 / ALXN2220:

[0470] LC:C23-LC:C88

[0471] LC:C134-LC:C194

[0472] LC:C214-HC:C223

[0473] HC:C22-HC:C97

[0474] HC:C147-HC:C203

[0475] HC1:229-HC2:229 and HC1:232-HC2:232

[0476] HC:C264-HC:C324

[0477] HC:C370-HC:C428

[0478] NI006 / ALXN2220 is a glycoprotein, and each heavy chain contains an N-linked glycan site at residue N300 in its constant region. During glycosylation profiling, the predominant N-glycan types were G0F (approximately 49.0%) and G1F (approximately 25.4%). More detailed glycosylation profiles of NI006 / ALXN2220 have been determined as follows (glycan type, location of glycosylation site, etc.):

[0479]

[0480] The naming of polysaccharides follows the order HexNac-hexose-fucose-NeuAc-NeuGc. For example, 23000 is HexNac(2)-hexose(3)-fucose(0)-NeuAc(0)-NeuGc(0). G1Fa and G1Fb are isomers and are grouped as G1F. G1F is calculated as the sum of G1Fa and G1Fb using the original, unrounded number.

[0481] Glycosylation profiles were drawn at Figure 6 The abundance of different polysaccharides in 11 individual batches of NI006 / ALXN2220 is listed in Table 2.

[0482] Table 2. Relative abundance of polysaccharides (in %)

[0483]

[0484] 0.2 pH dead zone (see Example 3)

[0485] 0.05 pH dead zone (see Example 3)

[0486] Glycosylation profiles were determined using the N-glycan method. Peptide mapping was performed using LCMS.

[0487] Furthermore, N-terminal glutamine was modified to pyroglutamic acid (abundance in samples: 99.9%) and C-terminal lysine cleavage of the heavy chain (abundance in samples: 95.8%) was identified as the major post-translational modification. In addition, as shown in Tables 3 and 4, small-ratio modifications such as methionine oxidation, asparagine deamidation, and asparagine succinimide formation have been experimentally determined (e.g., using LCMS-based methods) and further predicted.

[0488] Table 3. PTM determined experimentally

[0489]

[0490] Note: HC refers to the heavy chain and LC refers to the light chain. The underlined peptide sequences are identified as PTM sites. This refers to the N-terminal related peptide of the heavy chain, and # refers to the C-terminal related peptide of the heavy chain. pE(Q) refers to N-terminal glutamine modified with pyroglutamic acid. -K refers to the deletion of C-terminal lysine. -K-G amidation (P) refers to the amidation of C-terminal proline after the deletion of C-terminal lysine and glycine.

[0491] Table 4. Predicted PTM

[0492]

[0493] The abundance of post-translational modifications in the antibody NI006 / ALXN2220 sample is shown in Table 5:

[0494] Table 5. Relative abundance of PTM

[0495]

[0496] In summary, N-linked glycosylation of the heavy chain, N-terminal pyroglutamic acid obtained from N-terminal glutamine modification, and C-terminal lysine cleavage of the heavy chain are the main post-translational modifications of NI006 / ALXN2220.

[0497] In addition, charge variant analysis was performed, and the results are listed in Table 6:

[0498] Table 6. Charge Variants

[0499]

[0500] Example 3. ALXN2220 Manufacturing Process

[0501] In this embodiment, the scale-up of the ALXN2220 drug substance (DS) manufacturing process from 500L scale Stage I (process A1) to 2000L scale Stage III (process A2) is described, with the main upstream process changes summarized in Table 7.

[0502] The product quality results of ALXN2220 at DS release are summarized in Table 7. Notably, the Imaging Capillary Isoelectric Focusing (iCIEF) acidity% of the first 2000L DS batch (GMP 1 (A2)) was significantly higher than historical results from process A1 at the 500L scale. Figure 1 ).

[0503] To mitigate the risk of high acidity observed in the first 2000L DS batch (GMP 1 (A2)), several upstream process parameters were evaluated using a small-scale bioreactor. The most effective parameter for reducing acidity was identified as the effective pH controlled by pH setpoint and dead zone. When a 0.05 pH dead zone (culture ID: #1) was applied, the effective pH (as reflected by online pH) was approximately 0.05 and 0.15 lower, respectively, than the effective pH of a 0.10 pH dead zone (culture ID: #2) and a 0.20 pH dead zone (culture ID: #3) during most of the cell culture production phases (day 5 to day 14). Figure 5 ).

[0504] Table 7. Summary of upstream production processes for ALXN2220

[0505]

[0506] Cell culture samples from all three bioreactors were purified using a small-scale protein A assay followed by product quality testing (Tables 8 and 9). The small-scale data indicated that reducing the pH dead zone from 0.20 to 0.05 resulted in a significant reduction in acidic substances. Figure 3 Quite surprisingly, reducing the pH dead zone from 0.20 to 0.05 also led to a decrease in Man 5 (). Figure 4 - More robust control over high-mannose substances, and no meaningful adverse effects on other product quality attributes tested (Tables 8 and 9).

[0507] Following small-scale experiments (as mentioned above) demonstrating the effectiveness of pH control strategies in reducing acidity and Man 5, two additional upstream parameters (target seeding density and temperature-transfer viability cell density range) were further modified to confirm process robustness before implementing 2000L scale process A2 starting with the second 2000L batch. iCIEF at 2000L scale ( Figure 5 ) and N-glycans ( Figure 2 The DS release results confirm the effectiveness and robustness of the iCIEF acidity and Man 5 control strategy used for ALXN2220. Glycosylation profiles were determined using the N-glycan release method mentioned above.

[0508] Table 8. Summary of ALXN2220 pharmaceutical substance product quality (Phase 1)

[0509]

[0510] Table 9. Summary of ALXN2220 pharmaceutical substance product quality (Phase 2)

[0511]

[0512] Table 10. Summary of Product Quality Results from Small-Scale Studies

[0513]

[0514] In summary, reducing the pH dead zone from 0.20 to 0.05 surprisingly resulted in a reduction in Man 5, which not only... Figure 4 As can be seen in Table 9, batch GMP 1 (A2) (pH dead zone: 0.2) has a significantly higher Man5 content than all other batches GMP2 (A2) to GMP9 (A2) (pH dead zone: 0.05).

[0515] Example 4. Stability study of anti-TTR monoclonal antibody formulation

[0516] The formulation development of NI006 / ALXN2220 includes studies designed to select buffer systems and excipients to stabilize the protein. The formulation is developed to prevent product loss and to minimize degradation of purity and bioactivity to withstand stresses encountered during production, storage, transportation, and handling.

[0517] A pH buffer screening study was conducted to determine the optimal buffer system for the drug product formulation. A 20 mM histidine buffer at pH 5.8 was selected as the final buffer system.

[0518] Different types of excipients were evaluated through excipient studies, including disaccharides (such as sucrose and trehalose), amino acids (such as L-arginine hydrochloride), polyols (such as sorbitol), and surfactants (such as polysorbate 80). Samples were incubated at 40°C for up to 4 weeks. Thermal stability, formation of insoluble aggregates, and purity were monitored. Sucrose and polysorbate 80 were selected as the optimal excipients for the NI006 / ALXN2220 formulation because they were shown to minimize the reduction in SEC, cIEF, and caliper purity, thereby maintaining product purity.

[0519] In the excipient concentration screening study, three different polysorbate 80 concentrations (0.02%, 0.04%, and 0.06% (w / v)) and two sucrose concentrations (6.5% and 8% (w / v)) were ultimately tested. Three stress conditions were used in the screening: stirring, heat, and freeze-thaw. In the stirring study, samples were placed at 25°C with stirring at 200 rpm or without stirring for up to 7 days. In the heat study, samples were placed at 40°C for up to 2 weeks. In the freeze-thaw study, samples were subjected to up to 5 freeze-thaw cycles. Appearance, pH, protein concentration, number of subvisible particles, and purity were evaluated.

[0520] A concentration of 0.03% (w / v) polysorbate 80 was chosen as the surfactant strength because sub-visible particle formation was effectively suppressed and high SEC purity was maintained. Comparative studies were conducted to compare two formulations with 6.5% or 8% (w / v) sucrose concentrations. After incubation at 40°C for 1 month or at 25°C for 3 months, no significant differences were observed between the 6.5% and 8% (w / v) sucrose formulations in terms of DSC (differential scanning calorimetry), appearance, pH, protein concentration, SEC, cIEF, CE-SDS (non-reducing and reducing), sub-visible particles, and potency.

[0521] The final formulation developed was NI006 / ALXN2220 at a target concentration of 50 mg / mL in 20 mM histidine buffer, 8% (w / v) sucrose, 0.03% (w / v) polysorbate 80, and a pH of 5.8. Excipients were selected based on their stabilizing effect on the drug product. 20 mM L-histidine and L-histidine monohydrochloride stabilized the pH in the liquid state. 8% (w / v) sucrose changed the osmolality to isotonic, stabilized the NI006 / ALXN2220 protein to prevent aggregation in the liquid state, and acted as a cryoprotectant during freezing / thawing. 0.03% (w / v) polysorbate 80 was selected to stabilize the NI006 / ALXN2220 protein and prevent surface-induced protein denaturation or aggregation in the liquid state.

[0522] Manufacturing process development

[0523] The pharmaceutical product manufacturing process consists of thawing, combining and mixing of the drug substances, aseptic filtration, aseptic filling, capping, visual inspection, and batch packaging. Aseptic filtration was selected as the method to obtain a sterile pharmaceutical product and was performed using two aseptic filters (0.22 µm, PVDF) connected in series. The filters were subjected to water bubble point testing before and after aseptic filtration to ensure filter integrity. The compatibility of the pharmaceutical product with the components in contact with the filling line, the effects of shear stress caused by the peristaltic pump, and stability under light exposure were evaluated to mitigate potential adverse effects on product quality attributes during manufacturing. Non-clinical batches (batch 201901004) and three clinical batches (batch 201903038, batch 201904050, and 20200801) were filled. The non-clinical and clinical batches used the same filling volume, container closure system, unit operation sequence, and storage conditions. There were no significant changes in the pharmaceutical product manufacturing process between the non-clinical and clinical batches. Minor differences are described below:

[0524] • 2L size is used for non-clinical batches, and 14L size is used for clinical batches.

[0525] • The formulation used in non-clinical batch 201901004 contains 50 mg / mL in 20 mM histidine buffer, 8% sucrose (w / v), and 0.03% polysorbate 80 (w / v) (pH 5.8). The formulations used in clinical batches 201903038 and 201904050 contain 50 mg / mL in 20 mM histidine buffer, 6.5% sucrose (w / v), and 0.03% polysorbate 80 (w / v) (pH 5.8). The formulation used in clinical batch 20200801 contains 50 mg / mL in 20 mM histidine buffer, 8% sucrose (w / v), and 0.03% polysorbate 80 (w / v) (pH 5.8).

[0526] • 5L glass bottles are used for the merging and mixing of non-clinical batches of pharmaceutical substances, and 50L mixing bags are used for the merging and mixing of clinical batches of pharmaceutical substances.

[0527] Batch thawing, combining and mixing of pharmaceutical substances

[0528] Frozen drug substances stored in 2L PETG bottles were thawed at room temperature (18°C–24°C) in a dark room. After complete thawing, the drug substances were combined into a 50L mixing bag and stirred at an appropriate speed to allow movement to be observed without foaming. Mixing time was controlled at 15–20 minutes. Samples were taken for pH, protein concentration, osmolality, and bioload testing prior to aseptic filtration.

[0529] Aseptic filtration

[0530] In a Class A environment, batches of pharmaceutical materials were aseptically filtered into sterile 20L disposable bags via a peristaltic pump through two 0.22µm sterile filters connected in series. Filter integrity tests were performed on both filters before and after aseptic filtration.

[0531] Aseptic filling

[0532] Aseptic filling is performed inside a RABS unit, which completely encloses the filler and provides a Class A environment. The RABS unit separates the operator from the sterile interior. All filling components are autoclaved and aseptically assembled. Sterile, pyrogen-free 2mL (2R) glass vials are filled to the target volume of 2.25mL. Filling weight checks are performed periodically during filling to ensure a fill weight of 2.233–2.442 g / vial.

[0533] Cut in line

[0534] Inside the RABS unit, the filled vials are automatically sealed with 13mm rubber stoppers. The stoppers are then steam-sterilized at 122°C for 30 minutes.

[0535] Cap

[0536] Under Class A laminar flow protection, the stoppered vials are transferred to the capping machine via conveyor belt. The stoppered vials are then capped with 13mm plastic-aluminum flip caps. The caps are then steam-sterilized at 122°C for 30 minutes.

[0537] Visual inspection

[0538] Production personnel perform a 100% manual visual inspection of the capped vials, followed by a statistically based AQL (Acceptable Quality Limit) check by quality assurance. Release and stability samples are then removed after the visual inspection.

