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WO2025104243A1 - Anticorps anti-transthyrétine, compositions comprenant ledit anticorps et méthodes de traitement ou de prévention de l'amyloïdose à médiation par la transthyrétine - Google Patents

Anticorps anti-transthyrétine, compositions comprenant ledit anticorps et méthodes de traitement ou de prévention de l'amyloïdose à médiation par la transthyrétine Download PDF

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Publication number
WO2025104243A1
WO2025104243A1 PCT/EP2024/082486 EP2024082486W WO2025104243A1 WO 2025104243 A1 WO2025104243 A1 WO 2025104243A1 EP 2024082486 W EP2024082486 W EP 2024082486W WO 2025104243 A1 WO2025104243 A1 WO 2025104243A1
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antibody
seq
amyloid
composition
antibodies
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Aubin MICHALON
Michael Salzmann
Rahul GODAWAT
Siguang SUI
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Neurimmune AG
Alexion Pharmaceuticals Inc
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Neurimmune AG
Alexion Pharmaceuticals Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • TTR transthyretin
  • TTR transthyretin-mediated amyloidosis
  • a human anti-TTR antibody which is specific for the disease associated amyloidogenic form of TTR, featuring ALXN2220, also known as NI006 and which has been assigned the CAS Registry Number 2965214-50-8, has been manufactured, i.e., by recombinantly expressing the antibody’s heavy chain (HC) and light chain (LC) in the Chinese Ovary Hamster (CHO) K1 cell line, which recombinant antibody possesses a unique pattern of posttranslational modifications (PTMs) and in particular a unique glycosylation profile; see Examples 1 to 3.
  • PTMs posttranslational modifications
  • Antibody NI006/ALXN2220 is a fully human IgG1m3 allotype antibody and comprises the human constant heavy chain (HC) amino acid sequence as set forth in SEQ ID NO: 7, 9, and 39, respectively, and a corresponding human constant light chain (LC), here kappa light chain, as set forth in SEQ ID NO: 8.
  • HC human constant heavy chain
  • LC human constant light chain
  • the NI006/ALXN2220 IgG1 antibodies are made up as tetramers consisting of HC and two light LC chains linked by disulfide bridges.
  • the closest human gene/allele of the variable domains are IGKV1-3901 (93.3%) + IGKJ101 (100%) and IGHV4-30-201 (89.5%) + IGHJ302 (100%).
  • variable heavy (VH) and/or variable light (VL) chain CDRs comprising VHCDRs1-3 and VLCDRs1-3, including variable heavy and light chain domains of NI006/ALXN2220 has been first described in WO 2015/092077 A1 (designated as antibody NI-301.37F1) and in Michalon et al., Nat. Commun.12 (2021), 3142 (designated as antibody NI301A), the disclosure in these documents being incorporated by reference herein in parts pertinent thereto.
  • the present disclosure further provides full-length NI006/ALXN2220 antibody comprising PTMs in its heavy chain and/or light chain, which antibody is particularly suitable for providing a stable formulation that can be used as pharmaceutical composition in the treatment of ATTR.
  • the antibodies of the present disclosure comprise modified glutamine at N- terminal of the heavy chain sequence, wherein the modification comprises a pyro-glutamic acid (abundance in sample, e.g., recombinant formulation of the antibody about 99.9% or more, e.g., 100%).
  • the antibodies of the present disclosure comprise a clipped C- terminal lysine in the heavy chain (abundance in sample, e.g., recombinant formulation of the antibody, of about 95.8% or more, e.g., 96%, 97%, 98%, 99% or even 100%).
  • NI006/ALXN2220 antibody subtype containing both the N-terminal pyroglutamate and C- terminal lysine deletion have been identified as the major post-translational modifications via analysis of the antibody heavy chain sequence.
  • An N-terminal pyroglutamate modification is beneficial in therapeutic antibodies by enhancing stability, consistency, and reducing immunogenicity.
  • the N-terminal pyroglutamate modification helps protect the antibody’s N-terminus from proteolytic degradation by enzymes which increases its stability and extends the half-life of the antibody in circulation.
  • pyroglutamate formation enhances the homogeneity of the antibody preparation by ensuring a consistent N-terminal structure across molecules. This consistency can be beneficial for quality control and regulatory approval, as it results in a more uniform product with predictable properties.
  • it helps reduce the risk of immunogenicity. By cleaving the N-terminal glutamine or glutamic acid, it prevents unwanted immune responses that might be triggered by unmodified N-terminal residues, which could otherwise be recognized as foreign.
  • the enzyme responsible for converting N-terminal glutamine (or sometimes glutamic acid) into pyroglutamate is called glutaminyl cyclase.
  • the activity level of this enzyme can vary across cell types and can significantly influence the extent of pyroglutamate formation.
  • C-terminal lysine clipping is advantageous as it enhances charge homogeneity, reduces immunogenicity, improves consistency, and does not impact the antibody’s functionality.
  • the presence of C-terminal lysine adds a positive charge to the antibody, which can introduce charge heterogeneity across the antibody population.
  • the antibody preparation becomes more uniform in charge, simplifying downstream processes like purification and improving batch-to-batch consistency and homogeneity, which is beneficial for quality control.
  • antibodies naturally lack the C-terminal lysine due to proteolytic removal in vivo. Leaving the lysine on can make the antibody appear foreign to the immune system, potentially triggering an immune response. Removing this lysine reduces the risk of immunogenicity, making the therapeutic antibody more biocompatible.
  • the C-terminal lysine residue does not play a role in the antibody’s binding to its target antigen or its interaction with Fc receptors and thus, its removal does not impact the antibody’s therapeutic activity, so clipping it is beneficial without compromising efficacy.
  • C- terminal lysine clipping can depend on the cell line used for antibody production as different cell lines vary in their protease activity and the efficiency with which they remove C-terminal lysine residues from antibodies. Accordingly, an antibody having an N-terminal pyroglutamate modification and C-terminal lysine clipping is beneficial for therapeutic purposes and thus, is a preferred embodiment of the present invention. Furthermore, additional modifications, such as methionine oxidation, asparagine deamidation and asparagine succinimide formation, have been experimentally determined, as shown in Table 3.
  • a mature anti-TTR antibody which comprises a HC having the amino acid sequence of SEQ ID NO: 7, wherein X 1 is absent, glutamine, or a pyroglutamate (pE); X2 is methionine or oxidized methionine; X3 is asparagine, deamidated asparagine, or asparagine comprising a succinimide; X 4 is asparagine or deamidated asparagine; X5 is proline or amidated proline; X6 is absent or glycine; and X 7 is absent or lysine, and a LC having the amino acid sequence of SEQ ID NO: 8.
  • X 1 is absent, glutamine, or a pyroglutamate (pE)
  • X2 is methionine or oxidized methionine
  • X3 is asparagine, deamidated asparagine, or asparagine comprising a succinimide
  • X 4 is asparagine or deami
  • the antibody of the present disclosure comprises a HC having the amino acid sequence of SEQ ID NO: 7, wherein the N-terminal glutamine (Q1), in SEQ ID NO: 7, denoted by X1, is absent or is present, preferably absent.
  • X1 is present as glutamine, or as pyroglutamate (pE); preferably X1 is pE.
  • the antibody of the present disclosure comprises a HC having the amino acid sequence of SEQ ID NO: 7, wherein the C-terminal lysine (K450), in SEQ ID NO: 7, denoted by X 7 , is absent or is present.
  • X 7 is absent.
  • the antibody of the present disclosure comprises an HC having the amino acid sequence of SEQ ID NO: 39, wherein N-terminal residue X 1 is absent or present as a glutamine or a pE.
  • X2 is a methionine or an oxidized methionine.
  • X 3 is an asparagine, a deamidated asparagine, or an asparagine comprising a succinimide.
  • X4 is an asparagine, a deamidated asparagine, or an asparagine comprising a succinimide.
  • X5 is a proline or an amidated proline.
  • X 6 is absent or a glycine.
  • X 7 is absent or a lysine.
  • XI is an asparagine or a deamidated asparagine.
  • XII is a methionine or an oxidized methionine.
  • XIII is a methionine or an oxidized methionine.
  • X IV is an aspartate or an iso-aspartate.
  • X V is an asparagine or a glycosylated asparagine.
  • XVI is a methionine or an oxidized methionine.
  • XVII is a methionine or an oxidized methionine.
  • the antibody comprises an LC having the amino acid sequence of SEQ ID NO: 8. In embodiments, the antibody of the present disclosure comprises an HC having the amino acid sequence of SEQ ID NO: 7 and 39, respectively.
  • X1 is a pE. In some embodiments, X 6 is absent. In some embodiments, X 7 is absent. In some embodiments, X 5 is amidated. In some embodiments, X2 is an oxidized methionine.
  • X3 is a deamidated asparagine. In other embodiments, X3 is an asparagine comprising a succinimide. In some embodiments, X 4 is deamidated asparagine.
  • XI is a deamidated asparagine. In some embodiments, XII is an oxidized methionine. In some embodiments, X III is an oxidized methionine. In some embodiments, X IV is an iso-aspartate. In some embodiments, X V is a glycosylated asparagine. In some embodiments, XVI is an oxidized methionine. In some embodiments, XVII is an oxidized methionine.
  • X1 is a pE
  • X2 is an oxidized methionine
  • X3 is a deamidated asparagine or an asparagine comprising a succinimide
  • X 4 is a deamidated asparagine or an asparagine comprising a succinimide
  • X5 is a proline
  • X6 is a glycine
  • X7 is absent
  • XI is an asparagine
  • XII is a methionine
  • XIII is a methionine
  • XIV is an aspartate
  • XV is a glycosylated asparagine
  • XVI is a methionine
  • XVII is a methionine.
  • the C-terminal lysine (C450, numbering is based on the HC polypeptide sequence which comprises the N-terminal glutamine or pyroglutamate) of the HC of the antibody of the present disclosure may be clipped off (e.g., via proteolytic cleavage).
  • the antibody of the present disclosure comprises a HC having the amino acid sequence of SEQ ID NO: 7 and 39, respectively, wherein the C-terminal lysine, in SEQ ID NO: 7 and 39, respectively, denoted by X7, is either absent or present.
  • the C-terminal lysine at X7 of SEQ ID NO: 7 and 39, respectively is absent.
  • the antibody of the present disclosure comprises a HC having the amino acid sequence of SEQ ID NO: 7 and 39, respectively, wherein the C-terminal lysine at X7 and glycine at X6, are both absent, and the amino acid proximal thereto at the C-terminus of SEQ ID NO: 7 and 39, respectively, denoted by proline at X 5 , is either amidated or unmodified, preferably amidated.
  • the methionine at position 255 may be oxidized.
  • the antibody of the present disclosure comprises a HC having the amino acid sequence of SEQ ID NO: 7 and 39, respectively, wherein the M255, in SEQ ID NO: 7 and 39, respectively, denoted by methionine at X2, is either oxidized (modified) or unmodified.
  • the asparagine at position 318 (N318, numbering is based on the HC polypeptide sequence which comprises the N-terminal glutamine or pyroglutamate) may be unmodified or deamidated or may comprise a succinimide.
  • the asparagine at position 387 may be unmodified or modified, e.g., wherein the modified amino acid is deamidated.
  • the antibody of the present disclosure comprises a HC having the amino acid sequence of SEQ ID NO: 7 and 39, respectively, wherein the N387, in SEQ ID NO: 7 and 39, respectively, denoted by asparagine at X4, is either deamidated (modified) or is unmodified.
  • the antibodies of the present disclosure comprise N-glycosylation.
  • At least one amino acid in the heavy chain of the antibodies of the present disclosure is N-glycosylated.
  • Data in Example 2 show that N-glycosylation is a substantial PTM of the backbone amnio acid sequence making up the antibody of the present disclosure.
  • the N-glycosylation site was identified at position Asn300 (HC N300, numbering based on antibody HC polypeptide sequence of SEQ ID NO: 7 and 39, respectively having the N- terminal glutamine or pyrogluatmate (X1).
  • the disclosure relates to an antibody comprising a heavy chain polypeptide sequence set forth in SEQ ID NO: 7 and 39, respectively, wherein X1 is pyroglutamate and wherein X7 is cleaved off and wherein asparagine (N) in amino acid position 300 is N-glycosylated.
  • the disclosure relates to anti-TTR antibodies comprising a HC comprising the sequence set forth in SEQ ID NO: 7 and 39, respectively, wherein the glutamine (X1) is modified to pyroglutamate, the lysine (X7) is absent, and in which the amino acids at positions X 2 , X 3 , X 4 ,X 5 , and X 6 are either modified as indicated above or unmodified, preferably unmodified, and at least one amino acid is N-glycosylated.
  • the N-glycosylation site is at position Asn300 (N300, numbering based on antibody HC polypeptide sequence of SEQ ID NO: 7 and 39, respectively having the N-terminal glutamine or pyroglutamate (X 1 )).
  • the antibodies of the present disclosure comprise a pyro-glutamic acid at the N-terminal of the heavy chain sequence instead of glutamine, wherein the abundance in sample is about 99.9% or more, e.g., 100%.
  • the antibodies of the present disclosure comprise a C-terminal in which the C-terminal lysine in the heavy chain is clipped off, wherein the abundance in sample is about 95.8% or more, e.g., 96%, 97%, 98%, 99% or even 100%.
  • the antibodies of the present disclosure comprise deamidated asparagine at position 58 (N58 based on antibody HC polypeptide sequence of SEQ ID NO: 7 and 39, respectively having the N-terminal glutamine or pyroglutamate (X 1 )), wherein the abundance in sample is about 5% or less, e.g., 4%, 3%, 2%, 1%, or 0%.
  • the antibodies of the present disclosure comprise deamidated asparagine at position 318 (N318 based on antibody HC polypeptide sequence of SEQ ID NO: 7 and 39, respectively having the N-terminal glutamine or pyroglutamate (X1)), wherein the abundance in sample is about 9% or less, e.g., 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0%.
  • the antibodies of the present disclosure comprise deamidated asparagine at position 387 (N387 based on antibody HC polypeptide sequence of SEQ ID NO: 7 and 39, respectively having the N-terminal glutamine or pyroglutamate (X1)), wherein the abundance in sample is about 5% or less, e.g., 4%, 3%, 2%, 1%, or 0%.
  • the antibodies of the present disclosure comprise oxidized methionine at position 71 (M71 based on antibody HC polypeptide sequence of SEQ ID NO: 7 and 39, respectively having the N-terminal glutamine or pyroglutamate (X 1 )), wherein the abundance in sample is about 5% or less, e.g., 4%, 3%, 2%, 1%, or 0%.
  • the antibodies of the present disclosure comprise oxidized methionine at position 115 (M15 based on antibody HC polypeptide sequence of SEQ ID NO: 7 and 39, respectively having the N-terminal glutamine or pyroglutamate (X1)), wherein the abundance in sample is about 5% or less, e.g., 4%, 3%, 2%, 1%, or 0%.
  • the antibodies of the present disclosure comprise oxidized methionine at position 255 (M255 based on antibody HC polypeptide sequence of SEQ ID NO: 7 and 39, respectively having the N-terminal glutamine or pyroglutamate (X1)), wherein the abundance in sample is about 5% or less, e.g., 4%, 3%, 2%, 1%, or 0%.
  • the antibodies of the present disclosure comprise oxidized methionine at position 361 (M361 based on antibody HC polypeptide sequence of SEQ ID NO: 7 and 39, respectively having the N-terminal glutamine or pyroglutamate (X1)), wherein the abundance in sample is about 5% or less, e.g., 4%, 3%, 2%, 1%, or 0%.
  • the antibodies of the present disclosure comprise oxidized methionine at position 431 (M413 based on antibody HC polypeptide sequence of SEQ ID NO: 7 and 39, respectively having the N-terminal glutamine or pyroglutamate (X 1 )), wherein the abundance in sample is about 5% or less, e.g., 4%, 3%, 2%, 1%, or 0%.
  • the antibody of the present disclosure may comprise a glycated heavy chain and/or a glycated light chain.
  • an antibody having an N-terminal pyroglutamate modification and C- terminal lysine clipping is beneficial for therapeutic purposes, but it is preferred that the abundance of the other mentioned PTMs is rather low.
  • methionine oxidation, iso- aspartate formation, or deamidated asparagine in therapeutic antibodies is generally not favorable because it can negatively impact the stability, function, safety, and efficiency and thus, the abundance of methionine oxidation, iso-aspartate formation and asparagine deamidation should preferably around or below 5%.
  • X1 is a pE
  • X2 is a methionine
  • X3 is an asparagine
  • X4 is an asparagine
  • X5 is a proline
  • X6 is a glycine
  • X7 is absent
  • XI is an asparagine
  • XII is a methionine
  • X III is methionine
  • X IV is an aspartate
  • X V is a glycosylated asparagine
  • X VI is a methionine
  • XVII is a methionine.
  • the antibody of the present disclosure comprises two HCs, each having an amino acid sequence set forth in SEQ ID NO: 7 and 39, respectively, and two LCs, each having an amino acid sequence set forth in SEQ ID NO: 8, and wherein the glutamine at the N-terminus (X1) of the HC sequence, in each case, independently, is modified as pyro-glutamic acid (pE).
  • the antibody of the disclosure comprises two HCs comprising the amino acid sequence set forth in SEQ ID NO: 7 and 39, respectively, wherein the C-terminal lysine (X7) of the HC sequence, in each case, independently, is absent.
  • the antibody of the disclosure comprises two HCs comprising the amino acid sequence set forth in SEQ ID NO: 7 and 39, respectively, wherein the heavy chain, in each case, independently, is N-glycosylated, preferably wherein the N-glycosylation site is at position Asn300.
  • the antibody of the disclosure comprises two HCs comprising the amino acid sequence set forth in SEQ ID NO: 7, wherein the amino acids at position X2, X3, X4, and X5 of SEQ ID NO: 7 and 39, respectively are either modified or unmodified, preferably unmodified.
  • the antibody of the present disclosure comprises two HCs, each having an amino acid sequence set forth in SEQ ID NO: 7 and 39, respectively, and two LCs, each having an amino acid sequence set forth in SEQ ID NO: 8, and wherein the glutamine at the N-terminus (X1) of the HC sequence, in each case, independently, is modified as pyro-glutamic acid (pE); the C-terminal lysine (X7) of the HC sequence, in each case, independently, is absent; the heavy chain, in each case, independently, is N-glycosylated, preferably wherein the N-glycosylation site is at position Asn300 of SEQ ID NO: 7 and 39, respectively, the numbering of Asn corresponding to the polypeptide sequence of SEQ ID NO: 7 and 39, respectively comprising glutamine or pyroglutamate at X1; and the amino acids at position X2, X3, X4, and X5 of SEQ ID NO: 7 and 39, respectively are either modified unmodified, preferably
  • the antibody is glycosylated, in particular N-glycosylated.
  • the heavy chain of the antibody is glycosylated and even more particularly at N300 of the heavy chain (SEQ ID NO: 9).
  • the HC chain of the anti-TTR antibody of the present disclosure lacks the C-terminal lysine, has a modified glutamine at the N-terminus as pyro-glutamic acid and comprises at least one N-glycosylation site.
  • the antibody comprises an HC having the amino acid sequence of SEQ ID NO: 9 and an LC having the amino acid sequence of SEQ ID NO: 8, wherein the glutamine (Q) at position 1 of SEQ ID NO: 9 is modified to a pE, the lysine (K) at position 450 of SEQ ID NO: 9 is clipped off (e.g., via proteolytic digestion), the 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
  • the antibody comprises glycans, wherein the glycans are Man3+1F, G0-GN, G0F-GN, G0, G0F, Man5, G1F- GN/G1a, G1b, G1Fa, G1Fb, G2F, G2FS1, and G2FS2 type glycans.
  • the glycans are mostly G0F and G1F type glycans.
  • the HC set forth in SEQ ID NO: 9 further comprises one or more modifications: the asparagine (N) at position 58 is deamidated; the methionine (M) at position 71 is oxidized; the methionine (M) at position 115 is oxidized; the methionine (M) at position 225 is oxidized; the aspartate (D) at position 283 is isomerized; the asparagine (N) at position 318 is deamidated or comprises a succinimide; the methionine (M) at position 361 is oxidized; the asparagine (N) at position 387 is deamidated or comprises a succinimide; the methionine (M) at position 431 is oxidized; the glycine (G) at position 449 is absent; the proline (P) at position 448 is amidated after the loss of the C-terminal lysine and glycine.
  • each heavy chain of antibody NI006/ALXN2220 may contain a single N-linked glycosylation site at Asn300.
  • the N-linked glycosylation structure is predominantly a fucosylated, complex biantennary glycan with 0 galactose residues (G0F) (about 49 %) or with 1 galactose residue (G1F) (about 25 %).
  • the detailed glycosylation profile is shown in Examples 2 and 3.
  • Glycosylation plays a vital role in the stability, in vivo activity, solubility, serum half-life and immunogenicity of many therapeutic proteins.
  • the antibody of the present disclosure is an IgG antibody, particularly of the IgG1m3 subtype, and has a heavy chain which is N-glycosylated, preferably wherein the N-linked glycosylation site is Asn300.
  • a more detailed analysis of the glycan structure showed that more than 85% of the glycans are fucosylated, in particular about 89% to 94% of the glycans.
  • glycans are part of the major fucosylated glycan types, meaning that the majority of glycan structures attached to the antibody contain a fucose residue.
