FCRN Antibody Composition

Abstract

The present disclosure relates to compositions comprising the anti-FcRn antibody M281. The compositions include the complete, intact antibody and its size variants that do not contain two antibody heavy chains and two antibody light chains. Thus, an M281 pharmaceutical composition may contain an antibody containing a heavy chain comprising the amino acid sequence of SEQ ID NO:2 and a light chain comprising the amino acid sequence of SEQ ID NO:1, and the composition contains a major protein component with a molecular weight of 140,000 to 145,000 Da and a minor protein component with a molecular weight of 118,000 to 120,000 Da.

Description

[Technical field]

[0001] The present disclosure relates to compositions of Fc receptor (FcRn) antibodies. [Background technology]

[0002] Numerous autoimmune and alloimmune diseases are mediated by pathogenic antibodies. The stability, activity and transport of pathogenic antibodies depend on the neonatal Fc receptor (FcRn), a type I transmembrane protein that functions as an IgG-binding and serum albumin-binding intracellular vesicle transport protein. For example, many fetal and neonatal immune diseases are caused by the transfer of maternal antibodies from pregnant subjects, especially those with immunological diseases, to fetuses via human neonatal Fc receptor (FcRn) in the placenta. Summary of the Invention [Means for solving the problem]

[0003] The present disclosure relates to compositions comprising anti-FcRn antibodies ("M281 compositions"). The compositions include complete intact antibodies (i.e., antibodies having two antibody light chains and two antibody heavy chains) and size variants thereof that do not contain two antibody heavy chains and two antibody light chains, but instead contain two antibody heavy chains and only one antibody light chain. Thus, the M281 pharmaceutical composition may comprise an antibody (LHHL) comprising two heavy chains comprising or consisting of the amino acid sequence of SEQ ID NO:2 and two light chains comprising or consisting of the amino acid sequence of SEQ ID NO:1, the composition comprising a major protein component having a molecular weight of 140,000 to 145,000 Da (e.g., 140,500 to 143,000 Da) and a minor (molecular) component having a molecular weight of 118,000 to 120,000 Da (e.g., 119,000 to 120,000 Da or 119,150 to 119,350 Da). The major component comprises at least 80% (81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% by weight) of the protein in the composition, and the minor component comprises at least 0.8%, 1%, 2%, 3%, but not more than 20% (19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4% by weight) of the protein in the composition. Thus, for example, a minor protein component of molecular weight 118,000-120,000 Da may represent 0.8-2%, 1-2%, 0.8-3%, or 1-4% by weight of the protein in the composition.

[0004] In various embodiments, the major protein component is at least 99% by weight of the protein in the composition; the secondary protein component includes an antibody variant comprising two heavy chains and one light chain; the antibody variant comprises an unpaired heavy chain (comprising or consisting of the amino acid sequence of SEQ ID NO:2) and a paired heavy chain (comprising or consisting of the amino acid sequence of SEQ ID NO:2) and a light chain (comprising or consisting of the amino acid sequence of SEQ ID NO:1); the antibody variant comprises an unpaired heavy chain comprising a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:2, in which the C at position 219 is replaced by dehydroalanine; and the secondary protein component includes a) a first antibody variant comprising an unpaired heavy chain (comprising or consisting of the amino acid sequence of SEQ ID NO:2) and a paired heavy chain (comprising or consisting of the amino acid sequence of SEQ ID NO:2) and a light chain (comprising or consisting of the amino acid sequence of SEQ ID NO:); and b) a second antibody variant comprising an unpaired heavy chain comprising a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:2, in which the C at position 219 is replaced by dehydroalanine.

[0005] A method for preparing a pharmaceutical composition is described herein. The method includes the steps of: providing an antibody composition comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:2 and a light chain comprising the amino acid sequence of SEQ ID NO:1; determining whether the composition comprises a major protein component having a molecular weight of 140,000-145,000 Da (e.g., 140,500-143,000 Da) and a minor protein component having a molecular weight of 118,000-120,000 Da (e.g., 119,000-120,000 Da or 119,150-119,350 Da); determining that the composition contains a major protein component having a molecular weight of 140,000 to 145,000 Da (e.g., 140,500 to 143,000 Da) and a minor protein component having a molecular weight of 118,000 to 120,000 Da (e.g., 119,000 to 120,000 Da or 119,150 to 119,350 Da), and combining the composition with one or more pharma- ceutically acceptable excipients to prepare a pharmaceutical composition.

[0006] In various embodiments of the method, the prepared pharmaceutical composition comprises 28-32 mg / ml of an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:2 and a light chain comprising the amino acid sequence of SEQ ID NO:1; the prepared pharmaceutical composition comprises 9-11 mg / ml of an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:2 and a light chain comprising the amino acid sequence of SEQ ID NO:1; the method further comprises combining the composition with one or more pharma- ceutically acceptable excipients to prepare a pharmaceutical composition only if the major component is at least 90% by weight of the protein in the composition and the minor component is at least 3% by weight of the protein in the composition; the determining step comprises electrophoresis or chromatography; and the providing step comprises culturing cells expressing the heavy and light chains.

[0007] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials for use in the present invention are described herein, and other suitable methods and materials known in the art can also be used.Materials, methods, and examples are merely illustrative and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.

