Aqueous Compositions for Increasing Stability of Engineered Dimeric Proteins - Patent application

Abstract

The present application relates to an aqueous solution composition comprising a low buffer concentration and low ionic strength and neutral amino acids of an engineered dimeric protein comprising a monomer comprising at least one human serpin polypeptide operably linked to a human immunoglobulin Fc polypeptide or a polypeptide derived from an immunoglobulin Fc polypeptide, the aqueous solution composition increasing the stability of the Fc domain in the aqueous solution composition, in particular increasing the stability of the engineered dimeric protein.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of patent application No. 2102258.7, filed in the UK on February 17, 2021, the entire contents of which are incorporated herein by reference.

[0002] Incorporation by reference of sequence listing The contents of the text file named INHI-702_001WO Sequence_Listing.txt, created on February 16, 2022, and having a size of 148 kilobytes, are incorporated herein by reference in their entirety.

[0003] Shared ownership under joint research agreements The subject matter disclosed in this application was developed, and the claimed inventions made, by or on behalf of the parties to a joint research agreement that was in effect on or prior to the effective filing date of the claimed invention. The parties to the joint research agreement are: Arecor Limited and Inhibrx, Inc.

[0004] FIELD OF THEINVENTION The present invention relates to aqueous solution compositions of engineered dimeric proteins containing an Fc domain at low buffer concentrations and low ionic strength and containing neutral amino acids. [Background technology]

[0005] background Engineered proteins containing Fc domains are widely used in therapy. The Fc domain is the C-terminal region of an antibody that interacts with cell surface receptors called Fc receptors and with some proteins of the complement system, thereby activating the immune system. In IgG, IgA, and IgD antibody isotypes, the Fc domain is composed of two identical protein chain fragments, each of which originates from the second and third constant domains of the antibody heavy chain. In IgM and IgE antibody isotypes, the Fc domain is composed of two identical protein chain fragments, each of which originates from the second, third, and fourth constant domains of the antibody heavy chain. The molecular weight of the Fc domain is typically in the range of 25-40 kDa and can be larger in the presence of glycosylation. Activation of the immune system through antibody Fc domain binding results in a wide range of physiological effects, including cytolysis and degranulation of mast cells, basophils, and eosinophils.

[0006] A wide range of engineered antibody proteins have been developed, including bispecific and trispecific antibodies. Many engineered proteins have also been developed in which the Fc separated from the Fab portion of the antibody molecule (the portion that confers antigen-binding specificity) can serve a purpose other than its physiological purpose, in particular to extend the in vivo half-life of the engineered protein. WO 2013 / 003641 A2 and WO 2016 / 069574 A1 (INHIBRX) disclose engineered dimeric proteins comprising serpin polypeptides or amino acid sequences derived from serpins.

[0007] When formulated as an aqueous solution, during storage, proteins are susceptible to degradation resulting in loss of biological activity. Degradation is physical in nature and may include aggregation, precipitation, gel formation. Degradation may also be chemical in nature and may include hydrolytic cleavage, deamidation, cyclic imide formation, aspartic / glutamic acid isomerization or oxidation, etc.

[0008] The rate of degradation processes increases with increasing temperature, and protein therapeutic molecules are generally more stable at lower temperatures. However, it is often difficult to develop therapeutic protein products that are stable in liquid form for the intended storage period (usually 24 months), even under refrigeration. Furthermore, to ensure patient convenience, there is a need to develop products that are stable at elevated temperatures, such as up to 25°C or up to 30°C, for a certain period or the entire shelf life.

[0009] One of the most important parameters for controlling the stability of protein therapeutics is pH. Therefore, optimizing pH is a critical step in formulation development. Many therapeutic proteins are formulated at a selected pH between 4.0 and 8.5. It is considered important to maintain the pH at the selected value and to minimize pH fluctuations. It is therefore understood that the formulation needs to have some buffering capacity. Larger protein molecules usually have some self-buffering capacity due to the presence of ionizable groups between the amino acid side chains of the polypeptide backbone.

[0010] The present invention provides compositions that increase the stability of engineered proteins comprising an Fc domain in aqueous solution compositions, and in particular, compositions that increase the stability of engineered dimeric proteins, where each monomer of the dimeric protein comprises at least one human serpin polypeptide operably linked to a human immunoglobulin Fc polypeptide or a polypeptide derived from immunoglobulin Fc ("engineered dimeric proteins of the invention"). Summary of the Invention

[0011] The disclosure provides an aqueous solution composition having a pH range of 6.0-8.0 comprising: an engineered dimeric protein, wherein each monomer of the dimeric protein comprises at least one human serpin polypeptide operably linked to a human immunoglobulin Fc polypeptide, or a polypeptide derived from an immunoglobulin Fc polypeptide; optionally one or more buffers, the buffer being a substance having at least one ionizable group, having a pKa in the range of 4.0-10.0, the pKa being within 2 pH units of the pH of the composition; a neutral amino acid; and an uncharged tonicity modifier, wherein the buffer is present in the composition at a total concentration of 0-10 mM; and wherein the total ionic strength of the composition, excluding the contribution of the engineered dimeric protein, is less than 30 mM.

[0012] In some embodiments of the aqueous solution composition disclosed herein, the human serpin polypeptide is a human alpha 1 antitrypsin (AAT) polypeptide or is derived from a human AAT polypeptide. In some embodiments of the aqueous solution composition disclosed herein, each monomer of the dimeric protein comprises one human serpin polypeptide. In some embodiments of the aqueous solution composition disclosed herein, the human serpin polypeptide has the sequence of SEQ ID NO: 1 or 2. In some embodiments of the aqueous solution composition disclosed herein, the human immunoglobulin Fc polypeptide or the polypeptide derived from an immunoglobulin Fc polypeptide is a modified human IgG4Fc polypeptide. In some embodiments of the aqueous solution composition disclosed herein, the human immunoglobulin Fc polypeptide or the polypeptide derived from an immunoglobulin Fc polypeptide is a modified human IgG4Fc polypeptide and has the sequence of any one of SEQ ID NOs: 28-43. In some embodiments of the aqueous solution composition disclosed herein, each monomer of the dimeric protein has the sequence of SEQ ID NO: 56.

[0013] In some embodiments of the aqueous solution compositions disclosed herein, the protein is present at a concentration of 1-400 mg / ml, such as 10-200 mg / ml, such as 20-100 mg / ml, such as 30-60 mg / ml, for example, about 35 mg / ml or about 50 mg / ml.

[0014] In some embodiments of the aqueous solution compositions disclosed herein, the buffer is present at a total concentration of 0.1-10 mM, such as 0.5-10 mM, such as 1-10 mM, such as 1-8 mM, such as 1-6 mM, such as 2-6 mM, such as 2-5 mM, such as 3-5 mM. In some embodiments, the aqueous solution compositions disclosed herein are substantially free of buffer.

[0015] In some embodiments of the aqueous solution compositions disclosed herein, the buffer contains an ionizable group that has a pKa within 1 unit of the pH of the composition.

[0016] In some embodiments of the aqueous solution compositions disclosed herein, the one or more buffers are selected from the group consisting of citrate, histidine, maleate, sulfite, aspartame, aspartate, glutamate, tartrate, adenine, succinate, ascorbate, benzoate, phenyl acetate, gallate, cytosine, p-aminobenzoic acid, sorbate, acetate, propionate, alginate, urate, 2-(N-morpholino)ethanesulfonic acid, bicarbonate, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane, N-(2-acetamido)-2-iminodiacetic acid, 2-[(2-amino-2-oxoethoxy)phenyl]-2-propanediol ... The buffer is selected from the group consisting of N,N'-bis(2-ethanesulfonic acid), phosphate, N,N'-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-[N,N'-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid, triethanolamine, piperazine-N,N'-bis(2-hydroxypropanesulfonic acid), tris(hydroxymethyl)aminomethane (Tris), N-tris(hydroxymethyl)glycine, and N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, and salts thereof, and combinations thereof. In some embodiments of the aqueous solution composition disclosed herein, the buffer is selected from citrate, histidine, maleate, tartrate, benzoate, acetate, bicarbonate, phosphate, and tris(hydroxymethyl)aminomethane (Tris), for example, phosphate and Tris.

[0017] In some embodiments of the aqueous solution composition disclosed herein, the uncharged tonicity modifier is selected from the group consisting of polyols, sugars (e.g., monosaccharides and disaccharides), and sugar alcohols. In some embodiments of the aqueous solution composition disclosed herein, the uncharged tonicity modifier is selected from the group consisting of glycerol, 1,2-propanediol, mannitol, sorbitol, glucose, sucrose, trehalose, PEG300, and PEG400, particularly selected from the group consisting of glycerol, mannitol, sucrose, and trehalose. In some embodiments, the aqueous solution composition disclosed herein comprises a disaccharide as the uncharged tonicity modifier. In some embodiments, the aqueous solution composition disclosed herein comprises sucrose and / or trehalose as the uncharged tonicity modifier, particularly trehalose.

[0018] In some embodiments of the aqueous solution compositions disclosed herein, the total concentration of the uncharged tonicity modifier, or combination of tonicity modifiers, is 50-1000 mM, e.g., 200-600 mM, 200-500 mM, or wherein the total concentration of the uncharged tonicity modifier, or combination of tonicity modifiers, is 50-500 mM, e.g., 100-400 mM, 150-350 mM, 200-300 mM, or about 250 mM.

[0019] In some embodiments of the aqueous solution compositions disclosed herein, the osmolality of the composition is between 200-500 mOsm / L, e.g., about 300 mOsm / L, or wherein the osmolality of the composition is between 300-500 mOsm / L, e.g., about 400-460 mOsm / L.

[0020] In some embodiments, the aqueous solution composition disclosed herein comprises a neutral amino acid selected from glycine, methionine, proline, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, and glutamine. In some embodiments, the neutral amino acid is selected from glycine, methionine, and proline. In some embodiments, the aqueous solution composition disclosed herein comprises proline as a neutral amino acid. In some embodiments, the aqueous solution composition disclosed herein comprises glycine as a neutral amino acid. In some embodiments, the aqueous solution composition disclosed herein comprises proline and methionine as neutral amino acids. In some embodiments, the aqueous solution composition disclosed herein comprises glycine and methionine as neutral amino acids.

[0021] In some embodiments of the aqueous solution composition disclosed herein, the total concentration of one or more neutral amino acids in the composition is 20 to 600 mM, such as 20 to 500 mM, such as 20 to 400 mM, such as 20 to 300 mM, such as 50 to 300 mM. In some embodiments of the aqueous solution composition disclosed herein, the total concentration of one or more neutral amino acids in the composition is 50 to 200 mM, 100 to 200 mM, or 100 to 150 mM.

[0022] In some embodiments of the aqueous solution compositions disclosed herein, the total ionic strength of the composition excluding the contribution of the engineered dimeric protein is less than 20 mM. In some embodiments of the aqueous solution compositions disclosed herein, the total ionic strength of the composition excluding the contribution of the engineered dimeric protein is less than 10 mM.

[0023] In some embodiments of the aqueous solution compositions disclosed herein, the pH is between 6.8 and 7.8, e.g., between 7.0 and 7.8, between 7.1 and 7.6, between 7.1 and 7.5, between 7.2 and 7.5, between 7.1 and 7.4, between 7.2 and 7.3; or about 7.2 or about 7.3.

[0024] In some embodiments, the aqueous solution composition disclosed herein comprises a non-ionic surfactant. In some embodiments, the non-ionic surfactant is selected from the group consisting of alkyl glycosides, polysorbates, alkyl ethers of polyethylene glycol, block copolymers of polyethylene glycol and polypropylene glycol (poloxamers), and alkyl phenyl ethers of polyethylene glycol. In some embodiments, the non-ionic surfactant is a polysorbate, such as polysorbate 20 or polysorbate 80. In some embodiments, the non-ionic surfactant is a block copolymer of polyethylene glycol and polypropylene glycol (poloxamer), such as poloxamer 188.

[0025] In some embodiments of the aqueous solution composition disclosed herein, the non-ionic surfactant is present at a concentration of 10-2000 μg / ml, such as 50-1000 μg / ml, such as 50-500 μg / ml, such as about 200 μg / ml, or the non-ionic surfactant is present at a concentration of 250-1500 μg / ml, such as 750-1250 μg / ml, such as about 1000 μg / ml. In some embodiments, the aqueous solution composition disclosed herein includes a preservative, such as a phenolic or benzyl preservative. In some embodiments, the phenolic or benzyl preservative is selected from the group consisting of phenol, m-cresol, chlorocresol, benzyl alcohol, propylparaben, and methylparaben.

[0026] In some embodiments of the aqueous solution compositions disclosed herein, the preservative is present at a concentration of 10-100 mM, such as 20-80 mM, for example 25-50 mM.