[0539] Bulk packaging and storage

[0540] The filled drug vials are then bulk packaged and labeled. The bulk-packaged drug vials are stored at 2°C–8°C.

[0541] Container closure system

[0542] The container closure system used for pharmaceutical products is a 2 mL (2R) Type I glass vial sealed with a 13 mm rubber stopper and a 13 mm aluminum flip cap. Components with durability to sterilization and depyrogenation processes, as well as non-reactive contact surfaces with optimal protein compatibility, were selected. The compatibility of the container closure system with the pharmaceutical product was evaluated through accelerated and long-term stability studies given below.

[0543] The integrity of the container closure system is demonstrated through a dye penetration test. The drug product vials are immersed in a colored dye under vacuum, then depressurized and the dye penetration into the vials is examined. The container and closure system achieve 100% airtightness. A container closure integrity test (CCIT) is performed annually in the stability program using a non-destructive vacuum decay method. Product contact materials, glass vials, and rubber stoppers have been tested according to USP and European Pharmacopoeia requirements and are suitable for parenteral use. Conformity with each batch of vials and stoppers is verified through supplier-provided conformity certification.

[0544] Analysis program

[0545] color

[0546] The color conforms to the European Pharmacopoeia 2.2.2. Color measurement is performed using the color difference method.

[0547] transparency

[0548] Transparency conforms to European Pharmacopoeia 2.2.1. Transparency is measured using the light scattering method.

[0549] pH

[0550] Conforms to USP <791> pH measurement was performed using potentiometric method, as per European Pharmacopoeia 2.2.3.

[0551] osmolar concentration

[0552] Conforms to USP <785> And European Pharmacopoeia 2.2.35. Indirect determination of osmolality by measuring the decrease in the freezing point of a solution.

[0553] iCIEF

[0554] Full-column imaging capillary isoelectric focusing (iCIEF) is an analytical method for identifying and purifying proteins by separating them based on their isoelectric point (pI) and monitoring the percentage of charge variants in a protein sample. pI is an inherent property of a specific protein molecule and is the pH at which the protein molecule carries no net charge. Under an external electric field, charge variants move along a continuous pH gradient formed by amphoteric electrolytes and stop at a pH equal to their pI. At this pI, the protein carries no net charge and is not attracted by either electrode. Therefore, different monoclonal antibody substances with different pI values ​​are separated and focused at different locations. The pI values ​​and relative abundance of resolvable peaks can be identified and quantified using chromatographic software.

[0555] To meet the acceptance criterion of "conforming to the characteristics of the reference standard," the electrophoresis pattern should show a peak shape comparable to that of the reference standard. Furthermore, the difference (average) in the pI value of the main peak between the test sample and the reference standard should not exceed 0.2.

[0556] SEC-HPLC

[0557] Size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is a method for the purity analysis of proteins based on their size separation. The stationary phase consists of inert particles packed into a dense three-dimensional matrix. These particles have pores that allow only substances smaller than a certain size to enter. Larger molecules simply pass through the pores because they are too large to fit inside. Therefore, larger molecules flow through the column faster than smaller molecules; the smaller the molecule, the longer its retention time. After separation, the relative percentages of high molecular weight (HMW) substances, monomers, and low molecular weight (LMW) substances are quantified by UV detection.

[0558] CE-SDS (Reducible)

[0559] Reducing capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is a purity analysis method based on the electrophoretic mobility of proteins, where smaller proteins migrate faster than larger proteins. In this method, the test sample is denatured by heating in the presence of SDS. The sample is then reduced by adding the reducing agent β-mercaptoethanol (BME) to the sample solution. Separation is performed through an uncoated capillary, and the protein sample is detected at 220 nm using a photodiode array (PDA) detector. Results are reported as a purity percentage.

[0560] CE-SDS (Non-reducing)

[0561] CE-SDS (non-reducing) is a purity analysis method based on the electrophoretic mobility of proteins, where smaller proteins migrate faster than larger proteins. In this method, the test sample is denatured by heating in the presence of SDS. An alkylating agent, N-ethylmaleimide (NEM), is added to the sample solution to prevent thiol groups from binding to other thiol groups. Separation is performed through an uncoated capillary, and the protein sample is detected at 220 nm using a PDA detector. Results are reported as a percentage of purity.

[0562] biological load

[0563] Bioburden testing based on USP <61> In accordance with European Pharmacopoeia 2.6.12, 10 mL of the drug substance sample was filtered through a sterile surface of a 0.45 μm membrane. The filtered membrane was then transferred to a soybean-casein digestion agar plate for determination of total aerobic microbial count (TAMC). Another filtered membrane used for filtering the 10 mL drug substance sample was transferred to a Sabouraud dextrose agar plate for determination of total combined yeast and mold count (TYMC).

[0564] endotoxin

[0565] Bacterial endotoxin testing was performed using a kinetic turbidimetric method and based on USP. <85> And European Pharmacopoeia 2.6.14. Detection of endotoxins produced by Gram-negative bacteria using amoeboid cell lysates from horseshoe crabs, which coagulate with the endotoxins. Endotoxin concentration can be calculated by establishing a correlation between endotoxin concentration and the time or turbidity development rate required to reach a predetermined absorbance of the reaction mixture.

[0566] ELISA (binding assay)

[0567] The binding potency of the NI006 / ALXN2220 antibody was assessed using an ELISA method. Appropriately diluted samples, controls, and reference standards were loaded onto 96-well plates coated with misfolded TTRs (the antigen of NI006 / ALXN2220). After washing the plates, horseradish peroxidase (HRP)-conjugated goat anti-human IgG was added to the wells, allowing interaction with the bound NI006 / ALXN2220 antibody captured by the misfolded TTRs. After a final wash, a TMB substrate solution was loaded into the wells. TMB reacts specifically with peroxidase in the presence of peroxidase, producing a colorimetric signal proportional to the amount of NI006 / ALXN2220 protein bound to the well. Color development was stopped, and optical density was measured at 450 nm (minus 560 nm for wavelength correction). Dose-response curves for the samples and reference standards were plotted using a 4-parameter logistic (automatically estimated) regression model using SoftMax Pro GxP software. Calculate the EC50 values ​​for the sample and the reference standard. Calculate the relative binding activity of the sample using the following formula:

[0568] Relative binding activity of the sample (%) =

[0569] (EC50 of reference standard / EC50 of sample) × 100%

[0570] protein concentration

[0571] Because protein molecules contain aromatic amino acids, proteins in solution absorb ultraviolet light at a wavelength of 280 nm. According to Beer-Lambert's law, the absorbance (A) of a protein solution at a fixed wavelength is related to the protein concentration (C), cell path length (l), and protein extinction coefficient (ε), as follows: A = C / l / ε. Unlike traditional UV-Vis methods that rely on a single absolute absorbance value, slope spectroscopy uses cross-sectional data (absorbance versus path length) to determine the slope value, allowing for the quantification of sample concentration using the slope spectral equation (Slope = ε / C) derived from Beer-Lambert's law.

[0572] Cell-based assays

[0573] THP-1 is a human mononuclear cell line. NI006 / ALXN2220 is an antibody against misfolded TTR. The bioactivity of NI006 / ALXN2220 is to stimulate THP-1 cells to produce IL-8 by binding to mis-TTR in cell culture. In short, approximately 2 × 10⁴ cells / well of THP-1 cells in assay medium were seeded at 100 μL / well into 96-well cell culture plates. A mixture of serially diluted NI006 / ALXN2220 mAb standard (final concentration: 2000-0.039 g / mL) and misfolded TTR (final concentration: 10 μg / mL) was loaded into the wells in duplicate at 100 μL / well. After incubation at 37°C and 5% CO₂ for 20–24 hours, IL-8 production was measured using a human IL-8 ELISA kit.

[0574] Compatibility study

[0575] Evaluate the compatibility of NI006 / ALXN2220 with materials used in clinical applications. Assess compatibility with the following clinical application settings:

[0576] • Bags and infusion lines under PVC installation - IV bags, IV lines, and filters are constructed of PVC material.

[0577] • Bags and infusion lines in non-PVC configurations - IV bags, IV lines, and filters are constructed from non-PVC materials.

[0578] • The syringe, filling line, and filter under PVC configuration are made of PVC material.

[0579] • The syringe-infusion tubing and filter in non-PVC configurations are made of non-PVC materials.

[0580] The tests were conducted at 2°C–8°C for 24 hours, followed by 6 hours at 25°C (a total of 30 hours) at three concentrations (1.0 mg / mL, 20.0 mg / mL, and 50.0 mg / mL). Saline and glucose were used as diluents at concentrations of 1.0 mg / mL and 20.0 mg / mL. The results are shown in Tables 11 and 12.

[0581] Table 11. Results of the compatibility study during use .

[0582]

[0583] Cl: Colorless; SY: Pale yellow; C: Clear; FoP: Free of visible particles; SO: Light milky white

[0584] 1 The concentration is the same as that of the NI006 / ALXN2220 drug product.

[0585] Table 12. Results of the compatibility study in use (continued)

[0586]

[0587] 1 The concentration is the same as that of the NI006 / ALXN2220 drug product.

[0588] 2 The variability in the results is due to the variability in the measurements. All results from the in-use compatibility studies were within acceptable limits. Considering the short service life, the most likely incompatibilities with plastic materials would be adsorption (resolved by protein concentration) and visible particle formation (resolved by pharmacopoeia methods), and the results are not considered safety-related.

[0589] Visible particles were observed in the saline group, indicating that NI006 / ALXN2220 is relatively unstable in saline. Data showed no significant changes in appearance, protein concentration, subvisible particles, SEC-HPLC, and ELISA binding assays when glucose was used as a diluent. NI006 / ALXN2220 at concentrations of 1.0 mg / mL, 20.0 mg / mL, and 50.0 mg / mL was stable at 2℃–8℃ for 24 hours, followed by stability at 25℃ for 6 hours (total 30 hours). NI006 / ALXN2220 is compatible with the materials evaluated for clinical use.

[0590] A concentration of 0.15 mg / mL was also investigated, which is 1 / 10 of the lowest dose concentration used in clinical trials. Changes were observed in protein concentration and in ELISA tests. Changes in the ELISA test may have been caused by variations in protein concentration, possibly due to protein adsorption to the contact material. In conclusion, 5% glucose was chosen as the diluent for clinical use.

[0591] Table 13 below provides an example of the batch analysis data for non-clinical batch 201901004 and clinical batch 201903038.

[0592] Table 13. Batch Analysis

[0593]

[0594] 1 No acceptance criteria were set for non-clinical batches; the reported data are for informational purposes only.

[0595] 2 This result is the average of six injections from a follow-up examination. The initial result for (LC+HC) was 92.2%. The underlying cause of the initial result was not determined.

[0596] As a reference standard, a composition containing 50 mg / mL antibody was used, formulated in 20 mM histidine buffer, 8% (w / v) sucrose, and 0.03% (w / v) PS80 (pH 5.8), and stored at -70 ± 10 °C in 100 µl vials. Additional tests were performed to qualitatively characterize the reference standard, and the results are shown in Table 14.

[0597] Table 14. Qualitative results of the reference standard

[0598]

[0599] The protein concentration and potency of the reference standard are calibrated. The potency of the reference standard is specified as a value representing 100% relative potency.

[0600] Stability Study

[0601] Stability testing was performed on non-clinical batch 201901004 and clinical batch 201903038. Non-clinical batch (201901004) has 1-month stress stability data, 6-month accelerated stability data, and 18-month long-term stability data. Clinical batch (201903038) has 1-month stress stability data, 6-month accelerated stability data, and 12-month long-term stability data. Under stress conditions, both clinical batch (201903038) and non-clinical batch (201901004) showed a trend of significantly decreasing iCIEF peak (%) and increasing acidic peak (%), while no significant changes were observed in other purity assays and ELISA binding assays.

[0602] The shelf life of the pharmaceutical product is currently set at 24 months when stored protected from light at 5±3°C. The available in-use stability and compatibility data provided above indicate that, after dilution with 5% glucose solution, the ready-to-use solution for infusion is stable at 2°C–8°C for up to 24 hours, followed by stability at 25°C for 6 hours. From a microbiological perspective, the infusion solution should be used immediately. If not used immediately, the in-use shelf life is set at 4 hours at room temperature or 24 hours at 2°C–8°C. Tables 15 through 18 summarize the available stress data for non-clinical batch 201901004 and clinical batch 201903038.

[0603] Table 15. Stress stability data of non-clinical batch 201901004 at 40±2℃ / 75±5% RH

[0604]

[0605] Table 16. Stress stability of non-clinical batch 201901004 under exploratory specifications at 40±2℃ / 75±5% RH data

[0606]

[0607] Table 17. Stress stability data of clinical batch 201903038 at 40±2℃ / 75±5% RH

[0608]

[0609] Table 18. Stress stability numbers of clinical batch 201903038 under exploratory specifications at 40±2℃ / 75±5% RH according to

[0610]

[0611] Tables 19 to 22 summarize the available accelerated data for non-clinical batch 201901004 and clinical batch 201903038.