[0008] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. [Brief description of the drawings]

[0009] [Figure 1] Representative capillary electrophoresis (CE-SDS) electropherogram of M281 composition measured under non-reducing conditions; predominant intact IgG is labeled as (2) and size variants as (1). [Figure 2A-2B] Graphs showing representative data for M281 compositions analyzed by hydrophobic interaction liquid chromatography coupled with mass spectrometry (HILIC LC-MS); (A) UV chromatogram of M281 composition analyzed by hydrophobic interaction liquid chromatography with predominant light chain-heavy chain-heavy chain-light chain is labeled as (2) and size variants are labeled as (1); (B) Base peak chromatogram from mass spectrometry with predominant light chain-heavy chain-heavy chain-light chain is labeled as (2) and size variants are labeled as (1a, retention time 12.42 min; 1b, retention time 12.68 min). [Figure 3A-3B] Representative mass spectrometry results of selected fractions isolated by hydrophobic interaction liquid chromatography; (A) shows the mass spectrometry results of the fraction containing FIG. 2(1a); (B) shows the mass spectrometry results of the fraction containing FIG. 2(1b). [Figure 4] 1 is a cartoon representation of a peptide containing a disulfide bridge that has been subjected to trypsin digestion and then undergoes β-elimination in the presence of heat or reduction. [Diagram 5] Representative charge electropherogram (CE) of M281 composition; the major isoform eluted between 3.03 and 3.13 minutes, and the minor isoform eluted between 3.23 and 3.31 minutes. [Figure 6] FIG. 6 is a graph showing representative masses of isolated samples containing minor isoforms (fractions eluting at 3.23-3.31 min, as shown in FIG. 5) following analysis by microchip zone electrophoresis separation with direct electrospray ionization mass spectrometry. [Figure 7] FIG. 1 is a schematic flow chart of a method including the steps of separating compositions by SDS-PAGE gel electrophoresis, isolating select size variants in each composition by cutting out pages of the SDS-PAGE gel, digesting the isolated size variants with trypsin, and analyzing the mass distribution of the isolated and trypsin-digested size variants by nanoLC-MS. [Figure 8] FIG. 1 shows the chemical representation and molecular weights of predicted peptides resulting from trypsin digestion of (A) light chain-heavy chain-heavy chain-light chain (LC-HC-HC-LC); (B) heavy chain-heavy chain-light chain (HC-HC-LC); (C) heavy chain-heavy chain-light chain with dehydroalanine replacing Cys219 of the unpaired heavy chain. [Figure 9] Graph showing interchain disulfide bond quantification of LC-HC-HC-LC (data shown in blue) and HC-HC-LC (data shown in orange) assessed for isolated compositions containing LC-HC-LC-HC, Cys-A, DeHA-A, and Cys-HC-HC. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Detailed Description The present invention features novel compositions comprising antibodies targeted to the human neonatal Fc receptor (FcRn). These compositions are useful, for example, to promote clearance of autoantibodies in a subject, to suppress antigen presentation in a subject, to block an immune response (e.g., to block immune complex-based activation of an immune response in a subject), or to treat an immunological disease (e.g., an autoimmune disease) in a subject. Disclosed herein is a composition comprising the aforementioned isolated antibody and one or more size variants, wherein the final composition contains at least 80% of the total protein content comprising fully assembled light chain-heavy chain-heavy chain-light chain (LC-HC-HC-LC) having a molecular weight of about 140,000-143,000 (e.g., 141,750-141,800) Da, and up to 5% (4%, 3%, 2%, 1%, 0.8%) of the total protein content comprising a selected size variant of lower molecular weight (e.g., 118,000-120,000 Da).

[0011] Anti-FcRn antibody Antibodies that may be formulated as described herein include those having the light chain sequence

[0012] [ka] and heavy chain sequence

[0013] [ka] The antibody has the formula:

[0014] Variants of this antibody may also be formulated as described herein. Such variants include antibodies having a light chain sequence of a variant of SEQ ID NO: 1 with 1-5 single amino acid substitutions or deletions (and preferably including the CDR sequences of SEQ ID NO: 3-5) and a heavy chain sequence of a variant of SEQ ID NO: 24 with 1-5 single amino acid substitutions or deletions (and preferably including the CDR sequences of SEQ ID NO: 6-8). Antibodies consisting of a variant of SEQ ID NO: 1 and a variant of SEQ ID NO: 4 preferably retain the CDR sequences of M281: TGTGSDVGSYNLVS (light chain CDR1; SEQ ID NO: 3); GDSERPS (light chain CDR2; SEQ ID NO: 4); SSYAGSGIYV (light chain CDR3; SEQ ID NO: 5); TYAMG (heavy chain CDR1; SEQ ID NO: 6); SIGASGSQTRYADS (heavy chain CDR2; SEQ ID NO: 7); and LAIGDSY (heavy chain CDR3; SEQ ID NO: 8).