[0027] In some embodiments, the aqueous solution compositions disclosed herein are compositions for therapeutic use.

[0028] In some embodiments, the aqueous solution compositions disclosed herein are pharmaceutical compositions.

[0029] The present disclosure also provides a method of treating or alleviating a symptom of a disease or disorder associated with aberrant serine protease expression or activity in a subject in need thereof comprising administering an aqueous solution composition disclosed herein.

[0030] The present disclosure also provides a method of treating or alleviating inflammation or symptoms of an inflammatory disease or disorder in a subject in need thereof, while reducing the risk of infection, comprising administering to the subject an aqueous solution composition disclosed herein.

[0031] The present disclosure also provides a method of reducing the risk of infection in a subject in need thereof, comprising administering to the subject an aqueous solution composition disclosed herein.

[0032] The present disclosure also provides a method of treating or alleviating a symptom of AAT deficiency in a subject in need thereof, comprising administering to the subject an aqueous solution composition of the present disclosure, wherein the human serpin polypeptide is a human alpha-1 antitrypsin (AAT) polypeptide or is derived from a human AAT polypeptide.

[0033] Also provided herein are aqueous solution compositions of the present disclosure for use in a method of treating or wlv a symptom of a disease or disorder associated with aberrant serine protease expression or activity in a subject in need thereof, while reducing the risk of infection.

[0034] Also provided herein are aqueous solution compositions of the present disclosure for use in methods for treating or alleviating inflammation or symptoms of an inflammatory disease or disorder in a subject in need thereof, while reducing the risk of infection.

[0035] Also provided herein is an aqueous solution composition of the present disclosure for use in a method for reducing the risk of infection in a subject in need thereof.

[0036] Also provided herein is an aqueous solution composition of the present disclosure for use in a method of treating or alleviating a symptom of AAT deficiency in a subject in need thereof, wherein the human serpin polypeptide is a human alpha-1 antitrypsin (AAT) polypeptide or is derived from a human AAT polypeptide.

[0037] The present disclosure also provides use of an aqueous solution composition of the present disclosure for the manufacture of a medicament for treating or alleviating a symptom of a disease or disorder associated with aberrant serine protease expression or activity in a subject in need thereof.

[0038] The present disclosure also provides use of an aqueous solution composition of the present disclosure for the manufacture of a medicament for treating or alleviating inflammation or symptoms of an inflammatory disease or disorder, while reducing the risk of infection, in a subject in need thereof.

[0039] The present disclosure also provides a use of an aqueous solution composition of the present disclosure for the manufacture of a medicament for reducing the risk of infection in a subject in need thereof.

[0040] The present disclosure also provides for the use of an aqueous solution composition of the present disclosure for the manufacture of a medicament for treating or alleviating a symptom of AAT deficiency in a subject in need thereof, wherein the human serpin polypeptide is a human alpha-1 antitrypsin (AAT) polypeptide or is derived from a human AAT polypeptide.

[0041] In some embodiments of the aqueous solution composition or use of the present disclosure for use according to any of the methods of the present disclosure, the inflammatory disease or disorder is selected from the following: alpha-1 antitrypsin (AAT) deficiency, emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), allergic asthma, cystic fibrosis, lung cancer, ischemia-reperfusion injury, ischemia / reperfusion injury after heart transplantation, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, type I and / or type II diabetes, pneumonia, sepsis, graft-versus-host disease (GVHD), wound healing, systemic lupus erythematosus, and multiple sclerosis.

[0042] In some embodiments of the aqueous solution composition or use of the present disclosure for use with any of the methods of the present disclosure, the infection is selected from a bacterial infection, a fungal infection, and a viral infection.

[0043] In some embodiments of the disclosed aqueous solution composition or use for use according to any of the disclosed methods, the subject is a human.

[0044] 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 the present invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples described herein are for illustrative purposes only and are not intended to be limiting.

[0045] Other features and advantages of the invention will be apparent from and encompassed by the following detailed description and claims.

[0046] Detailed Description of the Invention Described herein are stable aqueous solutions of the engineered dimeric proteins of the present invention in the absence or presence of low buffer concentrations and low ionic strength.

[0047] Please note that all references to "pH" herein refer to the pH of the composition evaluated at 25° C. a All references to pK of ionizable groups evaluated at 25 °C. a (See CRC Handbook of Chemistry and Physics, 79th Edition, 1998, DR Lide). If necessary, the pK a Values ​​can be estimated using a suitable calculator.

[0048] The inventors believe that buffers have a detrimental effect on the engineered dimeric proteins of the present invention, therefore, the concentration of buffers in the composition should be limited as much as possible.

[0049] A buffer, if present, will have buffering capacity at the pH of the composition. A buffer will typically have a pK within 1 pH unit of the pH of the composition. a but which has a pK that is one pH unit greater or less than the pH of the composition. a Moieties having an ionizable group having a pK within 1 pH unit of the pH of the composition may also provide some buffering effect if present in sufficient amounts. a In another embodiment, the (or some) buffer contains an ionizable group having a pK within 1.5 pH units of the pH of the composition (e.g., between 1 and 1.5 pH units of the pH of the composition). a In further embodiments, the (or some) buffer comprises an ionizable group having a pK within 2 pH units of the pH of the composition (e.g., 1.5 to 2 pH units of the pH of the composition). a The ionizable group has the formula:

[0050] In some embodiments, the composition is substantially free of buffer. As used herein, "substantially free" means that the aqueous composition contains less than 0.1 mM buffer, e.g., no buffer. More preferably, a small amount of buffer is present to avoid or limit undesirable pH fluctuations. In some embodiments, the composition contains a single buffer. In some embodiments, the composition contains two buffers. Suitably, one or more buffers are present.

[0051] The total concentration of the buffer in the composition is 0-10 mM. In one embodiment, the total concentration of the buffer in the composition is 9.5 mM or less, such as 9 mM or less, such as 8 mM or less, such as 7 mM or less, such as 6 mM or less, such as 5.5 mM or less, such as 5 mM or less, such as 4.5 mM or less, such as 4 mM or less, such as 3 mM or less, such as 2 mM or less, such as 1 mM or less, such as 0.5 mM or less, such as 0.4 mM or less, such as 0.3 mM or less, such as 0.2 mM or less. In one embodiment, the total concentration of the buffer is 0.1 mM or more, such as 0.2 mM or more, such as 0.3 mM or more, such as 0.4 mM or more, such as 0.5 mM or more, such as 1 mM or more. Suitably, the total concentration of the buffer is 0.1 to 10 mM, for example, 0.5 to 10 mM, for example, 1 to 10 mM, for example, 1 to 8 mM, for example, 1 to 6 mM, for example, 2 to 6 mM, for example, 2 to 5 mM, for example, 3 to 5 mM.

[0052] When considering the concentration of the buffer in the solution, the buffering capacity of the engineered dimeric protein of the present invention itself should be excluded.

[0053] The pH of an aqueous solution decreases when an acid is added and increases when a base is added. At a given temperature and atmospheric pressure, the magnitude of the pH decrease due to the addition of an acid or the magnitude of the pH increase due to the addition of a base depends on (1) the amount of acid or base added, (2) the starting pH of the aqueous solution (i.e., before the addition of the acid or base), and (3) the presence of a buffer. Thus, (1) starting from a given pH, the addition of a larger amount of acid or base results in a larger pH change, (2) the addition of a given amount of acid or base results in the largest pH change at a neutral pH (i.e., pH 7.0), with the magnitude of the pH change decreasing as the starting pH moves away from pH 7.0, and (3) starting from a given pH, the magnitude of the pH change is smaller in the presence of a buffer than in the absence of a buffer. Thus, a buffer has the ability to reduce the change in pH when an acid or base is added to a solution.

[0054] Suitably, a substance is considered to be a buffer if, upon addition of either a strong acid or a strong base, the substance is capable of reducing the magnitude of the pH change of a solution by 75%, preferably 50%, and most preferably 25%, when compared to the same solution without the buffer, for a 0.1 mM increase in acid or base in the solution.

[0055] Conversely, a substance is not considered to be a buffer if it is unable to reduce the magnitude of the pH change of a solution by 75%, preferably 50%, and most preferably 25%, when either a strong acid or a strong base, as appropriate, is added to increase the acidity or base in the solution by 0.1 mM, compared to the same solution without the substance.

[0056] In one embodiment, the or a buffer is an amino acid. In another embodiment, the or a buffer is not an amino acid. In one embodiment, the composition does not include the amino acids lysine, arginine, histidine, glutamate, and aspartate. In one embodiment, the composition does not include cysteine.

[0057] Suitable buffers, if present, include, but are not limited to, citrate, histidine, maleate, sulfite, aspartame, aspartate, glutamate, tartrate, adenine, succinate, ascorbate, benzoate, phenyl acetate, gallate, cytosine, p-aminobenzoic acid, sorbate, acetate, propionate, alginate, urate, 2-(N-morpholino)ethanesulfonic acid, bicarbonate, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane, N-(2-acetamido)-2-iminodiacetic acid, 2-[(2-amino-2-oxo)phenyl]-2-propanediol ... N,N'-bis(2-ethanesulfonic acid), phosphate, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid, triethanolamine, piperazine-N,N'-bis(2-hydroxypropanesulfonic acid), tris(hydroxymethyl)aminomethane (tris), N-tris(hydroxymethyl)glycine, and N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, and salts thereof, and combinations thereof.

[0058] In one embodiment, the buffer is selected from the group consisting of citrate, histidine, maleate, tartrate, benzoate, acetate, bicarbonate, phosphate, and Tris, e.g., selected from the group consisting of histidine, maleate, tartrate, benzoate, acetate, bicarbonate, phosphate, and Tris, e.g., selected from the group consisting of histidine, acetate, phosphate, and Tris.

[0059] In one embodiment, the buffer is a phosphate (e.g., sodium phosphate) or Tris. In one embodiment, the buffer is a phosphate (e.g., sodium phosphate). Suitably, the buffer is Tris.

[0060] In one embodiment, the composition does not include sodium phosphate.

[0061] The primary solvent of the composition of the invention is water, such as water for injection. Other components of the composition (e.g., polyols) may contribute to solubilizing the engineered dimeric protein.

[0062] The composition comprises an uncharged tonicity modifier, such as a polyol, a sugar, for example a monosaccharide, a disaccharide, or a sugar alcohol. In one embodiment, the composition comprises an isotonicity modifier selected from the group consisting of glycerol, 1,2-propanediol, mannitol, sorbitol, glucose, sucrose, trehalose, PEG300, and PEG400, for example selected from the group consisting of glycerol, 1,2-propanediol, mannitol, sorbitol, sucrose, trehalose, PEG300, and PEG400. A mixture of uncharged tonicity modifiers, such as trehalose and sucrose, or trehalose and mannitol, is contemplated. In one embodiment, the uncharged tonicity modifier is selected from glycerol, mannitol, sucrose, and trehalose. Suitably, the uncharged tonicity modifier is a disaccharide. Thus, in another embodiment, the uncharged tonicity modifier is sucrose and / or trehalose, and in particular trehalose. When included, the uncharged tonicity modifier (or a combination of tonicity modifiers) is typically used in the composition at a total concentration of 50-1000 mM, such as 200-600 mM, for example about 200-500 mM. In one embodiment, the total concentration of the uncharged tonicity modifier (or a combination of tonicity modifiers) is 50-500 mM, for example 100-400 mM, 150-350 mM, 200-300 mM, or about 250 mM. Another concentration of interest is about 150 mM.

[0063] In one embodiment, when trehalose is the uncharged tonicity modifier, either alone or in combination with one or more other uncharged tonicity modifiers, the concentration of trehalose in the composition is 50-180 mM, for example 50-150 mM.

[0064] The composition suitably has an osmolality that is physiologically acceptable and therefore suitable for parenteral administration.The osmolality of the composition is therefore suitably 200-500 mOsm / L, for example about 300 mOsm / L.The composition is, for example, isotonic with human plasma.In another embodiment, the osmolality of the composition is 300-500 mOsm / L, for example 400-460 mOsm / L.The composition may also be hypotonic or hypertonic, for example intended to be diluted before administration.

[0065] The composition comprises neutral amino acid.As used herein, neutral amino acid is an amino acid whose side chain does not contain ionizable groups that are significantly ionized at the pH of the composition (e.g., more than 20% of the side chain, particularly more than 50% of the side chain has negative or positive charge).Examples of neutral amino acid are glycine, methionine, proline, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, and glutamine, and particularly their L-isomers.