[0612] Table 19. Accelerated stability data of non-clinical batch 201901004 at 25±2℃ / 60±5% RH

[0613]

[0614] Table 20. Accelerated stability of non-clinical batch 201901004 at 25±2℃ / 60±5% RH for exploratory testing data

[0615]

[0616] Table 21. Accelerated stability data of clinical batch 201903038 at 25±2℃ / 60±5% RH

[0617]

[0618] Table 22. Accelerated stability data of clinical batch 201903038 under exploratory specifications at 25±2℃ / 60±5% RH according to

[0619]

[0620] Tables 23 to 28 summarize the available long-term data for non-clinical batch 201901004 and clinical batch 201903038.

[0621] Table 23. Long-term stability data of non-clinical batch 201901004 at 5±3℃

[0622]

[0623] Table 24. Long-term stability data of non-clinical batch 201901004 at 5±3℃

[0624]

[0625] Table 25. Long-term stability data of non-clinical batch 201901004 at 5±3℃ for exploratory testing.

[0626]

[0627] Table 26. Long-term stability data of clinical batch 201903038 at 5±3℃

[0628]

[0629] Table 27. Long-term stability data of clinical batch 201903038 at 5±3℃

[0630]

[0631] Table 28. Long-term stability data of clinical batch 201903038 at 5±3℃ for exploratory testing.

[0632]

[0633] The stress stability, accelerated stability, and long-term stability data shown in Tables 11 to 28 demonstrate that the tested pharmaceutical formulations are long-term stable. For example, the formulations remained liquid without visible particles, the pH remained constant, and the monomer content, as measured by SEC-HPLC, did not decrease to below 96%, meaning that the contents of HMWS and LMWS remained below 4% under all test conditions. Furthermore, as measured by iCIEF, the amount of acidic substances did not exceed 40% during the long-term stability test, and ELISA binding assays showed that the antibody maintained its binding capacity (not decreasing to below 70% of the reference standard under all test conditions, and even not decreasing to below 95% during the long-term stability study).

[0634] Example 5: Antibody NI006 / ALXN2220 mediates amyloid depletion in patient's cardiac tissue, such as using high-resolution... High-resolution live-cell imaging has demonstrated

[0635] Extracellular deposition of thyroxine transporter amyloid (ATTR) is a hallmark of ATTR cardiomyopathy, and reducing cardiac ATTR load is a key therapeutic goal for improving cardiac function. In this study, high-resolution live-cell imaging was used to visualize the cellular mechanisms of ALXN2220-mediated ATTR depletion in patient-derived cardiac tissue.

[0636] method

[0637] Frozen myocardial tissue samples with ATTR amyloid deposits were prepared into sections 8 mm in diameter and 15 μm thick using a cryostat. The sections were mounted on 35 mm microplates specifically designed for imaging with the Nanolive system and stored at -20°C until use.

[0638] Two methods were used to visualize amyloid deposits in tissue sections. One method involved staining the deposits with the amyloid-specific dye Amytracker 680 (Ebba Biotech (1 mg / mL, in H2O)). A working solution of 1 μg / mL Amytracker 680 was prepared in H2O and applied to the tissue sections at room temperature for 5 min, followed by destaining in 70% ethanol and washing in H2O, then proceeding with amyloid depletion assays. The other method used an antibody NI006 / ALXN2220 labeled with the fluorescent dye Vivotag-680. The antibody labeling reaction was performed against free amines as indicated in the instruction manual, followed by dialysis to remove unbound dye.

[0639] Macrophages were generated by differentiating THP1 cells (THP1 Null2 cells: Invivogen, reference number thp-nullz, batch: T49-4101) for 3 days with phorbol 100 ng / mL 12-myristate-13-acetate (PMA, Sigma P8139), followed by 3 days of resting without PMA. Macrophages were dissociated by trypsin digestion and distributed to tissue sections at a density of 400,000 cells per well in the presence of 10 nM (1.5 μg / mL) NI006 / ALXN2220 or an isotype control (micro-culture dish: reference number 80136, μ-Dish 35 mm, low-type, polymer coverslip). This concentration has previously been identified as triggering maximal antibody-dependent phagocytosis of ATTR aggregates by macrophages in vitro.

[0640] Live-cell imaging was performed using a Nanolive CX-A instrument, employing refractive index imaging for cell visualization and fluorescence microscopy (Cy5 channel) for amyloid imaging. The Nanolive CX-A was housed in a temperature-controlled room and mounted on a vibration-proof table. During imaging, cell culture dishes were maintained at 37°C with controlled humidity and a 5% CO2 supply. For most experiments, imaging was performed every 15 minutes, and the experiments lasted approximately 20 hours.

[0641] Representative cells exhibiting tissue amyloid phagocytosis were selected to illustrate the mechanism of cardiac amyloid depletion in the presence of NI006 / ALXN220 and macrophages. The field of view was focused on cells and time points of interest, and the contrast settings for refractive index imaging and amyloid fluorescence were adjusted according to the internalized amyloid material.

[0642] result

[0643] Initially, ATTR deposits in myocardial tissue sections were identified using Amytracker 680, a red fluorescent dye specific for amyloid. NI006 / ALXN2220 was labeled with the green fluorescent dye A488, and selective binding of NI006 / ALXN2220 to ATTR was observed, as indicated by the overlap between the red and green fluorescence; see [link to related documentation]. Figure 7 (A) to Figure 7 (D)

[0644] This further demonstrates that NI006 / ALXN2220 triggers the phagocytosis of amyloid protein by macrophages. For example... Figure 8 Visualized punctate and intracellular red fluorescence patterns revealed the presence of ATTR amyloid protein in phagocytic vesicles. Macrophages exhibited a broad range of phagocytic activity indicated by the number of red fluorescent vesicles.

[0645] Furthermore, the experiment revealed that macrophages with two nuclei spontaneously formed under culture conditions. Figure 9 This illustrates a multinucleated cell that continuously dissociates amyloid protein fragments from a large sediment. Observations were made... Figure 10 The macrophages depicted in the study separate thin, elongated ATTR deposits from adjacent cardiomyocytes, allowing the deposits to move more than 20 µm. Macrophages also cleave protruding large amyloid deposits, thereby dissociating small and large amyloid fragments.

[0646] In summary, the use of fluorescently labeled NI006 / ALXN2220 demonstrated the binding of NI006 / ALXN2220 to ATTR amyloid protein in patient myocardial tissue sections. Furthermore, it was shown that NI006 / ALXN2220 triggers macrophage recruitment to amyloid deposits, subsequent amyloid fragmentation and internalization, and intracellular degradation, with multinucleated cells involved in the phagocytosis of large amyloid fragments. In the presence of an isotype control antibody, macrophages did not phagocytose cardiac ATTR amyloid protein.

[0647] Therefore, by developing this innovative high-resolution live-cell imaging method, it is possible to demonstrate NI006 / ALXN2220-mediated amyloid depletion in patient cardiac tissue, which further improves the understanding of the underlying cellular mechanisms of antibody-mediated amyloid depletion as a precision therapy for ATTR cardiomyopathy patients.

Claims

1. A composition comprising one or more antibodies, said antibodies comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO: 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO: 8, wherein SEQ ID NO: 9 comprises the following sequence: QLQLQESGPG LVKPSETLSL TCSVSGGSII SRSSYWGWIR QPPGKGLEWI GGIYHSGNTYDNPSLKSRLTMSVDTSKNQFSLNLRSVTAADTAVYYCARI VPGGDAFDIW GQGTMVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK, Among the antibodies, one or more of the antibodies are mentioned. Glutamine (Q) at position 1 was modified to pyroglutamic acid (pE). The lysine (K) at position 450 is absent, and The asparagine (N) at position 300 is glycosylated. And SEQ ID NO: 8 contains the following sequence: DIQMTQSPSSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, The antibodies described herein contain the following glycan types: Man3+1F, G0-GN, G0F-GN, G0, G0F, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1, and G2FS2.

2. A composition comprising one or more antibodies, said antibodies comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO: 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO: 8, wherein SEQ ID NO: 9 comprises the following sequence: QLQLQESGPG LVKPSETLSL TCSVSGGSII SRSSYWGWIR QPPGKGLEWI GGIYHSGNTYDNPSLKSRLTMSVDTSKNQFSLNLRSVTAADTAVYYCARI VPGGDAFDIW GQGTMVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK, Among the antibodies, one or more of the antibodies are mentioned. Glutamine (Q) at position 1 was modified to pyroglutamic acid (pE). The lysine (K) at position 450 is absent, and The asparagine (N) at position 300 is glycosylated. And SEQ ID NO: 8 contains the following sequence: DIQMTQSPSSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, Less than 3% of the polysaccharides are of the Man5 type.

3. A composition comprising one or more antibodies, said antibodies comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO: 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO: 8, wherein SEQ ID NO: 9 comprises the following sequence: QLQLQESGPG LVKPSETLSL TCSVSGGSII SRSSYWGWIR QPPGKGLEWI GGIYHSGNTYDNPSLKSRLTMSVDTSKNQFSLNLRSVTAADTAVYYCARI VPGGDAFDIW GQGTMVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK, Among the antibodies, one or more of the antibodies are mentioned. Glutamine (Q) at position 1 was modified to pyroglutamic acid (pE). The lysine (K) at position 450 is absent, and The asparagine (N) at position 300 is glycosylated. And SEQ ID NO: 8 contains the following sequence: DIQMTQSPSSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, The antibody comprises the following glycan types: Man3+1F, G0-GN, G0F-GN, G0, G0F, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1 and G2FS2, and less than 3% of the glycan is of type Man5.

4. The composition according to any one of claims 1 to 3, wherein the main polysaccharide type is G0F and G1F.

5. The composition according to claim 4, wherein about 49% of the polysaccharide is of the G0F type and about 25% of the polysaccharide is of the G1F type.

6. The composition according to any one of claims 1 to 5, wherein the composition comprises the following charge variant of the antibody: Main peak ≥ 50.0% Acid peak ≤ 40.0% The alkaline peak is ≤15.0%.

7. The composition according to any one of claims 1 to 6, wherein the composition comprises the following charge variant of the antibody: Main peak ≥ 63.0% Acid peak ≤ 32.0% The alkaline peak is ≤5.0%.

8. The composition according to any one of claims 1 to 7, wherein the antibody is contained in an aqueous formulation, wherein the formulation comprises the antibody at a concentration of about 50 mg / ml or about 100 mg / ml and histidine at a concentration of about 20 mM, sucrose at a concentration of about 6.5% by weight / volume (w / v) or about 8% (w / v) and PS80 at a concentration of about 0.03% w / v, wherein the formulation has a pH of about 5.

8.

9. The composition according to any one of claims 1 to 8, wherein the formulation The antibody, after being stored at 5±3°C for 6 months, has an acidic content of ≤40.0% and a basic content of ≤15.0%, as determined by iCIEF; and / or a high molecular weight content of ≤5.0%, as determined by SEC-HPLC, after being stored at 5±3°C for 6 months.

10. The composition according to any one of claims 1 to 9, wherein the antibody can be obtained by a method comprising the steps of: culturing CHO-K1 cells in Chinese hamster ovary (CHO)-K1 cells containing one or more polynucleotides or a vector containing said polynucleotides encoding an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO: 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO: 8, and isolating said antibody from the cell culture.

11. The composition of claim 10, wherein the method comprises culturing the CHO-K1 cells in a pH dead zone of less than 0.2, preferably ≤0.175, preferably ≤0.15, preferably ≤0.125, preferably ≤0.1, preferably ≤0.075, preferably ≤0.05, more preferably 0.05 or 0.1, and most preferably 0.

05.

12. The composition according to any one of claims 1 to 11, wherein one or more of the antibodies in the group of said antibodies: Asparagine (N) at position 58 is deamidinated. The methionine (M) at position 71 is oxidized. The methionine (M) at position 115 is oxidized. The methionine (M) at position 255 is oxidized. The aspartic acid (D) at position 283 is isoaspartic acid; The asparagine (N) at position 318 is deamidinated or contains succinimide; The methionine (M) at position 361 is oxidized. The asparagine (N) at position 387 is deamidinated or contains succinimide; The methionine (M) at position 431 is oxidized. The proline (P) at position 448 is amidated; and / or Glycine (G) at position 449 is absent.

13. The composition according to any one of claims 1 to 12, wherein in more than 90%, preferably in about 99% to 100% of the antibody, glutamine (Q) at position 1 of SEQ ID NO: 9 is modified with pyroglutamic acid, and / or wherein in more than 90%, preferably in about 95% to 100%, preferably in about 95% to 96% of the antibody, lysine (K) at position 450 of SEQ ID NO: 9 is absent.

14. The composition according to any one of claims 1 to 13, wherein more than 85% of the polysaccharide is fucoidylated, preferably wherein about 85% to 95% of the polysaccharide is fucoidylated.