  • the fucosylation profile is dependent on the cell line in which the antibody has been produced and thus, the fucosylation profile is specific for the antibody of the present invention. Furthermore, such a fucosylation profile is advantageous since fucosylation can improve the structural stability of the glycan structure on the antibody, which contributes to a longer in vivo half-life, which can reduce dosing frequency and improve patient compliance.
  • highly fucosylated antibodies are usually less likely to elicit an immune response in patients, as fucosylated glycans are common in human antibodies and less likely to be recognized as foreign.
  • High fucosylation levels are easier to achieve consistently with certain cell lines (like CHO cells) and are generally more reliable for large-scale manufacturing and a consistent product is obtained, which is crucial for regulatory approval and therapeutic efficacy.
  • the antibody of the present disclosure preferably 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 major glycan types are G0F and G1F. More preferably, the antibody of the present disclosure comprises glycans, wherein more than 85% of the glycans are fucosylated, preferably wherein about 85% to 95% of the glycans are fucosylated, more preferably wherein about 89% to 94% of the glycans are fucosylated.
  • the antibody of the present disclosure comprises glycans, wherein about 20% to 40% of the glycans are galactosylated preferably about 27% to 38%.
  • Mannose-containing glycans of the Man5 and Man31F type are mannose- containing glycans of the Man5 and Man31F type.
  • high levels of mannose structures like Man5 can indicate incomplete processing in the glycosylation pathway, so a low percentage (like in the present case) is generally favorable in therapeutic antibodies since it shows that the antibody production process is well-controlled, with most glycans processed to more complex structures.
  • mannose-rich glycans are less common in human antibodies and can sometimes increase immunogenicity, as the immune system might recognize them as foreign, a low mannose percentage is favorable for therapeutic applications of the antibody.
  • the antibody of the present disclosure comprises glycans, wherein about 1% to 4% of the glycans are mannose-containing glycans, preferably wherein the glycans are of the Man5 and Man31F type, and more preferably wherein about 1% to 4% of the glycans are of the Man5 type.
  • the mannose-containing glycans are present in an even lower amount, in particular between 1% and 2%.
  • the antibody of the present disclosure comprises glycans, wherein about 1% to 2% of the glycans are mannose-containing glycans, preferably wherein the glycans are of the Man5 and Man31F type, and more preferably wherein about 1% to 2% of the glycans are of the Man5 type.
  • glycan analysis showed that about 0.5% to 2% of the glycans are siaylated and thus, in one embodiment, the antibody of the present disclosure comprises glycans, wherein less than 2% of the glycans are siaylated, preferably wherein about 0.5% to 2% of the glycans are siaylated.
  • Mannose-containing glycans As regards the mannose-containing glycans, it has been surprisingly shown that reducing pH dead band from 0.20 to 0.05 (with a pH set to 6.9) led to reduction on Man 5 (see Example 3), which is favorable as explained above. In particular, the amount of Man5 type glycans is nearly halved.
  • less than 3% of the glycans are 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%.
  • embodiments of the disclosure further relate to methods and workflows for obtaining ALXN2220 with a desired product profile.
  • reducing the pH dead band from 0.20 to 0.05 led to considerable reduction in acidic species, e.g., from about 37% to about 29% in ALXN2220 process A2 and a general changed charged variant profile.
  • reducing pH dead band from 0.20 to 0.05 also led to appreciable reduction, e.g., about 50% reduction, in Man 5 levels (from about 3.8% to about 1.9%).
  • embodiments of the methods and workflows described herein provide a robust control of high mannose species in the antibody drug substance batches, without concomitant adverse impact on other product quality attributes, such as, for example, relative levels (in %) of main vs. HMW vs. LMW antibody species (as determined by SEC); levels (in %) of main antibody species, as determined using capillary electrophoresis sodium dodecyl sulfate under non-reduced conditions (CE-SDS-NR); and levels of antibody heavy and light chains (in %), as determined using capillary electrophoresis sodium dodecyl sulfate under reduced conditions (CE-SDS R).
  • relative levels in %) of main vs. HMW vs. LMW antibody species (as determined by SEC); levels (in %) of main antibody species, as determined using capillary electrophoresis sodium dodecyl sulfate under non-reduced conditions (CE-SDS-NR); and levels of antibody heavy and light
  • an antibody sample comprises basic and acid antibody variants, dependent on their post-translational modifications.
  • antibodies comprising deamidated asparagine residues at positions N58, N328 and/or N387 of their HC comprises sialyated N-glycosylation, galactosylated N-glycosylation, HC truncation between N58 and T59 as well as lysine glycation distributed evenly across all lysine in antibody’s HC and LC as referred to further above belong to the acidic species and antibodies comprising aspartate isomerization at D283 of their HC, lysine loss at K450 of the HC and proline amidation with glycine and lysine loss in their HC (P448) as referred to further above belong to the basic species.
  • the antibody of the present disclosure is an acidic antibody variant.
  • the antibody of the present disclosure is a basic antibody variant.
  • the antibody of the present disclosure is a neutral antibody variant.
  • the antibody of the present disclosure is an acidic antibody variant.
  • the disclosure relates to antibodies that are recombinant, e.g., generated via expression in a suitable host cell such as bacterial (e.g., E. coli) cells or cells of other microorganisms such as yeast cells; or mammalian cells such as CHO cells.
  • the recombinant antibody comprises an N-linked glycosylation structure which is predominantly a glycan with 0 galactose residues (G0F) (about 49 %) or with 1 galactose residue (G1F) (about 25 %).
  • the antibody comprises or consists essentially of the glycosylation profile as shown in Example 2 and referred to above.
  • the theoretical molecular weight (MW) of the antibody of the present disclosure is about 144.2 kDa
  • MS mass spectrometry
  • the molecular weight of the antibody of the disclsoure is between 147.0 and 147.6 kDa (intact IgG1).
  • Embodiments of the disclosure further relate to pharmaceutical compositions comprising the antibody of the present disclosure, as characterized above, which has a molecular weight of about 150 kDa, preferably a MW of about 147 kDa.
  • the experimentally determined pI of the antibody of the present disclosure is about 9.3 and the theoretical pI is about 8.4.
  • the experimentally determined extinction coefficient of the antibody of the present invention is 1.390 and the theoretically determined extinction coefficient is 1.438.
  • the antibodies of the present disclosure comprise human IgG antibodies comprising two identical heavy chains (HCs) and two identical light chains (LCs), wherein the identity may be based on sequence identity, i.e., wherein the primary amino acid sequence of the heavy chain polypeptide sequence(s), which make up the antibody tetramer, optionally or together with the primary amino acid sequence of the light chain polypeptide sequence(s), which make up the antibody tetramer, are identical.
  • sequence identity i.e., wherein the primary amino acid sequence of the heavy chain polypeptide sequence(s), which make up the antibody tetramer, optionally or together with the primary amino acid sequence of the light chain polypeptide sequence(s), which make up the antibody tetramer, are identical.
  • the four chains are stabilized by intra-chain and inter-chain disulfide bonds, wherein the positions of the disulfide bridges, which have been identified per Lys-C and trypsin digestion and subsequent LC-MS (see Example 2) are the following: 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, wherein the numbering of the cysteine residues (C) corresponds to their positions in the antibody HC sequence of SEQ ID NO: 7 and the antibody LC sequence of SEQ ID NO: 8, with SEQ ID NO: 7 having the N-terminal glutamine or pyroglutamate (X1).
  • the antibody is preferably a human or humanized antibody, typically human IgG and most preferably a human IgG1.
  • the antibody is a human IgG1m3 allotype and comprises preferably a kappa LC constant region.
  • the closest human gene/allele of the variable domains are IGKV1-3901 (93.3%) + IGKJ101 (100%) and IGHV4-30-201 (89.5%) + IGHJ302 (100%).
  • Antibodies, like many other proteins, are secreted from the cells via the co-translational translocation pathway.
  • a signal peptide that contains 5–30 amino acids present at the N-terminus of nascent proteins is recognized by the signal recognition particle (SRP) in the cytosol while the protein is still being synthesized on the ribosome.
  • SRP signal recognition particle
  • the SRP then delivers the SRP-ribosome-nascent chain (SRP-RNC) complex to the SRP-receptor (SR) in the endoplasmic reticulum (ER) membrane.
  • SRP-RNC SRP-ribosome-nascent chain
  • SR SRP-receptor
  • ER endoplasmic reticulum
  • WO 2014/058389 A1 discloses various signal peptides that can in principle be used for cloning.
  • the signal peptides as set forth in SEQ ID NOs: 17 and 18 have been used for the expression of the HC and LC, respectively, of the antibody of the present disclosure which resulted in an efficient expression of the HC and LC, thus being transported to the ER for proper folding, assembly and post-translational modification, leading to an antibody particularly suitable for formulating in a pharmaceutical composition.
  • the HC of the antibody of the present disclosure further comprises a signal peptide having an amino acid sequence of SEQ ID NO: 17, wherein the sequence including the HC and the signal peptide is set forth in SEQ ID NO: 19.
  • the LC of the antibody of the present disclosure further comprises a signal peptide having an amino acid sequence of SEQ ID NO: 18, wherein the sequence including the HC and the signal peptide is set forth in SEQ ID NO: 20.
  • SEQ ID NO: 18 the sequence including the HC and the signal peptide is set forth in SEQ ID NO: 20.
  • the above signal peptides for the heavy chain and light chain antibody sequences represent an optimal pair of signal peptides for the expression of NI006/ALXN2220 recombinantly, e.g., in CHO cells.
  • the present disclosure further relates to polynucleotides encoding the antibody of the present disclosure.
  • the present disclosure relates to one or more polynucleotides(s), preferably to two polynucleotides which encode the antibody of the present disclosure, i.e., the first polynucleotide comprises a nucleotide sequence encoding the HC and the second polynucleotides comprises a nucleotide sequence encoding the LC of the antibody.
  • the disclosure relates to polynucleotides encoding the antibody HC and the LC, wherein the HC has the amino acid sequence of SEQ ID NO: 7 (with no modifications, i.e., glutamine or pyroglutamate (X1), and wherein both lysine (X7) and glycine (X6) are present, and X2, X3, X4, and X5 are unmodified) and wherein the LC has the amino acid sequence of SEQ ID NO: 8.
  • SEQ ID NO: 7 with no modifications, i.e., glutamine or pyroglutamate (X1), and wherein both lysine (X7) and glycine (X6) are present, and X2, X3, X4, and X5 are unmodified
  • the LC has the amino acid sequence of SEQ ID NO: 8.
  • the disclosure further relates to polynucleotides encoding the antibody HC and the LC, wherein the HC has the amino acid sequence of SEQ ID NO: 9 (with modifications in the sequence of SEQ ID NO: 7 comprising deletion of C-terminal lysine and cyclization of N- terminal glutamine into pyroglutamate) and wherein the LC has the amino acid sequence of SEQ ID NO: 8.
  • the disclosure further relates to polynucleotides encoding the antibodies of the foregoing, wherein a first polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 13 which encodes the antibody HC and a second polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 14 which encodes the antibody LC.
  • the disclosure further relates to codon-optimized nucleic acids, for example, for efficient expression in the CHO cell line.
  • the first polynucleotide of the present disclosure comprises the nucleotide sequence encoding the antibody HC set forth in SEQ ID NO: 15 and a second polynucleotide comprises the nucleotide sequence encoding the antibody LC set forth in SEQ ID NO: 16.
  • the polynucleotides of the present disclosure further comprise a nucleotide sequence encoding a signal peptide.
  • the first nucleotide sequence comprises a nucleotide sequence encoding a signal peptide derived from a human immunoglobulin heavy chain and the second nucleotide sequences comprises a nucleotide sequence encoding a signal peptide derived from a human immunoglobulin kappa light chain.
  • the nucleotide sequence of the first signal peptide is set forth in SEQ ID NO: 21, wherein the first nucleotide sequence including the nucleotide sequence of the signal peptide has the sequence set forth in SEQ ID NO: 23.
  • nucleotide sequence of the second signal peptide is set forth in SEQ ID NO: 22, wherein the first nucleotide sequence including the nucleotide sequence of the signal peptide has the sequence set forth in SEQ ID NO: 24.
  • the above nucleic acids encoding the signal peptides for the heavy chain and light chain antibody sequences represent an optimal pair of signal peptide-encoding nucleic acids, e.g., for the expression of the respective heavy and light chain polypeptide sequences of NI006/ALXN2220 recombinantly, e.g., in CHO cells.
  • the present disclosure also relates to a nucleic acid molecule which is (a) polynucleotide(s) comprising a first nucleotide sequence set forth in SEQ ID NO: 13, and a second nucleotide sequence set forth in SEQ ID NO: 14; (b) messenger RNA (mRNA) equivalent of the first and the second nucleotide sequences of (a); (c) polynucleotide(s) comprising sequences which are complementary to the first and the second nucleotide sequences of (a) or the mRNA equivalent thereof of (b); or (d) polynucleotide(s) comprising sequences which are degenerates of the first and the second nucleotide sequences of (a) or the mRNA equivalent thereof of (b).
  • mRNA messenger RNA
  • the polynucleotide(s) of (d) comprise a first nucleotide sequence set forth in SEQ ID NO: 15, and a second nucleotide sequence set forth in SEQ ID NO: 16.
  • the degenerate sequences disclosed herein represent codon optimized nucleic acid sequences that confer optimal recombinant expression of the respective heavy and light chains of the antibody in a cell that is suitable for commercial manufacturing, e.g., CHO cells.
  • the polynucleotides of the present disclosure further comprise a nucleotide sequence encoding a signal peptide.
  • the first nucleotide sequence comprises a nucleotide sequence encoding a signal peptide derived from a human immunoglobulin heavy chain and the second nucleotide sequences comprises a nucleotide sequence encoding a signal peptide derived from a human immunoglobulin kappa light chain.
  • the nucleotide sequence of the first signal peptide is set forth in SEQ ID NO: 21, wherein the first nucleotide sequence including the nucleotide sequence of the signal peptide has the sequence set forth in SEQ ID NO: 23.
  • the nucleotide sequence of the second signal peptide is set forth in SEQ ID NO: 22, wherein the first nucleotide sequence including the nucleotide sequence of the signal peptide has the sequence set forth in SEQ ID NO: 24.
  • the disclosure further relates to both pre-processed (e.g., pre-protein) as well as processed (e.g., mature) forms for each of the antibody heavy and light chain polypeptides, which are encoded by the aforementioned nucleic acids, e.g., nucleic acid sequences set forth in SEQ ID NO: 13 or 15 (encoding the pre-protein antibody heavy chain) and SEQ ID NO: 14 or 16 (encoding the pre-protein antibody light chain).
  • the present disclosure further relates to one or more expression vectors comprising the polynucleotides/nucleic acid molecules of the present disclosure and to host cells comprising said vector(s) and/or polynucleotides/nucleic acid molecules of the present disclosure.
  • the antibody of the present disclosure is produced in CHO cells, in particular in the CHO cell line K1.
  • the host cell is a non-human cell, preferably a CHO cell, most preferably CHO-K1.
  • microbial for example E. coli cells, or insect cells can be used.
  • the present disclosure further relates to a method of manufacturing the antibody of the present disclosure which comprises at least culturing the host cell, preferably the CHO cell, most preferably the CHO K1 cell comprising the polynucleotide(s) of the present disclosure, preferably in the form of the vector(s) of the present disclosure and isolating the antibody from the culture.
  • a method of manufacturing an antibody as described herein produces an antibody comprising an HC having the amino acid sequence of SEQ ID NO: 7 and 39, respectively and an LC comprising the amino acid sequence of SEQ ID NO: 8.
  • the antibodies comprise an HC having the amino acid sequence of SEQ ID NO: 7 and 39, respectively, wherein N-terminal residue X1 is absent or present as glutamine or pE.
  • X2 is methionine or oxidized methionine.
  • X3 is asparagine, deamidated asparagine, or asparagine comprising a succinimide.
  • X4 is asparagine, deamidated asparagine, or asparagine comprising a succinimide.
  • X5 is proline or amidated proline.
  • X6 is absent or glycine.
  • X7 is absent or lysine.
  • XI is asparagine or deamidated asparagine.
  • X II is methionine or oxidized methionine.
  • X III is methionine or oxidized methionine.
  • X IV is aspartate or iso-aspartate.
  • X V is asparagine or glycosylated asparagine.
  • XVI is methionine or oxidized methionine.
  • XVII is methionine or oxidized methionine.
  • X 1 is pE and/or X 7 is absent.
  • the present disclosure further relates to a composition comprising the antibody of the present disclosure.
  • the composition comprises the antibody with or without the PTMs as defined hereinbefore.
  • the composition comprises a mixture of the antibodies as defined hereinbefore. As shown in Examples 2 and 3, about 99% to 100% of the antibodies present in a sample of a typical antibody composition have a N- terminal pyro-glutamic acid in the heavy chain and about 96% of the antibodies have a loss of the C-terminal lysine.
  • the antibodies in the formulation of the present disclosure have a heavy chain wherein the N-terminal pyro- glutamic acid is modified from N-terminal glutamine and/or about 96% (95% to 96%) of the antibodies are without (or devoid of) the C-terminal lysine.
  • Absence of C-terminal lysine may be a result of proteolytic cleavage of the antibody heavy chain containing said lysine or due to antibody engineering, e.g., via mutation or deletion of the triplet codon that encodes the HC C- terminal lysine in the nucleic acid encoding said HC.
  • the antibodies present in said composition further comprise the N-glycosylated HC as defined hereinbefore.
  • the composition comprises antibodies, wherein the antibodies comprise an HC having the amino acid sequence of SEQ ID NO: 7 and 39, respectively and a light chain having the amino acid sequence of SEQ ID NO: 8.
  • the antibodies comprise an HC having the amino acid sequence of SEQ ID NO: 7 and 39, respectively, wherein N-terminal residue X 1 is absent or present as glutamine or pE.
  • X 1 is PE for 99% to 100% of the antibodies of the composition, preferably wherein X1 is PE for 99.9% of the antibodies of the composition.
  • X2 is methionine or oxidized methionine.
  • X 3 is asparagine, deamidated asparagine, or asparagine comprising a succinimide.
  • X4 is asparagine, deamidated asparagine, or asparagine comprising a succinimide.
  • X5 is proline or amidated proline.
  • X 6 is absent or glycine.
  • X 7 is absent or lysine.
  • X I is asparagine or deamidated asparagine.
  • XII is methionine or oxidized methionine.
  • XIII is methionine or oxidized methionine.
  • XIV is aspartate or iso-aspartate.
  • X V is asparagine or glycosylated asparagine.
  • X VI is methionine or oxidized methionine.
  • X VII is methionine or oxidized methionine.
  • the composition comprises antibodies, wherein the antibodies comprise an HC having the amino acid sequence of SEQ ID NO: 7 and 39, respectively and a light chain having the amino acid sequence of SEQ ID NO: 8.
  • the antibodies comprise an HC having the amino acid sequence of SEQ ID NO: 7 and 39, respectively, wherein N-terminal residue X1 is absent or present as glutamine or pE.
  • X2 is methionine or oxidized methionine.
  • X3 is asparagine, deamidated asparagine, or asparagine comprising a succinimide.
  • X 4 is asparagine, deamidated asparagine, or asparagine comprising a succinimide.
  • X 5 is proline or amidated proline.
  • X6 is absent or glycine.
  • X7 is absent or lysine.
  • X 7 is absent in about 95% to about 96% of the antibodies of the composition, preferably wherein X 7 is absent for 95.8% of the composition.
  • X I is asparagine or deamidated asparagine.
  • X II is methionine or oxidized methionine.
  • X III is methionine or oxidized methionine.
  • XIV is aspartate or iso-aspartate.
  • XV is asparagine or glycosylated asparagine.
  • XVI is methionine or oxidized methionine.
  • X VII is methionine or oxidized methionine.
  • a portion of the antibodies of the composition are fragmented in the HC via clipping between asparagine at position 58 and threonine at position 59, as set forth in SEQ ID NO: 7 and 39, respectively.
  • about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the antibodies of the composition are fragmented.
  • some antibody species may be found in the composition that may have undergone other post-translational modifications (PTMs) such as partial cleavage, oxidation, deamidation, succinimide or pyroglutamate formation and isomerization.
  • PTMs post-translational modifications
  • the antibody may show methionine (M) oxidation, e.g., at HC position 255; asparagine (N) deamidation, e.g., at HC position 318 and/or at HC position 387; asparagine (N) succinimide formation, e.g., at HC position 318; and/or amidation of the C-terminal proline (P) after the loss of the C-terminal lysine and glycine.
  • M methionine
  • N asparagine
  • N asparagine
  • N asparagine
  • succinimide formation e.g., at HC position 318
  • P C-terminal proline
  • the composition of the present disclosure comprises in one embodiment 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 the antibodies the HC is preferably modified as follows: the glutamine (Q) at position 1 is modified to pyroglutamate (pE), the asparagine (N) at position 300 is glycosylated; and/or the lysine (K) at position 450 is absent, preferably wherein the glutamine (Q) at position 1 is modified to pyroglutamate (pE), the asparagine (N) at position 300 is glycosylated, and the lysine (K) at position 450 is absent,
  • the composition of the present disclosure comprises in one embodiment one or more antibodies with the following additional modifications in their HC: the methionine (M) at position 255 is oxidized the asparagine (N) as position 318 is
  • the composition of the present disclosure comprises one or more antibodies with the following additional modifications in their HC: the asparagine (N) at position 58 is deamidated 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 aspartate (D) at position 283 is iso-aspartate the methionine (M) at position 361 is oxidized, and/or the methionine (M) at position 431 is oxidized.