[0066] In one embodiment, the composition comprises a neutral amino acid selected from the group consisting of glycine, methionine, and proline. In one embodiment, the composition comprises proline as a neutral amino acid. In one embodiment, the composition comprises glycine as a neutral amino acid. A mixture of neutral amino acids is contemplated. In one embodiment, the composition comprises proline and methionine as neutral amino acids. In one embodiment, the composition comprises glycine and methionine as neutral amino acids. As can be seen from the examples, the presence of a neutral amino acid has been found to enhance the stability of the composition.

[0067] In one embodiment, the concentration of the neutral amino acid, for example, proline or glycine, is 20 to 600 mM, for example, 20 to 500 mM, for example, 20 to 400 mM, for example, 20 to 300 mM, or 50 mM to 300 mM. In one embodiment, the concentration of the neutral amino acid, for example, proline or glycine, is 50 to 200 mM, 100 to 200 mM, or 100 to 150 mM.

[0068] In one embodiment, methionine as a neutral amino acid, if present in the composition, is present at a concentration of 2-10 mM, for example about 2 mM.

[0069] In one embodiment, the concentration (i.e., total concentration) of all neutral amino acids, such as proline or glycine and methionine, in the composition is 20 to 600 mM, such as 20 to 500 mM, such as 20 mM to 400 mM, such as 20 to 300 mM, or 50 to 300 mM. In one embodiment, the concentration of all neutral amino acids, such as proline or glycine and methionine, in the composition is 50 to 200 mM, 100 to 200 mM, or 100 to 150 mM.

[0070] The composition may include a non-ionic surfactant, which may be selected from the group consisting of polysorbates, alkyl glycosides, alkyl ethers of polyethylene glycol, block copolymers of polyethylene glycol and polypropylene glycol, and alkyl phenyl ethers of polyethylene glycol.

[0071] A particularly suitable class of non-ionic surfactants are the polysorbates (fatty acid esters of ethoxylated sorbitan), such as polysorbate 20 or polysorbate 80. Polysorbate 20 is a monoester formed from lauric acid and polyoxyethylene (20) sorbitan, where the number 20 indicates the number of oxyethylene groups in the molecule. Polysorbate 80 is a monoester formed from oleic acid and polyoxyethylene (20) sorbitan, where the number 20 indicates the number of oxyethylene groups in the molecule. Polysorbate 20 is also known under various brand names, including, in particular, Tween 20, but also Alkest TW20. Polysorbate 80 is also known under various brand names, including, in particular, Tween 80, but also Alkest TW80. Other suitable polysorbates include polysorbate 40 and polysorbate 60.

[0072] Another suitable class of nonionic surfactants are the alkyl glycosides, in particular dodecyl maltoside. Other alkyl glycosides include dodecyl glucoside, octyl glucoside, octyl maltoside, decyl glucoside, decyl maltoside, tridecyl glucoside, tridecyl maltoside, tetradecyl glucoside, tetradecyl maltoside, hexadecyl glucoside, hexadecyl maltoside, sucrose monooctanoate, sucrose monodecanoate, sucrose monododecanoate, sucrose monotridecanoate, sucrose monotetradecanoate, and sucrose monohexadecanoate.

[0073] Another suitable class of nonionic surfactants are alkyl ethers of polyethylene glycols, in particular those known under the trade name Brij, such as polyethylene glycol (2) hexadecyl ether (Brij 52), polyethylene glycol (2) oleyl ether (Bri 93), and polyethylene glycol (2) dodecyl ether (Brij L4). Other suitable Brij surfactants include polyethylene glycol (4) lauryl ether (Brij 30), polyethylene glycol (10) lauryl ether (Brij 35), polyethylene glycol (20) hexadecyl ether (Brij 58), and polyethylene glycol (10) stearyl ether (Brij 78).

[0074] Another suitable class of nonionic surfactants are block copolymers of polyethylene glycol and polypropylene glycol (also known as poloxamers), particularly poloxamer 188, poloxamer 407, poloxamer 171, and poloxamer 185. Poloxamers are also known under the brand names Pluronics or Koliphors. For example, poloxamer 188 is sold as Pluronic® F-68.

[0075] Another suitable class of nonionic surfactants are the alkyl phenyl ethers of polyethylene glycol, particularly 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (also known under the trade name Triton X-100).

[0076] In one embodiment, the non-ionic surfactant is a polysorbate or a poloxamer. In one embodiment, the non-ionic surfactant is a polysorbate, such as polysorbate 80 or polysorbate 20. In one embodiment, the non-ionic surfactant is a poloxamer, such as poloxamer 188. The concentration of the non-ionic surfactant in the composition will usually be in the range of 10 to 2000 μg / ml. For example, for a polysorbate surfactant, an exemplary concentration is 50 to 1000 μg / ml, such as 100 to 500 μg / ml, such as about 200 μg / ml. For example, for a poloxamer surfactant, an exemplary concentration is 250 to 1500 μg / ml, such as 750 to 1250 μg / ml, such as about 1000 μg / ml.

[0077] In some embodiments, the concentration of the poloxamer surfactant is 0.025-0.15% by weight (w / v) per volume of the composition, such as 0.075-0.125% by weight (w / v) per volume of the composition, such as 0.1% by weight (w / v) per volume of the composition.

[0078] The composition of the present invention may further comprise a preservative, such as a phenolic or benzyl preservative. The preservative is suitably selected from the group consisting of phenol, m-cresol, chlorocresol, benzyl alcohol, propylparaben and methylparaben, in particular from the group consisting of phenol, m-cresol and benzyl alcohol. The concentration of the preservative is typically 10-100 mM, such as 20-80 mM, for example 25-50 mM. The optimum concentration of the preservative in the composition is determined by whether the composition meets the Pharmacopoeia Antimicrobial Effectiveness Test (USP <51> , Vol.32) are selected to ensure passing.

[0079] The inventors believe that the presence of ions adversely affects the stability of the engineered dimeric proteins of the present invention, therefore, the ionic strength of the composition should be limited as much as possible.

[0080] The total ionic strength of the composition, excluding the contribution of the engineered dimeric protein of the present invention, is less than 30 mM, suitably less than 25 mM, suitably less than 20 mM, suitably less than 15 mM, suitably less than 10 mM, for example less than 5 mM. The term "total ionic strength" is used herein as a function of the concentration of all ions in solution as follows: [ka] where c x is the molar concentration of ion x (molL -1 ), and z x is ion c x This sum covers all ions (n) present in the solution, excluding the contribution of the engineered dimeric proteins of the invention. It will be understood that optional neutral amino acids have a net charge of zero in the compositions of the invention and do not contribute to the total ionic strength. In any event, the contribution of neutral amino acids is not included.

[0081] The pH of the composition is between 6.0 and 8.0, such as between 6.8 and 7.8, for example between 7.0 and 7.8, between 7.1 and 7.6, between 7.1 and 7.5, between 7.2 and 7.5, between 7.1 and 7.4, between 7.2 and 7.3, or is about 7.2 or about 7.3.

[0082] In certain embodiments, the engineered dimeric protein of the present invention is substantially pure, i.e., the composition comprises a single protein and does not contain substantial amounts of additional proteins. In preferred embodiments, the engineered dimeric protein of the present invention comprises at least 99%, preferably at least 99.5%, more preferably at least about 99.9% of the total protein content of the composition. In preferred embodiments, the engineered dimeric protein of the present invention is sufficiently pure to be used in pharmaceutical compositions.

[0083] The engineered dimeric protein of the present invention is present in the composition at a concentration of about 1-400 mg / ml, suitably 10-200 mg / ml, more suitably 20-100 mg / ml, such as 30-60 mg / ml, for example about 35 mg / ml, or about 50 mg / ml.

[0084] In one embodiment, the present invention provides an aqueous solution composition having a pH in the range of 6.8 to 7.8, such as 7.0 to 7.8, such as 7.1 to 7.6, such as 7.1 to 7.5, such as about 7.2 or about 7.3, comprising: an engineered dimeric protein, wherein each monomer of the dimeric protein comprises at least one human serpin polypeptide operably linked to a human immunoglobulin Fc polypeptide or a polypeptide derived from an immunoglobulin Fc polypeptide; a buffer selected from phosphate (e.g., sodium phosphate) or Tris, such as Tris; a buffer selected from glycine, methionine, and and a neutral amino acid selected from the group consisting of glycine and methionine, or proline and methionine; and a non-charged tonicity modifier, such as a disaccharide, such as trehalose, sucrose, or a mixture of trehalose and sucrose; a non-ionic surfactant selected from a polysorbate and a poloxamer, such as a poloxamer such as poloxamer 188; wherein the buffer is present in the composition at a total concentration of 1 to 8 mM, such as 2 to 6 mM; and wherein the total ionic strength of the composition excluding the contribution of the engineered dimeric protein is less than 20 mM, such as less than 10 mM, such as less than 5 mM.

[0085] Suitably, the composition of the invention remains a clear solution after storage at 2-8° C. for an extended period of time, for example at least 4 weeks, 8 weeks, 12 weeks, 12 months, 18 months, or 24 months.

[0086] Suitably, the compositions of the invention remain a clear solution after storage at 25° C. for an extended period of time, for example at least 4 weeks, 8 weeks, 12 weeks, 12 months, 18 months, or 24 months.

[0087] Suitably, the compositions of the invention remain a clear solution after storage at 30° C. for an extended period of time, for example at least 4 weeks, 8 weeks, 12 weeks, 12 months, 18 months, or 24 months.

[0088] Suitably, the compositions of the present invention remain a clear solution after storage at 40° C. (i.e., a temperature suitable for accelerated stability testing) for at least 1 day, 3 days, 1 week, 2 weeks, or 4 weeks.

[0089] Suitably, the compositions of the invention have increased storage stability at either 2-8°C or at higher temperatures compared to a comparable composition comprising a higher concentration of the same buffer or buffers.

[0090] Suitably, the compositions of the invention have increased storage stability at either 2-8° C. or higher temperatures compared to comparable compositions having higher total ionic strength.

[0091] In one embodiment the composition of the invention comprises 8% or less high molecular weight species, such as 7% or less, such as 6% or less, such as 5% or less, such as 2% or less, such as 1% or less, such as 0.5% or less, for example 0.3% or less high molecular weight species (by weight of the engineered dimeric protein of the invention in the composition as measured by size exclusion chromatography or similar suitable technique) after storage at 2-8°C for at least 4 weeks, 8 weeks, 12 weeks, 12 months, 18 months or 24 months.

[0092] In one embodiment the composition of the invention comprises 18% or less high molecular weight species, such as 17% or less, such as 16% or less, for example 15% or less, such as 10% or less, such as 8% or less, for example 5% or less, such as 4% or less, for example 3% or less, such as 1% or less high molecular weight species (by total weight of engineered dimeric protein of the invention in the composition as measured by size exclusion chromatography or similar suitable technique) after storage at 25° C. for at least 4 weeks, 8 weeks, 12 weeks, 12 months, 18 months, or 24 months.

[0093] In one embodiment the composition of the invention comprises 25% or less high molecular weight species, such as 20% or less, such as 18% or less, such as 17% or less, such as 16% or less, such as 15% or less, such as 10% or less, such as 8% or less, such as 5% or less, such as 4% or less, such as 3% or less, such as 1% or less high molecular weight species (by weight of the engineered dimeric protein of the invention in the composition as measured by size exclusion chromatography or similar suitable technique) after storage at 30° C. for at least 4 weeks.

[0094] In one embodiment, the composition of the invention comprises 30% or less high molecular weight species (by total weight of engineered dimeric protein of the invention in the composition as measured by size exclusion chromatography or similar suitable technique), such as 25% or less, such as 20% or less, for example 18% or less, such as 17% or less, such as 16% or less, such as 15% or less, for example 10% or less, such as 8% or less, such as 6% or less, for example 4% or less high molecular weight species after storage at 40° C. for at least 1 day, 3 days, 1 week, 2 weeks, or 4 weeks.

[0095] As used herein, a high molecular weight species is a species resulting from aggregation of proteins that has an apparent molecular weight greater than that of the dimeric protein.

[0096] In one embodiment, the composition of the invention is a composition for use in therapy, hi one embodiment, the composition of the invention is a pharmaceutical composition.

[0097] In one embodiment, a method of inhibiting or downregulating abnormal serine protease expression or activity in a subject in need thereof is provided, comprising administering to said subject an aqueous solution composition as described herein. In some embodiments, abnormal serine protease expression or activity is associated with an inflammatory disease or disorder or risk of infection. In some embodiments, the inflammatory disease or disorder is selected from: alpha-1 antitrypsin (AAT) deficiency, emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), allergic asthma, cystic fibrosis, lung cancer, ischemia-reperfusion injury, ischemia / reperfusion injury after heart transplantation, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, type I and / or type II diabetes, pneumonia, sepsis, graft-versus-host disease (GVHD), wound healing, systemic lupus erythematosus, and multiple sclerosis.