[0015] In some instances, the light chain is

[0016] [ka] The sequence has at least 90%, 95%, or 98% identity to

[0017] In some instances, the heavy chain comprises:

[0018] [ka] The sequence has at least 90%, 95%, or 98% identity to

[0019] In some cases, the antibody contains amino acid substitutions, additions, and / or deletions in the constant region (e.g., Fc region) of the antibody that lead to, for example, reduced effector functions, such as reduced complement-dependent cytolysis (CDC), antibody-dependent cell-mediated cytolysis (ADCC), and / or antibody-dependent cell-mediated phagocytosis (ADCP), and / or reduced B-cell killing. The constant region is not directly involved in the binding of the antibody to its target, but exhibits various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity. In some cases, the antibody is characterized by reduced binding (i.e., lack of binding) to human complement factor C1q and / or human Fc receptors on natural killer (NK) cells. In other cases, the antibody is characterized by reduced binding (i.e., lack of binding) to human FcγRI, FcγRIIA, and / or FcγRIIIA. To alter or reduce antibody-dependent effector function, such as CDC, ADCC, ADCP, and / or B-cell killing, the antibody may be of the IgG class and may contain one or more amino acid substitutions E233, L234, G236, D265, D270, E318, K320, K322, A327, A330, P331, and / or P329 (EU numbering throughout, unless otherwise specified (Edelman et al., Proc. Natl. Acad. USA, 63:78-85 (1969)). In some cases, the antibody has the mutations L234A / L235A or D265A / N297A. In some cases, the antibody contains the amino acid substitution A297N relative to the sequence of SEQ ID NO:2, which changes the antibody to a glycosylated form. Antibodies with glycosylation at amino acid N297 are predicted to bind complement or Fc receptors (i.e., complement C1q binding), while antibodies with N297A (e.g., SEQ ID NO:2) are predicted to have little binding to complement or Fc receptors (i.e., complement C1q binding) and exhibit low CDC potential. In other cases, M281 contains a C-terminal lysine at residue 446 relative to SEQ ID NO:2. In some cases, the amino terminal Gln of the light chain is a pyroGln.

[0020] Vectors, host cells, and antibody production Anti-FcRn antibodies can be produced from host cells. Host cells refer to vehicles that contain the necessary cellular components, e.g., organelles, required for expressing the polypeptides and constructs described herein from their corresponding nucleic acids. Nucleic acids can be contained in nucleic acid vectors that can be introduced into host cells by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.). The selection of nucleic acid vectors depends in part on the intended host cells to be used. Generally, preferred host cells are of prokaryotic (e.g., bacterial) or eukaryotic (e.g., mammalian) origin.

[0021] Nucleic acid vector construction and host cells Nucleic acid sequences encoding the amino acid sequence of anti-FcRn antibodies can be prepared by a variety of methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. Nucleic acid molecules encoding anti-FcRn antibodies can be obtained using standard techniques, such as gene synthesis. Alternatively, nucleic acid molecules encoding wild-type anti-FcRn antibodies can be mutated to contain specific amino acid substitutions using standard techniques in the art, such as QuikChange™ mutagenesis. Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR techniques.

[0022] The nucleic acid sequence encoding anti-FcRn antibody can be inserted into a vector that can replicate and express the nucleic acid molecule in prokaryotic or eukaryotic host cells.Many vectors are available in the art and can be used.Each vector can contain various components that can be adjusted and optimized for compatibility with specific host cells.For example, vector components can include, but are not limited to, origin of replication, selection marker gene, promoter, ribosome binding site, signal sequence, nucleic acid sequence encoding protein of interest, and transcription termination sequence.

[0023] Mammalian cells can be used as host cells. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NS0, Sp2 / 0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells. Elsewhere, E. coli cells can be used as host cells. Examples of E. coli strains include E. coli 294 (ATCC® 31,446), E. coli λ1776 (ATCC® 31,537, E. coli BL21(DE3) (ATCC® BAA-1025), and E. The host cells include, but are not limited to, Escherichia coli RV308 (ATCC® 31,608). Various host cells have characteristic and specific mechanisms for post-translational processing and modification of protein products. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of the expressed anti-FcRn antibody. The above expression vectors can be introduced into suitable host cells using conventional techniques in the art, such as transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. Once the vector is introduced into the host cells for protein production, the host cells are cultured in conventional nutrient media modified as necessary to induce promoters, select transformants, or amplify genes encoding desired sequences.Methods for expression of therapeutic proteins are known in the art, see, e.g., Paulina Balbas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 (July 20, 2004) and Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed. 2012 (June 28, 2012).

[0024] Protein production, recovery, and purification The host cells used to produce anti-FcRn antibodies may be grown in media known in the art and suitable for culturing the selected host cells. Examples of suitable media for mammalian host cells include Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Expi293™ Expression Medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. Examples of suitable media for bacterial host cells include Luria Broth (LB) with the addition of necessary supplements such as a selection agent, e.g., ampicillin. The host cells are cultured at an appropriate temperature, such as about 20° C. to about 39° C., e.g., 25° C. to about 37° C., preferably 37° C., and at a CO2 level, such as 5-10% (preferably 8%). The pH of the medium is generally about 6.8-7.4, e.g., 7.0, depending mainly on the host organism. When an inducible promoter is used in the expression vector, protein expression is induced under conditions suitable for activation of the promoter.