  • the antibodies comprised in the composition of the present disclosure are produced via recombinant expression in CHO-K1 cells.
  • the composition of the present disclosure comprises one or more antibodies which comprise a pyro-glutamic acid at the N-terminal of the heavy chain sequence instead of glutamine, preferably wherein in more than 90%, preferably in about 99% to 100% of the antibodies the glutamine (Q) at position 1 of SEQ ID NO: 9 is modified to pyroglutamate.
  • the composition of the present disclosure comprises one or more antibodies in which the 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 the lysine (K) at position 450 of SEQ ID NO: 9 is absent.
  • the composition of the present disclosure comprises one or more antibodies in which the 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 the lysine (K) at position 450 of SEQ ID NO: 9 is absent and which comprise a pyro-glutamic acid at the N-terminal of the heavy chain sequence instead of glutamine, preferably wherein in more than 90%, preferably in about 99% to 100% of the antibodies the glutamine (Q) at position 1 of SEQ ID NO: 9 is modified to pyroglutamate.
  • the asparagine residues (N) at positions 58, 318, and 387 in the one or more antibodies are not deaminated and do not comprise a succinimide and the composition does comprise no antibodies or only a low amount of antibodies, for example less than about 5% of the antibodies, in which the asparagine residues (N) at positions 58, 318, and/or 387 are deamidated, respectively.
  • the methionine residues at positions 71, 115, 255, 361 and 431 in the one or more antibodies are not oxidized and the composition comprises no antibodies or only a low amount of antibodies, for example less than about 5% of the antibodies, in which the methionine residues at positions 71, 115, 255, 361 and/or 431 are oxidized.
  • the aspartate at position 283 in the one or more antibodies is not isomerized and the composition comprises no antibodies or only a low amount of antibodies, for example less than about 5% of the antibodies, in which the aspartate at position 283 is isomerized, respectively.
  • the glycine at position 449 in the one or more antibodies is not absent, i.e., the glycine is present and the composition comprises no antibodies or only a low amount of antibodies, for example less than about 5% of the antibodies, in which the glycine at position 449 is absent, respectively, i.e., the composition mainly comprises antibodies in which the glycine is present. Consequently, preferably, the proline at position 448 in the one or more antibodies is not amidated and the composition comprises no antibodies or only a low amount of antibodies, for example less than about 5% of the antibodies, in which the proline at position 448 is amidated.
  • the composition of the present disclosure comprises in embodiments one or more antibodies, preferably less than about 5%, preferably less than 2%, preferably less than about 1%, preferably about 0% to 1% in which the asparagine (N) at position 58 of SEQ ID NO: 9 is deamidated.
  • the composition of the present disclosure comprises one or more antibodies, preferably less than about 8%, preferably about 6% to 9% in which the asparagine (N) at position 318 of SEQ ID NO: 9 is deamidated.
  • the composition of the present disclosure comprises one or more antibodies, preferably less than about 3%, preferably about 1% to 3% in which the asparagine (N) at position 387 of SEQ ID NO: 9 is deamidated.
  • the composition of the present disclosure comprises one or more antibodies, preferably less than about 5%, preferably less than about 2%, preferably less than about 1%, preferably about 0% to 1% in which the methionine (M) at position 71 of SEQ ID NO: 9 is oxidized.
  • the composition of the present disclosure comprises one or more antibodies, preferably less than about 5%, preferably less than about 3%, preferably less than about 2, preferably about 1% to 3%, in which the methionine (M) at position 115 of SEQ ID NO: 9 is oxidized.
  • the composition of the present disclosure comprises one or more antibodies, preferably less than about 5%, preferably less than about 3%, preferably about 1% to 30%, in which the methionine (M) at position 255 of SEQ ID NO: 9 is oxidized.
  • the composition of the present disclosure comprises one or more antibodies, preferably less than about 5%, preferably less than about 2%, preferably less than about 1%, preferably about 0% to 1% in which the methionine (M) at position 361 of SEQ ID NO: 9 is oxidized.
  • the composition of the present disclosure comprises one or more antibodies, preferably less than about 5%, preferably less than about 3%, preferably less than about 2%, preferably about 1% to 2% in which the methionine (M) at position 431 of SEQ ID NO: 9 is oxidized.
  • composition of the present disclosure comprises one or more antibodies in which the aspartate (D) at position 283 of SEQ ID NO: 9 is modified to iso-aspartate.
  • composition of the present disclosure comprises one or more antibodies in which the proline (P) at position 448 of SEQ ID NO: 9 is amidated.
  • composition of the present disclosure comprises one or more antibodies in which the glycine (G) at position 449 of SEQ ID NO: 9 is absent.
  • composition of the present disclosure comprises one or more antibodies which are fragmented via clipping between asparagine at position 58 and threonine at position 59 of SEQ ID NO: 9.
  • the composition of the present disclosure comprises one or more antibodies, preferably less than about 6%, preferably less than about 5%, preferably about 2% to 5% in which the heavy chain is glycated.
  • the composition of the present disclosure comprises one or more antibodies, preferably less than about 4%, preferably less than about 3%, preferably about 1% to 3% in which the light chain is glycated.
  • the composition comprises one or more antibodies with a glycan profile as defined above in detail.
  • the composition of the present disclosure comprising the antibodies with the indicated PTMs is favorable for therapeutic purposes since such a composition is stable, safe, efficient, less immunogenic, functional and can be reliable reproducible manufactured (see Example 4).
  • the antibody of the present disclosure has been shown to be particularly stable in an aqueous formulation comprising about 50 mg/mL or 100 mg/mL of the antibody, 20 mM histidine, sucrose at a concentration of 65 mg/ml (6.5% (w/v)) or 80 mg/ml (8 % (w/v)), polysorbate 80 (PS) at a concentration of 0.03% (w/v) (0.3 mg/mL), at pH of 5.8.
  • the shelf-life for the drug product formulated in the above-mentioned aqueous formulation is currently set at 24 months stored at 5 ⁇ 3 ⁇ C, protected from light.
  • the present disclosure relates to a composition
  • a composition comprising from about 25 mg/mL to about 150 mg/mL of the antibody of the present disclosure, preferably comprising about 50 mg/mL or 100 mg/mL of the antibody of the present 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 of about 0.03% (w/v) (0.3 mg/mL), and having a pH of about 5.3 to 6.3, preferably of about 5.8.
  • the antibody is composed of two HCs having SEQ ID NO: 7 or 9 and two LCs having SEQ ID: 8, and wherein preferably in each HC the glutamine (X 1 of SEQ ID NO: 7 and Q at position 1 of SEQ ID NO: 9, respectively) at the N-terminus is modified as pyro-glutamic acid, the C-terminal lysine (X7 of SEQ ID NO: 7 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 of SEQ ID NO: 9, wherein the amino acids at position X2, X3, X4, and X5 of SEQ ID NO: 7 are either modified or unmodified, preferably unmodified or wherein the methionine at position M255 of SEQ ID NO: 9 is not oxidized, the asparagine at positions N318 and N387 of SEQ ID NO: 9 is not deamidated and does
  • the antibody is composed of two HCs having SEQ ID NO: 39 or 9 and two LCs having SEQ ID: 8, and wherein preferably in each HC the glutamine (X1 of SEQ ID NO: 39 and Q at position 1 of SEQ ID NO: 9, respectively) at the N-terminus is modified as pyro-glutamic acid, the C-terminal lysine (X 7 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 (XV of SEQ ID NO.
  • amino acids at position X 2 , X 3 , X 4 , and X 5 are either modified or unmodified, preferably unmodified or wherein the methionine at position M255 of SEQ ID NO: 9 is not oxidized, the asparagine at positions N318 and N387 of SEQ ID NO: 9 is not deamidated and does not comprise a succinimide, and the proline at position P448 of SEQ ID NO: 9 is not amidated, wherein the glycine (X6 of SEQ ID NO: 39 and G at position 449 of SEQ ID NO: 9) is present and not clipped off, and wherein the amino acids at positions XI, XII, XIII, XIV, XVI, XVI of SEQ ID NO: 39 are either modified or unmodified, preferably unmodified or wherein the methionine at positions M71, M115, M361, and M431 of SEQ ID NO:
  • the antibody is composed 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 glutamine at the N-terminus is modified as pyro- glutamic 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.
  • the composition comprises one or more antibodies as defined hereinbefore, i.e., the composition comprises one or more antibodies in which the 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 the lysine (K) at position 450 of SEQ ID NO: 9 is absent and which comprise a pyro-glutamic acid at the N-terminal of the heavy chain sequence instead of glutamine, preferably wherein in more than 90%, preferably in about 99% to 100% of the antibodies the glutamine (Q) at position 1 of SEQ ID NO: 9 is modified to pyroglutamate.
  • the 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 the lysine (K) at position 450 of SEQ ID NO: 9 is absent and which comprise
  • the asparagine residues (N) at positions 58, 318, and 387 in the one or more antibodies are not deaminated and do not comprise a succinimide and the composition does comprise no antibodies or only a low amount of antibodies, for example less than about 10%, preferably 5% of the antibodies, in which the asparagine residues (N) at positions 58, 318, and/or 387 are deamidated, respectively.
  • the methionine residues at positions 71, 115, 255, 361 and 431 in the one or more antibodies are not oxidized and the composition comprises no antibodies or only a low amount of antibodies, for example less than about 5% of the antibodies, in which the methionine residues at positions 71, 115, 255, 361 and/or 431 are oxidized.
  • the aspartate at position 283 in the one or more antibodies is not isomerized and the composition comprises no antibodies or only a low amount of antibodies, for example less than about 5% of the antibodies, in which the aspartate at position 283 is isomerized, respectively.
  • the glycine at position 449 in the one or more antibodies is not absent, i.e., the glycine is present and the composition comprises no antibodies or only a low amount of antibodies, for example less than about 5% of the antibodies, in which the glycine at position 449 is absent, respectively, i.e., the composition mainly comprises antibodies in which the glycine is present. Consequently, preferably, the proline at position 448 in the one or more antibodies is not amidated and the composition comprises no antibodies or only a low amount of antibodies, for example less than about 5% of the antibodies, in which the proline at position 448 is amidated.
  • the composition is an aqueous composition, also referred to as liquid formulation and thus, also comprises water for injection.
  • aqueous composition also referred to as liquid formulation and thus, also comprises water for injection.
  • RH relative humidity
  • several stability tests that have been performed with the formulation /pharmaceutical compositions of the present disclosure demonstrate that the antibody remains stable under various conditions, e.g., at 40 ⁇ 2 °C and 75 ⁇ 5% relative humidity (RH) for at least 1 month (stress stability studies); at 25 ⁇ 2 °C / 60 ⁇ 5% RH for at least 6 months (accelerated stability studies); and at 5 ⁇ 3 °C for at least 12 months to 18 months (long-term stability studies).
  • RH relative humidity
  • the composition can be characterized by the antibody remaining stable at 40 ⁇ 2 °C and 75 ⁇ 5% RH for at least 1 week, preferably for at least up to 1 month; at 25 ⁇ 2 °C / 60 ⁇ 5% RH for at least 1 month, and preferably for at least up to 6 months; and/or at 5 ⁇ 3 °C for at least 1 month, and preferably for at least up to 12 months, more preferably for at least up to 18 months.
  • the pharmaceutical composition has a shelf-life of 24 months at 2-8°C when protected from light.
  • the stability of the formulation may be at least partially attributed to the antibody’s constant region and more particularly, to the PTMs present in in the heavy chain of the antibody of the present disclosure as defined hereinbefore.
  • pharmaceutical formulations essentially characterized by a pH 5.3 to 6.3, preferably pH 5.8 ⁇ 0.1, typically comprising a histidine buffer and as excipients sucrose, polysorbate, preferably polysorbate 80, and water for infusion/injection have been developed for a recombinant human monoclonal antibody specific for the disease associated amyloidogenic form of TTR, featuring ALXN2220, also known as NI006, at a concentration of about 25 mg/ml to about 150 mg/ml, typically at about 50 mg/ml.
  • Histidine acts as a buffer agent.
  • the buffer capacity is related to the ability of histidine to maintain the pH near its pKa value. Since the pKa of histidine is approximately 6.0, at pH 5.8, it is slightly less efficient but still functional as a buffer.
  • the buffer capacity is also influenced by the concentration of the buffering agent and the proximity of the pH to the pKa.
  • Sucrose stabilizes proteins during freeze/thaw cycles and adds osmolality. Sucrose contributes significantly to the osmolality of the solution, which is important for maintaining the structural IgG antibody.
  • antibodies like all proteins, have buffering capacity due to the presence of ionizable groups in their amino acid side chains.
  • These groups include the carboxyl groups of aspartic acid and glutamic acid, the amino groups of lysine and arginine, the imidazole group of histidine, the hydroxyl group of tyrosine, the thiol group of cysteine, and the terminal amino and carboxyl groups.
  • composition of the present disclosure which comprises the above-referenced formulation fulfills at least the acceptance criteria as referred to in Tables 25 and 26 (which can also be used to determine whether a composition/formulation with changed concentration of components and/or substituted excipients are still long-term stable),
  • the composition of the present disclosure has a content of acidic species of the antibodies of ⁇ 40.0% and of basic species of the antibodies of ⁇ 15.0% after 6 months storage at 5 ⁇ 3 °C as determined by iCIEF; and/or has a content of high molecular weight species of ⁇ 5.0% after 6 months storage at 5 ⁇ 3 °C as determined by SEC-HPLC.
  • the amount of acidic species in the composition of the present disclosure is about 30%, preferably between 25% and 33%, more preferably between 25% and 32%. It has been further shown that the charged variant profile has varied in general. Thus, in a further preferred embodiment, the amount of acidic species in the composition of the present disclosure is equal or less than about 32%, and preferably the amount of basic species is equal to or less than about 5%, and the amount of neutral species is equal to or higher than about 63%.
  • Embodiments of the disclosure relate to methods for the production of 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, comprising (a) culturing a host cell, e.g., Chinese Hamster Ovary K1 (CHO-K1) cell, comprising one or more expression vectors or vector systems comprising the polynucleotides encoding the antibody HC and the LC under conditions sufficient for the expression of the antibody in the culture; (b) setting a pH setpoint and pH dead band of the culture to adjust level of acidic, basic, and/or neutral antibody species 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 formulation comprising the isolated antibody and a pharmaceutically acceptable carrier.
  • a host cell e.g., Chinese Hamster Ovary K1 (CHO-K1) cell,
  • the disclosure relate to methods for the production of 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, comprising (a) culturing a host cell, e.g., Chinese Hamster Ovary K1 (CHO-K1) cell, comprising one or more expression vectors or vector systems comprising the polynucleotides encoding the antibody HC and the LC under conditions sufficient for the expression of the antibody in the culture; (b) setting a pH setpoint and pH dead band of the culture to adjust level of acidic, basic, and/or neutral antibody species 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 formulation comprising the isolated antibody and a pharmaceutically acceptable carrier, wherein step (b) comprises setting a pH setpoint of about 6.90 and a pH deadband between 0.05 to 0.10
  • step (b) comprising setting the pH and pH deadband reduces the level of acidic species in cultured antibody preparation while also reducing levels of mannose 5 content.
  • step (b) comprising setting the pH at 6.90 and pH deadband between 0.05 and 0.10; preferably a pH at 6.90 and pH deadband of about 0.05, reduces levels of N-linked mannose-5 glycan (Man5) in the antibody preparation compared to a production method wherein step (b) is implemented at pH 6.90 and pH deadband of 0.20.
  • a reduction in Man5 levels is greater than >20%; more preferably the reduction in Man5 levels is about 40%-50% compared to levels of Man5 in an antibody preparation produced using an identical method except step (b) is implemented at pH 6.90 and pH deadband of 0.20.
  • step (b) comprising setting the pH at 6.90 and pH deadband between 0.05 and 0.10; preferably pH at 6.90 and pH deadband of about 0.05; is implemented in the production phase comprising day 5 to day 14 of the culture process, e.g., between day 5 to day 14 post-seeding of the CHO-K1 cells.
  • step (b) comprising setting the pH at 6.90 and pH deadband between 0.05 and 0.10; preferably pH at 6.90 and pH deadband of about 0.05, controls high mannose species in the antibody preparation, without concomitantly and adversely impact on secondary quality attributes of the antibody preparation, e.g., wherein secondary quality attributes are selected from (1) relative levels (in %) of main vs. high molecular weight (HMW) vs.
  • secondary quality attributes are selected from (1) relative levels (in %) of main vs. high molecular weight (HMW) vs.
  • LMW antibody species in the preparation e.g., as determined by size exclusion chromatography (SEC); (2) relative levels (in %) of main antibody species in the preparation, e.g., as determined using capillary electrophoresis sodium dodecyl sulfate under non-reduced conditions (CE-SDS-NR); and/or (3) relative levels (in %) of antibody heavy and light chains in the preparation, e.g., as determined using capillary electrophoresis sodium dodecyl sulfate under reduced conditions (CE-SDS R).
  • SEC size exclusion chromatography
  • CE-SDS-NR capillary electrophoresis sodium dodecyl sulfate under non-reduced conditions
  • CE-SDS R capillary electrophoresis sodium dodecyl sulfate under reduced conditions
  • Embodiments of the disclosure relate to antibodies produced according the above methods, e.g., 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, produced using a method comprising (a) culturing a host cell, e.g., Chinese Hamster Ovary K1 (CHO-K1) cell, comprising one or more expression vectors or vector systems comprising the polynucleotides encoding the antibody HC and the LC under conditions sufficient for the expression of the antibody in the culture; (b) setting a pH setpoint and pH dead band of the culture to adjust level of acidic, basic, and/or neutral antibody species in the culture, preferably wherein the adjustment comprises reduction of acidic species of the antibody culture by setting the pH at 6.90 and pH deadband between 0.05 and 0.10, preferably setting a pH deadband about 0.05; and (c) isolating the antibody HC and LC from the cell
  • the pH dead band is in embodiments set to less than 0.2, preferably of ⁇ 0.19, preferably of ⁇ 0.18, preferably of ⁇ 0.175, preferably of ⁇ 0.17, preferably of ⁇ 0.16, preferably of ⁇ 0.15, preferably of ⁇ 0.14, preferably of ⁇ 0.13, preferably of ⁇ 0.125, preferably of ⁇ 0.12, preferably of ⁇ 0.11, preferably of ⁇ 0.1, preferably of ⁇ 0.09, preferably of ⁇ 0.08, preferably of ⁇ 0.075, preferably of ⁇ 0.07, preferably of ⁇ 0.06, most preferably of ⁇ 0.05.
  • the pH dead band is set to about 0.1-0.05, preferably to 0.05 or 0.1, and most preferably to 0.05.
  • the antibodies of the disclosure are formulated, e.g., as high concentration pharmaceutical compositions for administration into a patient, at pH 5.8.
  • the pH is slightly below the pKa of histidine, which will still provide a buffering effect.
  • the pH is 5.8.
  • the concentration and/or nature of the osmolality agent here sucrose, may be adjusted, and the corresponding formulation tested in accordance with the Example.
  • the heavy and light chain complementarity determining regions VHCDRs and VLCDRs
  • the heavy and light antigen-binding domains of NI006/ALXN2220 has been described in WO 2015/092077 A1 (designated as antibody NI- 301.37F1) and in Michalon et al., Nat. Commun. 12 (2021), 3142 (designated as antibody NI301A).
  • NI006 (NI-301.37F1) is inter alia characterized by binding to aggregated human wild-type transthyretin (wtATTR), which is shown in Figures 2 to 4 and 7 and described in Examples 3 to 6, and further described at page 46, last paragraph.
  • wtATTR aggregated human wild-type transthyretin
  • WO 2015/092077 A1 discloses that NI006 (NI-301.37F1) does not bind to monomers and dimers of the human native transthyretin (TTR) as shown in Example 5 and Figure 4.
  • This binding profile is advantageous since the antibody binds selectively to aggregated wtTTR and thus allows prima facie to consider not only the treatment of hereditary transthyretin amyloidosis (hATTR) with polyneuropathy (formerly known as Familial Amyloid Polyneuropathy, FAP), which is due to mutations in the gene encoding TTR, but also the treatment of wild-type transthyretin amyloidosis (wtATTR), known as senile systemic amyloidosis (SSA). Moreover, the antibody is not at risk of interfering with native monomer assembly into physiological tetramers.
  • hATTR hereditary transthyretin amyloidosis
  • FAP Familial Amyloid Polyneuropathy
  • wtATTR wild-type transthyretin amyloidosis
  • SSA senile systemic amyloidosis
  • the antibody has been shown to remove ATTR deposits ex vivo from patient-derived myocardium by macrophages, as well as in vivo from mice grafted with patient-derived ATTR fibrils in a dose- and time-dependent fashion, wherein biological activity of ATTR removal involves antibody-mediated activation of phagocytic immune cells including macrophages; see Michalon et al., Nat. Commun.12 (2021), 3142.
  • the present disclosure relates to a composition, preferably a pharmaceutical composition, comprising the antibody of the present disclosure as defined hereinbefore at a concentration of about 50 mg/ml to about 100 mg/ml, preferably at a concentration of about 50 mg/mL or 100 mg/mL, preferably of about 50 mg/mL in an aqueous formulation further comprising L- histidine (L-histidine and L-histidine monohydrochloride), sucrose, polysorbate 80, and water for injection for use in a method of treating TTR amyloidosis (ATTR) in a subject.