[0098] In one embodiment, the risk of infection is a risk of infection selected from a bacterial infection, a fungal infection, and a viral infection.

[0099] In one embodiment, a method is provided for treating or alleviating symptoms of a disease or disorder associated with abnormal serine protease expression or activity in a subject in need thereof, the method comprising administering to said subject an aqueous solution composition as described herein. In another embodiment, a method is provided for treating or alleviating symptoms of inflammation or an inflammatory disease or disorder while reducing the risk of infection in a subject in need thereof, the method comprising administering to said subject an aqueous solution composition as described herein. In another embodiment, a method is provided for reducing the risk of infection in a subject in need thereof, the method comprising administering to said subject an aqueous solution composition as described herein.

[0100] In one embodiment, where the human serpin polypeptide is a human alpha-1 antitrypsin (AAT) polypeptide or is derived from a human AAT polypeptide, a method is provided for treating or alleviating a symptom of AAT deficiency in a cell, comprising administering to a subject in need thereof an aqueous solution composition described herein.

[0101] In one embodiment, there is provided an aqueous solution composition as described herein for use in a method for treating or alleviating symptoms of a disease or disorder associated with abnormal serine protease expression or activity in a subject in need thereof. In another embodiment, there is provided an aqueous solution composition as described herein for use in a method for treating or alleviating symptoms of inflammation or an inflammatory disease or disorder in a subject in need thereof while reducing the risk of infection. In another embodiment, there is provided an aqueous solution composition as described herein for use in a method for reducing the risk of infection in a subject in need thereof.

[0102] In certain embodiments, where the human serpin polypeptide is a human alpha-1 antitrypsin (AAT) polypeptide or is derived from a human AAT polypeptide, there is provided an aqueous solution composition as described herein for use in a method of treating or alleviating a symptom of AAT deficiency in a subject in need thereof.

[0103] In one embodiment, there is provided a use of the aqueous solution composition described herein for the manufacture of a medicament for treating or alleviating symptoms of a disease or disorder associated with abnormal serine protease expression or activity in a subject in need thereof.In another embodiment, there is provided a use of the aqueous solution composition described herein for the manufacture of a medicament for treating or alleviating symptoms of inflammation or an inflammatory disease or disorder while reducing the risk of infection in a subject in need thereof.In another embodiment, there is provided a use of the aqueous solution composition described herein for the manufacture of a medicament for reducing the risk of infection in a subject in need thereof.

[0104] In one embodiment, where the human serpin polypeptide is a human alpha-1 antitrypsin (AAT) polypeptide or is derived from a human AAT polypeptide, there is provided a use of an aqueous solution composition described herein for the manufacture of a medicament for treating or alleviating a symptom of AAT deficiency in a subject in need thereof.

[0105] Suitably the subject is a human.

[0106] Suitably, the inflammatory disease or disorder is selected from: alpha-1 antitrypsin (AAT) deficiency, emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), allergic asthma, cystic fibrosis, lung cancer, ischemia-reperfusion injury, ischemia / reperfusion injury after heart transplantation, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, type I and / or type II diabetes, pneumonia, sepsis, graft versus host disease (GVHD), wound healing, systemic lupus erythematosus, and multiple sclerosis.

[0107] Suitably the infection is selected from a bacterial infection, a fungal infection and a viral infection.

[0108] Compositions, such as those intended for intravenous administration, can be prepared as a concentrate for dilution prior to administration.

[0109] All embodiments described above with respect to the aqueous composition apply equally to the method and use of the present invention.

[0110] Also provided are containers, for example made of plastic or glass, that contain a single dose or multiple doses of the compositions described herein. The containers can be, for example, vials, pre-filled syringes, pre-filled infusion bags, or cartridges designed as replaceable items for use with injection devices.

[0111] The compositions of the invention may be suitably packaged for infusion or injection, particularly for intravenous, intravenous, subcutaneous, or intramuscular injection.

[0112] The compositions of the present invention may be suitably filled into vials as concentrates for intravenous infusion. Prior to use, the concentrates are removed from the vials and diluted in an infusion bag containing a suitable diluent, such as saline, glucose solution, or water for injection. The diluted compositions are then administered by intravenous infusion at a specific infusion rate (e.g., 8-16 mL / min).

[0113] One embodiment of the present invention is an injection or infusion device, particularly a device adapted for subcutaneous or intramuscular injection or infusion, for single or multiple use, comprising a container containing one or multiple doses of the composition of the present invention together with an injection needle.In some embodiments, the container is a replaceable cartridge containing multiple doses.In one embodiment, the injection device is in the form of a pen.In one embodiment, the injection device is in the form of a pre-filled syringe.In one embodiment, the injection or infusion device is in the form of a pump or another wearable injection or infusion device.

[0114] Compositions according to the present invention are expected to have good physical and chemical stability as described herein.

[0115] Description of sequence listing

[0116] SEQ ID NO:1 is the full-length human AAT polypeptide sequence.

[0117] SEQ ID NO:2 is the full-length human AAT polypeptide sequence.

[0118] SEQ ID NO:3 is the reactive site loop portion of the AAT protein.

[0119] SEQ ID NO:4 is the reactive site loop portion of the AAT protein.

[0120] SEQ ID NO:5 is the reactive site loop portion of the AAT protein.

[0121] SEQ ID NO:6 is a human IgG1 Fc polypeptide sequence.

[0122] SEQ ID NO:7 is a human IgG1 Fc polypeptide sequence including the hinge region at the N-terminus.

[0123] SEQ ID NO:8 is a modified IgG1Fc polypeptide sequence having mutations at residues M252, T256, and M428.

[0124] SEQ ID NO: 9 is a modified IgG1Fc polypeptide sequence comprising the N-terminal hinge region, with mutations at residues M252, T256, and M428.

[0125] SEQ ID NO:10 is a modified IgG1Fc polypeptide sequence in which residue G236 is deleted.

[0126] SEQ ID NO:11 is a modified IgG1Fc polypeptide sequence comprising the hinge region at the N-terminus, in which residue G236 is deleted.

[0127] SEQ ID NO: 12 is a modified IgG1Fc polypeptide sequence having mutations at residues L234 and L235.

[0128] SEQ ID NO: 13 is a modified IgG1Fc polypeptide sequence comprising the N-terminal hinge region, with mutations at residues L234 and L235.

[0129] SEQ ID NO: 14 is a modified IgG1 Fc polypeptide sequence having a deletion at residue G236 and mutations at residues L234 and L235.

[0130] SEQ ID NO: 15 is a modified IgG1 Fc polypeptide sequence comprising the N-terminal hinge region with a deletion at residue G236 and mutations at residues L234 and L235.

[0131] SEQ ID NO: 16 is a modified IgG1 Fc polypeptide sequence having a deletion at residue G236 and mutations at residues L234, L235, M252, T256, and M428.

[0132] SEQ ID NO: 17 is a modified IgG1 Fc polypeptide sequence comprising the N-terminal hinge region with a deletion at residue G236 and mutations at residues L234, L235, M252, T256, and M428.

[0133] SEQ ID NO:18 is a human IgG2 Fc polypeptide sequence.

[0134] SEQ ID NO:19 is a modified IgG2 Fc polypeptide sequence having a deletion at residue G236 and mutations at residues M252, T256, and M428.

[0135] SEQ ID NO:20 is a human IgG3 Fc polypeptide sequence.

[0136] SEQ ID NO:21 is a modified IgG3Fc polypeptide sequence having mutations at residues M252, T256, and M428.

[0137] SEQ ID NO:22 is a modified IgG3Fc polypeptide sequence having a deletion at residue G236.

[0138] SEQ ID NO:23 is a modified IgG3Fc polypeptide sequence having mutations at residues L234 and L235.

[0139] SEQ ID NO:24 is a modified IgG3Fc polypeptide sequence having a deletion at residue G236 and mutations at residues L234 and L235.

[0140] SEQ ID NO:25 is a modified IgG3 Fc polypeptide sequence having a deletion at residue G236 and mutations at residues L234, L235, M252, T256, and M428.

[0141] SEQ ID NO:26 is a human IgG4 Fc polypeptide sequence.

[0142] SEQ ID NO:27 is a human IgG4 Fc polypeptide sequence including the hinge region at the N-terminus.

[0143] SEQ ID NO:28 is a modified IgG4 Fc polypeptide sequence having mutations at residues M252, T256, and M428.

[0144] SEQ ID NO: 29 is a modified IgG4 Fc polypeptide sequence comprising the N-terminal hinge region, with mutations at residues M252, T256, and M428.

[0145] SEQ ID NO:30 is a modified IgG4Fc polypeptide sequence having a deletion at residue G236.

[0146] SEQ ID NO:31 is a modified IgG4Fc polypeptide sequence comprising the hinge region at the N-terminus, having a deletion at residue G236.

[0147] SEQ ID NO: 32 is a modified IgG4Fc polypeptide sequence having a mutation at residue L235.

[0148] SEQ ID NO: 33 is a modified IgG4Fc polypeptide sequence comprising the N-terminal hinge region, having a mutation at residue L235.

[0149] SEQ ID NO: 34 is a modified IgG4 Fc polypeptide sequence having mutations at residues L234 and L235.

[0150] SEQ ID NO: 35 is a modified IgG4 Fc polypeptide sequence comprising the N-terminal hinge region with mutations at residues L234 and L235.

[0151] SEQ ID NO: 36 is a modified IgG4 Fc polypeptide sequence having a mutation at residue S228.

[0152] SEQ ID NO: 37 is a modified IgG4 Fc polypeptide sequence comprising the N-terminal hinge region with mutations at residues S228 and L235.

[0153] SEQ ID NO: 38 is a modified IgG4 Fc polypeptide sequence comprising the N-terminal hinge region, with mutations at residues S228, L235, and M252.

[0154] SEQ ID NO: 39 is a modified IgG4 Fc polypeptide sequence comprising the N-terminal hinge region, with mutations at residues S228, L235, and M428.

[0155] SEQ ID NO: 40 is a modified IgG4 Fc polypeptide sequence comprising the N-terminal hinge region, with mutations at residues S228, L235, M252, and M428.

[0156] SEQ ID NO: 41 is a modified IgG4 Fc polypeptide sequence having mutations at residues L235, M252, T256, and M428.

[0157] SEQ ID NO: 42 is a modified IgG4 Fc polypeptide sequence comprising the N-terminal hinge region, with mutations at residues L235, M252, T256, and M428.

[0158] SEQ ID NO: 43 is a modified IgG4 Fc polypeptide sequence comprising the N-terminal hinge region, with mutations at residues S228, L235, M252, T256, and M428.

[0159] SEQ ID NO:44 is a human IgMFc polypeptide.

[0160] SEQ ID NO:45 is an AAT polypeptide-IgG-Fc polypeptide fusion protein comprising SEQ ID NO:1 and SEQ ID NO:6.

[0161] SEQ ID NO:46 is an AAT polypeptide-IgG-Fc polypeptide fusion protein comprising SEQ ID NO:1 and SEQ ID NO:18.

[0162] SEQ ID NO:47 is an AAT polypeptide-IgG-Fc polypeptide fusion protein comprising SEQ ID NO:48 and SEQ ID NO:6.

[0163] SEQ ID NO:48 is an AAT polypeptide.

[0164] SEQ ID NO:49 is an AAT polypeptide-IgG-Fc polypeptide fusion protein comprising SEQ ID NO:48 and SEQ ID NO:18.

[0165] SEQ ID NO:50 is an AAT polypeptide-IgG-Fc polypeptide fusion protein comprising SEQ ID NO:51 and SEQ ID NO:18.

[0166] SEQ ID NO:51 is an AAT polypeptide.

[0167] SEQ ID NO:52 is an AAT polypeptide-IgG-Fc polypeptide fusion protein comprising SEQ ID NO:1 and SEQ ID NO:6.

[0168] SEQ ID NO:53 is an AAT polypeptide-IgG-Fc polypeptide fusion protein comprising SEQ ID NO:2 and SEQ ID NO:17.

[0169] SEQ ID NO:54 is an AAT polypeptide-IgG-Fc polypeptide fusion protein comprising SEQ ID NO:2 and SEQ ID NO:43.

[0170] SEQ ID NO:55 is an AAT polypeptide-IgG-Fc polypeptide fusion protein comprising SEQ ID NO:51 and SEQ ID NO:7.

[0171] SEQ ID NO:56 is an AAT polypeptide-IgG4-Fc polypeptide fusion protein comprising SEQ ID NO:48 and SEQ ID NO:40.

[0172] Each monomer of the engineered dimeric protein of the present invention comprises at least one (e.g., one or two) human serpin polypeptides operably linked to a human immunoglobulin Fc polypeptide or a polypeptide derived from an immunoglobulin Fc polypeptide.