[0025] Protein recovery typically involves disrupting the host cells, generally by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris can be removed by centrifugation or filtration. The protein can be further purified. The anti-FcRn antibody can be purified by any method known in the art for protein purification, such as protein A affinity, other chromatography (e.g., ion exchange, affinity, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. (See Process Scale Purification of Antibodies, Uwe Gottschalk (ed.) John Wiley & Sons, Inc., 2009.) In some cases, the anti-FcRn antibody can be conjugated to a marker sequence, such as a peptide, that facilitates purification. An example of a marker amino acid sequence is a 6-histidine peptide (His tag), which binds to a nickel-functionalized agarose affinity column with micromolar affinity. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein.

[0026] Treatment methods and indications Blocking human FcRn by pharmaceutical compositions containing anti-FcRn antibodies described herein can be therapeutically beneficial in diseases driven by IgG autoantibodies.The ability of FcRn blockade to induce global IgG catabolism and the removal of multiple species of autoantibodies, small circulating metabolites, or lipoproteins provides a way to extend the practicality and availability of autoantibody removal strategies to patients with autoimmune disease pathology driven by autoantibodies.Without being bound by any theory, the predominant mechanism of action of anti-FcRn antibodies may be to increase the catabolism of pathogenic autoantibodies in circulation and reduce autoantibody and immune complex accumulation in affected tissues.

[0027] The pharmaceutical compositions are useful for promoting catabolism and clearance of pathogenic antibodies, such as IgG and IgG autoantibodies, reducing immune responses, such as blocking immune complex-based activation of immune responses in a subject, and treating immunological conditions or diseases in a subject. In particular, the pharmaceutical compositions are useful for reducing or treating immune complex-based activation of acute or chronic immune responses. The acute immune response may be activated by a medical condition selected from the group consisting of pemphigus vulgaris, lupus nephritis, myasthenia gravis, Guillain-Barre syndrome, antibody-mediated rejection antiphospholipid syndrome (e.g., fulminant antiphospholipid syndrome), immune complex-mediated vasculitis, glomerulitis, channelopathy, neuromyelitis optica, autoimmune deafness, idiopathic thrombocytopenic purpura (ITP), autoimmune hemolytic anemia (AIHA), immune neutropenia, dilated cardiomyopathy, and serum sickness. The chronic immune response may be activated by a medical condition selected from the group consisting of chronic inflammatory demyelinating polyneuropathy (CIDP), systemic lupus, chronic forms of disorders indicated for acute treatment, reactive arthropathy, primary biliary cirrhosis, ulcerative colitis, and antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis.

[0028] In some cases, the pharmaceutical compositions are useful for reducing or treating immune responses activated by autoimmune diseases, including but not limited to alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, Addison's disease, hemolytic anemia, autoimmune hepatitis, hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, localized scleroderma (CREST syndrome), cold agglutinin disease, Crohn's disease, dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia, fibromyositis, Graves' disease, Hashimoto's thyroiditis, hypothyroidism, inflammatory bowel disease, autoimmune lymphoproliferative syndrome, idiopathic rheumatoid arthritis, ... In one embodiment, the inflammatory bowel disease may be selected from the group consisting of chronic pulmonary fibrosis, IgA nephropathy, insulin-dependent diabetes mellitus, juvenile arthritis, lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, Takayasu's arteritis, temporal arteritis, ulcerative colitis, uveitis, vitiligo, and Wegener's granulomatosis.

[0029] In particular, the pharmaceutical compositions are useful for reducing or treating immune responses activated by systemic lupus erythematosus, antiphospholipid syndrome, pemphigus vulgaris / bullous pemphigoid, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, myasthenia gravis, or neuromyelitis optica.

[0030] In some cases, the pharmaceutical compositions are useful for reducing the risk of anemia or reducing the risk of developing anemia in the fetus. In some cases, the pharmaceutical compositions are useful for reducing or eliminating the need for IUT (intrauterine transfusion). In some cases, the pharmaceutical compositions and methods are useful for reducing or eliminating the need for prenatal PP+IVIg, postnatal transfusion, IVIg, and / or phototherapy.

[0031] In some cases, the pharmaceutical compositions are useful for reducing or treating immune responses activated by autoimmune diseases, including but not limited to alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, Addison's disease, hemolytic anemia, autoimmune hepatitis, hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, localized scleroderma (CREST syndrome), cold agglutinin disease, Crohn's disease, dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia, fibromyositis, Graves' disease, Hashimoto's thyroiditis, hypothyroidism, inflammatory bowel disease, autoimmune lymphoproliferative syndrome, idiopathic rheumatoid arthritis, ... In one embodiment, the inflammatory bowel disease may be selected from the group consisting of chronic pulmonary fibrosis, IgA nephropathy, insulin-dependent diabetes mellitus, juvenile arthritis, lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, Takayasu's arteritis, temporal arteritis, ulcerative colitis, uveitis, vitiligo, and Wegener's granulomatosis.

[0032] In some cases, the pharmaceutical compositions are useful for reducing or treating an immune response in a fetus or newborn. In some cases, the pharmaceutical compositions and methods are useful for reducing or treating an immune response in a fetus or newborn that has been activated by an autoimmune disease in a pregnant mother.