15. The composition according to any one of claims 1 to 14, wherein about 20% to 40% of the polysaccharide is galactosylated.

16. The composition according to any one of claims 1 to 15, wherein about 80% to 90% of the polysaccharide is part of a major fucoidylated polysaccharide type.

17. The composition according to any one of claims 1 to 16, wherein about 1% to 4% of the polysaccharide is a mannosaccharide, preferably wherein the polysaccharide is of the Man5 and Man31F type.

18. The composition according to any one of claims 1 to 17, wherein about 1% to 4% of the polysaccharide is of the Man5 type, preferably 1% to 3%, more preferably 1% to 2%.

19. The composition according to any one of claims 1 to 18, wherein less than 2% of the polysaccharide is sialylated, preferably wherein about 0.5% to 2% of the polysaccharide is sialylated.

20. The composition according to any one of claims 1 to 19, wherein in one or more of the antibodies, the asparagine (N) at position 58, position 318 and / or position 387 of SEQ ID NO: 9 is deamidinated.

21. The composition according to any one of claims 1 to 20, wherein in less than 5%, preferably less than 2%, preferably less than about 1%, preferably from about 0% to 1% of the antibody, the asparagine (N) at position 58 of SEQ ID NO: 9 is deamidinated.

22. The composition according to any one of claims 1 to 21, wherein in less than about 9%, preferably less than about 8%, preferably from about 6% to 9% of the antibody, the asparagine (N) at position 318 of SEQ ID NO: 9 is deamidinated.

23. The composition according to any one of claims 1 to 22, wherein in less than about 5%, preferably less than about 3%, preferably from about 1% to 43% of the antibody, the asparagine (N) at position 387 of SEQ ID NO: 9 is deamidinated.

24. The composition according to any one of claims 1 to 23, wherein in one or more of the antibodies, the methionine at position 71, position 115, position 255, position 361 and / or position 431 of SEQ ID NO: 9 is oxidized.

25. The composition according to any one of claims 1 to 24, wherein in less than about 5%, preferably less than about 2%, preferably less than about 1%, preferably from about 0% to 1% of the antibody, the methionine (M) at position 71 of SEQ ID NO: 9 is oxidized.

26. The composition according to any one of claims 1 to 25, wherein in less than about 5%, preferably less than about 3%, preferably less than about 2%, preferably from about 1% to 3% of the antibody, the methionine (M) at position 115 of SEQ ID NO: 9 is oxidized.

27. The composition according to any one of claims 1 to 26, wherein in less than about 5%, preferably less than about 3%, preferably from about 1% to 30% of the antibody, the methionine (M) at position 255 of SEQ ID NO: 9 is oxidized.

28. The composition according to any one of claims 1 to 27, wherein in less than about 5%, preferably less than about 2%, preferably less than about 1%, preferably from about 0% to 1% of the antibody, the methionine (M) at position 361 of SEQ ID NO: 9 is oxidized.

29. The composition according to any one of claims 1 to 28, wherein in less than about 5%, preferably less than about 3%, preferably less than about 2%, preferably from about 1% to 2% of the antibody, the methionine (M) at position 431 of SEQ ID NO: 9 is oxidized.

30. The composition according to any one of claims 1 to 29, wherein in one or more of the antibodies, the aspartic acid (D) at position 283 of SEQ ID NO: 9 is modified to isoaspartic acid.

31. The composition according to any one of claims 1 to 30, wherein in one or more of the antibodies, the proline (P) at position 448 of SEQ ID NO: 9 is amidated.

32. The composition according to any one of claims 1 to 31, wherein glycine (G) at position 449 of SEQ ID NO: 9 is absent in one or more of the antibodies.

33. The composition according to any one of claims 1 to 32, wherein one or more of the antibodies are fragmented by cleavage between asparagine at position 58 and threonine at position 59 of SEQ ID NO:

9.

34. The composition according to any one of claims 1 to 33, wherein in one or more of the antibodies, the light chain (LC) and / or the heavy chain (HC) is glycosylated, preferably wherein... (i) The LC is glycosylated in less than about 35%, preferably less than about 2%, preferably from about 1% to 3% of the antibody; and / or (ii) The HC is glycosylated in less than about 6%, preferably less than about 5%, preferably from about 2% to 5% of the antibody.

35. The composition according to any one of claims 1 to 32, wherein the antibody comprises a first HC (HC1), a first LC (LC1), a second HC (HC2), and a second LC (LC2).

36. The composition of claim 35, wherein the antibody comprises one or more of the following disulfide bridges: 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; and HC:C370-HC:C428 The cysteine ​​residues (C) are numbered according to their positions in SEQ ID NO: 8 and 9.

37. The composition according to any one of claims 1 to 36, wherein the antibody (i) Having a molecular weight of 147.0 kDa to 147.6 kDa: (ii) Having pI of 8.4 (theoretical) and 9.3 (determined), respectively; and / or (iii) It has extinction coefficients (A) of 1.39 (theoretical) and 1.438 (determined experimentally), respectively. 0.1% (mL / (mg)) cm).

38. The composition according to any one of claims 1 to 37, wherein the one or more antibodies are acidic variants.

39. The composition according to any one of claims 1 to 38, wherein the antibody is a recombinant antibody produced in Chinese hamster ovary (CHO) cells, preferably in CHO-K1 cells.

40. The composition according to any one of claims 1 to 39, wherein the HC of the antibody further comprises a signal peptide derived from the human immunoglobulin heavy chain, and wherein the LC further comprises a signal peptide derived from the human immunoglobulin κ light chain, preferably wherein the signal peptide of the HC has the amino acid sequence shown in SEQ ID NO: 17, and the signal peptide of the LC has the amino acid sequence shown in SEQ ID NO:

18.

41. The composition of claim 40, wherein the HC comprising the signal peptide has the amino acid sequence shown in SEQ ID NO: 19, and the LC comprising the signal peptide has the amino acid sequence shown in SEQ ID NO:

20.

42. A composition comprising one or more antibodies, said antibodies comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO: 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO: 8, wherein SEQ ID NO: 9 comprises the following sequence: QLQLQESGPG LVKPSETLSL TCSVSGGSII SRSSYWGWIR QPPGKGLEWI GGIYHSGNTYDNPSLKSRLTMSVDTSKNQFSLNLRSVTAADTAVYYCARI VPGGDAFDIW GQGTMVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK, Among the antibodies, one or more of the antibodies are mentioned. Glutamine (Q) at position 1 was modified to pyroglutamic acid (pE). The lysine (K) at position 450 is absent, and The asparagine (N) at position 300 is glycosylated. And SEQ ID NO: 8 contains the following sequence: DIQMTQSPSSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, The antibody comprises the following glycan types: Man3+1F, G0-GN, G0F-GN, G0, G0F, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1 and G2FS2, and the antibody is a recombinant antibody produced in Chinese hamster ovary (CHO)-K1 cells.

43. An antibody comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO: 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO: 8, wherein SEQ ID NO: 9 comprises the following sequence: QLQLQESGPG LVKPSETLSL TCSVSGGSII SRSSYWGWIR QPPGKGLEWI GGIYHSGNTYDNPSLKSRLTMSVDTSKNQFSLNLRSVTAADTAVYYCARI VPGGDAFDIW GQGTMVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK, SEQ ID NO: 8 contains the following sequence: DIQMTQSPSSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, The HC described therein includes the following modifications: Glutamine (Q) at position 1 was modified to pyroglutamic acid; The lysine (K) at position 450 is missing; and The asparagine (N) at position 300 is glycosylated, and The antibodies described herein contain the following glycan types: Man3+1F, G0-GN, G0F-GN, G0, G0F, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1, and G2FS2.

44. An antibody comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO: 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO: 8, wherein SEQ ID NO: 9 comprises the following sequence: QLQLQESGPG LVKPSETLSL TCSVSGGSII SRSSYWGWIR QPPGKGLEWI GGIYHSGNTYDNPSLKSRLTMSVDTSKNQFSLNLRSVTAADTAVYYCARI VPGGDAFDIW GQGTMVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK, SEQ ID NO: 8 contains the following sequence: DIQMTQSPSSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, The HC described therein includes the following modifications: Glutamine (Q) at position 1 was modified to pyroglutamic acid; The lysine (K) at position 450 is missing; and The asparagine (N) at position 300 is glycosylated, and Less than 3% of the polysaccharides are of the Man5 type.

45. The antibody according to claim 43 or 44, wherein the HC further comprises one or more of the following modifications: The asparagine (N) at position 58 is deamidinated; The methionine (M) at position 71 is oxidized; The methionine (M) at position 115 is oxidized; The methionine (M) at position 255 is oxidized; The aspartic acid (D) at position 283 is isomerized; The asparagine (N) at position 318 is deamidinated or contains succinimide; The methionine (M) at position 361 is oxidized; The asparagine (N) at position 387 is deamidinated or contains succinimide; The methionine (M) at position 431 is oxidized; Glycine (G) at position 449 is absent; The proline (P) at position 448 is amidated after losing the C-terminal lysine and glycine.

46. ​​The antibody according to any one of claims 43 to 45, wherein the main glycan type is G0F and G1F, preferably wherein about 49% of the glycan is of type G0F and about 25% of the glycan is of type G1F.

47. An antibody comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO: 8, wherein SEQ ID NO: 7 comprises the following sequence: X1LQLQESGPGLVKPSETLSLTCSVSGGSIISRSSYWGWIRQPPGKGLEWIGGIYHSGNTYDNPSLKSRLTMSVDTSKNQFSLNLRSVTAADTAVYYCARIVPGGDAFDIWGQG TMVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLX2ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLX3GKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESX4GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSX5X6X7 in: X1 is not present; it is either glutamine or pyroglutamic acid (pE). X2 is methionine or oxidized methionine; X3 is asparagine, deamidated asparagine, or asparagine containing succinimide; X4 is asparagine or deamidated asparagine; X5 is proline or amidated proline; X6 is absent or may be glycine; and X7 is absent or may be lysine; and SEQ ID NO: 8 contains the following sequence: DIQMTQSPSSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

48. The antibody according to claim 47, wherein the HC has an amino acid sequence comprising the sequence in SEQ ID NO: 39: X1LQLQESGPGLVKPSETLSLTCSVSGGSIISRSSYWGWIRQPPGKGLEWIGGIYHSGX I TYDNPSLKSRLTX I I SVDTSKNQFSLNLRSVTAADTAVYYCARIVPGGDAFDIWGQGTX III VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX2ISRTPEVTCVVVDVSHEDPEVKFNWYVX IV GVEVHNAKTKPREEQYX V STYRVVSVLTVLHQDWLX3GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEX VI TKNQVSLTCLVKGFYPSDIAVEWESX4GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVX VII HEALHNHYTQKSLSLSX5X6X7 in: X1 is not present; it is either glutamine or pyroglutamic acid (pE). X2 is methionine or oxidized methionine; X3 is asparagine, deamidated asparagine, or asparagine containing succinimide; X4 is asparagine, deamidated asparagine, or asparagine containing succinimide; X5 is proline or amidated proline; X6 is absent or may be glycine; X7 is absent or may be lysine; X I It is asparagine or deamidated asparagine; X II It is methionine or oxidized methionine; X III It is methionine or oxidized methionine; X IV It is aspartic acid or isoaspartic acid; X V It is asparagine or glycosylated asparagine; X VI It is methionine or oxymethionine; and X VII It is methionine or oxymethionine.

49. The antibody according to claim 47 or 48, wherein X1 is pyroglutamic acid (pE).

50. The antibody according to claim 47 or 48, wherein X1 is glutamine.

51. The antibody according to any one of claims 47 to 50, wherein X7 is absent.

52. The antibody according to any one of claims 47 to 50, wherein X7 is lysine.

53. The antibody according to claim 51, wherein X6 is absent.

54. The antibody according to any one of claims 47 to 52, wherein X6 is glycine.

55. The antibody according to any one of claims 47 to 54, wherein X5 is amidated proline.

56. The antibody according to any one of claims 47 to 54, wherein X5 is proline.

57. The antibody according to any one of claims 47 to 56, wherein X2 is oxymethionine.

58. The antibody according to any one of claims 47 to 56, wherein X2 is methionine.

59. The antibody according to any one of claims 47 to 58, wherein X3 is deamidated asparagine.

60. The antibody according to any one of claims 47 to 58, wherein X3 comprises asparagine containing succinimide.

61. The antibody according to any one of claims 47 to 58, wherein X3 is asparagine.

62. The antibody according to any one of claims 47 to 61, wherein X4 is deamidated asparagine.

63. The antibody according to any one of claims 47 to 61, wherein X4 comprises asparagine containing succinimide.

64. The antibody according to any one of claims 47 to 61, wherein X4 is asparagine.

65. The antibody according to any one of claims 48 to 64, wherein X I It is deamidated asparagine.

66. The antibody according to any one of claims 48 to 64, wherein X I It is asparagine.

67. The antibody according to any one of claims 48 to 66, wherein X II It is oxymethionine.

68. The antibody according to any one of claims 48 to 66, wherein X II It is methionine.

69. The antibody according to any one of claims 48 to 68, wherein X III It is oxymethionine.

70. The antibody according to any one of claims 48 to 68, wherein X III It is methionine.

71. The antibody according to any one of claims 48 to 70, wherein X IV It is isoflavone.

72. The antibody according to any one of claims 48 to 70, wherein X IV It is aspartic acid.

73. The antibody according to any one of claims 48 to 72, wherein X V It is glycosylated asparagine.

74. The antibody according to any one of claims 48 to 72, wherein X V It is asparagine.

75. The antibody according to any one of claims 48 to 74, wherein X VI It is oxymethionine.

76. The antibody according to any one of claims 48 to 74, wherein X VI It is methionine.

77. The antibody according to any one of claims 48 to 76, wherein X VII It is oxymethionine.

78. The antibody according to any one of claims 48 to 76, wherein X VII It is methionine.

79. The antibody according to any one of claims 47 to 78, wherein X1 is pyroglutamic acid (pE), X2 is methionine, X3 is asparagine, X4 is asparagine, X5 is proline, X6 is glycine, and X7 is absent.