[0173] As used herein, "operably linked" means linked as part of a continuous polypeptide chain, i.e., as a fusion protein. Such engineered proteins can be prepared by recombinant engineering techniques. As used herein, "operably linked" also means that at least one human serpin polypeptide fused to an immunoglobulin Fc polypeptide is functional with respect to inhibiting serine protease activity.

[0174] In certain embodiments, the human serpin polypeptide is a human alpha-1 antitrypsin (AAT) polypeptide or is derived from a human AAT polypeptide.

[0175] In certain embodiments, each monomer of the dimeric protein comprises one human serpin polypeptide.

[0176] In one embodiment, each monomer of the engineered dimeric protein comprises at least one (e.g., one) polypeptide selected from a human alpha-1 antitrypsin (AAT) polypeptide and a polypeptide derived from a human AAT polypeptide, wherein the at least one polypeptide is operably linked to the N-terminus of a human immunoglobulin Fc polypeptide or a polypeptide derived from an immunoglobulin Fc polypeptide.

[0177] For example, the or each polypeptide is a human alpha-1 antitrypsin (AAT) polypeptide or is derived from a human AAT polypeptide having the sequence of SEQ ID NO:1 or 2.

[0178] For example, the or each polypeptide that is human alpha-1 antitrypsin (AAT) or derived from a human AAT polypeptide has the sequence of SEQ ID NO: 1. For example, the or each polypeptide that is human alpha-1 antitrypsin (AAT) or derived from a human AAT polypeptide has a sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:1.

[0179] In some embodiments, the full-length human AAT polypeptide sequence has the following amino acid sequence: [ka] (SEQ ID NO:1)

[0180] For example, the or each polypeptide that is human alpha-1 antitrypsin (AAT) or is derived from a human AAT polypeptide has the sequence of SEQ ID NO: 2. For example, the or each polypeptide that is human alpha-1 antitrypsin (AAT) or is derived from a human AAT polypeptide has a sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:2.

[0181] In some embodiments, the full-length human AAT polypeptide sequence has the following amino acid sequence: [ka] (SEQ ID NO:2)

[0182] For example, the or each polypeptide which is human alpha-1 antitrypsin (AAT) or is derived from the human AAT polypeptide sequence is listed in GenBank Accession Nos. AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA34982. 1, NP_001002236.1, NP_000286.3, NP_001121179.1, NP_001121178.1, NP_001121177.1, NP_001121176.16, NP_001121175.1, NP_001121174.1, NP_001121172.1, and / or AAA51547.1.

[0183] For example, the or each polypeptide that is a human alpha-1 antitrypsin (AAT) polypeptide comprises a reactive loop sequence according to any one of the sequences of SEQ ID NOs: 3, 4, and 5.

[0184] In some embodiments, the reactive site loop portion of the AAT protein comprises at least the amino acid sequence: GTEAAGAMFLEAIPMSIPPEVKFNK (SEQ ID NO: 3). In some embodiments, the reactive site loop portion of the AAT protein comprises at least the amino acid sequence: GTEAAGAFLEAIPLSIPPEVKFNK (SEQ ID NO: 4). In some embodiments, the reactive site loop portion of the AAT protein comprises at least the amino acid sequence: GTEAAGALFLEAIPLSIPPEVKFNK (SEQ ID NO: 5).

[0185] In certain embodiments, the polypeptide comprises a human immunoglobulin Fc polypeptide, or the polypeptide derived from an immunoglobulin Fc polypeptide is a human IgG1 Fc polypeptide. In some embodiments, the human IgG1 Fc polypeptide sequence has the following amino acid sequence: [ka] (SEQ ID NO:6)

[0186] In some embodiments, the polypeptide comprises a hinge region attached to the N-terminus of an Fc polypeptide of the polypeptide, wherein the Fc polypeptide comprises a human IgG1 Fc polypeptide sequence having the following amino acid sequence: [ka] (SEQ ID NO:7)

[0187] In some embodiments where the polypeptide of the invention comprises an Fc polypeptide, the Fc polypeptide of the polypeptide comprises a human IgG1 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:6 or 7.

[0188] In some embodiments, the polypeptide of the invention comprises a modified IgG1Fc polypeptide, wherein the modified IgG1Fc polypeptide of the fusion protein comprises mutations at residues M252, T256, and M428, which correspond to residues 22, 26, and 198 of SEQ ID NO:6, or residues 32, 36, and 208 of SEQ ID NO:7 as described above, and has the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:8)

[0189] In some embodiments, the polypeptide of the invention comprises a hinge region attached to the N-terminus of a modified IgG1Fc polypeptide, wherein the modified IgG1Fc polypeptide of the fusion protein comprises mutations at residues M252, T256, and M428, which correspond to residues 22, 26, and 198 of SEQ ID NO:6, or residues 32, 36, and 208 of SEQ ID NO:7, as described above, and wherein the fusion protein comprises at least the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:9)

[0190] In some embodiments, the polypeptide of the invention comprises a modified IgG1Fc polypeptide, wherein the modified IgG1Fc polypeptide of the fusion protein comprises a modified human IgG1Fc polypeptide sequence in which residue G236, corresponding to residue 6 of SEQ ID NO:6 or residue 16 of SEQ ID NO:7 as described above, has been deleted, and has the following amino acid sequence: [ka] (SEQ ID NO:10)

[0191] In some embodiments, the polypeptide of the invention comprises a hinge region attached to the N-terminus of a modified IgG1Fc polypeptide, the modified IgG1Fc polypeptide of the fusion protein comprises a modified human IgG1Fc polypeptide sequence lacking residue G236, which corresponds to residue 6 of SEQ ID NO:6 or residue 16 of SEQ ID NO:7 as described above, and the fusion protein comprises at least the following amino acid sequence: [ka] (SEQ ID NO:11)

[0192] In some embodiments, the polypeptides of the invention comprise a modified IgG1Fc polypeptide, wherein the modified IgG1Fc polypeptide of the fusion protein comprises a mutation at residues L234 and L235, which correspond to residues 4 and 5 of SEQ ID NO:6 or residues 14 and 15 of SEQ ID NO:7 as described above, and has the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:12)

[0193] In some embodiments, a polypeptide of the invention comprises a hinge region attached to the N-terminus of a modified IgG1Fc polypeptide, wherein the modified IgG1Fc polypeptide of the polypeptide comprises mutations at residues L234 and L235, which correspond to residues 4 and 5 of SEQ ID NO:6 or residues 14 and 15 of SEQ ID NO:7 as described above, and the fusion protein comprises at least the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:13)

[0194] In some embodiments, the polypeptide of the invention comprises a modified IgG1Fc polypeptide, wherein the modified IgG1Fc polypeptide of the fusion protein comprises a deletion at residue G236 and mutations at residues L234 and L235, and the fusion protein comprises at least the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:14)

[0195] In some embodiments, a polypeptide of the invention comprises a hinge region attached to the N-terminus of a modified IgG1Fc polypeptide, wherein the modified IgG1Fc polypeptide of the fusion protein comprises a deletion at residue G236 and mutations at residues L234 and L235, and has the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:15)

[0196] In some embodiments, the polypeptide of the invention comprises a modified IgG1Fc polypeptide, wherein the modified IgG1Fc polypeptide of the fusion protein comprises a deletion at residue G236 and mutations at residues L234, L235, M252, T256, and M428, and wherein the fusion protein comprises at least the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:16)

[0197] In some embodiments, a polypeptide of the invention comprises a hinge region attached to the N-terminus of a modified IgG1Fc polypeptide, wherein the modified IgG1Fc polypeptide of the fusion protein comprises a deletion at residue G236 and mutations at residues L234, L235, M252, T256, and M428, and has the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:17)

[0198] In some embodiments, the polypeptide of the present invention comprises a modified IgG1Fc polypeptide, wherein the modified IgG1Fc polypeptide of the fusion protein comprises a modified human IgG1Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17.

[0199] In some embodiments, the polypeptide of the invention comprises an Fc polypeptide, and the Fc polypeptide of the fusion protein comprises a human IgG2 Fc polypeptide sequence having the following amino acid sequence: [ka] (SEQ ID NO:18)

[0200] In some embodiments where a fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein comprises a modified human IgG2 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:18.

[0201] In some embodiments, a polypeptide of the invention comprises a modified IgG2Fc polypeptide, wherein the modified IgG2Fc polypeptide of the fusion protein comprises a deletion at residue G236 and mutations at residues M252, T256, and M428, and has the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:19)

[0202] In some embodiments, the polypeptide of the invention comprises an Fc polypeptide, and the Fc polypeptide of the fusion protein comprises a human IgG3 Fc polypeptide sequence having the following amino acid sequence: [ka] (SEQ ID NO:20)

[0203] In some embodiments, a polypeptide of the invention comprises an Fc polypeptide, wherein the Fc polypeptide of the fusion protein comprises a human IgG3 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:20.

[0204] In some embodiments, a polypeptide of the invention comprises a modified IgG3Fc polypeptide, wherein the modified IgG3Fc polypeptide of the fusion protein comprises mutations at residues M252, T256, and M428 and has the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:21)

[0205] In some embodiments, the polypeptide of the invention comprises a modified IgG3Fc polypeptide, wherein the modified IgG3Fc polypeptide of the fusion protein comprises a modified human IgG3Fc polypeptide sequence in which residue G236, corresponding to residue 6 of SEQ ID NO:20 described above, is deleted, and has the following amino acid sequence: [ka] (SEQ ID NO:22)

[0206] In some embodiments, the polypeptide of the invention comprises a modified IgG3Fc polypeptide, wherein the modified IgG3Fc polypeptide of the fusion protein comprises mutations at residues L234 and L235, which correspond to residues 4 and 5 of SEQ ID NO:20 described above, and has the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:23)

[0207] In some embodiments in which a fusion protein of the invention comprises a modified IgG3Fc polypeptide, the modified IgG3Fc polypeptide of the fusion protein comprises a deletion at residue G236 and mutations at residues L234 and L235, and has the following amino acid sequence: [ka] (SEQ ID NO:24)

[0208] In some embodiments, the polypeptide of the invention comprises a modified IgG3Fc polypeptide, wherein the modified IgG3Fc polypeptide of the fusion protein comprises a deletion at residue G236 and mutations at residues L234, L235, M252, T256, and M428, and has the following amino acid sequence: [ka] (SEQ ID NO:25)

[0209] In some embodiments, the polypeptide of the present invention comprises a modified IgG3Fc polypeptide, wherein the modified IgG3Fc polypeptide of the fusion protein comprises a modified human IgG3Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21, 22, 23, 24, or 25.

[0210] In some embodiments, the polypeptide of the invention comprises an Fc polypeptide, and the Fc polypeptide of the fusion protein comprises a human IgG4 Fc polypeptide sequence having the following amino acid sequence: [ka] (SEQ ID NO:26)

[0211] In some embodiments, the polypeptides of the invention comprise an Fc polypeptide, wherein the Fc polypeptide of the fusion protein comprises a hinge region attached to the N-terminus of the Fc polypeptide of the fusion protein, wherein the Fc polypeptide comprises a human IgG4 Fc polypeptide sequence having the following amino acid sequence: [ka] (SEQ ID NO:27)

[0212] In some embodiments, when a fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein comprises a human IgG4 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26 or 27.