[0033] In particular, the pharmaceutical compositions are useful for reducing or treating immune responses activated by systemic lupus erythematosus, antiphospholipid syndrome, pemphigus vulgaris / bullous pemphigoid, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, myasthenia gravis, or neuromyelitis optica. In some cases, the pharmaceutical compositions are useful for reducing or treating immune responses in fetuses or newborns. In some cases, the pharmaceutical compositions and methods are useful for reducing or treating immune responses activated by systemic lupus erythematosus, antiphospholipid syndrome, pemphigus vulgaris / bullous pemphigoid, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, myasthenia gravis, or neuromyelitis optica in pregnant mothers.

[0034] The pharmaceutical composition is useful in methods for decreasing pathogenic antibody transport (e.g., pathogenic maternal IgG antibody transport) across the placenta of a pregnant subject, increasing pathogenic antibody catabolism in a pregnant subject, and treating antibody-mediated enhancement of viral disease in a fetus or newborn by administering an isolated antibody that binds to human FcRn to the pregnant subject. Diseases and disorders that can benefit from FcRn inhibition by the pharmaceutical compositions described herein include diseases and disorders in the fetus and / or newborn caused by the transfer of maternal pathogenic antibodies (e.g., maternal pathogenic IgG antibodies) across the placenta from the pregnant subject to the fetus and / or newborn.

[0035] In some cases, the disease and disorder that can benefit from the treatment with pharmaceutical compositions described herein is fetal and neonatal alloimmune and / or autoimmune disorder.Fetal and neonatal alloimmune disorder is the disorder in fetus and / or neonatal caused by pathogenic antibody in pregnant subject.Pathogenic antibody in pregnant subject can attack the antigen of fetus (e.g., antigen that fetus inherits from its father), causing fetus or neonatal to have fetal and neonatal alloimmune and / or autoimmune disorder.

[0036] Examples of fetal and neonatal alloimmune and / or autoimmune disorders that may be treated include, but are not limited to, fetal and neonatal alloimmune thrombocytopenia (FNAIT), hemolytic disease of the fetus and newborn (HDFN), alloimmune pantithrombocytopenia, congenital heart block, fetal arthrogryposis, neonatal myasthenia gravis, neonatal autoimmune hemolytic anemia, neonatal antiphospholipid syndrome, neonatal polymyositis, dermatomyositis, neonatal lupus, neonatal scleroderma, Behcet's disease, neonatal Graves' disease, neonatal Kawasaki disease, neonatal autoimmune thyroid disease, and neonatal type I diabetes.

[0037] In some cases, the diseases and disorders that can benefit from treatment with the pharmaceutical compositions described herein are viral diseases, where antibodies promote virus entry into host cells, leading to increased or enhanced infectivity in cells, such as antibody-mediated enhancement of viral diseases.In some cases, antibodies can bind to virus surface proteins, and the antibody / virus complex can bind to FcRn on the cell surface through the interaction between the antibody and the receptor.The antibody / virus complex can then be internalized into the cell.For example, the virus can enter fetal cells and / or tissues by forming a complex with maternal IgG antibodies.The maternal IgG antibodies can bind to virus surface proteins, and the IgG / virus complex can bind to FcRn in the syncytiotrophoblast of the placenta, which then transfers the complex to the fetus.

[0038] In some cases, the pharmaceutical compositions described herein may be used to treat antibody-mediated enhancement of viral diseases. In some cases, viral diseases enhanced by pathogenic antibodies (e.g., pathogenic IgG antibodies) include alphavirus infection, flavivirus infection, Zika virus infection, chikungunya virus infection, Ross River virus infection, severe acute respiratory syndrome coronavirus infection, Middle East respiratory syndrome, avian influenza infection, influenza virus infection, human respiratory syncytial virus infection, Ebola virus infection, yellow fever virus infection, dengue virus infection, human immunodeficiency virus infection, respiratory syncytial virus infection, Hantavirus infection, Getah virus infection, Sindbis virus infection, Bunyamwera virus infection, West Nile virus infection, etc. These include, but are not limited to, viral diseases caused by, for example, rabies virus infection, Japanese encephalitis virus B infection, rabbit pox virus infection, lactate dehydrogenase elevated virus infection, reovirus infection, rabies virus infection, foot and mouth disease virus infection, porcine reproductive and respiratory syndrome virus infection, simian hemorrhagic fever virus infection, equine infectious anemia virus infection, caprine arthritis virus infection, African swine fever virus infection, lentivirus infection, BK papovavirus infection, Murray Valley encephalitis virus infection, enterovirus infection, cytomegalovirus infection, pneumovirus infection, morbillivirus infection, and measles virus infection.

[0039] Blockade of human FcRn by anti-FcRn antibodies may be therapeutically beneficial in diseases driven by pathogenic antibodies (e.g., pathogenic IgG antibodies). The ability of FcRn blockade to induce global pathogenic antibody catabolism and removal of multiple species of pathogenic antibodies, small circulating metabolites, or lipoproteins without perturbing serum albumin provides a way to extend the utility and availability of pathogenic antibody removal strategies to patients with autoimmune disease pathology driven by pathogenic antibodies. Without being bound by theory, the predominant mechanism of action of anti-FcRn antibodies may be to increase the catabolism of pathogenic antibodies in the circulation and reduce pathogenic antibodies and immune complex accumulation in affected tissues.