80. The antibody according to any one of claims 48 to 79, Where X1 is pyroglutamic acid (pE), X2 is methionine, X3 is asparagine, X4 is asparagine, X5 is proline, X6 is glycine, X7 is absent, and X... I It's asparagine, X II It's methionine, X III It's methionine, X IV It is aspartic acid, X V It is glycosylated asparagine, X VI It is methionine, and X VII It is methionine; or Where X1 is pyroglutamic acid (pE), X2 is oxidized methionine, X3 is asparagine, X4 is asparagine, X5 is proline, X6 is glycine, X7 is absent, and X... I It's asparagine, X II It's methionine, X III It is oxidized methionine, X IV It is aspartic acid, X V It is glycosylated asparagine, X VI It is methionine, and X VII It is methionine.

81. The antibody according to any one of claims 42 to 80, wherein the antibody comprises a glycosylated light chain (LC).

82. The antibody according to any one of claims 42 to 81, wherein the antibody comprises glycosylated heavy chain (HC).

83. The antibody according to any one of claims 42 to 82, wherein the antibody comprises a first HC (HC1), a first LC (LC1), a second HC (HC2), and a second LC (LC2).

84. The antibody of claim 83, wherein the antibody comprises one or more of the following disulfide bridges: 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; and HC:C370-HC:C428 The cysteine ​​residues (C) are numbered to their positions in SEQ ID NO: 7 and 8, where glutamine (X1) is present in SEQ ID NO: 7 and SEQ ID NO: 9 and 8, respectively.

85. The antibody according to any one of claims 42 to 84, wherein the antibody has a molecular weight of 147.0 kDa to 147.6 kDa.

86. The antibody according to any one of claims 42 to 85, wherein the antibody has pI of 8.4 (theoretical) and 9.3 (determined experimentally), respectively.

87. The antibody according to any one of claims 42 to 86, wherein the antibody has extinction coefficients (A values) of 1.39 (theoretical) and 1.438 (determined experimentally), respectively. 0.1% (mL / (mg)) cm).

88. The antibody according to any one of claims 42 to 87, wherein the antibody is an acidic antibody variant.

89. The antibody according to any one of claims 42 to 88, wherein the constant region of each HC contains an N-linked glycan site at residue N300 as shown in SEQ ID NO:7 or 9.

90. The antibody of claim 89, wherein the antibody comprises one or more of the following glycan types: Man3+1F, GO-GN, GOF-GN, GO, GOF, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1 and G2FS2, preferably wherein the primary glycan type is GOF and G1F.

91. The antibody according to claim 89 or 90, wherein more than 85% of the glycan is fucoidylated, preferably wherein about 85% to 95% of the glycan is fucoidylated.

92. The antibody according to any one of claims 89 to 91, wherein about 20% to 40% of the polysaccharide is galactosylated.

93. The antibody according to any one of claims 89 to 92, wherein about 80% to 90% of the glycan is part of a major fucosylated glycan type, preferably wherein the major glycan type is G0F and G1F.

94. The antibody according to any one of claims 89 to 94, wherein about 1% to 4% of the polysaccharide is a mannose-containing polysaccharide, preferably wherein the polysaccharide is of the Man5 and Man31F type.

95. The antibody according to any one of claims 89 to 94, wherein about 1% to 4% of the polysaccharide is of the Man5 type.

96. The antibody according to any one of claims 89 to 95, wherein less than about 3% of the glycan is of the Man5 type, preferably about 1% to 2.5%, and more preferably about 1% to 2% of the glycan is of the Man5 type.

97. The antibody according to any one of claims 89 to 96, wherein less than 2% of the glycan is sialylated, preferably wherein about 0.5% to 2% of the glycan is sialylated.

98. The antibody according to any one of claims 42 to 87, wherein the antibody is a recombinant antibody produced in Chinese hamster ovary (CHO) cells, preferably wherein the antibody is produced in CHO-K1 cells.

99. An antibody comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO: 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO: 8, wherein SEQ ID NO: 9 comprises the following sequence: QLQLQESGPG LVKPSETLSL TCSVSGGSII SRSSYWGWIR QPPGKGLEWI GGIYHSGNTYDNPSLKSRLTMSVDTSKNQFSLNLRSVTAADTAVYYCARI VPGGDAFDIW GQGTMVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK in: Glutamine (Q) at position 1 was modified to pyroglutamic acid; The lysine (K) at position 450 is missing; and The asparagine (N) at position 300 is glycosylated; and The antibody described herein contains the following disulfide bridges: 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; and HC:C370-HC:C428 The cysteine ​​residues (C) are numbered to correspond to their positions in SEQ ID NO: 8 and 9; and the antibody comprises the following glycan types: Man3+1F, G0-GN, G0F-GN, G0, G0F, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1 and G2FS2, preferably wherein the main glycan types are G0F and G1F; SEQ ID NO: 8 contains the following sequence: DIQMTQSPSSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

100. The antibody of claim 99, wherein less than 3% of the glycan is of the Man5 type.

101. The antibody according to claim 99 or 100, wherein the HC further comprises one or more of the following modifications: The asparagine (N) at position 58 is deamidinated; The methionine (M) at position 71 is oxidized; The methionine (M) at position 115 is oxidized; The methionine (M) at position 255 is oxidized; The aspartic acid (D) at position 283 is isomerized; The asparagine (N) at position 318 is deamidinated or contains succinimide; The methionine (M) at position 361 is oxidized; The asparagine (N) at position 387 is deamidinated or contains succinimide; The methionine (M) at position 431 is oxidized; Glycine (G) at position 449 is absent; The proline (P) at position 448 is amidated after losing the C-terminal lysine and glycine; Lysine (K) residues are glycosylated.

102. The antibody according to any one of claims 42 to 101, wherein the HC further comprises a signal peptide derived from the human immunoglobulin heavy chain, and wherein the LC further comprises a signal peptide derived from the human immunoglobulin κ light chain, preferably wherein the signal peptide of the HC has the amino acid sequence shown in SEQ ID NO: 17, and the signal peptide of the LC has the amino acid sequence shown in SEQ ID NO: 18, preferably the HC containing the signal peptide has the amino acid sequence shown in SEQ ID NO: 19, and the LC containing the signal peptide has the amino acid sequence shown in SEQ ID NO:

20.

103. An antibody comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO: 8, wherein SEQ ID NO: 7 comprises the following sequence: X1LQLQESGPGLVKPSETLSLTCSVSGGSIISRSSYWGWIRQPPGKGLEWIGGIYHSGNTYDNPSLKSRLTMSVDTSKNQFSLNLRSVTAADTAVYYCARIVPGGDAFDIWGQG TMVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLX2ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLX3GKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESX4GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSX5X6X7 in: X1 is not present; it is either glutamine or pyroglutamic acid (pE). X2 is methionine or oxidized methionine; X3 is asparagine, deamidated asparagine, or asparagine containing succinimide; X4 is asparagine or deamidated asparagine; X5 is proline or amidated proline; X6 is absent or may be glycine; and X7 is absent or may be lysine; and SEQ ID NO: 8 contains the following sequence: DIQMTQSPSSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, The HC further comprises a signal peptide derived from the human immunoglobulin heavy chain, and the LC further comprises a signal peptide derived from the human immunoglobulin κ light chain. Preferably, the signal peptide of the HC has the amino acid sequence shown in SEQ ID NO: 17, and the signal peptide of the LC has the amino acid sequence shown in SEQ ID NO:

18.

104. The antibody according to claim 103, wherein the HC has an amino acid sequence comprising the following sequence: SEQ ID NO: 39 X1LQLQESGPGLVKPSETLSLTCSVSGGSIISRSSYWGWIRQPPGKGLEWIGGIYHSGX I TYDNPSLKSRLTX I I SVDTSKNQFSLNLRSVTAADTAVYYCARIVPGGDAFDIWGQGTX III VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX2ISRTPEVTCVVVDVSHEDPEVKFNWYVX IV GVEVHNAKTKPREEQYX V STYRVVSVLTVLHQDWLX3GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEX VI TKNQVSLTCLVKGFYPSDIAVEWESX4GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVX VII HEALHNHYTQKSLSLSX5X6X7 in: X1 is not present; it is either glutamine or pyroglutamic acid (pE). X2 is methionine or oxidized methionine; X3 is asparagine, deamidated asparagine, or asparagine containing succinimide; X4 is asparagine, deamidated asparagine, or asparagine containing succinimide; X5 is proline or amidated proline; X6 is absent or may be glycine; X7 is absent or may be lysine; X I It is asparagine or deamidated asparagine; X II It is methionine or oxidized methionine; X III It is methionine or oxidized methionine; X IV It is aspartic acid or isoaspartic acid; X V It is asparagine or glycosylated asparagine; X VI It is methionine or oxymethionine; and X VII It is methionine or oxymethionine.

105. The antibody according to claim 103 or 104, wherein the HC comprising the signal peptide has the amino acid sequence shown in SEQ ID NO: 19, and the LC comprising the signal peptide has the amino acid sequence shown in SEQ ID NO:

20.

106. One or more polynucleotides comprising nucleotide sequences of an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC) encoding an anti-thyroxine transporter (TTR) antibody, wherein the HC has an amino acid sequence of SEQ ID NO: 7, 9 or 39, and the LC has an amino acid sequence of SEQ ID NO:

8.

107. A nucleic acid molecule, said nucleic acid molecule is (a) A polynucleotide comprising the first nucleotide sequence shown in SEQ ID NO: 13 of an immunoglobulin heavy chain (HC) encoding an anti-thyroxine transporter (TTR) antibody and the second nucleotide sequence shown in SEQ ID NO: 14 of an immunoglobulin light chain (LC) encoding an anti-thyroxine transporter (TTR) antibody; (b)(a) messenger RNA (mRNA) equivalents of the first nucleotide sequence and the second nucleotide sequence; (c) A polynucleotide comprising a sequence complementary to the first nucleotide sequence and the second nucleotide sequence of (a) or its mRNA equivalent in (b); or (d) A polynucleotide comprising the first nucleotide sequence of (a) and the second nucleotide sequence or (b) a degenerate form of its mRNA equivalent.

108. The nucleic acid molecule of claim 107, wherein the sequence of the degenerate of the first nucleotide sequence is shown in SEQ ID NO: 15 and the sequence of the degenerate of the second nucleotide sequence is shown in SEQ ID NO:

16.

109. The polynucleotide of claim 106 or the nucleic acid molecule of claim 107 or 108, wherein the first nucleotide sequence comprises a nucleotide sequence encoding a first signal peptide derived from the human immunoglobulin heavy chain, and the second nucleotide sequence comprises a nucleotide sequence encoding a second signal peptide derived from the human immunoglobulin κ light chain, preferably wherein the nucleotide sequence of the first signal peptide is shown in SEQ ID NO: 21, and the nucleotide sequence of the second signal peptide is shown in SEQ ID NO:

22.

110. The polynucleotide or nucleic acid molecule of claim 109, wherein the first nucleotide sequence comprising the nucleotide sequence of the signal peptide has the sequence shown in SEQ ID NO: 23, and the second nucleotide sequence comprising the nucleotide sequence of the signal peptide has the sequence shown in SEQ ID NO:

24.

111. One or more polynucleotides comprising a first nucleotide sequence encoding an immunoglobulin heavy chain (HC) of an anti-thyroxine transporter (TTR) antibody and a second nucleotide sequence encoding an immunoglobulin light chain (LC), wherein the HC has an amino acid sequence of SEQ ID NO: 7, 9 or 39, and the LC has an amino acid sequence of SEQ ID NO: 8, wherein the first nucleotide sequence comprises a nucleotide sequence encoding a first signal peptide derived from the human immunoglobulin heavy chain, and the second nucleotide sequence comprises a nucleotide sequence encoding a second signal peptide derived from the human immunoglobulin κ light chain, preferably wherein the nucleotide sequence of the first signal peptide is shown in SEQ ID NO: 21, and the nucleotide sequence of the second signal peptide is shown in SEQ ID NO:

22.