[0213] In some embodiments in which a fusion protein of the invention comprises a modified IgG4Fc polypeptide, the modified IgG4Fc polypeptide of the fusion protein comprises mutations at residues M252, T256, and M428, which correspond to residues 22, 26, and 19 of SEQ ID NO:26 or residues 34, 38, and 210 of SEQ ID NO:27 as described above, and has the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:28)

[0214] In some embodiments, the polypeptide of the invention comprises a hinge region attached to the N-terminus of a modified IgG4Fc polypeptide, wherein the modified IgG4Fc polypeptide of the fusion protein comprises mutations at residues M252, T256, and M428, which correspond to residues 22, 26, and 19 of SEQ ID NO:26 or residues 34, 38, and 210 of SEQ ID NO:27 as described above, and wherein the fusion protein comprises at least the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:29)

[0215] In some embodiments, the polypeptide of the invention comprises a modified IgG4Fc polypeptide, wherein the modified IgG4Fc polypeptide of the fusion protein comprises a modified human IgG4Fc polypeptide sequence in which residue G236, corresponding to residue 6 of SEQ ID NO:26 or residue 19 of SEQ ID NO:27 as described above, is deleted, and has the following amino acid sequence: [ka] (SEQ ID NO:30)

[0216] In some embodiments, the polypeptide of the invention comprises a hinge region attached to the N-terminus of a modified IgG4Fc polypeptide, the modified IgG4Fc polypeptide of the fusion protein comprises a modified human IgG4Fc polypeptide lacking residue G236, which corresponds to residue 6 of SEQ ID NO:26 or residue 19 of SEQ ID NO:27 as described above, and the fusion protein has at least the following amino acid sequence: [ka] (SEQ ID NO:31)

[0217] In some embodiments, the polypeptide of the invention comprises a modified IgG4Fc polypeptide, wherein the modified IgG4Fc polypeptide of the fusion protein comprises a mutation at residue L235, which corresponds to residue 5 of SEQ ID NO:26 or residue 17 of SEQ ID NO:27 as described above, and has the following amino acid sequence, where the mutated amino acid residue is boxed: [ka] (SEQ ID NO:32)

[0218] In some embodiments, a polypeptide of the invention comprises a hinge region attached to the N-terminus of a modified IgG4Fc polypeptide, wherein the modified IgG4Fc polypeptide of the fusion protein comprises a mutation at residue L235, which corresponds to residue 5 of SEQ ID NO:26 or residue 17 of SEQ ID NO:27 as described above, and has the following amino acid sequence, where the mutated amino acid residue is boxed: [ka] (SEQ ID NO:33)

[0219] In some embodiments, the polypeptide of the invention comprises a modified IgG4Fc polypeptide, wherein the modified IgG4Fc polypeptide of the fusion protein comprises a mutation at residues L234 and L235, which correspond to residues 4 and 5 of SEQ ID NO:26 or residues 16 and 17 of SEQ ID NO:27 as described above, and has the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:34)

[0220] In some embodiments, the polypeptide of the invention comprises a hinge region attached to the N-terminus of a modified IgG4Fc polypeptide, the modified IgG4Fc polypeptide of the fusion protein comprises mutations at residues L234 and L235, which correspond to residues 4 and 5 of SEQ ID NO:26 or residues 16 and 17 of SEQ ID NO:27 as described above, and the fusion protein comprises at least the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:35)

[0221] In some embodiments, a polypeptide of the invention comprises a modified IgG4Fc polypeptide, wherein the modified IgG4Fc polypeptide of the fusion protein comprises a mutation at residue S228, which corresponds to residue 10 of SEQ ID NO:27 as described above, and has the following amino acid sequence, where the mutated amino acid residue is boxed: [ka] (SEQ ID NO:36)

[0222] In some embodiments, the polypeptide of the invention comprises a hinge region attached to the N-terminus of a modified IgG4Fc polypeptide, the modified IgG4Fc polypeptide of the fusion protein comprises mutations at residues S228 and L235, which correspond to residues 10 and 17 of SEQ ID NO:27 as described above, and the fusion protein comprises at least the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:37)

[0223] In some embodiments, the polypeptide of the invention comprises a hinge region attached to the N-terminus of a modified IgG4Fc polypeptide, the modified IgG4Fc polypeptide of the fusion protein comprises mutations at residues S228, L235, and M252, which correspond to residues 10, 17, and 34 of SEQ ID NO:27 as described above, and the fusion protein comprises at least the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:38)

[0224] In some embodiments, the polypeptide of the invention comprises a hinge region attached to the N-terminus of a modified IgG4Fc polypeptide, the modified IgG4Fc polypeptide of the fusion protein comprises mutations at residues S228, L235, and M428, which correspond to residues 10, 17, and 34 of SEQ ID NO:27 as described above, and the fusion protein comprises at least the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:39)

[0225] In some embodiments, the polypeptide of the invention comprises a hinge region attached to the N-terminus of a modified IgG4Fc polypeptide, the modified IgG4Fc polypeptide of the fusion protein comprises mutations at residues S228, L235, M252, and M428, which correspond to residues 10, 17, 34, and 210 of SEQ ID NO:27 as described above, and the fusion protein comprises at least the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:40)

[0226] In some embodiments, the polypeptide of the invention comprises a modified IgG4Fc polypeptide, wherein the modified IgG4Fc polypeptide of the fusion protein comprises mutations at residues L235, M252, M256, and M428, which correspond to residues 5, 22, 26, and 197 of SEQ ID NO:26 or residues 17, 34, 38, and 210 of SEQ ID NO:27 as described above, and comprises the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:41)

[0227] In some embodiments, the polypeptide of the invention comprises a hinge region attached to the N-terminus of a modified IgG4Fc polypeptide, wherein the modified IgG4Fc polypeptide of the fusion protein comprises mutations at residues L235, M252, M256, and M428, which correspond to residues 5, 22, 26, and 197 of SEQ ID NO:26 or residues 17, 34, 38, and 210 of SEQ ID NO:27 as described above, and wherein the fusion protein comprises at least the following amino acid sequence, where the mutated amino acid residues are boxed: [ka] (SEQ ID NO:42)

[0228] In some embodiments, the polypeptide of the invention comprises a hinge region attached to the N-terminus of a modified IgG4Fc polypeptide, the modified IgG4Fc polypeptide of the fusion protein comprises mutations at residues S228, L235, M252, T256, and M428, which correspond to residues 10, 17, 34, 38, and 210 of SEQ ID NO: 27 as described above, and the fusion protein comprises at least the following amino acid sequence, with the mutated amino acid residues boxed: [ka] (SEQ ID NO:43)

[0229] In some embodiments where a fusion protein of the invention comprises a modified IgG4Fc polypeptide, the modified IgG4Fc polypeptide of the fusion protein comprises a modified human IgG4Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43.

[0230] For example, the human immunoglobulin Fc polypeptide or a polypeptide derived from an immunoglobulin Fc polypeptide is a modified human IgG4 Fc polypeptide having the sequence of SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43.

[0231] In some embodiments, the polypeptides of the invention comprise an Fc polypeptide derived from a modified human IgG4 Fc polypeptide, wherein the modified human IgG4 Fc polypeptide comprises mutations at positions S228, L235, M252, T256, and M428.

[0232] In some embodiments, a polypeptide of the invention comprises a modified human IgG4Fc polypeptide, wherein the modified human IgG4Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 27, 36, or 37, wherein the modified human IgG4Fc polypeptide comprises a mutation at position M252 (residue 34 of SEQ ID NO: 27) and / or position M428 (residue 210 of SEQ ID NO: 27).

[0233] In certain embodiments, the modified human IgG4 Fc polypeptide further comprises a mutation at position T256 (residue 38 of SEQ ID NO:27).

[0234] In some embodiments, the polypeptide of the invention comprises an Fc polypeptide, and the Fc polypeptide of the fusion protein comprises a human IgM Fc polypeptide sequence having the following amino acid sequence: [ka] (SEQ ID NO:44)

[0235] In certain embodiments, the human immunoglobulin Fc polypeptide or a polypeptide derived from an immunoglobulin Fc polypeptide is a modified human IgG4 Fc polypeptide.

[0236] In one embodiment, each monomer of the dimeric protein has the sequence of SEQ ID NO: 45. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 1) and the IgG-Fc polypeptide portion of the fusion protein is shown in italics (SEQ ID NO: 6). [ka] (SEQ ID NO:45)

[0237] In one embodiment, each monomer of the dimeric protein has the sequence of SEQ ID NO: 46. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 1) and the IgG-Fc polypeptide portion of the fusion protein is shown in italics (SEQ ID NO: 18). [ka] (SEQ ID NO:46)

[0238] In one embodiment, each monomer of the dimeric protein has the sequence of SEQ ID NO: 47. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 48), the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 6), and the Met351Glu mutation is boxed and the Met358Leu mutation is shaded gray. [ka] (SEQ ID NO:47)

[0239] In one embodiment, each monomer of the dimeric protein has the sequence of SEQ ID NO: 49. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 48), the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 18), the Met351Glu mutation is boxed, and the Met358Leu mutation is shaded gray. [ka] (SEQ ID NO:49)

[0240] In one embodiment, each monomer of the dimeric protein has the sequence of SEQ ID NO: 50. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 51), the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 18), the Met351Glu mutation is shaded in black, and the Met358Leu mutation is shaded in gray. [ka] (SEQ ID NO:50)

[0241] In one embodiment, each monomer of the dimeric protein has the sequence of SEQ ID NO: 52. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 1) and the IgG-Fc polypeptide portion of the fusion protein is shown in italics (SEQ ID NO: 6). [ka] (SEQ ID NO:52)

[0242] In one embodiment, each monomer of the dimeric protein has the sequence of SEQ ID NO: 53. As shown below, the AAT polypeptide portion of the fusion protein is underlined, and the Met351Glu and Met358Leu mutations are boxed (SEQ ID NO: 2). The IgG1-Fc polypeptide portion of the fusion protein is shown in italics (SEQ ID NO: 17), with Gly236 deleted, and the mutations Met252Ile, Thr256Asp, and Met428Leu boxed. [ka] (SEQ ID NO:53)

[0243] In one embodiment, each monomer of the dimeric protein has the sequence of sequence number 54. As shown below, the AAT polypeptide portion of the fusion protein is underlined and the Met351Glu and Met358Leu mutations are boxed (SEQ ID NO:2). The IgG1-Fc polypeptide portion of the fusion protein is shown in italics (SEQ ID NO:43) and the mutations Ser228Pro, Leu235Glu, Met252Ile, Thr256Asp, and Met428Leu are boxed. [ka] (SEQ ID NO:54)

[0244] In one embodiment, each monomer of the dimeric protein has the sequence of SEQ ID NO: 55. As shown below, the AAT polypeptide portion of the fusion protein is underlined, the AAT reactive loop sequence of SEQ ID NO: 51, the Met351Leu mutation is shaded in black, and the Met358Leu mutation is shaded in grey, and the IgG1-Fc polypeptide portion of the fusion protein is shown in italics (SEQ ID NO: 7). [ka] (SEQ ID NO:55)

[0245] In one embodiment, each monomer of the dimeric protein has the sequence of SEQ ID NO: 56. As shown below, the AAT polypeptide portion of the fusion protein is underlined, the Met351Leu mutation is shaded in black, and the Met358Leu mutation is shaded in grey (SEQ ID NO: 48): and the IgG4-Fc polypeptide portion of the fusion protein is shown in italics, the mutations S228P, L235E, M252Y, and M428L are boxed, and the GS linker is shown in bold (SEQ ID NO: 40). [ka] (SEQ ID NO:56)

[0246] In some embodiments of the monomer of the dimeric protein of the present disclosure, the IgG polypeptide portion of the monomer can be connected to the AAT polypeptide portion without a GS linker. In some embodiments, the IgG polypeptide portion of the monomer of the dimeric protein of the present disclosure can be connected to the AAT polypeptide portion by a covalent bond.

[0247] The monomers of the dimeric protein can be linked to each other by disulfide bridges. In particular, a pair of human immunoglobulin Fc polypeptides, or polypeptides derived from immunoglobulin Fc polypeptides, are linked to form a functional Fc domain. Other embodiments

[0248] Although the present invention has been described in conjunction with its detailed description, the above description is for illustrative purposes only and is not intended to limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. Working Example

[0249] General method

[0250] Methods for assessing the stability of engineered dimeric proteins

[0251] a) Visual evaluation

[0252] Visible particles are suitably detected using 29.9.20 European Pharmacopoeia Monograph (Particle contamination: visible particles). The required equipment consists of an observation station equipped with: A good sized matte black panel held in a vertical position An appropriately sized non-glare white panel placed vertically next to a black panel An adjustable lamp holder fitted with a suitable light source of shaded white light and a suitable light diffuser (an observation illuminator containing two 13W fluorescent tubes, each 525mm long, is suitable). Illuminance at the viewpoint is maintained between 2000 lux and 3750 lux.

[0253] Remove any attached labels from the container and clean and dry the exterior. Gently rotate or invert the container to avoid trapping air bubbles and observe in front of a white panel for approximately 5 seconds. Repeat this procedure in front of a black panel. Record the presence of particles.

[0254] The visual scores are ranked as follows: Visual score 1: A clear solution with no visible particles Visual score 2: Slight particle formation (up to 20 very small particles) Visual score 3: More significant precipitation (more than 20 particles, including larger particles)

[0255] Particles in samples with a visual score of 3 are clearly detectable under normal light with a simple visual evaluation, while samples with visual scores of 1 and 2 usually appear as clear solutions with the same evaluation. Samples with a visual score of 1 and 2 are considered "passes" and samples with a visual score of 3 are considered "fails."

[0256] (b) Size Exclusion Chromatography (SEC)

[0257] Method 1

[0258] The amount of high molecular weight species is measured using a 300 x 7.8 mm TSK Gel G3000SWXL (or equivalent) size exclusion column. The mobile phase is 250 mM potassium chloride and 200 mM potassium phosphate buffer (pH 6.2) with a flow rate of 0.5 ml / min, an injection volume of 4 μl (corresponding to 200 μg protein) and detection at 280 nm. The run time is 30 min. Results are expressed as % High Molecular Weight Species (HMWS), i.e., the sum of all peak areas corresponding to aggregated protein relative to the sum of all protein-related peaks on the chromatogram.