[0040] The pharmaceutical composition described herein can be administered to a pregnant subject who has or is at risk of having a medical condition that activates immune response in the pregnant subject. In some cases, the pregnant subject may have a medical condition that activates immune response in the pregnant subject in the past. In some cases, the pregnant subject has a medical history of having a previous fetus or newborn with fetal and newborn alloimmune and / or autoimmune disorder. In some cases, when a pathogenic antibody associated with immune disease is detected in a biological sample (e.g., blood or urine sample) obtained from the pregnant subject, the anti-FcRn antibody described herein can be administered to the pregnant subject. In some cases, the pathogenic antibody detected in the biological sample of the pregnant subject is known to bind to an antigen derived from the fetus in the pregnant subject (e.g., an antigen that the fetus inherits from the father of the fetus).

[0041] In some cases, the pharmaceutical composition can be administered to a subject who is planning to become pregnant, has or is at risk of having a medical condition that activates immune response in the pregnant subject, and / or has previously had a medical condition that activates immune response in the pregnant subject.In some cases, the subject is planning to become pregnant and has a medical history of having a previous fetus or newborn with fetal and newborn alloimmune and / or autoimmune disorder.In some cases, the anti-FcRn antibody described herein can be administered to a subject who is planning to become pregnant and whose biological sample contains pathogenic antibodies associated with immune disease.

[0042] In some cases, the pharmaceutical compositions described herein may be administered to a subject (e.g., a pregnant subject) to reduce or treat immune complex-based activation of an acute or chronic immune response in the subject. The acute immune response may be activated by a medical condition (e.g., pemphigus vulgaris, lupus nephritis, myasthenia gravis, Guillain-Barre syndrome, antibody-mediated rejection, fulminant antiphospholipid syndrome, immune complex-mediated vasculitis, glomerulitis, channelopathy, neuromyelitis optica, autoimmune hearing loss, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, immune neutropenia, dilated cardiomyopathy, serum sickness, chronic inflammatory demyelinating polyneuropathy, systemic lupus, reactive arthropathy, primary biliary cirrhosis, ulcerative colitis, or antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis).

[0043] In some cases, the formulations described herein may be administered to a subject (e.g., a pregnant subject) to reduce or treat an immune response activated by an autoimmune disease. Autoimmune diseases include, for example, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome (e.g., antiphospholipid antibody syndrome), epidermolysis bullosa, membranous nephropathy, Addison's disease, hemolytic anemia, warm autoimmune hemolytic anemia (wAIHA), antifactor antibodies, heparin-induced thrombocytopenia (HICT), sensitized transplantation, autoimmune hepatitis, hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, localized scleroderma (CREST syndrome), cold agglutinin disease, Crohn's disease, dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia, fibromyositis, Gleicher's disease, and others. The condition may be rheumatoid arthritis, Hashimoto's thyroiditis, hypothyroidism, inflammatory bowel disease, autoimmune lymphoproliferative syndrome, idiopathic pulmonary fibrosis, IgA nephropathy, insulin-dependent diabetes mellitus, juvenile arthritis, lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, Takayasu's arteritis, temporal arteritis, ulcerative colitis, uveitis, vitiligo, or Wegener's granulomatosis. EXAMPLES

[0044] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0045] The following materials and methods were used in the examples presented herein.

[0046] material M281 production: Light and heavy chain producing cell cultures were clarified by centrifugation and filtration followed by viral inactivation using detergent treatment. After viral inactivation, the material was applied to a Protein A column to remove process related impurities (e.g., host cell proteins (HCPs), DNA, and media additives). The eluate was applied to an anion exchange column and a virus removal filter. After viral removal and prior to concentration and diafiltration using a buffer of 25 mM sodium phosphate and 25 mM sodium chloride at pH 6.5, further filtration was performed through a polyethersulfone membrane with a nominal molecular weight cut-off of 30 kDa. The material was formulated by the addition of trehalose to a final concentration of 8.7% w / w and polysorbate 80 to a final concentration of 0.01% weight / volume (w / v). Dilute M281 to a target of 30 mg / mL (27-33 mg / mL range) using formulation buffer (25 mM sodium phosphate, 25 mM sodium chloride, 8.7% trehalose, 0.01% w / v polysorbate 80, pH 6.5).

[0047] [Example 1] Detection of size variant species Size variant species were reproducibly detected and analyzed by non-reducing capillary electrophoresis with SDS (NR CE-SDS) in compositions containing M281 antibody produced using the methods described herein.

[0048] result A composition of M281 was prepared and analyzed by NR CE-SDS. The electropherogram read of the M281 composition analyzed by NR CE-SDS showed two separate peaks, one with a retention time of 26-27 min and the other with a retention time of 27.5-29 min (Figure 1). The latter peak corresponded to fully assembled light chain-heavy chain-heavy chain-light chain (LC-HC-HC-LC), while the former peak corresponded to a size variant molecule with a lower mass compared to the heavy chain-heavy chain-light chain (referred to as HC-HC-LC or HHL). The level of size variant molecules detected by NR CE-SDS remained at approximately 4.0-4.5 percent of the total protein content throughout all downstream processing steps, GMP DS and DP lots, and during stability studies (data not shown). In addition, there was no significant change in the level of this size variant as detected by NR CE-SDS during scale-up from the 250 L pilot level to the GMP 2000 L scale. The level of this size variant. The formation of size variants in M281 compositions increased when the compositions were exposed to denaturing conditions, such as increasing the incubation temperature (e.g., from 37° C. to 70° C.) during sample preparation or prior to CE-SDS analysis.