112. A nucleic acid molecule comprising a first nucleotide sequence of an immunoglobulin heavy chain (HC) encoding an anti-thyroxine transporter (TTR) antibody as shown in SEQ ID NO: 13 and a second nucleotide sequence of an immunoglobulin light chain (LC) encoding an anti-thyroxine transporter (TTR) antibody as shown in SEQ ID NO: 14, wherein the first nucleotide sequence comprises a nucleotide sequence encoding a first signal peptide derived from the human immunoglobulin heavy chain, and the second nucleotide sequence comprises a nucleotide sequence encoding a second signal peptide derived from the human immunoglobulin κ light chain, preferably wherein the nucleotide sequence of the first signal peptide is shown in SEQ ID NO: 21 and the nucleotide sequence of the second signal peptide is shown in SEQ ID NO:

22.

113. One or more expression vectors, said expression vectors comprising a polynucleotide according to any one of claims 106 and 109 to 111, or a nucleic acid molecule according to any one of claims 107 to 110 and 112.

114. A host cell comprising a polynucleotide according to any one of claims 106 and 109 to 111, or a nucleic acid molecule according to any one of claims 107 to 110 and 112, or a vector according to claim 114.

115. The host cell according to claim 114, wherein the host cell is a non-human cell, preferably Escherichia coli, insect, or CHO cell.

116. The host cell according to claim 114 or 115, wherein the host cell is a CHO cell, preferably a CHO-K1 cell.

117. A method for manufacturing an anti-TTR antibody, the method comprising: (a) Culturing cells according to any one of claims 114 to 116, and (b) Isolate the antibody or its immunoglobulin chain from the culture.

118. A method for generating an antibody or an antigen-binding fragment thereof according to any one of claims 42 to 105, wherein the antibody comprises at least the following post-translational modifications: The glutamine (Q) at position X1 and position 1 of SEQ ID NO: 9 is pyroglutamic acid (pE) and / or The lysine (K) at position X7 and position 450 of SEQ ID NO: 9 is absent. The method includes a) Cloning a nucleic acid molecule containing the first nucleotide sequence according to any one of claims 107 to 110 and 112 into an expression vector, and cloning a nucleic acid molecule containing the second nucleotide sequence according to any one of claims 107 to 110 and 112 into an expression vector, wherein the first nucleotide sequence and the second nucleotide sequence can be provided in the same or different expression vectors; b) Transform the expression vector into CHO-K1 cells; c) Culture host cells under conditions that allow the expression of immunoglobulin chains containing both heavy and light chains; d) Separate the immunoglobulin chains and the resulting IgG antibodies from the culture, and optionally digest the IgG antibodies to generate antigen-binding fragments of the antibodies, for example using enzymatic digestion including papain cleavage to generate Fab fragments.

119. A method for generating an antibody or an antigen-binding fragment thereof according to any one of claims 42 to 105, wherein the antibody comprises at least the following post-translational modifications: The glutamine (Q) at position 1 of SEQ ID NO: 9 is pyroglutamic acid (pE). The lysine (K) at position 450 of SEQ ID NO: 9 is absent, and The asparagine (N) at position 300 of SEQ ID NO: 9 is glycosylated, wherein less than 3% of the said polysaccharide is of the Man5 type. The method includes a) Cloning a nucleic acid molecule containing the first nucleotide sequence according to any one of claims 107 to 110 and 112 into an expression vector, and cloning a nucleic acid molecule containing the second nucleotide sequence according to any one of claims 107 to 110 and 112 into an expression vector, wherein the first nucleotide sequence and the second nucleotide sequence can be provided in the same or different expression vectors; b) Transform the expression vector into CHO-K1 cells; c) Culture host cells under conditions that allow expression of immunoglobulin chains containing heavy and light chains, wherein the culture is carried out with a pH dead zone of 0.1 to 0.05, preferably 0.05; d) Separate the immunoglobulin chains and the resulting IgG antibodies from the culture, and optionally digest the IgG antibodies to generate antigen-binding fragments of the antibodies, for example using enzymatic digestion including papain cleavage to generate Fab fragments.

120. An antibody that can be encoded by a polynucleotide according to any one of claims 106 and 109 to 111 or by a vector according to claim 113, or can be obtained by any one of claims 117 to 119.

121. A composition comprising one or more antibodies according to any one of claims 42 to 105 and 120.

122. The composition according to claim 121, wherein in one or more of the antibodies, the glutamine (Q) at position 1 of SEQ ID NO: 9 is modified with pyroglutamic acid and wherein X1 in SEQ ID NO: 7 or 39 is pyroglutamic acid, respectively.

123. The composition according to claim 121 or 122, wherein in more than 90%, preferably in about 99% to 100% of the antibody, glutamine (Q) at position 1 of SEQ ID NO: 9 is modified with pyroglutamic acid and wherein X1 in SEQ ID NO: 7 or 39 is pyroglutamic acid, respectively.

124. The composition according to any one of claims 121 to 122, wherein in one or more of the antibodies, lysine (K) at position 450 of SEQ ID NO: 9 and X7 in SEQ ID NO: 7 or 39 are respectively absent.

125. The composition according to any one of claims 121 to 124, wherein in more than 90%, preferably in about 95% to 100%, preferably in about 95% to 96% of the antibody, lysine (K) at position 450 of SEQ ID NO: 9 and X7 in SEQ ID NO: 7 or 39 are respectively absent.

126. The composition according to any one of claims 121 to 125, wherein the antibody comprises a polysaccharide, preferably an N-polysaccharide, preferably wherein, respectively, the asparagine (N) at position 300 of SEQ ID NO: 9 is glycosylated and the X in SEQ ID NO: 39 is glycosylated. V It is glycosylated asparagine.

127. The composition according to any one of claims 121 to 126, wherein the antibody comprises the following glycan types: Man3+1F, G0-GN, G0F-GN, G0, G0F, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1 and G2FS2, preferably wherein the main glycan type is G0F and G1F.

128. The composition according to claim 126 or 127, wherein more than 85% of the polysaccharide is fucoidylated, preferably wherein about 85% to 95% of the polysaccharide is fucoidylated.

129. The composition according to any one of claims 126 to 128, wherein about 20% to 40% of the polysaccharide is galactosylated.

130. The composition according to any one of claims 126 to 129, wherein about 80% to 90% of the polysaccharide is part of a major fucoidylated polysaccharide type, preferably wherein the major polysaccharide type is G0F and G1F.

131. The composition according to any one of claims 125 to 130, wherein about 1% to 4% of the polysaccharide is a mannose-containing polysaccharide, preferably wherein the polysaccharide is of the Man5 and Man31F type.

132. The composition according to any one of claims 125 to 131, wherein less than about 3%, preferably about 1% to 2.5%, preferably 1% to 2% of the polysaccharide is of the Man5 type.

133. The composition according to any one of claims 125 to 132, wherein less than 2% of the polysaccharide is sialylated, preferably wherein about 0.5% to 2% of the polysaccharide is sialylated.

134. The composition according to any one of claims 121 to 133, wherein, in one or more of the antibodies, the asparagine (N) at positions 58, 318 and / or 387 of SEQ ID NO: 9 is deamidinated and X in SEQ ID NO: 39 is... I X3 and / or X4 are deamidated asparagines.

135. The composition according to any one of claims 121 to 134, wherein in less than 5%, preferably less than about 2%, preferably less than about 1%, preferably from about 0% to 1% of the antibody, respectively, the asparagine (N) at position 58 of SEQ ID NO: 9 is deamidinated and X in SEQ ID NO: 39 I It is deamidated asparagine.

136. The composition according to any one of claims 121 to 135, wherein in less than about 9%, preferably less than about 8%, preferably from about 6% to 9% of the antibody, respectively, the asparagine (N) at position 318 of SEQ ID NO: 9 is deamidated and X3 in SEQ ID NO: 39 is deamidated asparagine.

137. The composition according to any one of claims 121 to 136, wherein in less than about 5%, preferably less than about 3%, preferably from about 1% to 4% of the antibody, respectively, the asparagine (N) at position 387 of SEQ ID NO: 9 is deamidinated and wherein X4 in SEQ ID NO: 39 is deamidinated asparagine.

138. The composition according to any one of claims 121 to 137, wherein in one or more of the antibodies, respectively, the methionine at positions 71, 115, 255, 361 and / or 431 of SEQ ID NO: 9 is oxidized and X in SEQ ID NO: 39 is oxidized. II X III X2, X VI and / or X VII It is oxymethionine.

139. The composition according to any one of claims 121 to 138, wherein in less than about 5%, preferably less than about 2%, preferably less than about 1%, preferably from about 0% to 1% of the antibody, respectively, the methionine (M) at position M71 of SEQ ID NO: 9 is oxidized and X in SEQ ID NO: 39 is oxidized. II It is oxymethionine.

140. The composition according to any one of claims 121 to 139, wherein in less than about 5%, preferably less than about 3%, preferably less than about 2%, preferably from about 0% to 2% of the antibody, respectively, the methionine (M) at position M115 of SEQ ID NO: 9 is oxidized and X in SEQ ID NO: 39 is oxidized. III It is oxymethionine.

141. The composition according to any one of claims 121 to 140, wherein in less than about 5%, preferably less than about 3%, preferably from about 1% to 4% of the antibody, respectively, the methionine (M) at position M255 of SEQ ID NO: 9 is oxidized and X2 in SEQ ID NO: 39 is oxidized methionine.

142. The composition according to any one of claims 121 to 141, wherein in less than about 5%, preferably less than about 2%, preferably less than about 1%, preferably from about 0% to 1% of the antibody, respectively, the methionine (M) at position M361 of SEQ ID NO: 9 is oxidized and X in SEQ ID NO: 39 is oxidized. VI It is oxymethionine.

143. The composition according to any one of claims 121 to 142, wherein in less than about 5%, preferably less than about 3%, preferably less than about 2%, preferably from about 0% to 2% of the antibody, respectively, the methionine (M) at position M431 of SEQ ID NO: 9 is oxidized and X in SEQ ID NO: 39 is oxidized. VII It is oxymethionine.

144. The composition according to any one of claims 121 to 143, wherein in one or more of the antibodies, respectively, the aspartic acid (D) at position 283 of SEQ ID NO: 9 is modified to isoaspartic acid and X in SEQ ID NO: 39 IV It is isoflavone.

145. The composition according to any one of claims 121 to 144, wherein in one or more of the antibodies, respectively, the proline (P) at position 448 of SEQ ID NO: 9 is amidated and X5 of SEQ ID NO: 7 or 39 is amidated proline.

146. The composition according to any one of claims 121 to 145, wherein in one or more of the antibodies, glycine (G) at position 449 of SEQ ID NO: 9 and X5 of SEQ ID NO: 7 or 39 are respectively absent.

147. The composition according to any one of claims 121 to 146, wherein one or more of the antibodies are fragmented by cleavage between asparagine at position 58 of SEQ ID NO: 9 and SEQ ID NO: 7 or 39 and threonine at position 59.

148. The composition according to any one of claims 121 to 147, wherein the light chain (LC) is glycosylated in one or more of the antibodies.

149. The composition according to any one of claims 121 to 148, wherein the LC is glycosylated in less than about 5%, preferably less than about 2%, preferably from about 0% to 2% of the antibody.

150. The composition according to any one of claims 121 to 149, wherein the heavy chain (HC) is glycosylated in one or more of the antibodies.

151. The composition according to any one of claims 121 to 150, wherein the HC is glycosylated in less than about 6%, preferably less than about 5%, preferably from about 2% to 5% of the antibody.

152. The composition according to any one of claims 121 to 151, wherein the antibody comprises a first HC (HC1), a first LC (LC1), a second HC (HC2), and a second LC (LC2).

153. The composition of claim 152, wherein the antibody comprises one or more of the following disulfide bridges: 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; and HC:C370-HC:C428 The cysteine ​​residues (C) are numbered according to their positions in SEQ ID NO: 8 and 9.

154. The composition according to any one of claims 121 to 153, wherein the antibody (i) Having a molecular weight of 147.0 kDa to 147.6 kDa; (ii) Having pI of 8.4 (theoretical) and 9.3 (determined), respectively; and / or (iii) It has extinction coefficients (A) of 1.39 (theoretical) and 1.438 (determined), respectively. 0.1% (mL / (mg)) cm).

155. The composition according to any one of claims 121 to 154, wherein the composition comprises an acidic substance of the antibody, preferably about 25% to 33% of an acidic antibody substance.