  • L- histidine L-histidine and L-histidine monohydrochloride
  • sucrose sucrose
  • polysorbate 80 preferably of about 50 mg/mL
  • water for injection for use in a method of treating TTR amyloidosis (ATTR) in a subject.
  • TTR amyloidosis TTR amyloidosis
  • the composition is the composition of the present disclosure, i.e., the composition comprising the antibodies of the present disclosure at a concentration of about 50 mg/ml to about 100 mg/ml, preferably at a concentration of about 50 mg/mL or 100 mg/mL, preferably of about 50 mg/mL in an aqueous formulation further comprising L-histidine (L-histidine and L-histidine monohydrochloride), sucrose, polysorbate 80, and water for injection for use in a method of treating TTR amyloidosis (ATTR) in a subject.
  • the formulation of the composition of the present disclosure and the composition for use in accordance with the present disclosure respectively, has a pH of 5.8.
  • the formulation of the composition of the present disclosure and the composition for use in accordance with the present disclosure respectively, has as an osmolality of ⁇ 240 mOsm/Kg.
  • the formulation of the composition is stable and thus, in one embodiment, the pharmaceutical composition for use in accordance with the present disclosure has a shelf-life of 24 months at 2-8°C and protected from light and shows preferably the above- mentioned stability criteria.
  • the composition of the present disclosure and the composing for use in accordance with the present disclosure comprises a formulation, which when added up to about 2 ml essentially consists of: 50 mg or 100 mg of the antibody, 2.12 mg L-Histidine, 5.56 mg L-Histidine monohydrochloride 160 mg sucrose, 0.6 mg PS80, and water for injection.
  • the corresponding amounts of the antibody and the excipients can be calculated in case it is intended to provide a formulation of another volume, for example 2.25 ml, 20 ml, or 22.5 ml.
  • antibody NI006/ALXN2220 can be produced in Chinese hamster ovary (CHO) cells.
  • CHO cells are the most widely used mammalian cells for the production of recombinant monoclonal antibodies due to their ability to perform post-translational modifications (PTMs) on the antibody molecules, which typically take place in the human body as well.
  • PTMs post-translational modifications
  • CHO-K1 CHO-S
  • CHO- DXB11 CHO-DG44
  • the antibody used in the composition of the present disclosure and in the composition for use in accordance with the present disclosure, respectively is produced in CHO cells, preferably in CHO-K1 cells and is purified from the cell culture medium for further use. Furthermore, the compatibility of the antibody formulation with clinical in-use materials was evaluated.
  • NI006/ALXN2220 is less stable in saline, wherein the data show that when diluted in a glucose solution, no substantial changes in the appearance, protein concentration, sub-visible particle, SEC-HPLC, and ELISA binding assay were observed.
  • NI006/ALXN2220 at concentrations of 1.0 mg/mL, 20.0 mg/mL, and 50.0 mg/mL was shown to be stable at 2 - 8 °C for 24 hours followed by 25 °C for 6 hours (30 hours in total) and being compatible with the clinical in-use materials evaluated.
  • the most common route for the administration of monoclonal antibodies in therapy is intravenous (IV) infusion.
  • Intravenous administration is particularly important for monoclonal antibodies due to their large molecular size, which generally prevents them from being effectively absorbed through the gut or skin. This means that oral or transdermal delivery methods are not suitable for these types of drugs. Additionally, IV administration bypasses the first-pass metabolism in the liver, which can significantly alter the drug's efficacy and safety profile. Therefore, the composition of the present disclosure and the composition for use in accordance with the present disclosure, respectively has been particularly developed for IV administration and is thus, preferably the formulation suitable for intravenous administration.
  • the composition of the present disclosure and the composition for use in accordance with the present disclosure respectively has not been reconstituted from a lyophilized anti-TTR antibody or antigen-binding fragment thereof and/or is not further lyophilized. Since in the composition of the present disclosure and the composition for use in accordance with the present disclosure, respectively, sucrose is used as tonicity modifier and additional stabilizer of the antibody in the formulation, there is no need for NaCl, especially if the primary purpose of NaCl is to stabilize the protein, which sucrose can accomplish without increasing the ionic strength of the solution, Therefore, the pharmaceutical composition of the present disclosure is preferably essentially free of sodium chloride.
  • composition of the present disclosure and the composition for use in accordance with the present disclosure, respectively is essentially free of (or, e.g., completely lacks) a poloxamer.
  • the formulation of the composition of the present disclosure and the composition for use in accordance with the present disclosure, respectively, is preferably provided in vials.
  • the formulation is provided in 2 ml glass vials, preferably type I clear glass vials, preferably with a 12.5% overfill (2.25 ml)
  • other pharmaceutical containers with different fill volumes are also encompassed by the disclosure, for example 20 ml vials, e.g., 20 ml glass vials, preferably with a 12.5% overfill (22.5 ml), or other container/vials which are designed such that the vials/container and amount of antibody provided, respectively, add up to the flat doses, optionally plus about 12.5%, provided for example by the overfill.
  • the formulation is provided in 2 mL glass vial with an aluminum flip-off seals over a rubber stopper, to which preferably a volume of 2.25 ml is added.
  • formulation of the composition of the present disclosure and the composition for use in accordance with the present disclosure, respectively is a preservative- free concentrate for solution for infusion that is presented as sterile, colorless to slightly yellow, clear to slightly opalescent liquid, essentially free of visible particles, wherein preferably the concentrate for solution is diluted in sterile glucose before administration, as an intravenous infusion.
  • the present disclosure further relates to corresponding pharmaceutical containers comprising the composition/formulation of the present disclosure.
  • the present disclosure further relates to a stable aqueous formulation for use in a method of treating TTR amyloid cardiomyopathy (ATTR-CM), wherein the formulation (i) essentially consists of: (a) the antibody of the present disclosure, preferably the antibody is composed of two HCs having SEQ ID NO: 7 or 9 and two LCs having SEQ ID: 8, and wherein preferably in each HC the glutamine (X 1 of SEQ ID NO: 7 and Q at position 1 of SEQ ID NO: 9, respectively) at the N-terminus is modified as pyro-glutamic acid, the C-terminal lysine (X7 of SEQ ID NO: 7 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 of SEQ ID NO: 9, wherein the amino acids at position X2, X3, X4, and X5 of SEQ ID NO: 7 are either modified or unmodified,
  • the antibody is composed of two HCs having SEQ ID NO: 39 or 9 and two LCs having SEQ ID: 8, and wherein preferably in each HC the glutamine (X 1 of SEQ ID NO: 39 and Q at position 1 of SEQ ID NO: 9, respectively) at the N-terminus is modified as pyro-glutamic 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 V of SEQ ID NO.
  • amino acids at position X2, X3, X4, and X5 are either modified or unmodified, preferably unmodified or wherein the methionine at position M255 of SEQ ID NO: 9 is not oxidized, the asparagine at positions N318 and N387 of SEQ ID NO: 9 is not deamidated and does not comprise a succinimide, and the proline at position P448 of SEQ ID NO: 9 is not amidated, wherein the glycine (X6 of SEQ ID NO: 39 and G at position 449 of SEQ ID NO: 9) is preferably present and not clipped off, and wherein the amino acids at positions X I , X II , X III , X IV , X VI , X VI of SEQ ID NO: 39 are either modified or unmodified, preferably unmodified or wherein the methionine at positions M71, M115, M361, and M431 of SEQ ID NO:
  • the present disclosure further relates to a kit comprising one or more containers of the present disclosure and means for delivering the formulation/composition to a human subject, optionally wherein the means comprise an infusion bag or a syringe.
  • the formulation is a preservative-free, clear to opalescent and colorless to pale yellow solution, provided as 100 mg/2 mL in a vial.
  • the present disclosure further relates to an article of manufacture comprising one or more container(s) of the present disclosure, and a label, wherein the label prescribes that the antibody is indicated for the treatment of ATTR, especially ATTR cardiomyopathy (ATTR-CM), e.g., due to wild-type or hereditary transthyretin-mediated amyloid cardiomyopathy (wtATTR-CM or hATTR-CM).
  • the kit comprises a leaflet prescribing that the antibody is to be administered intravenously, which may contain, e.g., instructions for IV dosing.
  • the container(s) are glass vials as defined hereinbefore.
  • the kits may further comprise a diluent.
  • the diluent comprises a 5 % glucose solution, wherein the glucose solution can be provided in a further container.
  • the formulation of the antibody is a preservative- free, clear to opalescent and colorless to pale yellow solution, provided as 100 mg/2 mL in a vial.
  • the present disclosure further relates to a method of treating ATTR, preferably ATTR cardiomyopathy (ATTR-CM) and/or ATTR polyneuropathy (ATTR-PN), wherein the method comprises administering the antibody and the composition, respectively, of the present disclosure to a subject in need thereof, preferably wherein administration is performed intravenously.
  • ATTR-CM ATTR cardiomyopathy
  • ATTR-PN ATTR polyneuropathy
  • the antibody/composition is diluted and administered as an intravenous infusion over approximately one to two hours, wherein dilution is preferably performed in 5% glucose solution.
  • the patient to be treated with the antibodies of the present disclosure or pharmaceutical compositions comprising the same have been pre-screened, e.g., for identification that they have wtATTR-CM or hATTR-CM and the treatment is provided selectively to such patients who have been positively identified.
  • the subject has been previously treated with and/or is concurrently receiving a disease modifying agent, for example a TTR tetramer stabilizer like tafamidis (VYNDAQEL® or VYNDAMAX®), or Acoramidis (AG10).
  • a disease modifying agent for example a TTR tetramer stabilizer like tafamidis (VYNDAQEL® or VYNDAMAX®), or Acoramidis (AG10).
  • tafamidis oral disease-modifying treatment acting
  • ATTR-CM wild-type and hereditary forms
  • the present disclosure further provides a method for manufacturing the antibody of the present disclosure and a corresponding drug product, respectively.
  • the method of producing the antibody or an antigen-binding fragment thereof comprises a) cloning the nucleic acid molecule and the polynucleotide, respectively, of the present d isclosure which comprises the first nucleotide sequence and preferably the nucleotide sequence of the first signal peptide into an expression vector, and cloning the nucleic acid molecule and the polynucleotide, respectively, of the present disclosure which comprises the second nucleotide sequence and preferably the nucleotide sequence of the second signal peptide into an expression vector, wherein the first and the second nucleotide sequences may be provided in the same or different expression vectors, preferably in different expression vectors; b) transforming the expression vector(s) into a non-human host cell, preferably into a CHO cells, more preferably CHO-K1 cells; c) culturing the host cell under conditions allowing for the expression of the i mmunoglobulin chains comprising heavy chain and light chain; and
  • the method of the present disclosure comprises cultivation of the host cell, in particular the CHO-K1 cells with a pH dead band of less than 0.2, preferably of ⁇ 0.19, preferably of ⁇ 0.18, preferably of ⁇ 0.175, preferably of ⁇ 0.17, preferably of ⁇ 0.16, preferably of ⁇ 0.15, preferably of ⁇ 0.14, preferably of ⁇ 0.13, preferably of ⁇ 0.125, preferably of ⁇ 0.12, preferably of ⁇ 0.11, preferably of ⁇ 0.1, preferably of ⁇ 0.09, preferably of ⁇ 0.08, preferably of ⁇ 0.075, preferably of ⁇ 0.07, preferably of ⁇ 0.06, most preferably of ⁇ 0.05.
  • the pH dead band is set to about 0.1-0.05, preferably to 0.05 or 0.1, and most preferably to 0.05.
  • the method of the present disclosure comprises cultivation of the host cell, in particular the CHO-K1 cells at pH 6.9 with a pH dead band of less than 0.2, preferably of ⁇ 0.19, preferably of ⁇ 0.18, preferably of ⁇ 0.175, preferably of ⁇ 0.17, preferably of ⁇ 0.16, preferably of ⁇ 0.15, preferably of ⁇ 0.14, preferably of ⁇ 0.13, preferably of ⁇ 0.125, preferably of ⁇ 0.12, preferably of ⁇ 0.11, preferably of ⁇ 0.1, preferably of ⁇ 0.09, preferably of ⁇ 0.08, preferably of ⁇ 0.075, preferably of ⁇ 0.07, preferably of ⁇ 0.06, most preferably of ⁇ 0.05.
  • the pH dead band is set to about 0.1-0.05, preferably to 0.05 or 0.1, and most preferably to 0.05. In embodiment, the pH is set to pH 6.9 with the above indicated pH dead band.
  • the method may be further used to produce the pharmaceutical composition of the present disclosure and the corresponding drug product.
  • the method further comprises e) formulating the antibody in an aqueous solution comprising 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% weight per 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 generate a pharmaceutical composition comprising the antibody; and optionally f) filing the composition into a vial, and further optionally g) packing the pharmaceutical composition and vial, respectively, in a kit together with instructions for administration of the antibody, e.g., intravenously in a human patient.
  • the antibody of the present invention has been shown to remove ATTR deposits ex vivo from patient-derived myocardium by macrophages, as well as in vivo from mice grafted with patient-derived ATTR fibrils in a dose- and time-dependent fashion, wherein the biological activity of ATTR removal involves antibody-mediated activation of phagocytic immune cells including macrophages; see Michalon et al., Nat. Commun.12 (2021), 3142.
  • a high-resolution live-cell imaging assay has been developed.
  • This assay includes the incubation of cardiac tissue sections that comprise ATTR with THP-1 derived macrophages in the presence of the anti-TTR antibody and subsequent live-cell imaging.
  • the method includes the staining of amyloid deposits in the tissue sections with the amyloid specific red fluorescent dye Amytracker 680 and the labelling ALXN2220 with the green fluorescent dye Vivotag-680. After incubation of the stained tissue sections with the macrophages in the presence of labelled ALXN2220, high-resolution live-cell imaging was performed as described in Example 5.
  • the method includes the staining of amyloid deposits in the tissue sections with the amyloid specific red fluorescent dye Amytracker 680. ALXN2220 was not labelled. After incubation of the stained tissue sections with the macrophages in the presence of the antibody, high-resolution live-cell imaging was performed as described in Example 5.
  • the method includes labelling of ALXN2220 with the green fluorescent dye Vivotag-680. After incubation of the unstained tissue sections with the macrophages in the presence of the labelled antibody, high-resolution live-cell imaging was performed as described in Example 5 and the fluorescent signal visible in Fig. 9 and 10 represents amyloid staining due to the antibody binding to ATTR. As shown in Fig.
  • the present invention relates in one aspect to a method of validating an amyloid depleting drug, also referred to as amyloid depleting compound, comprising incubating tissue sections comprising amyloid deposits with macrophages in the presence of the amyloid depleting drug, wherein the tissue sections are stained with an amyloid specific fluorescent dye and/or the amyloid depleting drug is labeled with a fluorescent dye, and performing high- resolution live-cell imaging, wherein (a) an overlap of the antibody fluorescence signal and the amyloid fluorescence signal indicates binding of the amyloid depleting drug to the amyloid; (b) the presence of a punctuate and intracellular fluorescence signal in phagocytic vesicles indicates macrophage mediated internalization of amyloid; and (c) the successively separation of a punctuate fluorescence signal from the fluorescence signal of the amyloid indicates macrophage mediated amyloid fragmentation.
  • an amyloid depleting drug also referred to as am
  • the live-cell imaging can be used to visualize binding of the amyloid depleting drug to the amyloid deposits, macrophage mediated internalization of amyloid, and macrophage mediated amyloid fragmentation, wherein these mechanisms led to intracellular amyloid degradation. Accordingly, if any one of (a) to (c), preferably all of (a) to (c), more preferably at least any one of (b) and (c), or (b) and (c) is/are observed during live- cell imaging, it is verified that the amyloid depleting drug has indeed amyloid depleting activity. To confirm that the results are specific, i.e.
  • the observed mechanisms should not be observed when the tissue sections having amyloid deposits and macrophages are not incubated in the presence of the amyloid depleting drug, but for example in the presence of a control compound.
  • the control compound belongs preferably to the same class of substances as the amyloid depleting drug, and preferably in the same substance class as antibodies, but shows unspecific binding. Same regards to the methods described further below.
  • the present invention relates to a screening method for identifying and optionally obtaining an amyloid depleting drug from a plurality of test compounds comprising incubating tissue sections having 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) an overlap of the test compound fluorescence signal and the amyloid fluorescence signal indicates binding of the amyloid depleting drug to the amyloid; (b) the presence of a punctuate and intracellular fluorescence signal in phagocytic vesicles indicates macrophage mediated internalization of amyloid; and (c) the successively separation of a punctuate fluorescence signal from the fluorescence signal of the amyloid indicates macrophage mediated amyloid fragmentation, wherein the presence of any one of items (a) to (c), preferably of all of (a) to
  • the present invention further relates to a method of screening of an amyloid depleting drug for its capability to bind to amyloid, and to mediate macrophage recruitment to amyloid deposits, followed by amyloid fragmentation and internalization, i.e., for its capability to activate macrophage mediated amyloid fragmentation and internalization, leading to intracellular degradation of amyloid, wherein the method comprises incubating tissue sections comprising amyloid deposits with macrophages in the presence of the amyloid depleting drug, wherein the tissue sections are stained with an amyloid specific fluorescent dye and/or the amyloid depleting drug is labeled with a fluorescent dye, and performing high-resolution live-cell imaging, wherein (a) an overlap of the antibody fluorescence signal and the amyloid fluorescence signal indicates binding of the amyloid depleting drug to the amyloid; (b) the presence of a punctuate and intracellular fluorescence signal in phagocytic vesicles indicates macrophage mediated internalization of am
  • the methods of the present invention comprise the testing of a control sample, e.g.., the tissue sections are incubated with macrophages in the presence of a control.
  • the test substance used for the method of screening should have a an enhanced binding of the amyloid depleting drug to the amyloid, an enhanced internalization of amyloid, and/or an enhanced amyloid fragmentation in comparison to the control and/or in order to be suitable in the treatment of an amyloidosis or amyloid-related disease, the amyloid depleting drug should have a an enhanced binding of the amyloid depleting drug to the amyloid, an enhanced internalization of amyloid, and/or an enhanced amyloid fragmentation in comparison to the control.
  • the control sample can be any control showing unspecific binding to the amyloid and/or the macrophages.
  • the present invention also relates to a method of producing a pharmaceutical composition of an amyloid depleting drug comprising (i) providing, optionally producing the amyloid depleting drug; (ii) subjecting the amyloid depleting drug to the method as defined hereinbefore; (iii) using the information obtained in step (ii) as part of an assessment of whether the amyloid depleting drug may be used as a pharmaceutical composition or not; and optionally formulating the amyloid depleting drug found to be useful as a pharmaceutical composition in step (iii) with a pharmaceutically acceptable carrier, such as a buffer, a tonicity agent and/or a surfactant, most preferably all three components.
  • a pharmaceutically acceptable carrier such as a buffer, a tonicity agent and/or a surfactant
  • the present invention relates to a method for characterization, validation, development and/or quality control, for example batch control of an amyloid depleting drug comprising (i) providing, optionally producing the amyloid depleting drug; (ii) subjecting the amyloid depleting drug to the method as described hereinbefore; (iii) communicating the information obtained in (i) to a client, contracting party or cooperation partner and/or selecting the drug that has been determined to be a suitable amyloid depleting drug; and optionally (iv) using the amyloid depleting drug or a pharmaceutical composition comprising the amyloid depleting drug for the treatment of an amyloidosis or amyloid-related disease.
  • Combination therapies are also of interest for the treatment of amyloidosis.
  • a TTR tetramer stabilizer such as tafamidis and diflunisal
  • a TTR gene silencer is a promising approach for the treatment of TTR amyloidosis.
  • the impact of the drugs on each other must be carefully tested to avoid unwanted effects, like lowering of treatment efficiency.
  • the present invention also refers to a method of analyzing the impact of an agent, for example a second drug, a tracer, radiation, a label, preferably wherein the agent is a second drug, and in particular a second drug for the treatment of an amyloidosis or amyloid-related disease or a pain killer, e.g., a non-steroidal anti-inflammatory drug such as ibuprofen, or paracetamol, on the amyloid depleting activity of the amyloid depleting drug comprising incubating tissue sections having amyloid deposits with macrophages, preferably THP-1 derived macrophages, in the presence of the amyloid depleting drug and the agent; and performing high-resolution live-cell imaging, where (a) an overlap of the antibody fluorescence signal and the amyloid fluorescence signal indicates binding of the amyloid depleting drug to the amyloid; (b) the presence of a punctuate and intracellular fluorescence signal in phagocytic ve
  • An enhanced fluorescence signal emitted from the amyloid deposit indicates that the binding of the amyloid depleting drug is more efficient in the presence of the agent and thus, indicates a synergistic effect on the amyloid depleting activity of the amyloid depleting drug.
  • a reduced fluorescence signal emitted from the amyloid deposit indicates that the binding of the amyloid depleting drug is less efficient in the presence of the agent and thus, indicates a detrimental effect on the amyloid depleting activity of the amyloid depleting drug.
  • an enhanced intracellular fluorescence signal in the phagocytic vesicles indicates an enhanced macrophage mediated internalization of amyloid and thus, indicates a synergistic effect on the amyloid depleting activity of the amyloid depleting drug.
  • a reduced intracellular fluorescence signal in the phagocytic vesicles indicates a reduced macrophage mediated internalization of amyloid and thus, indicates a detrimental effect on the amyloid depleting activity of the amyloid depleting drug.