[0049] Example 2. Molecular weight characterization of size variant species detected in M281 compositions To determine the molecular masses of size variant species in the M281 composition, the M281 composition was analyzed by three methods including hydrophobic interaction liquid chromatography coupled to mass spectrometry (HILIC LC-MS), microchip zone electrophoretic separation with direct electrospray ionization mass spectrometry, and non-reducing tryptic digestion of proteins separated by gel electrophoresis followed by nano-liquid chromatography coupled to mass spectrometry.

[0050] result Analysis of the M281 composition by HILIC LC-MS revealed a peak centered at a retention time of 12.5 min in the UV chromatogram of the HILIC separation (Figure 2A), which resolved into two species by mass analysis, one centered at a retention time of 12.42 min and the other centered at a retention time of 12.68 min (Figure 2B). The distribution of molecular masses in the peaks at retention times of 12.42 min and 12.68 min was determined by deconvolution of the mass spectra for both peaks. Deconvolution of the mass spectrum of the species in the peak at retention time 12.42 min revealed multiple size variants, including a predominant species at 119,277 Da (Figure 3A). The aforementioned predominant species of 119,277 Da was close to the theoretical mass of 119,177 Da for a heavy-heavy-light chain (designated HC-HC-LC or HHL) with a dehydroalanine replacing the cysteine ​​at position 219 in the unpaired heavy chain, the expected product of β-elimination of the disulfide bond (Figure 4). Deconvolution of the mass spectrum of the species in the peak at retention time 12.68 min revealed multiple size variants, including a predominant species of 119,329 Da (Figure 3B). The aforementioned predominant species of 119,329 Da was close to the theoretical mass of 119,329 Da for a heavy-heavy-light chain (designated HC-HC-LC or HHL) with a cysteinylated unpaired heavy chain.

[0051] To further characterize the molecular weights of the size variant species in the M281 composition, the M281 composition was analyzed by microchip zone electrophoresis (MZE) separation with direct electrospray ionization mass spectrometry (ESI-MS). The electropherogram of the MZE separation showed a minor acidic species with a migration time of approximately 3.23 min (Figure 5). Deconvolution of the mass spectrum of the species in the peak eluting around 3.23 min revealed multiple size variants, including two predominant species with molecular masses of 119,178 Da and 119,332 Da. The aforementioned predominant species were close to the theoretical masses of 119,177 Da for heavy-heavy-light chain (HHL) with a dehydroalanine replacing the cysteine ​​at position 219 in the unpaired heavy chain, and 119,329 Da for heavy-heavy-light chain (referred to as HC-HC-LC or HHL) with a cysteinylated unpaired heavy chain, respectively.

[0052] To further characterize the molecular weights of the size variant species in the M281 composition, the M281 composition was separated by SDS-PAGE gel electrophoresis and two bands corresponding to light-heavy-heavy-light and heavy-heavy-light chains were excised from the gel (detailed in FIG. 7). The isolated gel fragments were subjected to non-reducing in-gel trypsin digestion and the resulting digestion fragments were analyzed by nano-liquid chromatography coupled with mass spectrometry (detailed in FIG. 7). The predicted tryptic digest peptides and molecular masses are shown in FIG. 8 for light-heavy-heavy-light, heavy-heavy-light with a cysteinylated unpaired heavy chain, and heavy-heavy-light with a dehydroalanine replacing the cysteine ​​at position 219 (of SEQ ID NO:2) in the unpaired heavy chain. Tryptic peptides corresponding to the latter two species were detected as predominant species in the band from SDS-PAGE gel electrophoresis corresponding to the heavy-heavy-light chain and as minor species in the band from SDS-PAGE gel electrophoresis corresponding to the light-heavy-heavy-light chain band (Table 1 and Figure 9).

[0053] [Table 1]

[0054] In conclusion, the three analytical methods identified size variant species in the M281 composition as having similar molecular masses as cysteinylated heavy-heavy-light and dehydroalanine heavy-heavy-light chains.

[0055] In another example, M281 preparation was prepared and subjected to a stability study upon storage at 5±3° C. The weight percent of protein was in the 119,150-119,350 Da range (HHL is shown in Table 2).