156. The composition according to any one of claims 121 to 155, wherein the composition comprises the following charge variant of the antibody: Main peak ≥ 63.0% Acid peak ≤ 32.0% The alkaline peak is ≤5.0%.

157. The composition according to any one of claims 121 to 156, wherein the antibody is a recombinant antibody produced in Chinese hamster ovary (CHO) cells, preferably in CHO-K1 cells.

158. The composition of claim 157, wherein the method comprises culturing the CHO-K1 cells in a pH dead zone of less than 0.2, preferably ≤0.175, preferably ≤0.15, preferably ≤0.125, preferably ≤0.1, preferably ≤0.075, preferably ≤0.05, more preferably 0.05 or 0.1, and most preferably 0.

05.

159. The composition according to any one of claims 121 to 158, wherein the HC of the antibody further comprises a signal peptide derived from the human immunoglobulin heavy chain, and wherein the LC further comprises a signal peptide derived from the human immunoglobulin κ light chain, preferably wherein the signal peptide of the HC has the amino acid sequence shown in SEQ ID NO: 17, and the signal peptide of the LC has the amino acid sequence shown in SEQ ID NO:

18.

160. The composition of claim 159, wherein the HC comprising the signal peptide has the amino acid sequence shown in SEQ ID NO: 19, and the LC comprising the signal peptide has the amino acid sequence shown in SEQ ID NO:

20.

161. The composition according to any one of claims 1 to 41 and 121 to 160, wherein the composition is a stable formulation comprising the following: The antibody is present at a concentration of approximately 25 mg / ml to approximately 150 mg / ml; Histidine at a concentration of approximately 20 mM; Sucrose with a concentration of approximately 50 mg / ml to approximately 80 mg / ml; Polysorbate 80 (PS) at a concentration of about 0.01% (w / v) to about 0.5% (w / v); Water for injection The formulation described therein has a pH of about 5.3 to about 6.

3.

162. The composition according to any one of claims 1 to 41 and 121 to 161, wherein the composition is a pharmaceutical composition comprising, in an aqueous formulation, a concentration of about 50 mg / ml to about 100 mg / ml of the antibody, for use in a method of treating a subject with TTR amyloidosis (ATTR), wherein the formulation comprises, as an excipient, L-histidine, L-histidine hydrochloride sucrose, Polysorbate 80 (PS80), and Water for injection.

163. The composition according to claim 161 or the composition used according to claim 162, wherein the pH of the formulation is about 5.

8.

164. The composition according to claim 161 or 163, or the composition used according to claim 162 or 163, wherein the formulation has an osmolar concentration of ≥240 mOsm / Kg.

165. The composition according to claim 161, 163 or 164, or the composition used according to any one of claims 162 to 164, wherein the formulation has a shelf life of 24 months at 2°C-8°C and protected from light.

166. The composition according to any one of claims 161 and 163 to 165, or the composition used according to any one of claims 162 to 165, wherein the formulation comprises the antibody at a concentration of about 50 mg / ml or about 100 mg / ml and histidine at a concentration of about 20 mM, sucrose at a concentration of about 6.5% by weight / volume (w / v) or about 8% (w / v) and PS80 at a concentration of about 0.03% w / v, wherein the formulation has a pH of about 5.

8.

167. The composition according to any one of claims 161 and 163 to 166, or the composition used according to any one of claims 162 to 166, wherein when the total volume is about 2 ml, the formulation consists essentially of the following: The antibody, in doses of 50 mg or 100 mg, 2. 12 mg of L-histidine, 5.56 mg of L-histidine hydrochloride 160mg sucrose, 0.6mg PS80, and Water for injection.

168. The composition according to any one of claims 161 and 163 to 167, or the composition used according to any one of claims 162 to 167, wherein the antibody is produced in Chinese hamster ovary (CHO) cells, preferably in the CHO cell line K1.

169. The composition according to any one of claims 161 and 163 to 168, or the composition used according to any one of claims 162 to 168, wherein the formulation is suitable for intravenous administration.

170. The composition according to any one of claims 161 and 163 to 169, or the composition used according to any one of claims 162 to 169, wherein the formulation is provided in a 2-mL or 20-mL single-dose vial.

171. The composition according to any one of claims 161 and 163 to 170, or the composition used according to any one of claims 162 to 170, wherein the formulation is supplied in a 2 mL glass vial having an aluminum flip-top seal above a rubber stopper.

172. The composition according to any one of claims 161 and 163 to 171, or the composition used according to any one of claims 162 to 171, wherein the formulation is a preservative-free concentrate of an infusion solution that is sterile, colorless to pale yellow, clear to pale milky white liquid and substantially free of visible particles.

173. The composition used according to any one of claims 162 to 172, wherein the formulation is diluted in sterile glucose prior to administration as an intravenous infusion.

174. A pharmaceutical container comprising the composition according to any one of claims 161 and 163 to 172.

175. The drug container according to claim 174, wherein the drug container is a 2ml or 20ml vial.

176. The vial according to claim 175, wherein the vial is a glass vial having an aluminum flip cap above a 13mm rubber stopper.

177. The vial according to claims 175 to 176, wherein the vial contains approximately 12.5% ​​volume overfill or a total volume of 2.25 ml or 22.5 ml of the pharmaceutical preparation.

178. A stable aqueous formulation for use in a method for treating trans-transmitted triglyceride amyloid cardiomyopathy (ATTR-CM), wherein the formulation (i) It is basically composed of the following items: (a) An anti-TTR antibody comprising an immunoglobulin heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 7 or 39 or SEQ ID NO: 9 and an immunoglobulin light chain (LC) containing the amino acid sequence of SEQ ID NO:

8. (b) Histidine at a concentration of approximately 20 mM (c) Sucrose at a concentration of approximately 8% (w / v), (d) PS80 at a concentration of approximately 0.03% w / v (e) Water for injection, (ii) It has a pH of approximately 5.8; (iii) Contain in a 2 mL disposable vial containing approximately 100 mg of antibody or in a 20 mL disposable vial containing 1000 mg of antibody: (iv) Dilute with sterile glucose solution to a concentration of not less than 1 mg / mL of antibody before administration; and (v) Administered via intravenous infusion.

179. The formulation used according to claim 178, wherein the N-terminal glutamine of the HC amino acid sequence is converted to pyroglutamic acid, the C-terminal lysine of the HC amino acid sequence is deleted, and wherein the HC is N-glycosylated.

180. The formulation used according to claim 179, wherein the HC has the amino acid sequence of SEQ ID NO:

15.

181. A therapeutic kit, said therapeutic kit comprising: (i) One or more containers according to claims 174 to 177; (ii) A device for delivering the formulation to a human subject, particularly wherein the device comprises an infusion bag or syringe for intravenous administration of the antibody.

182. The therapeutic kit of claim 181, wherein the formulation is a preservative-free, clear to milky white and colorless to pale yellow solution provided in a vial at 100 mg / 2 mL.

183. An article comprising: (i) one or more containers according to any one of claims 174 to 177, and (ii) A label, wherein the label specifies that the antibody is indicated for the treatment of ATTR, particularly wild-type or hereditary thyroxine transporter-mediated amyloid cardiomyopathy (ATTR-CM) and / or (iii) Instructions for use specifying that the antibody shall be administered intravenously.

184. The article of claim 183, wherein the container comprises a 2 mL or 20 mL glass vial.

185. The article of claim 183 or 184, wherein the antibody in the container is diluted prior to administration, wherein the dilution is performed with a 5% glucose solution, optionally wherein the glucose solution for dilution is provided in a second container in the article.

186. The product according to any one of claims 183 to 185, wherein the formulation of the antibody is a preservative-free, clear to milky white and colorless to pale yellow solution provided in a vial at 100 mg / 2 mL.

187. A method for treating TTR amyloidosis (ATTR), preferably ATTR cardiomyopathy (ATTR-CM), and / or ATTR polyneuropathy (ATTR-PN), the method comprising administering to a patient in need an antibody according to any one of claims 42 to 105 and 119, or a composition according to any one of claims 1 to 41, 121 to 161, and 163 to 171.

188. The method of claim 187, wherein the method comprises administering the antibody to a patient suffering from ATTR-CM, thereby treating the patient's ATTR-CM.

189. The method of claim 187, wherein the method comprises administering the antibody to a patient suffering from ATTR-PN to treat the patient's ATTR-PN.

190. The method according to any one of claims 187 to 189, wherein the antibody is diluted and administered as an intravenous infusion.

191. The method of claim 190, wherein dilution is carried out in a 5% glucose solution.

192. The method according to any one of claims 187 to 191, wherein the patient has previously been treated with a TTR tetramer stabilizer and / or is receiving the TTR tetramer stabilizer concurrently.

193. The method of claim 192, wherein the TTR tetramer stabilizer is selected from chlorobenzazole acid and aclomid.

194. Use of the antibody according to any one of claims 42 to 105 and 119 or the composition according to any one of claims 1 to 41, 121 to 161 and 163 to 171 for the manufacture of a medicament for the treatment of TTR amyloidosis (ATTR).

195. The antibody according to any one of claims 42 to 105 and 119, wherein the antibody is prepared in CHO cells, preferably in CHO-K1 cells, and named ALXN2220.

196. The composition used according to any one of claims 162 to 173, wherein the subject has been treated with a TTR tetramer stabilizer, preferably with chlorpromazine or aclomid, or simultaneously treated with the TTR tetramer stabilizer, preferably with chlorpromazine or aclomid.

197. A method for producing an antibody or an antigen-binding fragment thereof according to any one of claims 42 to 105 and 109, the method comprising: a) Cloning a nucleic acid molecule containing the first nucleotide sequence according to any one of claims 107 to 110 and 112 into an expression vector, and cloning a nucleic acid molecule containing the second nucleotide sequence according to any one of claims 107 to 110 and 112 into an expression vector, wherein the first nucleotide sequence and the second nucleotide sequence can be provided in the same or different expression vectors; b) Transform the expression vector into non-human host cells, preferably into CHO cells, more preferably into CHO-K1 cells; c) Culture host cells under conditions that allow the expression of immunoglobulin chains containing both heavy and light chains; d) Separate the immunoglobulin chains and the resulting IgG antibodies from the culture, and optionally digest the IgG antibodies to generate antigen-binding fragments of the antibodies, for example using enzymatic digestion including papain cleavage to generate Fab fragments, and optionally... e) The antibody is formulated in an aqueous solution comprising the antibody at a concentration of about 50 mg / ml or about 100 mg / ml, histidine at a concentration of about 20 mM, sucrose at a concentration of about 6.5% by weight / volume (w / v) or about 8% (w / v) sucrose, and PS80 at a concentration of about 0.03% w / v, wherein the formulation has a pH of about 5.8 to produce a pharmaceutical composition comprising the antibody; and f) Optionally, the composition is filled into vials, and g) Optionally, the pharmaceutical composition and vial are packaged together with instructions for administering the antibody, for example, intravenously to human patients, in the kit.

198. The method of claim 197, wherein step (c) comprises culturing the cells in a pH dead zone of less than 0.2, preferably ≤0.175, preferably ≤0.15, preferably ≤0.125, preferably ≤0.1, preferably ≤0.075, preferably ≤0.05, more preferably 0.05 or 0.1, and most preferably 0.

05.

199. A method for producing an antibody composition according to any one of claims 1 to 42 and 121 to 173, the method comprising: a) Cloning a nucleic acid molecule containing the first nucleotide sequence according to any one of claims 107 to 110 and 112 into an expression vector, and cloning a nucleic acid molecule containing the second nucleotide sequence according to any one of claims 107 to 110 and 112 into an expression vector, wherein the first nucleotide sequence and the second nucleotide sequence can be provided in the same or different expression vectors; b) Transform the expression vector into CHO-K1 cells; c) CHO-K1 cells are cultured under conditions that allow the expression of immunoglobulin chains containing heavy and light chains, wherein the culture is carried out in a pH dead zone of less than 0.2, preferably ≤0.175, preferably ≤0.15, preferably ≤0.125, preferably ≤0.1, preferably ≤0.075, preferably ≤0.05, more preferably 0.05 or 0.1, and most preferably 0.05; d) Separate the immunoglobulin chains and the resulting IgG antibodies from the culture to prepare the composition, and optionally digest the IgG antibodies to generate antigen-binding fragments of the antibodies, for example using enzymatic digestion including papain cleavage to generate Fab fragments; and optionally... e) The antibody is formulated in an aqueous solution comprising the antibody at a concentration of about 50 mg / ml or about 100 mg / ml, histidine at a concentration of about 20 mM, sucrose at a concentration of about 6.5% by weight / volume (w / v) or about 8% (w / v) sucrose, and PS80 at a concentration of about 0.03% w / v, wherein the formulation has a pH of about 5.8 to produce a pharmaceutical composition comprising the antibody; and f) Optionally, the composition is filled into vials, and g) Optionally, the pharmaceutical composition and vial are packaged together with instructions for administering the antibody, for example, intravenously to human patients, in the kit.