  • an enhanced separation of the punctuate fluorescence signal from the fluorescence signal of the amyloid indicates an enhanced macrophage mediated amyloid fragmentation and thus, indicates a synergistic effect on the amyloid depleting activity of the amyloid depleting drug.
  • a reduced separation of the punctuate fluorescence signal from the fluorescence signal of the amyloid indicates a reduced macrophage mediated amyloid fragmentation and thus, indicates a detrimental effect on the amyloid depleting activity of the amyloid depleting drug.
  • the amyloid depleting drug is an anti-amyloid antibody and most preferably an anti-TTR antibody and thus, the amyloid deposit is preferably a TTR amyloid deposit, and the amyloidosis and the amyloid-related disease are preferably TTR amyloidosis or an TTR amyloid-related disease, most preferably cardiac TTR amyloidosis.
  • the tissue section is preferably a cardiac tissue section.
  • the agent is preferably a second drug, which is preferably a drug useful in the treatment of TTR amyloidosis, for example a TTR tetramer stabilizer, such as tafamidis, or diflunisal, or a TTR gene silencer.
  • the amyloid depleting drug to be validated with the method of to the present invention, to be characterized with the method of the present invention, to be subjected to quality control using the method of the present invention, or to be analyzed regarding the impact of an agent, for example a second drug on its amyloid depleting activity is preferably the anti-TTR antibody of the present invention, most preferably antibody ALXN2220 as characterized hereinbefore.
  • Said antibody can also be used as positive control in the methods of the present invention, in particular in the methods of screening and identifying. Accordingly, the methods of the present invention comprise the use of a positive control, which is preferably the antibody of the present invention as defined hereinbefore, and most preferably antibody ALXN2220.
  • a positive control which is preferably the antibody of the present invention as defined hereinbefore, and most preferably antibody ALXN2220.
  • the high-resolution live-cell imaging uses in a preferred embodiment refractive index imaging for cell visualization and fluorescence microscopy for amyloid imaging. If the amyloid in the tissue sections is stained with an amyloid specific fluorescent dye and the antibody is also labelled with a fluorescent dye, preferably two different dyes are used, preferably dyes with a different colour, e.g., a red and a green fluorescent dye.
  • the tissue section comprising the amyloid is preferably derived from a subject suffering from an amyloidosis or an amyloid-related disease, preferably from TTR amyloidosis, most preferably from cardiac TTR amyloidosis.
  • the present invention further relates to a kit useful for performing the method of the present invention which comprises at least a first fluorescent dye for amyloid staining and/or a second fluorescent dye for labelling of the amyloid depleting drug, and optionally instructions for use or consumables useful for performing the method, e.g. microdishes.
  • the kit may also comprise a negative and/ or positive control, wherein the positive control can be for example the antibody as defined hereinbefore, preferably antibody ALXN2220.
  • the method of the present invention may be performed together with a corresponding method using a patient-derived amyloid xenograft (PDAX) non-human animal model, wherein the animal is characterized by an implant of amyloid fibrils derived from the tissue or organ of a patient suffering from an amyloidosis or amyloid-related disease, wherein the amyloid and amyloid fibrils, respectively, comprise amyloid transthyretin (ATTR), and wherein the amyloid fibrils are subcutaneously or subcapsularly implanted or implanted in the kidney, the peritoneum, the muscles, the brain, the ventricles, the nerves, the eyes, the tongue, or the heart, wherein the corresponding method comprises administering the amyloid depleting drug or a test substance to the PDAX non-human animal model and determining amyloid fibrils in the model, wherein the accelerated elimination or reduction of the amyloid fibrils upon administration of the drug or the test substance compared to a control is indicative for the
  • “about 8%” can mean any percentage between 7.2% and 8.8%, preferably any percentage between 7.6% and 8.4%.
  • “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).
  • the term "about” refers to concentrations ranging from 45 mg/mL to 50 mg/mL, and preferably from 48 mg/mL to 52 mg/mL and to concentrations ranging from 90 mg/mL to 113 mg/mL, preferably from 96 mg/mL to 113 mg/mL.
  • the term "and/or” is understood to mean that all members of a group which are connected by the term “and/or” are disclosed cumulatively in any combination, both alternatively to each other and in each case to each other.
  • an "antibody” as used herein refers to a population of immunoglobulin molecules that specifically bind to a target antigen.
  • the term “antibody” is understood to encompass not only a singular molecular entity but also variants of the antibody that may arise from post- translational modifications, such as glycosylation, deamidation, lysine clipping, and pyroglutamate formation.
  • the term “antibody” refers to this population as a whole, including such modified and unmodified forms.
  • the phrases "essentially free of NaCl” and “substantially free of NaCl” in the context of the formulation/pharmaceutical composition of the present disclosure mean that the pharmaceutical composition/formulation contains either no NaCl or only trace amounts that are considered negligible for the intended use of the product and, i.e., that NaCl is present at such a low level that it does not affect the formulation’s performance, stability, safety, or efficacy.
  • phrases may also refer to formulations/pharmaceutical compositions to which NaCl is not intentionally added, but which may comprise either Na 2+ or Cl- due to the presence of salts of other excipients, for example histidine-HCl.
  • the phrases "essentially free of” and “substantially free of” in the context of the formulation/pharmaceutical composition of the present disclosure mean that the pharmaceutical composition/formulation does not contain the substance which is referred to or only trace amounts that are considered negligible for the intended use of the product and, i.e., that the substance is present at such a low level that it does not affect the formulation’s performance, stability, safety, or efficacy.
  • binding potency refers to a characteristic corresponding to a quantitative measure of biological activity (e.g., TTR-binding).
  • a binding potency assay e.g., an ELISA assay
  • a binding potency assay can be used to measure the ability of an anti-TTR antibody or antigen-binding fragment thereof of the disclosure to elicit a specific response in a disease-relevant system (e.g., a subject having ATTR-CM, such as WT-ATTR-CM).
  • the activity measured in the assay is a surrogate for an intended biological effect and can be used to assess the maintenance of that effect over time (e.g., following storage).
  • the expressions "is capable of binding” and “binds to” refers to the capability of the antibody or antigen-binding fragment thereof to bind to, for example aggregated TTR, under experimental conditions (for example in an ELISA assay).
  • pharmaceutical composition refers to a mixture containing a therapeutic agent (e.g., an anti-TTR antibody 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, e.g., a human, in order to prevent, treat or control a particular disease or condition affecting, or that may affect, the subject (e.g., ATTR-CM, such as WT-ATTR-CM).
  • a subject such as a mammal, e.g., a human
  • ATTR-CM a particular disease or condition affecting, or that may affect, the subject
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • RT room temperature
  • Percent (%) sequence identity with respect 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 the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST.
  • the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, is calculated as follows: 100 multiplied by (the fraction X/Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B.
  • the percent sequence identity of A to B will not equal the percent sequence identity of B to A.
  • the terms "treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, in which the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development of cardiac deficiency.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival (e.g., prolonging survival of a human subject having ATTR for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more years, e.g., for the lifetime of the subject) as compared to expected survival if not receiving treatment.
  • pH dead band means a small range around the setpoint within which pH fluctuations are tolerated before correction occurs.
  • a “pH dead band of 0.05” means that the pH is allowed to fluctuate by ⁇ 0.05 units around the target pH before corrective action, like adjusting with acid or base, is applied.
  • isolated in the context of a polypeptide or an antibody, refers to a molecule that has been removed from its native environment, e.g., cell culture. An “isolated” antibody is substantially free of material from the cellular source from which it is derived.
  • isolated molecules e.g., polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells
  • isolated molecules are those that have been purified to a degree that they are no longer in a form in which they are found in nature.
  • the isolated molecule is substantially pure.
  • substantially purified refers to material that is at least 50% pure (e.g., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.
  • the purified molecule is of pharmaceutical grade.
  • polynucleotide is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA).
  • mRNA messenger RNA
  • pDNA plasmid DNA
  • a polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).
  • PNA peptide nucleic acids
  • nucleic acid refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.
  • isolated nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
  • a recombinant polynucleotide encoding an antibody contained in a vector is considered isolated for the purposes of the present disclosure.
  • Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present disclosure.
  • Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules produced synthetically.
  • polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
  • Fig. 1 cIEF Acidic % of ALXN2220 GMP Drug Substance Batches.
  • Fig. 2 Amount of mannose-containing glycans (Man5) of ALXN2220 GMP Drug Substance Batches.
  • Fig. 3 iCIEF Acidic % of Small-Scale Study.
  • Fig. 4 Man 5 profile of Small-Scale Study.
  • Fig. 1 cIEF Acidic % of ALXN2220 GMP Drug Substance Batches.
  • Fig. 2 Amount of mannose-containing glycans (Man5) of ALXN2220 GMP Drug Substance Batches.
  • Fig. 3 iCIEF Acid
  • Fig. 5 Online pH of Small-Scale Bioreactors.
  • Fig. 6 Glycosylation profile of ALXN2220 as determined by LCMS.
  • Fig. 7 ATTR deposits in myocardium tissue sections were identified using Amytracker 680, a red fluorescent dye specific for amyloid.
  • ALXN2220 labelled with the green f luorescent dye A488 selectively binds to ATTR as indicated by the overlap between red and green fluorescence.
  • FIG. 8 ALXN2220 triggered amyloid phagocytosis by macrophages. The punctate and intracellular red fluorescence pattern revealed the presence of ATTR amyloid in phagocytic vesicles. Macrophages presented a broad range of phagocytic activity i ndicated by the number of red fluorescent vesicles. Fig.
  • FIG. 9 Multinucleated macrophage successively detaches fragments of amyloid from a large deposit. The fluorescence images shown the process of fragment detachment over time (after 2.5 h, 3 h, 4 h, 12 h, 13 h, and 13.5 h).
  • Fig. 10 The macrophage was observed separating a thin and elongated ATTR deposit from the adjacent cardiomyocytes, moving the deposit by more than 20 ⁇ m. The macrophage also severed a protruding large amyloid deposit, detaching small and large fragments of amyloid.
  • the fluorescence images show the movement of the deposits over time (after 1 h, 4 h, 5.5 h, 6 h.11 h, 13.5 h, and 16 h).
  • TTR anti-transthyretin
  • compositions e.g., pharmaceutical compositions
  • TRR transthyretin-mediated amyloidosis
  • kits suitable for use in said methods are provided herein including kits suitable for use in said methods.
  • Antibodies and antibody composition of the present disclosure The present disclosure relates to a monoclonal human-derived anti-TTR antibody, which comprises an immunoglobulin light chain (LC) and an immunoglobulin heavy chain (HC) with the amino acid sequences set forth in Table 1, below.
  • LC immunoglobulin light chain
  • HC immunoglobulin heavy chain
  • the antibody may have a HC having the amino acid sequence of SEQ ID NO: 7 and a LC having the amino acid sequence of SEQ ID NO: 8, wherein X1 is absent, glutamine, or a pyroglutamate (pE); X 2 is methionine or oxidized methionine; X 3 is asparagine, deamidated asparagine, or asparagine comprising a succinimide; X 4 is asparagine or deamidated asparagine; X5 is proline or amidated proline; X6 is absent or glycine; and X 7 is absent or lysine, and a LC having the amino acid sequence of SEQ ID NO: 8.
  • X1 is absent, glutamine, or a pyroglutamate (pE)
  • X 2 is methionine or oxidized methionine
  • X 3 is asparagine, deamidated asparagine, or asparagine comprising a succinimide
  • the antibody of the present disclosure comprises a HC as shown in SEQ ID NO: 7, wherein the glutamine (X1), the glycine (X6) and lysine (X7) are present, and wherein the amino acids at positions X2, X3, X4 and X5 are either modified as indicated in Table 1 (e.g., oxidized, deamidated, succinimide comprising, and/or amidated) or unmodified.
  • the antibody of the present disclosure comprises a HC as shown in SEQ ID NO: 7, wherein the glutamine (X1) is absent or modified to pyroglutamate, preferably modified to pyroglutamate, and wherein glycine (X 6 ) and lysine (X 7 ) are present, and wherein the amino acids at positions X2, X3, X4 and X5 are either modified as indicated above or unmodified.
  • the antibody of the present disclosure comprises a HC as shown in SEQ ID NO: 7, wherein the lysine (X7) is absent, wherein the glycine (X6) and the glutamine (X1) are present, and wherein the amino acids at positions X 2 , X 3 , X 4 and X 5 are either modified as indicated above or unmodified.
  • the antibody of the present disclosure comprises a HC as shown in SEQ ID NO: 7, in which the lysine (X 7 ) and the glycine (X 6 ) are absent, wherein the glutamine (X 1 ) is present, and wherein the amino acids at positions X2, X3, X4 and X5 are either modified as indicated above or unmodified.
  • the antibody of the present disclosure comprises a HC as shown in SEQ ID NO: 7, wherein the glutamine (X1) is absent or modified to pyroglutamate, preferably modified to pyroglutamate, wherein the lysine (X 7 ) is absent, wherein glycine (X 6 ) is present, and wherein the amino acids at positions X 2 , X 3 , X 4 and X 5 are either modified as indicated above or unmodified.
  • the antibody of the present disclosure comprises a HC as shown in SEQ ID NO: 7, wherein the glutamine (X1) is absent or modified to pyroglutamate, preferably modified to pyroglutamate, wherein the lysine (X7) and the glycine (X6) are absent, and wherein the amino acids at positions X 2 , X 3 , X 4 and X 5 are either modified as indicated above or unmodified.
  • the antibody of the present disclosure comprises a HC as shown in SEQ ID NO: 7, wherein the glutamine (X1) is modified to pyroglutamate, wherein the lysine (X7) is absent, and wherein the amino acids at positions X2, X3, X4 and X5 are modified as indicated above or unmodified. In a preferred embodiment, X2, X3, X4 and X5 are unmodified.
  • the antibody of the present disclosure comprises a HC as shown in SEQ ID NO: 7, wherein the glutamine (X 1 ) is modified to pyroglutamate, wherein the lysine (X 7 ) is absent, and in which the amino acids at positions X2, X3, X4 and X5 are unmodified.
  • the antibody of the present disclosure is preferably composed of two HCs, each having an amino acid sequence set forth in SEQ ID NO: 7, and two LCs, each having an amino acid sequence set forth in SEQ ID NO: 8, and wherein in each HC the glutamine (X1) at the N- terminus is modified as pyro-glutamic acid, the C-terminal lysine (X 7 ) 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 position X2, X3, X4, and X5 are either modified or unmodified, preferably unmodified.
  • the antibody has a HC having the amino acid sequence of SEQ ID NO: 39 with the PTMs as detailed in the summary of the invention section, and a LC having the amino acid sequence of SEQ ID NO: 8.
  • the antibody may have a HC having the amino acid sequence of SEQ ID NO: 39 and a LC having the amino acid sequence of SEQ ID NO: 8, wherein X 1 is absent, glutamine, or a pyroglutamate (pE), preferably a pE; X 2 is methionine or oxidized methionine, preferably methionine; X3 is asparagine, deamidated asparagine, or asparagine comprising a succinimide, preferably asparagine; X 4 is asparagine or deamidated asparagine, preferably asparagine; X 5 is proline or amidated proline, preferably proline; X6 is absent or glycine, preferably glycine; X7 is absent or
  • the antibody may have a HC having the amino acid sequence of SEQ ID NO: 9 and a LC having the amino acid sequence of SEQ ID NO: 8.
  • the heavy chain of the antibody of the present disclosure has lost the C- terminal lysine, i.e., the antibody has undergone C-terminal lysine clipping and the C-terminal lysine is cleaved or absent, respectively.
  • the C-terminal lysine as shown in SEQ ID NO: 9 is absent, preferably from each heavy chain of the antibody.
  • Said sequence i.e., the sequence of the heavy chain with an absent C-terminal lysine is set forth in SEQ ID NO: 10.
  • the glutamine at the N-terminal is modified as pyro-glutamic acid, i.e., the heavy chain of the antibody as shown in SEQ ID NO: 9 has undergone N-terminal glutaminyl cyclization.
  • Said sequence i.e., the sequence of the heavy chain without N-terminal glutamine is set forth in SEQ ID NO: 11. Instead the N-terminus is cyclized and comprises pyroglutamate, respectively.
  • the heavy chain of the anti-TTR antibody of the present disclosure has lost the C-terminal lysine as shown in SEQ ID NO: 9, i.e., the C-terminal lysine is cleaved or absent, respectively and the glutamine at the N-terminal as shown in SEQ ID NO: 9 is modified as pyroglutamate.
  • Said sequence i.e., the sequence of the heavy chain without C- terminal lysine and without N-terminal glutamine is set forth in SEQ ID NO: 12. Instead the N- terminus is cyclized and comprises pyroglutamate, respectively.
  • the antibody is glycosylated, in particular N-glycosylated.
  • the heavy chain of the antibody is glycosylated and even more particularly at N300 of the heavy chain (SEQ ID NO: 9).
  • the present disclosure provides different species of the antibodies, i.e., antibodies being encoded by the same gene and comprise the same primary amino acid sequence, but which have been posttranslational modified to a different extent.
  • the most prominent PTMs are the modification of the C-terminus by clipping off the lysine and the pyroglutamate formation at the N-terminus as well as N-glycosylation.
  • the theoretical molecular weight of the antibody of the present disclosure is 144.2 kDa
  • the weight determined by mass spectrometry (MS) is 144.2 kDa (deglycosylated) and between 147.0 and 147.6 kDa (intact IgG1), respectively.
  • the antibody comprised in the pharmaceutical composition of the present disclosure has a molecular weight of about 150 kDa, preferably of about 147 kDa.
  • the antibody of the present disclosure comprises at least 8 disulfide bridges, preferably at the following positions: 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, wherein the numbering of the cysteine residues (C) corresponds to their positions in SEQ ID NOs: 7 and 8, with SEQ ID NO: 7 having the N-terminal glutamine (X1) and to SEQ ID NOs: 9 and 8, respectively, with SEQ ID NO: 9 having the N-terminal glutamine (Q).
  • C cysteine residues
  • the HC of the antibody of the present disclosure my further comprises a signal peptide having an amino acid sequence of SEQ ID NO: 17, wherein the sequence including the HC and the signal peptide is set forth in SEQ ID NO: 19.
  • the LC of the antibody of the present disclosure further comprises a signal peptide having an amino acid sequence of SEQ ID NO: 18, wherein the sequence including the HC and the signal peptide is set forth in SEQ ID NO: 20.
  • compositions comprising either one species of the above-described antibodies, preferably antibodies comprising a LC as shown in SEQ ID NO: 8 and a HC as shown in SEQ ID NO: 7, in which the glutamine (X1) is modified to pyroglutamate and in which the lysine (X 7 ) is absent, and in which the amino acids at positions X 2 , X 3 , X 4 and X5 are unmodified, and in which X6 is present, or antibodies comprising a LC as shown in SEQ ID NO: 8 and a HC as shown in SEQ ID NO: 9 from which the C-terminal lysine (K) is clipped off and in which the N-terminal glutamine is modified to pyroglutamate, or antibodies comprising a HC as shown in SEQ ID NO: 39 in which the glutamine (X1) is modified to pyroglutamate and in which the lysine (X7) is absent, and in which the amino acids at positions X2,
  • the composition comprises a mixture of the above-described antibody species, 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 antibodies present in the composition, the N-terminal is modified as pyro-glutamic 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 antibodies present in the composition, the C-terminal lysine is clipped of. Table 1.
  • Anti-TTR Antibody Sequences SEQ Domain/ ID Amino Acid Sequence/Nucleotide Sequence Region NO: V H-CDR1 1/38 SRSSY SRSSYWG (according to Kabat et al., U.S. Dept.
  • V H-CDR2 2 GIYHSGNTYDNPSLKS VH-CDR3 3 IVPGGDAFDI VL-CDR1 4 RASQSVGTYLN VL-CDR2 5 AASSLQS SEQ Domain/ ID Amino Acid Sequence/Nucleotide Sequence Region NO: VL-CDR3 6
  • QQSYSSPPT HC 7 X1LQLQESGPGLVKPSETLSLTCSVSGGSIISRSSYWGWIRQPPG KGLEWIGGIYHSGNTYDNPSLKSRLTMSVDTSKNQFSLNLRSV TAADTAVYYCARIVPGGDAFDIWGQGTMVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDK
  • VHCDR1 ends just before a tryptophan (W), typically preceding the motif W-valine (WV), W-isoleucine (WI), or W-alanine (WA). Since the heavy chain CDR1 of NI006/ALXN2220 contains the sequence 31-SRSSYWGWI-39 (SEQ ID NO: 37), the boundary for this CDR may end in Y or G, both of which precede W, depending on how the rule is applied. For avoidance of doubt, the broadest definition of VHCDR1 comprising SRSSY is used herein. In embodiments, the VHCDR1 comprises SRSSYWG (SEQ ID NO: 38).
  • Polynucleotides and nucleic acid molecules of the present disclosure can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • a polynucleotide encoding an antibody can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single-stranded and double-stranded regions.
  • a polynucleotide encoding an antibody can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • a polynucleotide encoding an antibody may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • a variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
  • An isolated polynucleotide encoding a non-natural variant of a polypeptide derived from an immunoglobulin can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues.