[0259] Method 2

[0260] The amount of high molecular weight species is measured using a 300 x 7.8 mm TSK Gel G3000SWXL (or equivalent) size exclusion column. The mobile phase is 250 mM potassium chloride and 200 mM potassium phosphate buffer (pH 6.2) with a flow rate of 0.5 ml / min, an injection volume of 10 μl (corresponding to 500 μg protein), and detection at 280 nm. The run time is 30 min. Results are expressed as % High Molecular Weight Species (HMWS), i.e., the sum of all peak areas corresponding to aggregated protein relative to the sum of all protein-related peaks on the chromatogram.

[0261] In either method, an increase in %HMWS refers to the change observed in %HMWS at a given time point compared to the %HMWS value at time zero (i.e., immediately prior to incubation at the storage temperature).

[0262] (c) Subvisible Particles Assessment (SVP)

[0263] The number of sub visible particles per container in liquid samples is assessed using a HIAC9703+ liquid particle counter. Blank specimens and particle count sets are used for system suitability. Samples are degassed at 75 torr for 10 minutes before measurement and are tested undiluted. Results are reported as the average of triplicate measurements of 5 mL each.

[0264] Example 1 - Formulation Examples

[0265] The following example formulations can be prepared:

[0266] Example A: 50mg / ml of engineered dimeric protein of the present invention Tris 3mM Trehalose 300mM Proline 100mM Polysorbate 20 0.1mg / ml Water for injection (appropriate amount) Adjust pH to 7.3 using hydrochloric acid or sodium hydroxide Ionic strength 2.6mM

[0267] Example B: 50mg / ml of engineered dimeric protein of the present invention Tris 3mM Trehalose 300mM Methionine 2mM Polysorbate 20 0.1mg / ml Water for injection (appropriate amount) Adjust pH to 7.3 using hydrochloric acid or sodium hydroxide Ionic strength 2.6mM

[0268] Example C: 50mg / ml of engineered dimeric protein of the present invention Tris 3mM Trehalose 300mM Proline 100mM Methionine 2mM Polysorbate 20 0.1mg / ml Water for injection (appropriate amount) Adjust pH to 7.3 using hydrochloric acid or sodium hydroxide Ionic strength 2.6mM

[0269] Example D: 50mg / ml of engineered dimeric protein of the present invention Tris 5mM Trehalose 300mM Proline 100mM Polysorbate 20 0.1mg / ml Water for injection (appropriate amount) Adjust pH to 7.3 using hydrochloric acid or sodium hydroxide Ionic strength 4.3mM

[0270] Example E: 50mg / ml of engineered dimeric protein of the present invention Tris 3mM Trehalose 300mM Proline 100mM Poloxamer 188 1mg / ml Water for injection (appropriate amount) Adjust pH to 7.3 using hydrochloric acid or sodium hydroxide Ionic strength 2.6mM

[0271] Example F: 50mg / ml of engineered dimeric protein of the present invention Tris 5mM Trehalose 300mM Proline 100mM Poloxamer 188 1mg / ml Water for injection (appropriate amount) Adjust pH to 7.3 using hydrochloric acid or sodium hydroxide Ionic strength 4.3mM

[0272] Example G: 50mg / ml of engineered dimeric protein of the present invention Tris 5mM Trehalose 150mM Sucrose 100mM Proline 100mM Polysorbate 20 0.1mg / ml Water for injection (appropriate amount) Adjust pH to 7.3 using hydrochloric acid or sodium hydroxide Ionic strength 4.3mM

[0273] Example H: 50mg / ml of engineered dimeric protein of the present invention Tris 5mM Trehalose 150mM Sucrose 100mM Proline 100mM Methionine 2mM Poloxamer 188 1mg / ml Water for injection (appropriate amount) Adjust pH to 7.3 using hydrochloric acid or sodium hydroxide Ionic strength 4.3mM

[0274] Example I: 50mg / ml of engineered dimeric protein of the present invention Tris 5mM Trehalose 150mM Sucrose 100mM Glycine 100mM Methionine 2mM Poloxamer 188 1mg / ml Water for injection (appropriate amount) Adjust pH to 7.3 using hydrochloric acid or sodium hydroxide Ionic strength 4.3mM

[0275] Example J: 50mg / ml of engineered dimeric protein of the present invention Tris 5mM Trehalose 150mM Sucrose 100mM Glycine 100mM Poloxamer 188 1mg / ml Water for injection (appropriate amount) Adjust pH to 7.3 using hydrochloric acid or sodium hydroxide Ionic strength 4.3mM

[0276] Example K: 50mg / ml of engineered dimeric protein of the present invention Sodium phosphate 5mM Trehalose 150mM Sucrose 100mM Proline 100mM Methionine 2mM Polysorbate 0.1mg / ml Water for injection (appropriate amount) Adjust pH to 7.3 using hydrochloric acid or sodium hydroxide Ionic strength 11.1mM

[0277] Example L: 50mg / ml of engineered dimeric protein of the present invention Sodium phosphate 5mM Trehalose 150mM Sucrose 100mM Proline 100mM Methionine 2mM Poloxamer 188 1mg / ml Water for injection (appropriate amount) Adjust pH to 7.3 using hydrochloric acid or sodium hydroxide Ionic strength 11.1mM

[0278] The stability of the formulations can be determined using visual assessment and SEC (see General Methods) after incubation at 40° C. for 2, 4, and 8 weeks.

[0279] The stability of the formulations can be determined using visual assessment and SEC (see General Methods) after incubation at 25° C. for 2, 4, 8, 12, and 26 weeks.

[0280] The stability of the formulations can be determined using visual assessment and SEC (see General Methods) after incubation at 2-8° C. for 2, 4, 8, 12, and 26 weeks.

[0281] In the following examples, an engineered dimeric protein ("PROTEIN-1") having SEQ ID NO:56 as the monomer sequence was used.

[0282] Example 2 - Effect of ionic strength on storage stability of PROTEIN-1

[0283] The effect of ionic strength on the stability of PROTEIN-1 (50 mg / ml) was investigated by comparing a charged tonicity modifier (sodium chloride, 150 mM) with an uncharged tonicity modifier (glycerol, 300 mM).

[0284] All formulations contained polysorbate 20 (0.1 mg / mL) and either Tris (2 mM) or sodium phosphate (2 mM) as a buffer. Formulations containing Tris were adjusted to pH 8.0 and those containing sodium phosphate were adjusted to pH 7.0. Table 1 summarizes the formulations tested. [Table 1]

[0285] All formulations were stored for 7 weeks at 25° C. The stability of PROTEIN-1 was assessed by following the rate of formation of high molecular weight species using SEC (Method 1) as described in General Methods, and by visual assessment.

[0286] The HMWS generation rates for formulations 1-01 through 1-04 are shown in Table 2, where it can be seen that the HMWS generation rates are lowest for the low ionic strength formulations containing the uncharged tonicity modifier glycerol (compare formulations 1-01 and 1-03, and 1-02 and 1-04). The HMWS generation rates were observed to be higher for the equivalent formulations at pH 7.0 using sodium phosphate buffer compared to the formulations at pH 8.0 using Tris buffer (compare formulations 1-01 and 1-02, and 1-03 and 1-04). [Table 2]

[0287] Example 3 - Optimal pH for storage stability of PROTEIN-1

[0288] The effect of pH on the stability of PROTEIN-1 (50 mg / ml) was investigated. All formulations contained polysorbate 20 (0.1 mg / ml), Tris (1 mM) as a buffer, and either trehalose (300 mM) or a mixture of trehalose (150 mM) and mannitol (250 mM) as uncharged tonicity modifiers. Some formulations contained methionine (2 mM). Table 3 summarizes the formulations tested. [Table 3]

[0289] Formulations were stored for 26 weeks at 25° C. or 26 weeks at 2–8° C. The stability of PROTEIN-1 was assessed by following the rate of formation of high molecular weight species using SEC (Method 1) as described in General Methods, and by visual assessment.

[0290] The HMWS generation rates in formulations 2-01 through 2-06 are shown in Table 4, where it can be seen that at 25° C., the HMWS generation rate was lower in the pH 7.5 formulations compared to the pH 7.0 formulations at both 25° C. and 2-8° C. (comparing formulations 2-01 and 2-02 using trehalose as the uncharged tonicity modifier). Comparing formulations 2-03 and 2-06 (using a mixture of trehalose and mannitol as the uncharged tonicity modifier), it can be seen that the HMWS generation rate was slower at pH 7.5 compared to pH 7.0 (at both 25° C. and 2-8° C.), although the lowest rate of HMWS generation was observed at pH 7.2.

[0291] In summary, a pH of 7.2–7.5 appears to be particularly suitable for preventing the formation of HMWS. [Table 4]

[0292] Example 4 - Effect of buffer concentration on storage stability of PROTEIN-1

[0293] The effect of buffer concentration on the stability of PROTEIN-1 (50 mg / ml) was examined. All formulations contained polysorbate 20 (0.1 mg / ml). Table 5 summarizes the formulations tested. [Table 5]

[0294] Formulations were stored for 7 and 27 weeks at 25° C. or for 27 weeks at 2-8° C. The stability of PROTEIN-1 was assessed by following the rate of formation of high molecular weight species using SEC (Method 1) as described in General Methods, and by visual assessment.

[0295] The rate of HMWS formation in formulations 3-01 through 3-07 is shown in Table 6, where it can be seen that after 7 weeks at 25° C., the rate of HMWS formation was lower in formulations containing 2 mM Tris buffer compared to the corresponding formulations containing 50 mM Tris buffer (compare formulations 3-01 and 3-02 at pH 8). The same trend was observed at pH 7.0: the rate of HMWS was lower in formulations without Tris buffer compared to the corresponding formulations containing 50 mM Tris buffer (compare formulations 3-04 and 3-03 at pH 7.0, both of which contain glycerol as the uncharged tonicity modifier). After 27 weeks, the same trend was observed at both 25°C and 2-8°C, where decreasing the Tris buffer concentration from 5 mM to 2 mM to 1 mM reduced the rate of HMWS formation (comparing formulations 3-05 to 3-07, both containing a mixture of trehalose and mannitol as uncharged tonicity modifiers, at pH 7.5).

[0296] In summary, when using Tris as a buffer in the pH range of 7.0-7.5, the concentration should be less than 50 mM, and as low as possible to reduce the production of HMWS. [Table 6]

[0297] Example 5 - Effect of non-charged tonicity modifiers on storage stability of PROTEIN-1

[0298] The effect of different uncharged tonicity modifiers on the storage stability of PROTEIN-1 (50 mg / ml) was investigated. All formulations contained polysorbate 20 (0.1 mg / ml). Table 7 summarizes the formulations tested. [Table 7]

[0299] Formulations were stored for 12 weeks at 25° C., 26 weeks at 25° C., 16 weeks at 2-8° C., or 26 weeks at 2-8° C. The stability of PROTEIN-1 was assessed by following the rate of formation of high molecular weight species using SEC (Method 1) as described in General Methods, and by visual assessment.

[0300] The percentage of HMWS formed in formulations 4-01 through 4-07 is shown in Table 8, where it can be seen that when comparing the use of glycerol, trehalose, and sucrose as uncharged tonicity modifiers after 12 weeks at 25° C. and 16 weeks at 2-8° C., the percentage of HMWS formed was lowest in the formulations containing trehalose (compare formulations 4-01 through 4-03 with 2 mM Tris buffer at pH 7). The same was observed at the longer storage period of 26 weeks, where comparing the use of trehalose and sucrose as uncharged tonicity modifiers, the formulations containing trehalose had a lower percentage of HMWS (compare formulations 4-04 through 4-07 with 1 mM Tris buffer at pH 7.5). Of the two trehalose concentrations tested (200 mM and 300 mM), a lower HMWS generation rate was observed at the higher concentration of 300 mM (formulations 4-04 and 4-05). However, in some cases, especially at higher doses of the composition, the concentration of trehalose may be limited by pharmacologically acceptable limits (e.g., about 150 mM). In such cases, a mixture of trehalose with another uncharged tonicity modifier, such as sucrose, may be optimal.

[0301] In summary, the use of trehalose as the uncharged tonicity modifier gave the best stability profile for the concentrations and tonicity modifiers tested. [Table 8]

[0302] Example 6 - Effect of neutral amino acids on storage stability of PROTEIN-1

[0303] The effect of various neutral amino acids on the stability of PROTEIN-1 (50 mg / ml) was investigated. All formulations contained polysorbate 20 (0.1 mg / ml), Tris buffer (2 mM), and glycerol (300 mM) as uncharged tonicity modifiers. Table 9 summarizes the formulations tested. [Table 9]

[0304] The formulations were stored for 16 weeks at 25° C. The stability of PROTEIN-1 was assessed by following the rate of formation of high molecular weight species using SEC (Method 1) as described in General Methods, and by visual assessment.