200. A method for manufacturing an antibody according to any one of claims 42 to 105 and 120 or a composition according to any one of claims 1 to 42 and 121 to 173. (a) Culturing cells according to any one of claims 114 to 116, wherein the culturing is carried out in a pH dead zone of less than 0.2, preferably ≤0.175, preferably ≤0.15, preferably ≤0.125, preferably ≤0.1, preferably ≤0.075, preferably ≤0.05, more preferably 0.05 or 0.1, and most preferably 0.05; and (b) Isolate the antibody or its immunoglobulin chain from the culture.

201. A method for verifying amyloid depletion drugs, the method comprising: (i) In the presence of the amyloid depletion drug, tissue sections with amyloid deposits are incubated together with macrophages, preferably THP-1 derived macrophages. The tissue sections are stained with an amyloid-specific fluorescent dye and / or the amyloid-depleting drug is labeled with a fluorescent dye; and (ii) Perform high-resolution live-cell imaging. in (a) The overlap of the fluorescence signal of the amyloid depletion drug and the fluorescence signal of amyloid indicates the binding of the amyloid depletion drug to the amyloid. (b) The presence of punctate and intracellular fluorescent signals in phagocytic vesicles indicates macrophage-mediated amyloid internalization; and (c) The sequential separation of punctate fluorescent signals from the fluorescent signals of said amyloid protein indicates macrophage-mediated amyloid protein fragmentation.

202. The method of claim 201, wherein items (a) to (c) were not observed for the control sample, wherein the tissue sections and macrophages with amyloid deposits were not incubated together in the presence of the amyloid depletion drug, but optionally in the presence of a control compound that does not bind amyloid and / or cannot mediate phagocytosis.

203. The method of claim 201 or 202, wherein the amyloid depletion drug or a pharmaceutical composition comprising the amyloid depletion drug is used to treat amyloidosis or amyloid-related diseases.

204. A screening method for identifying and optionally obtaining amyloid depletion drugs from a variety of test compounds, the screening method comprising: (i) In the presence of the test compound, tissue sections with amyloid deposits were incubated with macrophages, preferably THP-1-derived macrophages. The tissue sections are stained with an amyloid-specific fluorescent dye and / or the test compound is labeled with a fluorescent dye; and (ii) Perform high-resolution live-cell imaging. in (a) The overlap of the fluorescence signal of the test compound and the fluorescence signal of amyloid protein indicates the binding of the test compound to the amyloid protein; (b) The presence of punctate and intracellular fluorescent signals in phagocytic vesicles indicates macrophage-mediated amyloid internalization; and (c) Sequential separation of punctate fluorescent signals from the fluorescent signals of said amyloid protein indicates macrophage-mediated amyloid protein fragmentation. The presence of any one of items (a) to (c) indicates the suitability of the test compound as an amyloid depletion agent, and optionally the amyloid depletion agent or a pharmaceutical composition containing the amyloid depletion agent is used to treat amyloidosis or amyloid-related diseases.

205. A method for analyzing the effect of a drug on the amyloid depletion activity of an amyloid depletion drug, the method comprising: (i) In the presence of the amyloid depletion drug and the agent, samples of tissue sections with amyloid deposits are incubated with macrophages, preferably THP-1-derived macrophages, and iii) Perform high-resolution live-cell imaging on each sample. in (a) The overlap of the fluorescence signal of the amyloid depletion drug and the fluorescence signal of amyloid indicates the binding of the amyloid depletion drug to the amyloid. (b) The presence of punctate and intracellular fluorescent signals in phagocytic vesicles indicates macrophage-mediated amyloid internalization; and (c) Sequential separation of punctate fluorescent signals from the fluorescent signals of said amyloid protein indicates macrophage-mediated amyloid protein fragmentation. in, Compared to the control sample, in which the tissue sections and macrophages containing amyloid deposits were incubated together in the presence of the amyloid depletion drug but without the drug, the substantially unchanged fluorescence patterns of items (a) to (c) indicate the suitability of the combination of the amyloid depletion drug and the drug in the treatment of amyloidosis or amyloid-related diseases, and the substantially changed fluorescence patterns of any of items (a) to (c) indicate the effect of the drug on the amyloid depletion activity of the amyloid depletion drug, optionally wherein (a) Enhanced fluorescence signal; (b) Enhanced intracellular fluorescence signal in the phagocytic vesicles; as well as (c) The punctate fluorescence signal is separated and enhanced from the fluorescence signal of the amyloid protein. The synergistic effect indicating the amyloid depletion activity of the amyloid depletion drug, and (a) Reduced fluorescence signal; (b) Reduced intracellular fluorescence signal in the phagocytic vesicles; and (c) The separation of the punctate fluorescence signal from the fluorescence signal of the amyloid protein is reduced. Indicates the impairing effect on the amyloid depletion activity of the amyloid depletion drug.

206. A method for screening amyloid depletion drugs by means of their ability to bind to amyloid and mediate macrophage recruitment to amyloid deposits, followed by amyloid fragmentation and internalization, the method comprising: (i) In the presence of the amyloid depletion drug, tissue sections with amyloid deposits are incubated together with macrophages, preferably THP-1 derived macrophages. The tissue sections are stained with an amyloid-specific fluorescent dye and / or the amyloid-depleting drug is labeled with a fluorescent dye; and (ii) Perform high-resolution live-cell imaging. in (a) The overlap of the fluorescence signal of the amyloid depletion drug and the fluorescence signal of amyloid indicates the binding of the amyloid depletion drug to the amyloid. (b) The presence of punctate and intracellular fluorescent signals in phagocytic vesicles indicates macrophage-mediated amyloid internalization; and (c) Sequential separation of punctate fluorescent signals from the fluorescent signals of said amyloid protein indicates macrophage-mediated amyloid protein fragmentation. And optionally, the amyloid depletion drug or a pharmaceutical composition containing the amyloid depletion drug can be used to treat amyloidosis or amyloid-related diseases.

207. The method of any one of claims 201 to 206, wherein the high-resolution live-cell imaging uses refractive index imaging for cell visualization and fluorescence microscopy for amyloid imaging.

208. The method according to any one of claims 201 to 207, wherein the amyloid depletion drug is labeled with a fluorescent dye, the fluorescent dye being different from the fluorescent dye used to stain the amyloid deposits.

209. The method according to any one of claims 201 to 208, wherein the tissue sections are obtained from a patient suffering from amyloidosis or amyloid-related diseases.

210. The method according to any one of claims 205 to 209, wherein the amyloidosis or the amyloid-associated disease is thyroxine transporter (TTR) amyloidosis or TTR amyloid-associated disease, preferably cardiac TTR amyloidosis.

211. The method according to any one of claims 201 to 210, wherein the amyloid depletion agent is an anti-amyloid antibody or contains an anti-amyloid antibody fragment.

212. The method according to any one of claims 201 to 211, wherein the amyloid depletion agent is an anti-TTR antibody or contains an anti-TTR antibody fragment and / or the control compound is an isotype control antibody.

213. The method of claim 210 or 212, wherein the antibody is human, preferably a human memory B cell-derived antibody or a variant thereof containing a heterologous constant domain, or a humanized antibody, or a chimeric antibody, preferably wherein the antibody is an IgG1 antibody, such as an IgG1 λ antibody or an IgG1 κ antibody.

214. The method according to any one of claims 205 to 213, wherein the agent is a second drug for treating amyloidosis or an analgesic, preferably wherein the second drug is a TTR tetramer stabilizer or a silencer.

215. A method for producing a pharmaceutical composition of an amyloid depletion drug, the method comprising: (i) Providing, optionally, the amyloid depletion drug; (ii) subjecting the amyloid depletion drug to the method according to any one of claims 201 to 202 and 205 to 214; (iii) Using the information obtained in step (ii) as part of an evaluation of whether the amyloid depletion drug can be used as a pharmaceutical composition; and optionally (iv) Formulating the amyloid depletion drug, which was found to be usable as a pharmaceutical composition in step (iii), together with a pharmaceutically acceptable carrier, said carrier preferably comprising a buffer, a tensioning agent, and / or a surfactant, most preferably all three components, and optionally... (v) Using the pharmaceutical composition to treat amyloidosis or amyloid-related diseases.

216. A method for the characterization, validation, development, and / or quality control of an amyloid depletion drug, the method comprising: (i) Providing, optionally, the amyloid depletion drug; (ii) subjecting the amyloid depletion drug to the method according to any one of claims 201 to 202 and 207 to 214; (iii) to communicate the information obtained in (ii) to customers, contracting parties, or partners and / or to select the drug that has been identified as a suitable amyloid depletion drug; and optionally... (iv) Treating amyloidosis or amyloid-related diseases with the amyloid depletion drug or a pharmaceutical composition containing the amyloid depletion drug.

217. A kit for use in performing the method according to any one of claims 201 to 216, the kit comprising a first fluorescent dye for staining amyloid and / or a second fluorescent dye for labeling the amyloid depletion drug, preferably negative and / or positive controls and optionally instructions for use, wherein preferably the positive control is an antibody according to any one of claims 42 to 105 and 120.

218. The method according to any one of claims 201 to 214, wherein the method is performed in addition to methods using a patient-derived amyloid xenograft (PDAX) nonhuman animal model, wherein the animal is characterized by an implant of amyloid fibrils derived from tissues or organs of a patient suffering from amyloidosis or amyloid-related diseases, wherein the amyloid protein and amyloid fibrils each comprise amyloid-thyroxine transporter (ATTR), and wherein the amyloid fibrils are implanted subcutaneously or subcapsularly, or implanted in the kidney, peritoneum, muscle, brain, ventricle, nerve, eye, tongue, or heart. The method includes (a) administering the amyloid depletion drug or test substance to the PDAX nonhuman animal model; and (b) Identify the amyloid fibrils in the model. Compared with the control, the accelerated elimination or reduction of amyloid fibrils after administration of the drug or the test substance respectively indicates the suitability of the amyloid depletion drug for the treatment of amyloidosis or amyloid-related diseases and the amyloid depletion activity of the test substance.

219. The method according to any one of claims 201 to 216 and 218, wherein the amyloid depletion agent is an antibody according to any one of claims 42 to 105 and 120.

220. A method for generating an antibody comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO: 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO: 8, the method comprising (a) culturing host Chinese hamster ovary K1 (CHO-K1) cells comprising one or more expression vectors or vector systems containing polynucleotides encoding the antibody HC and the LC under conditions sufficient to express the antibody in a culture; (b) setting a pH set point and a pH dead zone of the culture to regulate the levels of acidic, basic, and / or neutral antibody substances in the culture; and (c) isolating the antibody HC and LC from the cell culture; and (d) optionally formulating the isolated antibody into a pharmaceutical preparation.

221. The method of claim 220, wherein step (b) comprises setting the pH setpoint to about 6.90 and setting the pH dead zone to less than 0.2, preferably ≤0.175, preferably ≤0.15, preferably ≤0.125, preferably ≤0.1, preferably ≤0.075, preferably ≤0.05, more preferably between 0.05 and 0.1 to reduce the level of acidic antibody substances; preferably, the pH dead zone is set to about 0.

05.

222. The method of claim 221, wherein the implementation of step (b) reduces the level of acidic substances in the cultured antibody preparation compared to a control, said control being, for example, an antibody preparation produced using the same production method except that step (b) is performed at pH 6.90 and a pH dead zone of about 0.

20.

223. The method according to claim 221 or 222, wherein the implementation of step (b) reduces the level of N-linked mannose-5-glycan (Man5); preferably wherein the reduction in Man5 level is greater than >20%; more preferably wherein the reduction in Man5 level is about 40% to 50% compared to a control, said control being, for example, an antibody preparation produced using the same production method except that step (b) is performed at pH 6.90 and a pH dead zone of about 0.

20.

224. The method according to any one of claims 220 to 223, wherein step (b) is performed during the production phase, which includes days 5 to 14 of the culture process, for example, between days 5 and 14 after seeding of host cells, such as CHO-K1 cells.

225. The method according to any one of claims 220 to 224, wherein step (b) controls the high mannose content in the antibody preparation without consequently and adversely affecting secondary quality properties selected from (1) the relative levels (in %) of the major antibody substance in the preparation relative to high molecular weight (HMW) antibody substance relative to low molecular weight (LMW) antibody substance, for example, as determined by size exclusion chromatography (SEC); (2) the relative levels (in %) of the major antibody substance in the preparation, as determined by capillary electrophoresis of sodium dodecyl sulfate (CE-SDS-NR) under non-reducing conditions; and / or (3) the relative levels (in %) of the antibody heavy and light chains in the preparation, for example, as determined by capillary electrophoresis of sodium dodecyl sulfate (CE-SDS-NR) under reducing conditions.

226. An antibody produced using the production method according to any one of claims 220 to 225.

227. The antibody of claim 226, wherein the antibody comprises the heavy chain and light chain of ALXN2220 / NI006.

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