  • RNA may be isolated from the original B cells, hybridoma cells or from other transformed cells by standard techniques, such as a guanidinium isothiocyanate extraction and precipitation followed by centrifugation or chromatography. Where desirable, mRNA may be isolated from total RNA by standard techniques such as chromatography on oligo dT cellulose. Suitable techniques are familiar in the art.
  • cDNAs that encode the light and the heavy chains of the antibody may be made, either simultaneously or separately, using reverse transcriptase and DNA polymerase in accordance with well-known methods. PCR may be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences.
  • PCR also may be used to isolate DNA clones encoding the antibody light and heavy chains.
  • the libraries may be screened by consensus primers or larger homologous probes, such as human constant region probes.
  • DNA typically plasmid DNA
  • DNA may be isolated from the cells using techniques known in the art, restriction mapped and sequenced in accordance with standard, well known techniques set forth in detail, e.g., in the foregoing references relating to recombinant DNA techniques.
  • the DNA may be synthetic according to the present disclosure at any point during the isolation process or subsequent analysis.
  • the present disclosure also relates to a polynucleotide encoding the antibody of the present disclosure.
  • the present disclosure relates to a polynucleotide encoding at least one of the immunoglobulin chains of the antibody of the present disclosure.
  • the polynucleotide comprises, consists essentially of, or consists of a nucleic acid having a polynucleotide sequence of the HC or LC as depicted in Table 1.
  • the polynucleotides may be produced or manufactured by any method known in the art.
  • a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides, e.g., as described in Kutmeier et al., BioTechniques 17 (1994), 242, which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • a polynucleotide encoding an antibody, or antigen-binding fragment, variant, or derivative thereof may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the antibody may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably polyA + RNA, isolated from, any tissue or cells expressing the TTR-specific antibody, such as hybridoma cells selected to express an antibody) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody.
  • Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art. Accordingly, in one embodiment of the present disclosure the cDNA encoding an antibody, immunoglobulin chain, or fragment thereof is used for the production of an anti-TTR antibody.
  • the polynucleotide or nucleic acid is DNA.
  • a polynucleotide comprising a nucleic acid which encodes a polypeptide normally may include a promoter and/or other transcription or translation control elements operable associated with one or more coding regions.
  • An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s).
  • Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operable associated” or “operable linked” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
  • a promoter region would be operable associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid.
  • the promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells.
  • Other transcription control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operable associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art.
  • transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus).
  • Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit ß-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells.
  • Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
  • a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
  • a polynucleotide of the present disclosure is RNA, for example, in the form of messenger RNA (mRNA).
  • mRNA messenger RNA
  • Polynucleotide and nucleic acid coding regions of the present disclosure may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present disclosure.
  • proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
  • polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full-length" polypeptide to produce a secreted or "mature" form of the polypeptide.
  • the native signal peptide e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operable associated with it.
  • a heterologous mammalian signal peptide, or a functional derivative thereof may be used.
  • the signal peptides as shown as depicted in Table 1 are used in the context of the present disclosure. Expression of Antibody Polypeptides
  • the polynucleotides encoding the antibodies are typically inserted in an expression vector for introduction into host cells that may be used to produce the desired quantity of antibody.
  • the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art as also described in appended Example 1.
  • methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • the disclosure thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the disclosure, or a heavy or light chain thereof, operable linked to a promoter.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
  • vector or "expression vector” is used herein to mean vectors used in accordance with the present disclosure as a vehicle for introducing into and expressing a desired gene in a host cell.
  • such vectors may be selected from the group consisting of plasmids, phages, viruses, and retroviruses.
  • vectors compatible with the instant disclosure will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.
  • numerous expression vector systems may be employed.
  • one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV), or SV40 virus.
  • Others involve the use of polycistronic systems with internal ribosome binding sites.
  • cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells.
  • the marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper.
  • the selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by co-transformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals as also described in appended Example 1.
  • the cloned light and heavy chain genes are inserted into an expression vector, preferably into two different expression vectors (one for the heavy chain gene and one for the light chain gene) along with the signal peptide sequences as explained above.
  • 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).
  • multiple gene products of interest such as heavy and light chains of antibodies may be produced from a single polycistronic construct.
  • These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of antibodies.
  • IRES internal ribosome entry site
  • Compatible IRES sequences are disclosed in US patent no.6,193,980 which is also incorporated herein. Those skilled in the art will appreciate that such expression systems may be used to effectively produce the full range of antibodies disclosed in the instant application. Therefore, in one embodiment the present disclosure provides a vector comprising the polynucleotide encoding at least the binding domain or variable region of an immunoglobulin chain of the antibody, optionally in combination with a polynucleotide that encodes the variable region of the other immunoglobulin chain of said binding molecule.
  • the expression vector may be introduced into an appropriate host cell.
  • Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection including lipotransfection using, e.g., Fugene® or lipofectamine, protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus.
  • transfection including lipotransfection using, e.g., Fugene® or lipofectamine, protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus.
  • plasmid introduction into the host is via standard calcium phosphate co- precipitation method.
  • the host cells harboring the expression construct are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy and/or light chain protein synthesis.
  • Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorter analysis (FACS), immunohistochemistry and the like.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence-activated cell sorter analysis
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody for use in the methods described herein.
  • the disclosure includes host cells comprising a polynucleotide encoding an antibody of the disclosure, or a heavy or light chain thereof, which preferably are operable linked to a heterologous promoter.
  • the disclosure also includes host cells comprising a vector, as defined hereinabove, comprising a polynucleotide encoding at least the binding domain or variable region of an immunoglobulin chain of the antibody, optionally in combination with a polynucleotide that encodes the variable region of the other immunoglobulin chain of said binding molecule.
  • a single vector or vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • the host cell may be co-transfected with two expression vectors of the disclosure, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes both heavy and light chain polypeptides.
  • the light chain is advantageously placed before the heavy chain to avoid an excess 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.
  • host cells refers to cells which harbor vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene.
  • the terms “cell” and “cell culture” are used interchangeably to denote the source of antibody unless it is clearly specified otherwise.
  • recovery of polypeptide from the “cells” may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.
  • a variety of host-expression vector systems may be utilized to express antibody molecules for use in the methods described herein.
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the disclosure in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., Escherichia coli, Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid
  • bacterial cells such as E. coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
  • mammalian cells such as Chinese Hamster Ovary (CHO) cells, in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies; see, e.g., Foecking et al., Gene 45 (1986), 101; Cockett et al., Bio/Technology 8 (1990), 2.
  • the host cell line used for protein expression is often of mammalian origin; those skilled in the art are credited with ability to preferentially determine particular host cell lines which are best suited for the desired gene product to be expressed therein.
  • Exemplary host cell lines include, but are not limited to, CHO (Chinese Hamster Ovary), DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte) and 2
  • CHO and 293 cells are particularly preferred.
  • Host cell lines are typically available from commercial services, the American Tissue Culture Collection or from published literature.
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • CHO cells and most preferably the CHO cell line K1 as shown in Example 1 is used for the expression of the polynucleotides of the present disclosure to generate the antibodies and immunoglobulin chains, respectively.
  • stable expression is preferred.
  • cell lines which stably express the antibody molecule may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which stably express the antibody molecule.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11 (1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48 (1992), 202), and adenine phosphoribosyltransferase (Lowy et al., Cell 22 (1980), 817) genes can be employed in tk-, hgprt- or aprt-cells, respectively.
  • anti-metabolite resistance can be used as the basis of selection for 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.
  • Genes encoding antibodies, or antigen-binding fragments, variants or derivatives thereof of the disclosure can also be expressed in non-mammalian cells such as bacteria or insect or yeast or plant cells.
  • Bacteria which readily take up nucleic acids include members of the enterobacteriaceae, such as strains of E. coli or Salmonella; Bacillaceae, such as B. subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that, when expressed in bacteria, the heterologous polypeptides typically become part of inclusion bodies. The heterologous polypeptides must be isolated, purified and then assembled into functional molecules.
  • tetravalent forms of antibodies are desired, the subunits will then self-assemble into tetravalent antibodies; see, e.g., international application WO 02/096948.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix of glutathione- agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • eukaryotic microbes may also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms although a number of other strains are commonly available, e.g., Pichia pastoris.
  • the plasmid YRp7 for example, (Stinchcomb et al., Nature 282 (1979), 39; Kingsman et al., Gene 7 (1979), 141; Tschemper et al., Gene 10 (1980), 157) is commonly used.
  • This plasmid already contains the TRP1 gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No.44076 or PEP4-1 (Jones, Genetics 85 (1977), 12).
  • the presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is typically used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present disclosure can be purified according to standard procedures of the art, including for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, e.g. ammonium sulfate precipitation, or by any other standard technique for the purification of proteins; see, e.g., Scopes, "Protein Purification", Springer Verlag, N.Y. (1982).
  • the present disclosure also provides a method for preparing an anti-TTR antibody or an antibody recognizing mutated, misfolded, misassembled or aggregated TTR species and/or fragments thereof or immunoglobulin chain(s) thereof, said method comprising: (a) culturing the host cell as defined hereinabove, which cell comprised a polynucleotide or a vector as defined hereinbefore; and (b) isolating said antibody or immunoglobulin chain(s) thereof from the culture.
  • the present disclosure also relates to an antibody or immunoglobulin chain(s) thereof encoded by a polynucleotide as defined hereinabove or obtainable by said method for preparing an anti-TTR antibody or an antibody recognizing mutated, misfolded, misassembled or aggregated TTR species and/or fragments thereof or immunoglobulin chain(s) thereof.
  • Pharmaceutical compositions/Formulations The present disclosure provides pharmaceutical compositions containing the anti-TTR antibody and a mixture of the anti-TTR antibody species, respectively, as defined hereinbefore.
  • the pharmaceutical compositions of the present disclosure may be formulated as described below.
  • a pharmaceutical composition containing the anti-TTR antibody may be formulated to include sucrose, polysorbate 80, and histidine.
  • the pharmaceutical composition may include the anti-TTR antibody of the present disclosure at a concentration of about 50 mg/mL, a histidine buffer with a pH of about 5.8, 8.0% w/v of sucrose, and 0.03 % w/v of polysorbate 80 all in a volume of 2.0 mL.
  • the antibody comprises a HC chain having the amino acid sequence set forth in SEQ ID NO: 7, 39 or 9 and a LC having the amino acid sequence set forth in SEQ ID NO: 8.
  • about 99% to 100% of the antibodies present in the pharmaceutical formulation have a N-terminal pyro-glutamic acid in the heavy chain and about 96% of the antibodies have a loss of the C-terminal lysine.
  • the antibodies in the formulation of the present disclosure have a heavy chain wherein the N- terminal pyro-glutamic acid is modified from N-terminal glutamine and/or about 96% of the antibodies have a loss of the C-terminal lysine.
  • the antibody of the present disclosure is composed of two HCs having an amino acid sequence set forth in SEQ ID NO: 7, and two LCs having an amino acid sequence set forth in SEQ ID NO: 8, and wherein in each HC the glutamine (X1) at the N- terminus is modified as pyro-glutamic 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 position X2, X3, X4, and X5 are either modified or unmodified, preferably unmodified.
  • the antibody of the present disclosure is composed of two HCs having an amino acid sequence set forth in SEQ ID NO: 39, and two LCs having an amino acid sequence set forth in SEQ ID NO: 8, and wherein in each HC the glutamine (X1) at the N-terminus is modified as pyro-glutamic 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 (XV), and wherein the amino acids at position X2, X3, X4, and X5 are either modified or unmodified, preferably unmodified, and wherein the amino acids at positions XI, XII, XIII, XIV, XVI, XVI are either modified or unmodified, preferably unmodified.
  • the antibody of the present disclosure is composed of two HCs having SEQ ID NO: 9, and two LCs having SEQ ID: 8, wherein in the HC the glutamine at the N-terminus is modified as pyro-glutamic acid, the C-terminal lysine is lost, i.e., C- terminal lysine is cleaved or absent, respectively, and the heavy chain is N-glycosylated, preferably wherein the N-glycosylation site is at position Asn300.
  • the antibody is composed of two heavy chains having SEQ ID NO: 12 (preferably wherein the N-terminus is cyclized), and two light chains having SEQ ID NO: 8, and wherein the heavy chain is N- glycosylated, preferably wherein the N-glycosylation site is at position Asn300.
  • some antibody species may be found in the analyzed antibody composition that may have undergone other post- translational modifications (PTMs) such as partial cleavage, oxidation, deamidation, succinimide or pyroglutamate formation and isomerization.
  • PTMs post- translational modifications
  • the antibody may show methionine (M) oxidation at HC position 255; asparagine (N) deamidation at HC position 318 and/or at HC position 387; asparagine (N) succinimide formation at HC position 318; and/or amidation of the C-terminal proline (P) after the loss of the C-terminal lysine and glycine.
  • M methionine
  • N deamidation at HC position 318 and/or at HC position 387
  • asparagine (N) succinimide formation at HC position 318
  • amidation of the C-terminal proline (P) after the loss of the C-terminal lysine and glycine.
  • the antibody of the present disclosure may be formulated into a pharmaceutic composition described herein at a concentration of about 25 mg/mL to about 150 mg/m (e.g., about 25 mg/mL, about 50 mg/mL, about 75 mg/mL, about 100 mg/mL, or about 125 mg/mL), preferably of 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), most preferably 50 mg/mL or 100 mg/mL of the anti-TTR antibody of the present disclosure.
  • a concentration of about 25 mg/mL to about 150 mg/m e.g., about 25 mg/mL, about 50 mg/mL, about 75 mg/mL, about 100 mg/mL, or about 125 mg/mL
  • the pharmaceutical composition also includes 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% weight per unit volume (w/v) (e.g., about 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, or 9% w/v sucrose).
  • sucrose for example, in an amount of about 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, or 9% w/v sucrose.
  • the pharmaceutical composition may include 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.
  • the pharmaceutical composition includes about 6.5% w/v sucrose or about 8% w/v sucrose, most preferably 8% w/v sucrose.
  • Polysorbate The pharmaceutical composition also includes a polysorbate for example 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)).
  • the pharmaceutical composition may include 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.
  • the pharmaceutical composition includes about 0.03% w/v PS(80).
  • Buffer The pharmaceutical composition also includes a histidine buffer.
  • the pharmaceutical composition may include the 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, mM, 90 mM, or 100 mM).
  • 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, mM, 90 mM, or 100 mM.
  • the pharmaceutical may include 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 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM,
  • the pharmaceutical composition includes about 20 mM of histidine, preferably L-histidine and L-histidine monohydrochlorid.
  • pH The pharmaceutical composition may have a pH of from about 5.0 to about 8.0 (e.g., about 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0).
  • 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.
  • the pharmaceutical composition has a pH of about 5.8.
  • Volumes The pharmaceutical composition may be provided (e.g., in a vial or other container, as described herein) in a volume of 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, about 10 mL to about 20
  • the pharmaceutical composition may be present in a volume of 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 2.1mL to about 2.25 mL) or about 1 mL to about 100 mL (e.g., about 1 mL, about 1.8 mL, about 1.9 mL, about 2 mL, about 2.1 mL, about 2.2 mL about 2.25 mL, about 2.3 mL, about 2.4 mL, about 2.5 mL about 3 mL, about 4 mL
  • 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. Additional formulations
  • the pharmaceutical composition may be prepared so that it has not been reconstituted from a lyophilized anti-TTR antibody and/or is not further lyophilized. Also, the pharmaceutical composition may be prepared so that it is essentially free of sodium chloride and/or essentially free of a poloxamer.
  • the pharmaceutical composition can also be prepared as a sterile composition.
  • the pharmaceutical composition comprises or consist of 50 mg/mL of the antibody of the present disclosure, 20 mM histidine (L-Histidine 1.06 mg/mL and L-Histidine monohydrochloride 2.78 mg/mL), 65 mg/mL or 80 mg/mL sucrose, preferably 80 mg/mL sucrose, 0.3 mg/mL polysorbate 80, and water for infusion/injection at pH 5.8.
  • the disclosure in PCT publication WO2024105092 A1 is incorporated by reference herein in all parts pertinent thereto. Characteristics The disclosure features the disclosure of a pharmaceutical composition with improved characteristics (e.g., stability, solubility, storage, etc.), as described herein (e.g., see Example 4).
  • the pharmaceutical composition can be characterized by having a shelf-life of 24 months at 2 to 8°C, preferably when protected from light.
  • the pharmaceutical composition of the present disclosure may be characterized by one, two, three or all four stability criteria (i) to (iv) in any combination: (i) the main peak decline (representing the monomer content) under heat stress conditions for 1 month at about 40°C, or for 6 months at about 25°C, or during long-term storage for 18 months at about 5°C is less than 5%, preferably less than 4%, more preferably less than 3%, more preferably less than 2% as measured by SEC-HPLC; (ii) the pharmaceutical composition shows a content of acidic species of the anti-TTR antibody or antigen-binding fragment thereof under heat stress conditions for 2 weeks at about 40°C, or for 3 months at about 25°C of less than or about equal to about 40% as measured by cIEF; (iii) the pharmaceutical composition shows a content of acidic species of the anti-TTR antibody or antigen-binding
  • the pharmaceutical composition shows any one or all of the characteristics as set forth in the Tables of Example 4.
  • the pharmaceutical composition has an osmolality of ⁇ 240 mOsm/Kg.
  • the pharmaceutical composition is stable upon freezing and thawing.
  • the term “stable” or “stability”, as used in the context of a pharmaceutical composition described herein, refers to the maintenance of the physical and functional characteristics of the composition over time.
  • 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 osmolarity after long term storage.
  • the pharmaceutical composition of the present disclosure has been shown to remain stable at 40 ⁇ 2 °C and 75 ⁇ 5% RH for at least 1 month (stress stability studies); at 25 ⁇ 2 °C / 60 ⁇ 5% RH for at least 6 months (accelerated stability studies); and/or at 5 ⁇ 3 °C for at least 12 months to 18 months (long-term stability studies). Furthermore, the extinction coefficient of the pharmaceutical composition has been determined to be 1.438 (mg/mL) -1 cm -1 and the pharmaceutical formulation has been shown to be a sterile, colorless to slightly yellow, clear to slightly opalescent solution, essentially free of visible particles with a pH of 5.8.
  • the antibody of the present disclosure, the polynucleotide(s) of the present disclosure, the vector(s) of the present disclosure, the host cell of the present disclosure, or the compositions of the present disclosure, containing the anti-TTR antibody of the present disclosure can be used in the prophylactic or therapeutic treatment of a disease associated with TTR amyloidosis, such as ATTR, e.g., ATTR-CM, ATTR polyneuropathy(ATTR-PN), Familial Amyloid Polyneuropathy (FAP), Familial Amyloid Cardiomyopathy (FAC), Senile Systemic Amyloidosis (SSA), systemic familial amyloidosis, leptomeningeal / Central Nervous System (CNS) amyloidosis including Alzheimer disease, TTR-related ocular amyloidosis, TTR-related renal amyloidosis, TTR-related hyperthyroxinemia, TTR- related ligament amyloidosis including carpal tunnel syndrome, rotator cuff
  • the antibody of the present disclosure can be used in in vivo detection or imaging of or targeting a therapeutic and/or diagnostic agent to TTR in the human or animal body, preferably wherein said in vivo imaging comprises scintigraphy, positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR), optical imaging or magnetic resonance imaging (MRI).
  • said in vivo imaging comprises scintigraphy, positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR), optical imaging or magnetic resonance imaging (MRI).
  • PET positron emission tomography
  • SPECT single photon emission tomography
  • NIR near infrared
  • MRI optical imaging or magnetic resonance imaging
  • the pharmaceutical compositions described herein can be used in a method of treating or preventing transthyretin-mediated amyloidosis (ATTR).
  • the pharmaceutical compositions described herein can be used in a method of treating or preventing ATTR amy
  • a pharmaceutical composition containing a human anti-TTR antibody of the present disclosure at a concentration of about 50 mg/mL, a histidine buffer with a pH of about 5.8, 6.5% or 8.0% w/v of sucrose, and 0.03 % w/v of polysorbate 80 can be administered to treat ATTR or ATTR- CM, such as WT-ATTR-CM.
  • the pharmaceutical composition containing the anti-TTR antibody can be administered to a human subject to treat, prevent, or control transthyretin-mediated amyloidosis (ATTR), including ATTR amyloidosis with cardiomyopathy (ATTR-CM, such as WT-ATTR-CM).
  • the pharmaceutical composition is preferably be administered by intravenous injection or infusion
  • the composition and methods provided herein may be used to treat a subject that has ATTR, ATTR-CM, ATTR polyneuropathy (ATTR-PN), Familial Amyloid Polyneuropathy (FAP), Familial Amyloid Cardiomyopathy (FAC), Senile Systemic Amyloidosis (SSA), systemic familial amyloidosis, leptomeningeal/Central Nervous System (CNS) amyloidosis, Alzheimer disease, TTR-related ocular amyloidosis, TTR-related renal amyloidosis, TTR-related hyperthyroxinemia, TTR-related ligament amyloidosis, carpal tunnel syndrome, rotator cuff tears, lumbar spinal stenosis, preeclampsia, or a known pathogenic TTR mutation (e.g., one that causes amyloidosis).
  • ATTR-PN ATTR polyneuropathy
  • FAP Familial Amy
  • the subject may have sporadic, WT-ATTR-CM and a negative genetic testing for a TTR mutation.
  • the pharmaceutical composition may be ready-to use for administration to a subject in need thereof, preferably via intravenous infusion.