[0056] [Table 2] The inventions described in the original claims of this application are listed below. [Invention 1] A pharmaceutical composition comprising an antibody (LHHL) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:2 and a light chain comprising the amino acid sequence of SEQ ID NO:1, said composition comprising a major protein component having a molecular weight of 140,500 to 143,000 Da, and a minor protein component having a molecular weight of 119,150 to 119,350 Da, said major component comprising at least 80% by weight of the protein in said composition, and said minor component comprising at least 1% by weight of the protein in said composition. [Invention 2] The pharmaceutical composition of claim 1, wherein the major protein component is at least 93% by weight of the protein in the composition. [Invention 3] The pharmaceutical composition according to claim 1, wherein the secondary protein component comprises an antibody variant comprising two heavy chains and one light chain. [Invention 4] The pharmaceutical composition of invention 3, wherein the antibody variant comprises an unpaired heavy chain and a paired heavy and light chain. [Invention 5] The pharmaceutical composition of invention 3, wherein said antibody variant comprises an unpaired heavy chain comprising a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein C at position 219 is replaced by dehydroalanine. [Invention 6] The pharmaceutical composition of invention 1, wherein said secondary protein components comprise: a) a first antibody variant comprising an unpaired heavy chain and a paired heavy and light chain; and b) a second antibody variant comprising an unpaired heavy chain comprising a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein C at position 219 is replaced by dehydroalanine. [Invention 7] providing a composition of antibody (LHHL) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:2 and a light chain comprising the amino acid sequence of SEQ ID NO:1; determining whether the composition contains a major protein component having a molecular weight of 145,000 to 160,000 Da and a minor protein component having a molecular weight of 119,150 to 119,350 Da; only if said composition comprises a major protein component having a molecular weight of 145,000 to 160,000 Da and a minor protein component having a molecular weight of 119,150 to 119,350 Da, combining said composition with one or more pharma- ceutically acceptable excipients to prepare a pharmaceutical composition. 7. A method for preparing a pharmaceutical composition according to any one of claims 1 to 6, comprising: [Invention 8] The method according to Invention 7, wherein the prepared pharmaceutical composition contains 28 to 32 mg / ml of an antibody (LHHL) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 and a light chain comprising the amino acid sequence of SEQ ID NO: 9. [Invention 9] The method according to claim 7, wherein the prepared pharmaceutical composition comprises 9 to 11 mg / ml of an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 24 and a light chain comprising the amino acid sequence of SEQ ID NO: 19. [Invention 10] The method of invention 7, further comprising the step of combining said composition with one or more pharma- ceutically acceptable excipients to prepare a pharmaceutical composition only when said major component is at least 90% by weight of protein in said composition and said minor component is at least 3% by weight of protein in said composition. [Invention 11] The method of claim 7, wherein the determining step comprises electrophoresis or chromatography. [Invention 12] The method of claim 7, wherein the providing step comprises a step of culturing cells expressing the heavy chain and the light chain.

Claims

1. 1. A pharmaceutical composition, comprising: A major protein antibody component comprising a light-heavy-heavy-light chain (LHHL) having a molecular weight of 140,500-143,000 Da; and A secondary protein antibody component comprising a heavy-heavy-light (HHL) chain with a molecular weight of 119,150-119,350 Da Including, said major protein antibody component being 95-99% by weight of the total protein in said composition and said minor protein antibody component being 1-3% by weight of the total protein in said composition; each of the heavy chains comprises the amino acid sequence of SEQ ID NO:2 and each of the light chains comprises the amino acid sequence of SEQ ID NO:1; The pharmaceutical composition.

2. 2. The pharmaceutical composition of claim 1, wherein the major protein antibody component is 95-98% by weight of the total protein in the composition and the minor protein antibody component is 2-2.5% by weight of the total protein in the composition.

3. providing a composition comprising said major protein antibody components including said LHHL, wherein said heavy chains each comprise the amino acid sequence of SEQ ID NO:2 and said light chains each comprise the amino acid sequence of SEQ ID NO:1; determining whether said composition comprises a major protein antibody component comprising said LHHL and having a molecular weight of 140,500-143,000 Da, and a minor protein antibody component comprising said HHL and having a molecular weight of 119,150-119,350 Da; and only if said composition comprises a major protein antibody component having a molecular weight of 140,500-143,000 Da and a minor protein antibody component having a molecular weight of 119,150-119,350 Da, combining said composition with one or more pharma- ceutically acceptable excipients to prepare a pharmaceutical composition.

3. A method for preparing the pharmaceutical composition of claim 1 or 2, comprising:

4. The method of claim 3, wherein the prepared pharmaceutical composition comprises 28-32 mg / ml of the major protein antibody component.

5. The method of claim 3, wherein the prepared pharmaceutical composition comprises 9-11 mg / ml of the major protein antibody component.

6. 4. The method of claim 3, further comprising the step of combining said composition with one or more pharma- ceutically acceptable excipients to prepare a pharmaceutical composition only when said major protein antibody component is 95-99% by weight of the total protein in said composition and said minor protein antibody component is 1-3% by weight of the total protein in said composition.

7. The method of claim 3 , wherein the determining step comprises electrophoresis or chromatography.

8. 4. The method of claim 3, wherein the providing step comprises culturing a cell expressing the heavy chain comprising the amino acid sequence of SEQ ID NO:2 and the light chain comprising the amino acid sequence of SEQ ID NO:

1. (1) each heavy chain of said major protein antibody component comprises the amino acid sequence of SEQ ID NO:2 and each light chain of said major protein antibody component comprises the amino acid sequence of SEQ ID NO:1; and (2) the secondary protein antibody component comprises (i) a first heavy chain having the amino acid sequence of SEQ ID NO:2 paired with a light chain having the amino acid sequence of SEQ ID NO:1, and (ii) a second heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:2, in which the C at position 219 is replaced by dehydroalanine; 2. The pharmaceutical composition of claim 1.

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