[0305] The HMWS formation rates for formulations 5-01 through 5-06 are shown in Table 10, where it can be seen that after 16 weeks at 25° C., the addition of both glycine (300 mM) and proline (at all concentrations tested) reduced the HMWS formation rate (compare formulations 5-01 through 5-05 with the control formulation 5-06). When comparing glycine and proline directly, at a given concentration of neutral amino acid, the HMWS formation rate was lower in the formulations containing proline (compare formulation 5-01 with formulation 5-03 and formulation 5-02 with formulation 5-04). Of the three proline concentrations tested (100 mM, 200 mM, and 500 mM), the lowest HMWS formation rate was observed at the highest concentration of 500 mM (compare formulations 5-03, 5-04, and 5-05). In practice, however, the concentrations of proline used in commercial therapeutic formulations would ideally be lower than this (eg, about 100-150 mM) due to osmolarity limitations.

[0306] In summary, the addition of neutral amino acids (eg, proline or glycine, especially proline) resulted in more stable formulations. [Table 10]

[0307] Example 7 - Effect of combination of methionine with other neutral amino acids on storage stability of PROTEIN-1

[0308] The effect of methionine in combination with either the neutral amino acid glycine or proline on the stability of PROTEIN-1 (50 mg / ml) was examined. All formulations contained polysorbate 20 (0.1 mg / ml), Tris buffer (2 mM or 1 mM), and glycerol (300 mM) or trehalose (300 mM) as uncharged tonicity modifiers. Table 11 summarizes the formulations tested. [Table 11]

[0309] Formulations were stored for 16 weeks at 25° C., 26 weeks at 25° C., or 26 weeks at 2-8° C. The stability of PROTEIN-1 was assessed by following the rate of formation of high molecular weight species using SEC (Method 1) as described in General Methods, and by visual assessment.

[0310] The percentage of HMWS produced in formulations 6-01 through 6-04 is shown in Table 12, where it can be seen that the addition of methionine to the formulations containing glycine reduced the percentage of HMWS produced after 16 weeks at 25° C. (Compare formulations 6-01 and 6-02). The addition of methionine to the formulations containing proline also reduced the percentage of HMWS produced after 26 weeks at 25° C. or 2-8° C. (Compare formulations 6-03 and 6-04). [Table 12]

[0311] Example 8 - Storage stability testing of formulations of PROTEIN-1 with poloxamer surfactants

[0312] The effect of poloxamer surfactants on the stability of PROTEIN-1 (50 mg / ml) was investigated. The batch of PROTEIN-1 used in this example was different from the batch used in Examples 2-7. Table 13 summarizes the formulations tested. [Table 13]

[0313] Formulations were stored for 6, 9, 12, and 24 months at 25° C. and 2-8° C. The stability of PROTEIN-1 was assessed by following the rate of formation of high molecular weight species using SEC (Method 1) as described in General Methods, and by visual assessment.

[0314] As can be seen from Tables 14A and 14B, under storage conditions the formulations produced a lower rate of HMWS.

[0315] As shown in Tables 15A and 15B, the formulations tested passed the visual test after storage at 25°C and 2-8°C. [Table 14] [Table 15] [Table 16] [Table 17]

[0316] Example 9 - Evaluation of Subvisible Particle Size Limits of PROTEIN-1 Formulations

[0317] The stability of the PROTEIN-1 formulations described in Table 16A was evaluated after storage at 25° C. for 6 months by visual assessment and by determining the number of subvisible particles (SVPs) (both as described in General Methods). [Table 18]

[0318] After six months of storage at 25° C., formulation 9-01 contained very few visible particles. The number of subvisible particles is shown in Table 16B. [Table 19]

[0319] USP <788> The Particulate Matter in Parenteral Drugs11 (harmonized with Ph.Eur.2.9.19) requires that preparations less than 100 mL contain no more than 6000 particles per container ≥10 μm and no more than 600 particles per container ≥25 μm, as measured by light obscuration (such as the common method HIAC). Thus, after long-term storage, formulation 9-01 is well below the upper limit required for subvisible particles, indicating excellent storage stability.

[0320] Example 10 - Design of Experiments (DOE) Study Evaluating Formulation Component Concentrations on PROTEIN-1 Stability

[0321] The effect of varying protein, buffer, and poloxamer surfactant concentrations on the stability of PROTEIN-1 was assessed by following the rate of formation of high molecular weight species in the formulations using SEC (Method 2), as described in General Methods. The rate of HMWS formation in the various formulations after 12 months at 5° C. is shown in Table 17. [Table 20]

[0322] All formulations tested showed good storage stability. Comparing formulations RF08 with RF02, RF01 with RF11, RF15 with RF09, and RF16 with RF05, it can be seen that lowering the protein concentration slightly improves stability. Comparing formulations RF08 and RF01 with RF15 and RF16, and formulations RF02 and RF11 with RF09 and RF05, it can be seen that lower buffer (Tris) concentrations slightly improve stability, but even higher buffer (Tris) concentrations of 10 mM result in formulations with good stability. Finally, increasing the poloxamer concentration from 0.03% to 0.17% had minimal impact on storage stability.

[0323] Comparative Example 11-Effect of Buffer Concentration, Charge of Tonicity Modifier, and Neutral Amino Acid on Storage Stability of Immunoglobulin G1 (IgG1) at 30°C

[0324] The effect of buffer concentration and charge of tonicity modifier on the stability of IgG1 (100 mg / ml) was investigated. Citrate buffer was tested. Sodium chloride (150 mM) was used as the charged tonicity modifier and glycerol (300 mM) was used as the uncharged tonicity modifier. The effect of proline and glycine (50 mM) on the stability of IgG1 in the presence of 1 mM buffer and glycerol (300 mM) was also investigated. All formulations tested contained polysorbate 80 (0.2 mg / ml) and were adjusted to pH 6.0. Table 18 summarizes the formulations tested. All formulations were stressed at 30° C. for 8 weeks. The stability of IgG1 was followed by following the generation rate of high molecular weight species using SEC (Method 1). [Table 21]

[0325] All formulations tested passed visual inspection after storage at 30° C. The HMWS generation ratios for formulations 8-01 through 8-08 after storage at 30° C. are shown in Table 19. The HMWS generation ratios decreased with increasing buffer concentration in both the presence of sodium chloride and glycerol (compare formulations 8-01 and 8-03, and 8-04 and 8-06). At lower citrate concentrations (1 or 5 mM), there was a trend toward slightly greater stability at higher ionic strengths of the formulations (compare formulations 8-01 and 8-04, and 8-02 and 8-05). This order was reversed at higher citrate concentrations (20 mM) (compare formulations 8-03 and 8-06), where the ionic strength of both formulations was greater than 70 mM. The presence of neutral amino acids (proline or glycine) only slightly increased the rate of HMWS production (compare formulation 8-04 with formulations 8-07 and 8-08). [Table 22]

[0326] Overview of the Example

[0327] The data in Examples 2-10 show that compositions of engineered dimeric proteins of the invention as defined herein, such as PROTEIN-1, are stable when formulated at low ionic strength with neutral amino acids. The ionic strength of the compositions is kept appropriately low by using low buffer concentrations (or no buffer at all) and by using uncharged tonicity modifiers instead of charged tonicity modifiers.

[0328] Comparing these data with Comparative Example 11, it can be seen that this behavior is significantly different from that exhibited by the tested four-chain antibody (IgG1 type), which turned out to be more stable at higher buffer concentrations and in some cases at higher ionic strengths, and was also found to be destabilized in the presence of neutral amino acids.

[0329] Without being limited by theory, the present invention combines compositional features that are believed to work in concert to screen for unnatural hydrophobic patches as well as minimize the rate of proton exchange at unnaturally exposed unstable sites, resulting in stability of engineered dimeric proteins of the present invention, such as PROTEIN-1.

[0330] Throughout this specification and the claims that follow, unless the context indicates otherwise, the word "comprise" and variations such as "comprises" and "comprising" are understood to mean the inclusion of a stated integer, step, group of integers, or group of steps, but not the exclusion of other integers, steps, groups of integers, or groups of steps.

[0331] All patents, patent applications, and literature references mentioned throughout the present specification are hereby incorporated by reference in their entirety.

[0332] The present invention includes all combinations of preferred and more preferred groups, suitable groups and more suitable groups, and embodiments of the groups described above.

Claims

1. An aqueous solution composition comprising an engineered dimeric protein, Each monomer of the dimeric protein comprises an AAT polypeptide comprising SEQ ID NO: 48 and a modified human IgG4 Fc polypeptide; The aqueous solution composition has a pH in the range of 6.0 to 8.0 and is (i) 0.1 to 10 mM tris(hydroxymethyl)aminomethane (Tris) (ii) 50 to 1000 mM trehalose (iii) 20 to 600 mM proline or 20 to 600 mM glycine and the total ionic strength of the composition, excluding the contribution of the engineered dimeric protein, is less than 30 mM; Aqueous composition.

2. 10. The aqueous solution composition of claim 1, wherein the engineered dimeric protein is present at a concentration of 1-400 mg / ml, 10-200 mg / ml, 20-100 mg / ml, or 30-60 mg / ml, or wherein the engineered dimeric protein is present at a concentration of about 35 mg / ml, or about 50 mg / ml.

3. The aqueous solution composition described in claim 1, wherein the concentration of Tris is 3 mM.

4. The aqueous solution composition of claim 1, wherein the concentration of Tris is 5 mM.

5. The aqueous solution composition of claim 1, wherein the concentration of the trehalose is about 150 mM.

6. The aqueous solution composition of claim 1, wherein the concentration of the trehalose is 300 mM.

7. 18. The aqueous solution composition according to any one of claims 1 to 17, wherein the aqueous solution composition has an osmolality of 200 to 500 mOsm / L, 300 to 500 mOsm / L, or 400 to 460 mOsm / L, or the aqueous solution composition has an osmolality of about 300 mOsm / L.

8. The aqueous solution composition according to claim 1, wherein the concentration of proline is 20 to 600 mM, 20 to 500 mM, 20 to 400 mM, 20 to 300 mM, or 50 to 300 mM, or the concentration of proline is 50 to 200 mM, 100 to 200 mM, or 100 to 150 mM.

9. The aqueous solution composition according to claim 1, wherein the concentration of glycine is 20 to 600 mM, 20 to 500 mM, 20 to 400 mM, 20 to 300 mM, or 50 to 300 mM, or the concentration of glycine is 50 to 200 mM, 100 to 200 mM, or 100 to 150 mM.

10. The aqueous solution composition of claim 1, wherein the total ionic strength is 2.6 mM.

11. 2. The aqueous solution composition of claim 1, wherein the total ionic strength is 4.3 mM.

12. The aqueous solution composition of claim 1, wherein the total ionic strength is 11.1 mM.

13. 2. The aqueous solution composition of claim 1, wherein the pH is about 7.

3.

14. The aqueous solution composition of claim 1, further comprising 10 to 2000 μg / ml of polysorbate 20.

15. The aqueous solution composition of claim 1, further comprising 10 to 2000 μg / ml of poloxamer 188.

16. The aqueous solution composition of claim 1, further comprising 2 to 10 mM methionine.

17. The aqueous solution composition of claim 1, further comprising 50 to 1000 mM sucrose.

18. The aqueous solution composition of claim 1, further comprising a preservative, said preservative being a phenolic or benzyl preservative, and said phenolic or benzyl preservative being selected from the group consisting of phenol, m-cresol, chlorocresol, benzyl alcohol, propylparaben, and methylparaben.

19. The aqueous solution composition of claim 1, wherein the modified human IgG4Fc polypeptide is a mutant human IgG4Fc polypeptide.

20. The aqueous solution composition of claim 1, wherein the modified human IgG4 Fc polypeptide comprises the mutations S228P, L235E, M252Y, and M428L.

21. The aqueous solution composition of claim 1, wherein the modified human IgG4 Fc polypeptide comprises a hinge region at the N-terminus.

22. The aqueous solution composition of claim 1, wherein the modified human IgG4 Fc polypeptide comprises a hinge region at the N-terminus and contains the mutations S228P, L235E, M252Y, and M428L.

23. The aqueous solution composition of claim 1, wherein the AAT polypeptide is operably linked to the modified human IgG4 Fc polypeptide via a GS linker.

24. A pharmaceutical composition comprising the aqueous solution composition according to any one of claims 1 to 23, for use in inhibiting or downregulating abnormal serine protease expression or activity. Pharmaceutical compositions.