  • the pharmaceutical composition may be diluted prior to infusion in glucose or a polymer thereof, preferably wherein the polymer is dextran.
  • the concentration of glucose or a polymer thereof may be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% w/v, preferably in 5% glucose.
  • Containers Provided herein are also containers comprising the pharmaceutical composition of the disclosure.
  • Suitable containers include, for example, bottles (e.g., infusion bottles), vials (e.g., a Type I clear glass vial), syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle)
  • 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 capable of accommodating about a 10% to about 15% (e.g., 10% or 12.5%) volume overfill of the pharmaceutical composition.
  • the container may include a volume of a pharmaceutical composition described herein, such as a volume of, e.g., 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, e.g., 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.
  • the pharmaceutical composition i.e., the drug product described herein is provided as a concentrate for solution for infusion that is presented as sterile, colorless to slightly yellow, clear to slightly opalescent liquid, essentially free of visible particles and supplied in a 2 mL (2R) glass vial with an aluminum flip-off cap over a 13 mm rubber stopper.
  • the product is preferably diluted in sterile glucose before administration, which is a commercial product and in one embodiment not accompanying the drug product.
  • the container is a 2 ml or 20 ml vial and most preferably, a glass vial with an aluminum flip-off cap over a 13 mm rubber stopper, which preferably contains an approximate 12.5% volume overfill, or a total volume of 2.25 ml or 22.5 ml of the pharmaceutical formulation.
  • Articles of Manufacture/Therapeutic kits The present disclosure also provides an article of manufacture (e.g., a kit, and in particular a therapeutic kit) containing materials useful for the treatment or prevention of transthyretin- mediated amyloidosis (ATTR) in a human subject.
  • an article of manufacture e.g., a kit, and in particular a therapeutic kit
  • the article of manufacture includes 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, 4950, or 50 or more) containers of the disclosure as described hereinbefore and a label or package insert on or associated with the one or more container, wherein the container comprise the composition of the present disclosure.
  • the label or package insert indicates that the composition is used for treating ATTR.
  • the article of manufacture may include at least a first container with the pharmaceutical composition
  • the article of manufacture may further include a second container with a second therapeutic agent, like a TTR tetramer stabilizer, for example tafamidis.
  • the article of manufacture in this embodiment of the disclosure may further include a package insert indicating that the compositions can be used to treat ATTR.
  • the article of manufacture may further include a second (or third) container including a pharmaceutically acceptable buffer, such as BWFI, PBS, Ringer’s solution, and dextrose solution.
  • the article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • the article of manufacture may include a second container with a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer’s solution, and/or a glucose or a polymer thereof, such as dextran (e.g., at a concentration of about 5% w/v).
  • a pharmaceutically acceptable buffer such as phosphate-buffered saline, Ringer’s solution
  • a glucose or a polymer thereof such as dextran (e.g., at a concentration of about 5% w/v).
  • the article of manufacture may include other materials, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • the article of manufacture includes vials, preferably clear glass vials sealed with a (grey) rubber stopper and a (blue) aluminum-plastic cover flip-off cap.
  • the antibody is presented in vials at a concentration of 50 mg/mL and is provided as a concentrate for solution for infusion that is presented as sterile, colorless to slightly yellow, clear to slightly opalescent liquid, essentially free of visible particles.
  • the article of manufacture includes a dosing syringe in a dosing pump or an infusion bag comprising the diluted antibody formulation. Live-cell imaging methods of the present disclosure Due to the findings made during the experiments performed within the scope of the present invention, methods for screening, validation, characterization, and quality control of an amyloid depleting drug have been developed.
  • the method of validating an amyloid depleting drug comprises the steps of incubating tissue sections comprising amyloid deposits with macrophages in the presence of the amyloid depleting drug or a test compound in case of the screening method and performing high-resolution live-cell imaging.
  • the live-cell imaging uses refractive index imaging for cell visualization and fluorescence microscopy for amyloid imaging. Accordingly, either the tissue sections are stained with an amyloid specific fluorescent dye which results in the staining of the amyloid deposits in the tissue sections; the amyloid depleting drug and the test compound, respectively, is labeled with a fluorescent dye, which results in labeling of the amyloid deposits in the tissue sections upon binding of the amyloid depleting drug and the test compound, respectively, to the amyloid; or the tissue sections are stained with an amyloid specific dye and the amyloid depleting drug and the test compound, respectively, is labelled.
  • the tissue sections are stained with an amyloid specific fluorescent dye before addition of the macrophages and the amyloid depleting drug (or in case of the screening method, a test compound), and the amyloid depleting drug and the test compound, respectively, is labeled with a fluorescent dye, wherein this approach is preferably used for visualizing and detecting the binding of the amyloid depleting drug and the test compound, respectively, to the amyloid deposits via generating an overlap image of the fluorescence signal of the amyloid depleting drug and the test compound, respectively and the fluorescence signal emitted by the amyloid specific fluorescent dye.
  • the amyloid depleting drug and the test compound, respectively is labeled with a fluorescent dye which is different from the fluorescent dye used to stain the amyloid deposits.
  • the tissue sections are stained with an amyloid specific fluorescent dye before addition of the macrophages and the amyloid depleting drug and the test compound, respectively, and the amyloid depleting drug and the test compound, respectively, is not labelled, wherein this approach is preferably used to visualize and determine macrophage mediated internalization of amyloid via detecting intracellular fluorescence signal in phagocytic vesicles.
  • a fluorescence signal is usually a punctuate fluorescence signal.
  • the amyloid depleting drug and the test compound, respectively is labeled with a fluorescent dye and the tissue sections are not stained before addition of the macrophages and the amyloid depleting drug and the test compound, respectively, wherein this approach is preferably used to visualize and determine macrophage mediated amyloid fragmentation via detecting the successive separation of a fluorescence signal, preferably a punctuate fluorescence signal, from the fluorescence signal of the amyloid deposit, which is generated via binding of the labelled amyloid depleting drug and the test compound, respectively, to the amyloid deposit.
  • a fluorescence signal preferably a punctuate fluorescence signal
  • any fluorescent dye can be used in the methods of the present invention, which is either suitable for staining amyloid, i.e., an amyloid specific fluorescent dye, or suitable to label a drug.
  • the amyloid depleting drug and the test compound respectively is labelled with the fluorescent dye Vivotag-680.
  • the amyloid deposits are preferably stained with the amyloid specific dye Amytracker 680 or any other Amytracker variant, for example Amytracker 480, 520, 540, or 630.
  • Thioflavin T staining can be performed.
  • live-cell imaging is performed with the Nanolive CX-A instrument using 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 installed on an anti-vibration table.
  • the cell culture dishes comprising the tissue sections were maintained at 37°C with controlled humidity and 5% CO2 supply.
  • imaging frequency is every 15 min and experiment duration is preferably about 20 hours.
  • binding of the amyloid depleting drug and the test compound, respectively, to the amyloid deposit can be observed as well as macrophage recruitment to amyloid deposits leading to internalization and fragmentation of the amyloid and amyloid deposit, respectively, which is followed by intracellular degradation of the amyloid. If any one of (a) binding of the amyloid depleting drug and the test compound, respectively, to the amyloid, (b) amyloid internalization, and (c) amyloid fragmentation is observed in the method of the present invention, this verifies that the amyloid depleting compound has indeed amyloid depleting activity and that the test compound has amyloid depleting activity, respectively.
  • amyloid depleting compound has indeed amyloid depleting activity and that the test compound has amyloid depleting activity, respectively. More preferably, if (b) amyloid internalization and (c) amyloid fragmentation is observed, this indicates that the amyloid depleting compound has indeed amyloid depleting activity and that the test compound has amyloid depleting activity, respectively.
  • Macrophages are immune cells that originate from embryogenesis or from the differentiation of monocytes. They can adopt numerous phenotypes depending on their origin, tissue distribution and in response to different stimuli and tissue environment.
  • macrophages are endowed with a continuum of phenotypes that are rarely strictly pro-inflammatory or anti-inflammatory and exhibit a broad expression profile that sweeps over the whole polarization spectrum.
  • three main macrophage subpopulations coexist in human tissues: na ⁇ ve macrophages also called M0, pro- inflammatory macrophages referred as M1 macrophages, and anti-inflammatory macrophages also known as M2 macrophages.
  • M0 na ⁇ ve macrophages also called M0
  • pro- inflammatory macrophages referred as M1 macrophages
  • anti-inflammatory macrophages also known as M2 macrophages.
  • Na ⁇ ve macrophages display phagocytic functions, recognize pathogenic agents, and rapidly undergo polarization towards pro or anti-inflammatory macrophages to acquire their full panel of functions.
  • Pro-inflammatory macrophages are widely involved in inflammatory response, during which they exert anti-microbial and anti-tumoral functions.
  • anti-inflammatory macrophages are implicated in the resolution of inflammation, the phagocytosis of cell debris and tissue reparation following injuries. Macrophages also play important deleterious or beneficial roles in the initiation and progression of different pathophysiological settings including solid and hematopoietic cancers.
  • any kind of macrophage can be used as long as they are able to perform phagocytosis on amyloidogenic proteins.
  • the macrophages used in the methods of the present invention are THP1 cell-derived macrophages.
  • the macrophages were generated by differentiation of THP1 cells with phorbol 12-myristate-13-acetate (PMA), which is preferably used at a concentration of at 100 ng/mL and wherein incubation was preferably performed for 3 days, preferably followed by 3 days of rest without PMA.
  • PMA phorbol 12-myristate-13-acetate
  • Macrophages are preferably distributed to the tissue sections at a density of 400000 cells in the presence of the amyloid depleting drug.
  • amyloid depleting drug to be analysed is an antibody, preferably an anti-TTR antibody and most preferably the anti-TTR antibody as defined hereinbefore, the antibody is preferably used at a concentration of 10 nM (1.5 ug/mL). This concentration was previously identified as triggering maximum antibody-dependent phagocytosis of ATTR aggregates by macrophages in vitro.
  • the amyloid depleting drug also referred to as amyloid depleting compound is any compound which is capable to remove or reduce amyloid from tissue, preferably via recruitment of macrophages which internalize and degrade the amyloid.
  • the compound is an anti-amyloid antibody.
  • ADCP antibody-dependent cell-mediated phagocytosis
  • the amyloid depleting drug is preferably an antibody or a fragment or derivative thereof comprising an Fc domain or an antibody fragment fused to domain which is able to induce phagocytosis, like an Fc domain.
  • the suitability of the live-cell imaging method has been verified with tissue sections comprising TTR amyloid and with an anti-TTR antibody, in particular with the anti-TTR antibody ALXN2200 as described hereinbefore.
  • the amyloid depleting drug is preferably an anti- TTR antibody, most preferably the anti-TTR antibody as defined hereinbefore, which is capale to deplete amyloid TTR, wherein the TTR is wildtype or mutated TTR, preferably wildtype TTR.
  • the methods of the present invention can not only be used for the validation, screening, quality control, characterization, etc. of an anti-TTR antibody, but also for the validation, screening, quality control, characterization, etc. of any compound which is able to deplete TTR amyloid by recruitment of macrophages and phagocytosis, respectively.
  • the methods can be applied to any amyloid depleting compound, for example to compounds depleting amyloidogenic ⁇ -synuclein ( ⁇ -syn), tau, prion protein (PrP), amyloid beta (A ⁇ ), ⁇ 2-microglobulin ( ⁇ 2-m), Immunoglobulin light chain (LC), Immunoglobulin heavy chain (HC), serum amyloid A (SAA), amylin (IAPP), Chromosome 9 open reading frame 72 (C9orf72), TAR DNA-binding protein 43 (TDP-43), superoxide dismutase 1 (SOD1), RNA- binding protein fused in sarcoma (FUS), huntingtin (htt), optineurin (OPTN), neuroserpin, ABri, Adan, ubiquilin,optineurin, leucocyte chemotactic factor 2 (LECT)
  • amyloid fibril-forming proteins can be derived from AmyPro, an open- access database providing a collection of amyloid fibril-forming proteins (Varadi et al., Nucleic Acids Research 46 (2016), D387–D392, DOI: 10.1093/nar/gkx950), and/or can be those listed in Table 1 of Benson et al., Amyloid 25 (2016), 215–219.
  • the amyloidogenic protein is involved in systemic amyloidosis, and more preferably selected from the following list: transthyretin (TTR), in particular wild type TTR and variant TTR, preferably wild type TTR, immunoglobulin light chain (LC), immunoglobulin heavy chain (LH), serum amyloid A (SAA), leucocyte chemotactic factor 2 (LECT2), gelsolin, apolipoprotein AI (ApoAI), apolipoprotein AII (ApoAII), apolipoprotein AIV (ApoAIV), apolipoprotein CII (ApoCII), apolipoprotein CIII (ApoCIII), fibrinogen, ⁇ 2 microglobulin, in particular wild type and variant ⁇ 2 microglobulin, cystatin C, ABriPP, prion protein, and lysozyme; see for example Benson et al., Amyloid 25 (2016), 215–219 and
  • the tissue section is preferably a section of cardiac tissue, kidney tissue, liver tissue, gastro- intestinal tissue, skin tissue, muscle tissue, tongue tissue, fat tissue, salivary gland tissue, lymph node tissue, brain tissue, pancreatic tissue or any amyloidoma.
  • the tissue section is a cardiac tissue section.
  • the tissue sections are obtained from a subject suffering from an amyloidosis or amyloid-related disease, preferably form TTR amyloidosis, most preferably of cardiac TTR amyloidosis.
  • the tissue sections obtained from the patient are tissue sections which comprise the amyloid deposit.
  • the methods of the present invention can also comprise the analysis of a control compound, which is for example an isotype control antibody, in particular if the amyloid depleting compound is an antibody.
  • a control compound which is for example an isotype control antibody, in particular if the amyloid depleting compound is an antibody.
  • an amyloid depleting drug is analyzed which is suitable for TTR amyloid depletion and thus, suitable for the treatment of transthyretin (TTR) amyloidoses or a TTR amyloid-related disease, preferably cardiac TTR amyloidosis.
  • TTR transthyretin
  • a TTR amyloid-related disease preferably cardiac TTR amyloidosis.
  • the drug comprises an anti-amyloid protein antibody or amyloid binding molecule.
  • the drug is an antibody
  • the control is a corresponding isotype control antibody.
  • Suitable antibody candidates are known from the prior art, e.g. anti-transthyretin (TTR) antibodies are disclosed in WO 2015/092077 A1, WO 2014/124334 A2, WO 2018/007923 A3, WO 2016/120810 Al, US 2017/0058023 A1 and US 9,879,080 B2.
  • TTR anti-transthyretin
  • the method the present invention enables obtaining and selection of amyloid depleting compounds, in particular antibodies which can be reasonably expected to be as specific as to selectively bind to the target amyloid protein in the patient to be treated at the desired and necessary location of the for example toxic amyloid deposits and mediate the depletion of the amyloid.
  • the candidate antibody is a humanized, human-like or human antibody, preferably a human-derived antibody, most preferably isolated from human memory B cells, and recombinant variants thereof, which typically substantially comprise the variable heavy and light chain of the original human-derived antibody and a human constant domain which is preferably of the IgG1 or IgG4 subtype but not necessarily identical with the constant domain of the original human-derived antibody.
  • a further aspect of the present invention relates to a process for the manufacture of a pharmaceutical composition which comprises an amyloid depleting drug and a pharmaceutically acceptable carrier.
  • amyloid depleting drug that has been determined to be a suitable amyloid depleting drug by the method of the present invention as described, supra, is mixed with a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers and administration routes can be taken from corresponding literature known to the person 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) by the University of Sciences in Philadelphia, ISBN 0-683-306472, Vaccine Protocols. 2nd Edition by Robinson et al., Humana Press, Totowa, New Jersey, USA, 2003; Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems. 2nd Edition by Taylor and Francis.
  • compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways. Examples include administering a composition containing a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods.
  • Aerosol formulations such as nasal spray formulations include purified aqueous or other solutions of the active agent with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucous membranes.
  • Pharmaceutical compositions for oral administration such as single domain antibody molecules (e.g., "nanobodiesTM") etc are also envisaged in the present invention.
  • Such oral formulations may be in tablet, capsule, powder, liquid or semi-solid form.
  • a tablet may comprise a solid carrier, such as gelatin or an adjuvant.
  • Formulations for rectal or vaginal administration may be presented as a suppository with a suitable carrier; see also O'Hagan et al., Nature Reviews, Drug Discovery 2(9) (2003), 727- 735. Further guidance regarding formulations that are suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17 th ed. (1985) and corresponding updates. For a brief review of methods for drug delivery see Langer, Science 249 (1990), 1527-1533.
  • Preferred carrier in accordance with the present invention are buffers, a tonicity agents and/or a surfactants, most preferably all three components.
  • the pharmaceutical composition is designed for the treatment of an ATTR or ATTR-related disease.
  • the amyloidosis is characterized by the ATTR deposit in the patient, and especially the respective precursor protein.
  • the pharmaceutical composition is a pharmaceutical composition comprising the amyloid depleting drug, in particular the anti-amyloid antibody, most preferably the anti-TTR antibody as defined hereinbefore in an aqueous formulation as defined hereinbefore.
  • the present invention relates to a method for characterization, validation, development and/or quality control, which also included batch control, of an amyloid depleting drug, which is suitable for the treatment of an amyloidosis or amyloid-related disease.
  • an amyloid depleting drug is characterized in the live-cell imaging method and the information about the drug that has been subjected to the method of validating an amloid depleting drug of the present invention as described, supra, is communicated to a client, contracting party or cooperation partner.
  • the drug that has been determined to be a suitable amyloid depleting drug may be selected, and optionally the amyloid depleting drug or a pharmaceutical composition comprising the amyloid depleting drug is used for the treatment of an amyloidosis or amyloid-related disease.
  • the method of the present invention may be performed in addition to another method useful for verifying the therapeutic applicability of a candidate drug and/or useful to detect the capability of a drug to deplete amyloidogenic proteins and amyloid deposits, respectively.
  • a method is for example a method employing the patient-derived amyloid xenograft (PDAX) non-human animal model as disclosed in WO 2020/094883 A1, which content is herein incorporated by reference.
  • PDAX patient-derived amyloid xenograft
  • NI006/ALX2220 is produced in the CHO K1 cell line (ATCC No. CCL 61). Construction of Expression Plasmids and Sequence Confirmation First, signal peptides were added to the sequences encoding the heavy chain gene (set forth in SEQ ID NO: 15) and light chain gene (set forth in SEQ ID NO: 16) of NI006/ALXN2220 and each expression cassette (heavy chain gene including signal peptide and light chain including signal peptide) was cloned into an individual expression vector.
  • the expression vectors comprise typical elements, like a promoter to control gene expression, a ribosome entry site, two selectable markers, one for maintenance in mammalian cells and one for maintenance in E. coli, a terminator, and a replication origin for propagation in E. coli.
  • the CHO K1 host cell line was thawed and cultured before used for transfection.
  • the linearized light and heavy chain expression vectors were transfected with a ratio of 1:1 into the CHO-K1 host cell line using Freestyle Max reagent.48 hours after transfection, cells were plated in four 96-well plates in selection media comprising the corresponding antibiotics for maintenance in mammalian cells.
  • NI006/ALXN220 Characterization of mature NI006/ALXN220 Antibody NI006/ALXN2220 was produced in the CHO-K1 cell line (ATCC No. CCL 61) as described in Example 1 and obtained from the cell culture.
  • the amino acid sequence of NI006/ALXN2220’s mature heavy chain (HC) and light chain (LC) is set forth in SEQ ID NOs: 7 and 8, with the below-mentioned modifications.
  • the total number of amino acids, number of amino acids of the heavy chain, and number of amino acids of the light chain are 1328, 450, and 214, respectively.
  • Further characterization of antibody NI006/ALXN2022 was mainly performed by standard procedures, for example by mass spectroscopy analysis. For example, liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis of fragments of NI006/ALXN2220 obtained from Lys-C and trypsin sequential digestion as well as free sulfhydryl analysis was used to identify post-translational modifications of NI006/ALXN2220.
  • LC-MS/MS liquid chromatography with tandem mass spectrometry
  • characterization of antibody-based therapeutics via LC-MS analysis is a standard procedure and can be performed by a skilled artisan; see for example Robotham and Kelly, Approaches to the Purification, Analysis and Characterization of Antibody-Based Therapeutics (2020), 1-33.
  • Exemplary Released N-Glycan Method for determination of glycosylation profile Glycan method is used to profile N- N-glycans of ALXN2220 are released using PNGase F and labeled with 2-AB, followed by HILIC (Hydrophilic Interaction Chromatography) separation and fluorescence detection (FLD) with a UPLC system. Individual N-glycans are quantified by their peak area percentages relative to the total peak area.
  • Exemplary Peptide Mapping Studies using LCMS Analysis of the primary structure and post-translational modifications is accomplished by peptide mapping with high resolution mass spectrometry (MS).
  • MS mass spectrometry
  • ALXN2220 is reduced, denatured, and alkylated. Reduced and alkylated protein undergoes sequential digestion with endoproteinase Lys-C/trypsin. The resulting digested peptides are separated by reversed phase C18 column followed by detection with high resolution mass spectrometer. The identity of the peptides is confirmed against the theoretical mass of the peptides and MS/MS.
  • Post- translational modification is quantitated by the peak area percentages of the modified peptide relative to the sum of the peak area of modified and unmodified form of the peptide.
  • Exemplary icIEF Method (charge method) for determining charge variants Imaging Capillary Isoelectric Focusing (iCIEF) is used to separate charge variants of protein based on their isoelectric points (pI). Samples are prepared by mixing with master mixture composed of pharmalytes, pI markers, urea, and water. ALXN2220 is separated into main peak, acidic region, and basic region through a pH gradient formed by carrier ampholytes inside the capillary, separating the isoforms according to their isoelectric points. The focused protein zones are detected by whole-capillary UV detection. Acidic, main, and basic species are quantified by their peak area percentages relative to the total of all protein peak detected.
  • iCIEF Imaging Capillary Isoelectric Focusing
  • the molecular weight of antibody NI006/ALXN2220 as determined by standard mass spectroscopy is approximately 147.1 kDa for the intact IgG1 and 144.2 kDa for the deglycosylated variant.
  • the pI of antibody NI006/ALXN2220 is 8.4 (theoretical) and 9.3 (experimentally determined).
  • the Theoretical Extinction Coefficient (A0.1%) (mL/(mg * cm)) of antibody NI006/ALXN2220 is 1.390 (theoretical) and 1.438 (experimentally determined).
  • the monoclonal antibody NI006/ALXN2220 is an IgG1 subclass antibody, which is composed of two heavy chains of the IgG1 subclass and two light chains of the kappa subclass. The four chains are stabilized by multiple disulfide bonds.
  • NI006/ALXN2220 is a glycoprotein, and the constant region of each heavy chain contains one N-linked glycan site at residue N300.
  • glycosylation profile (the types of sugar, the location of glycosylation site(s), etc.) has been determined for NI006/ALXN2220: Glycan Glycan Type 23100 Man3+1F 33000 G0-GN 33100 G0F-GN 43000 G0 43100 G0F 25000 Man5 34100/44000 G1F-GN/G1a 44000 G1b 44100 G1Fa 44100 G1Fb 45100 G2F 45110 G2FS1 45120 G2FS2
  • the nomenclature of the glycans follows the order of HexNac-Hexose-Fucose-NeuAc-NeuGc.
  • G1Fa and G1Fb are isomers and are grouped into G1F.
  • G1F is calculated as the sum of G1Fa and G1Fb using the original unrounded numbers.
  • the glycosylation profile is depicted in Figure 6 and the abundance of the different glycans in 11 separate batches of NI006/ALXN2220 is listed in Table 2. Table 2.
  • Peptide mapping has been carried out using LCMS. Furthermore, glutamine at N-terminal modified as pyro-glutamic (abundance in sample: 99.9) acid and C-terminal lysine clipping of the heavy chain (abundance in sample: 95.8%) have been identified as the major post-translational modifications. In addition, 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 as shown in Tables 3 and 4: Table 3. Experimentally determined PTMs No. Seq. No. Location Sequence SEQ PTMs ID NO.
  • Peptide sequences in underlined font were identified as the site of the PTMs. * refers to N-terminal related peptide of heavy chain and # refers to C-terminal related peptide of heavy chain.
  • pE(Q) refers to the N-terminal glutamine modified as pyro-glutamic acid.
  • -K refers to loss of the C-terminal lysine.
  • -K -G Amidation(P) refers to the amidation of the C-terminal proline after the loss of the C-terminal lysine and glycine. Table 4.
  • the consensus N-linked glycosylation site is defined by four residues N-X-S/T-Z.
  • the glycosylation site is essentially fully occupied if both X and Z residues are not proline. If either X or Z residues are proline, there is only trace level glycosylation.
  • HC N62 may contain trace glycosylation.
  • N300 is not likely to be deamidated due to N-linked glycans occupancy.
  • Table 5 The abundance of the post translational modifications in a sample of antibody NI006/ALXN2220 is shown in Table 5: Table 5.
  • ALXN2220 manufacturing process In this Example, scale up of ALXN2220 drug substance (DS) manufacturing process, from phase I at 500L scale (Process A1) to phase III at 2000L scale (Process A2) is described, with the major upstream process changes summarized in Table 7.
  • ALXN2220 product quality results at DS release are summarized in Table 7.
  • the imaged capillary isoelectric focusing (iCIEF) acidic % of 1 st 2000L DS batch (GMP 1 (A2)) was considerably higher than historical results from process A1 at 500L scale ( Figure 1).
  • Example 4 Stability Studies of the anti-TTR monoclonal antibody formulation
  • Formulation development of NI006/ALXN2220 included studies designed to select a buffer system and excipients to stabilize the protein. The formulation was developed to prevent product loss, as well as minimize the purity and bioactivity decline against stresses encountered during production, storage, shipping, and handling.
  • a pH buffer screening study was performed to determine the optimal buffer system for drug product formulation.20 mM histidine buffer, at pH 5.8 was chosen as the final buffer system.
  • excipients including disaccharides (such as sucrose and trehalose), amino acids (such as L-arginine HCl), polyhydric alcohols (such as sorbitol), and surfactants (such as polysorbate 80) were evaluated though excipient studies. Samples were incubated at 40oC for up to 4 weeks. The thermal stability, formation of insoluble aggregates, and purity were monitored. Sucrose and polysorbate 80 were chosen as the optimal excipients for NI006/ALXN2220 formulation, as they were shown to minimize the decrease of SEC, cIEF and caliper purity, thus retaining product purity.
  • disaccharides such as sucrose and trehalose
  • amino acids such as L-arginine HCl
  • polyhydric alcohols such as sorbitol
  • surfactants such as polysorbate 80
  • a polysorbate 80 concentration of 0.03% (w/v) was chosen as the surfactant strength, since subvisible particle formation was effectively suppressed and high SEC purity was maintained.
  • a comparison study was conducted to compare two formulations with 6.5% or 8% (w/v) sucrose concentration. No substantial differences, between formulations of 6.5% and 8% (w/v) sucrose, were observed in DSC (Differential Scanning Calorimeter), appearance, pH, protein concentration, SEC, cIEF, CE-SDS (non-reduced & reduced), sub-visible particles, and potency after incubation at 40oC for 1 month or incubation at 25 °C for 3 months.
  • the final formulation developed is 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.
  • the excipients were selected according to their stabilizing effect on the drug product.
  • L-Histidine and L-Histidine monohydrochloride at a concentration of 20 mM, stabilize the pH in the liquid state.
  • Sucrose at a concentration of 8% (w/v), modifies the osmolality to be isotonic, stabilizes the NI006/ALXN2220 protein against aggregate formation in the liquid state, and serves as a cryo-protectant during freeze/thaw.
  • Polysorbate 80 at a concentration of 0.03% (w/v), was chosen to stabilize NI006/ALXN2220 protein against surface-induced protein denaturation or aggregation in the liquid state.
  • Manufacturing Process Development The drug product manufacturing process consists of drug substance thawing, pooling and mixing, sterile filtration, aseptic filling, stoppering, capping, visual inspection, and bulk packaging. Sterile filtration was chosen as the method to obtain the sterile drug product and is performed by using two series connected sterile filters (0.22 ⁇ m, PVDF). Prior to and after sterile filtration, water bubble point testing for the filters is performed to assure filter integrity.
  • a non-clinical lot (lot 201901004) and three clinical lots (lot 201903038, lot 201904050 and 20200801) have been filled.
  • the non-clinical lot and clinical lots used the same fill volume, container closure system, sequence of unit operations, and storage conditions. No significant changes were made to the drug product manufacturing process between the non- clinical lot and clinical lots. Minor differences are described below: • A 2 L scale was used for the non-clinical lot and 14 L scale was used for the clinical lots.
  • the formulation used for the non-clinical lot 201901004 was 50 mg/mL in 20 mM histidine buffer, 8% sucrose (w/v), and 0.03% polysorbate 80 (w/v), pH 5.8.
  • the formulation used for clinical lots 201903038 and 201904050 was 50 mg/mL in 20mM histidine buffer, 6.5% sucrose (w/v), and 0.03% polysorbate 80 (w/v), pH 5.8.
  • the formulation used for the clinical lots 20200801 was 50 mg/mL in 20mM histidine buffer, 8% sucrose (w/v), and 0.03% polysorbate 80 (w/v), pH 5.8.
  • a 5 L glass bottle was used for pooling and mixing of the drug substance for the non- clinical lot, and a 50 L mixing bag was used for pooling and mixing of the drug substance for the clinical lots.
  • Bulk Drug Substance Thawing, Pooling and Mixing Frozen drug substance stored in 2 L PETG bottles is thawed at room temperature (18 - 24 °C) in a room protected from light. After complete thawing, the drug substance is pooled into a 50 L mixing bag and stirred at an appropriate speed so that movement is observed without generating foam. The mixing time is controlled at 15 - 20 minutes. Prior to sterile filtration, samples are taken for pH, protein concentration, osmolality, and bioburden.
  • the bulk drug substance is aseptically filtered in a Grade A environment via a peristaltic pump through two series connected 0.22 ⁇ m sterile filters into a sterile 20 L single use bag. Prior to and after sterile filtration, filter integrity testing is performed on both filters. Aseptic Filling Aseptic filling is performed inside the RABS unit, which fully encloses the filler and provides a Grade A environment. The RABS unit separates the operator from the aseptic interior. All filling components are autoclaved and aseptically assembled. Sterile, depyrogenated 2 mL (2R) glass vials are filled to a target volume of 2.25 mL.
  • Stoppering Filled vials are automatically stoppered with 13 mm rubber stoppers inside the RABS unit. The stoppers are steam sterilized at 122 °C for 30 minutes. Capping The stoppered vials are transferred to the capping machine via conveyor belt under Grade A laminar flow protection. The stoppered vials are capped with 13 mm plastic aluminum flip-off caps. The caps are steam sterilized at 122 °C for 30 minutes. Visual Inspection A manual 100% visual inspection is performed on the capped vials by production personnel, followed by a statistically based AQL (acceptable quality limit) inspection by Quality Assurance.
  • AQL accepted quality limit
  • Container Closure System The container closure system for the drug product is a 2 mL (2R) Type I glass vial sealed with a 13 mm rubber stopper and a 13 mm flip-off aluminum cap. The components were selected for their durability to sterilization and depyrogenation processes, as well as non-reactive contact surfaces that are optimally compatible with proteins. The compatibility of the container closure system with the drug product is evaluated by the accelerated and long-term stability studies presented below. The integrity of the container closure system was demonstrated by the dye ingression test.
  • Drug product vials were immersed under colored dye and held under vacuum before depressurized and the infiltration of the dye into the vials examined.
  • the container and closure system achieved 100% air tightness.
  • Container closure integrity testing (CCIT) is performed annually in the stability program using a non-destructive vacuum decay method.
  • the product contact materials, the glass vial and rubber stopper have been tested per USP and Ph.Eur. requirements and are suitable for parenteral use. Compliance is verified on the Certificate of Conformance provided by the vendor with each lot of vials and stoppers.
  • Analytical procedures Color Color complies with Ph. Eur. 2.2.2. Chromatic aberration method is used for color measurement. Clarity Clarity complies with Ph. Eur.2.2.1. Light scattering method is used for clarity measurement.
  • iCIEF Whole column Imaging Capillary Isoelectric Focusing
  • pI is an intrinsic property of a specific protein molecule and is the pH at which the protein molecule does not carry any net electrical charge.
  • charge variants move along a continuous pH gradient formed by ampholytes and stop where the pH equals its pI.
  • the protein carries no net electrical charge and is not drawn by either of the electrodes. Therefore, different monoclonal antibody species with different pI values are separated and focused at different positions.
  • the pI values and relative abundance of the resolved peaks can be identified and quantified using chromatographic software.
  • the electropherogram should show comparable peak profile to the electropherogram of the reference standard.
  • the difference of pI values of the main peak between tested sample and the reference standard should be no more than 0.2.
  • SEC-HPLC Size Exclusion Chromatography-High Performance Liquid Chromatography is a purity analysis method that separates proteins based on their sizes.
  • the stationary phase is composed of inert particles packed into a dense three-dimensional matrix. The particles have small pores which only allow species below a certain size to enter. Larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules; the smaller the molecule, the longer the retention time. Following separation, the relative percentages of High Molecular Weight (HMW) species, monomer, and Low Molecular Weight (LMW) species are quantified via UV detection.
  • HMW High Molecular Weight
  • LMW Low Molecular Weight
  • CE-SDS Reduced Capillary Electrophoresis-Sodium Dodecyl Sulfate
  • BME reducing agent beta- mercaptoethanol
  • CE-SDS (Non-reduced)
  • CE-SDS (Non-reduced) is a purity analysis method that separates proteins based on their electrophoretic mobility, where smaller size proteins move faster than larger size proteins.
  • the test samples are denatured by heating in the presence of SDS.
  • the alkylating reagent N-Ethylmaleimide (NEM) is added to the sample solution in order to prevent the sulfhydryls from binding to other sulfhydryls. Separation is performed through an uncoated capillary and the protein samples are detected with a PDA detector at 220 nm. Results are reported as percent purity.
  • Bioburden Bioburden testing is performed by membrane filtration based on USP ⁇ 61> and Ph.
  • the endotoxin generated by gram-negative bacteria is detected using amebocyte lysate from the horseshoe crab, which coagulates with endotoxin.
  • the endotoxin concentration can be calculated.
  • ELISA Binding Assay
  • An ELISA method is used to assess the binding potency of the NI006/ALXN2220 antibody. Samples, control, and reference standard at appropriate dilutions are loaded onto misfolded- TTR (antigen for NI006/ALXN2220) coated half-area 96 well plate(s).
  • HRP horseradish peroxidase
  • TMB substrate solution is loaded into the wells. TMB specifically reacts with peroxide in the presence of peroxidase and produces a colorimetric signal that is proportional to the amount of NI006/ALXN2220 protein bound to the wells. The color development is stopped and optical density is measured at 450 nm (minus 560 nm for wavelength correction). Sample and reference standard dose response curves are plotted according to a 4 parameter logistic (auto-estimate) regression model using SoftMax Pro GxP software.
  • Cell-based Assay THP-1 is a human monocytic cell line.
  • NI006/ALXN2220 is an antibody against misfolded TTR.
  • the bioactivity of NI006/ALXN2220 is to stimulate THP-1 cell to produce IL-8 by binding the mis-TTR in cell culture.
  • NI006/ALXN2220 mAb standard final concentration: 2000 - 0.039 g/mL
  • misfolded-TTR final concentration: 10 ⁇ g/mL
  • IL-8 production is measured using human IL-8 ELISA kit.
  • the variability of the result is due to assay variability. All results from the in-use compatibility study are within the acceptance criteria. The result is not considered safety relevant considering that, within the short in-use timeframe, the most probable incompatibility with plastics materials would be adsorption (addressed by protein concentration) and visible particle formation (addressed by the compendial method). In the saline group, visible particles were observed, indicating that NI006/ALXN2220 is less stable in saline. The data show that when using glucose as the diluent, no substantial changes in the appearance, protein concentration, sub-visible particle, SEC-HPLC, and ELISA binding assay were observed.
  • NI006/ALXN2220 at concentrations of 1.0 mg/mL, 20.0 mg/mL, and 50.0 mg/mL is stable at 2 - 8 °C for 24 hours followed by 25 °C for 6 hours (30 hours in total).
  • NI006/ALXN2220 is compatible with the clinical in-use materials evaluated.
  • the potency of the reference standard is assigned a value of 100% relative potency. Stability Studies Non-clinical lot 201901004 and clinical lot 201903038 were placed on stability testing.
  • the non-clinical lot (201901004) has 1 month of stress stability data, 6 months of accelerated stability data, and 18 months of long-term stability data.
  • the clinical lot (201903038) has 1 month of stress stability data and 6 months of accelerated stability data, and 12 months of long- term stability data. Under stress conditions, both clinical lot (201903038) and nonclinical lot (201901004) show trend of significant decrease in iCIEF main peak (%) and increase in acidic peaks (%), while no significant changes were observed in other purity assays as well as ELISA binding assay.
  • the shelf life for the drug product is currently set at 24 months stored at 5 ⁇ 3 ⁇ C, protected from light.
  • the available in-use stability and compatibility data, presented above, indicate that the ready-to-use solution for infusion is stable for up to 24 hours at 2-8°C followed by 6 hours at 25°C after dilution with 5% glucose solution. From a microbiological perspective, the solution for infusion should be used immediately. If not used immediately, the in-use shelf life is set to 4 hours at room temperature or 24 hours at 2 - 8 °C.
  • Tables 15 to 18 summarize the available stress data for the non-clinical lot 201901004 and clinical lot 201903038. Table 15.
  • Accelerated Stability Data for Exploratory Specification for Non-Clinical Lot 201901004 at 25 ⁇ 2 °C / 60 ⁇ 5% RH T est T0 1 Month 3 Months 6 Months Cell-based Assay 129% 117% 140% 94% Table 21. Accelerated Stability Data for Clinical Lot 201903038 at 25 ⁇ 2 °C / 60 ⁇ 5% RH Acceptance Test T0 1 Month 3 Months 6 Months Criteria Report result (refer Color B7 B7 B7 B7 to Ph.
  • the tested pharmaceutical formulation is long-term stable.
  • the formulation remains liquid with no visible particles, the pH remains constant, and the monomer content as measured by SEC-HPLC does not drop below 96%, meaning that the content of HMWS and LMWS remains under 4% under all tested conditions.
  • Example 5 Antibody NI006/ALXN2220-mediates amyloid depletion from patient cardiac tissue as demonstrated using high-resolution live-cell imaging Extracellular deposition of transthyretin amyloid (ATTR) is the hallmark of ATTR cardiomyopathy and reducing cardiac ATTR load is a key therapeutic goal to achieve improvements in cardiac function.
  • TRR transthyretin amyloid
  • Amytracker 680 at 1 ug/mL was prepared in H 2 O and applied on the tissue sections for 5 min at RT, followed by destaining in 70% alcohol and washing in H 2 O before continuing with the amyloid depletion assay.
  • Another approach was to use the antibody NI006/ALXN2220 labelled with the fluorescent dye Vivotag-680. The antibody labelling reaction was directed to free amines, performed as indicated in the instruction manual, and followed by dialysis to remove unbound dyes.
  • Macrophages were generated by differentiation of THP1 cells (THP1 Null2 cells: Invivogen, reference #thp-nullz, Lot: T49-4101) with phorbol 12-myristate-13-acetate (PMA, Sigma P8139) at 100 ng/mL for 3 days, followed by 3 days of rest without PMA. Macrophages were detached by trypsinization and distributed to the tissue sections at a density of 400000 cells per well (Micro-dishes: Reference # 80136, ⁇ -Dish 35 mm, low, polymer coverslip) in presence of NI006/ALXN2220 or isotype control at 10 nM (1.5 ug/mL).
  • NI006/ALXN2220 was labelled with the green fluorescent dye A488 and selective binding of NI006/ALXN2220 to ATTR could be shown as indicated by the overlap between red and green fluorescence; see Fig. 7 (A) to (D). It was further shown that NI006/ALXN2220 triggered amyloid phagocytosis by macrophages.
  • Fig. 8 The punctate and intracellular red fluorescence pattern as visualized in Fig. 8 revealed the presence of ATTR amyloid in phagocytic vesicles. Macrophages presented a broad range of phagocytic activity indicated by the number of red fluorescent vesicles. Furthermore, the experiments revealed that macrophages with two nuclei formed spontaneously under the culture conditions. Fig. 9 shows one such multinucleated cell successively detaching fragments of amyloid from a large deposit. The macrophage depicted in Fig. 10 was observed separating a thin and elongated ATTR deposit from the adjacent cardiomyocytes, moving the deposit by more than 20 ⁇ m.
  • the macrophage also severed a protruding large amyloid deposit, detaching small and large fragments of amyloid.
  • NI006/ALXN2220 binding to ATTR amyloid in patient myocardium tissue sections was demonstrated using fluorescently labelled NI006/ALXN2220.
  • NI006/ALXN2220 triggered the process of macrophage recruitment to amyloid deposits, followed by amyloid fragmentation and internalization, and intracellular degradation, whereas multinucleated cells participated in the phagocytosis of large amyloid fragments.
  • macrophages did not phagocytize cardiac ATTR amyloid.
  • NI006/ALXN2220-mediated amyloid depletion from patient cardiac tissue could be demonstrated, which further improved the understanding of the cellular mechanism underlying antibody-mediated depletion of amyloid as a precision therapy for patients with ATTR cardiomyopathy.

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Abstract

L'invention concerne des anticorps anti-transthyrétine (TTR), des polynucléotides et des vecteurs d'expression correspondants ainsi que des compositions (par exemple, des compositions pharmaceutiques) et des articles manufacturés associés qui contiennent l'anticorps anti-TTR en tant que médicament. L'invention concerne également des méthodes de traitement ou de prévention de l'amyloïdose à médiation par la transthyrétine (ATTR) chez un sujet en ayant besoin à l'aide des compositions pharmaceutiques. De plus, l'invention concerne des procédés de validation, d'identification et de criblage d'un médicament à déplétion d'amyloïdes à l'aide d'une imagerie de cellules vivantes à haute résolution ainsi que des procédés de production d'une composition pharmaceutique d'un médicament à déplétion d'amyloïdes et des kits appropriés pour une utilisation dans lesdits procédés.
PCT/EP2024/082486 2023-11-15 2024-11-15 Anticorps anti-transthyrétine, compositions comprenant ledit anticorps et méthodes de traitement ou de prévention de l'amyloïdose à médiation par la transthyrétine Pending WO2025104243A1 (fr)

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