Recombinant intravenous immunoglobulin (rIVIG) compositions and methods of production and use thereof

Abstract

Compositions of recombinant intravenous immunoglobulin (rIVIG) proteins and methods for purifying and using rIVIG proteins. The compositions comprise oligomeric Fc molecules that bind Fc receptors with high avidity. The rIVIG proteins are useful as immunomodulatory molecules to treat immune disorders, including autoimmune diseases, such as refractory immune thrombocytopenia, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, lupus, Graves' disease, Kawasaki disease, dermatomyositis, myasthenia gravis, Guillain-Barre syndrome, autoimmune hemolytic anemia, and other immune and inflammatory conditions. The rIVIG proteins are also useful as immunomodulatory agents for patients to reduce immune rejection of organ transplants, stem cell transplants, and bone marrow transplants. In addition, the present invention provides rIVIG proteins of non-human origin for use in veterinary immune disorders, such as canine rIVIG proteins for treating dogs with autoimmune hemolytic anemia, immune thrombocytopenic purpura, rheumatoid arthritis, or other canine immune disorders.

Description

[0001] Related application statement

[0002] This application is a continuation-in-part of U.S. Provisional Patent Application Serial No. 62 / 315,483, filed on March 30, 2016, and claims priority thereto, the entire contents of which are hereby incorporated herein by reference. Technical Field

[0003] This invention relates to compositions and methods for producing recombinant proteins that can be used as alternatives to currently used human IVIG formulations (an acronym for intravenous immunoglobulin). The invention also relates to methods for using such compositions to treat immune disorders and other disorders and diseases. Background Technology

[0004] The clinical use of immunoglobulins as therapeutic agents dates back over a century, when Emil Behring and colleagues discovered that immune serum could improve toxin-mediated diseases (1). Sixty-two years later, Ogden Bruton administered intravenous human immunoglobulins as immunoglobulin replacement therapy in patients with agammaglobulinemia (2). Prior to that, only limited doses of immunoglobulins could be administered intramuscularly, and because these formulations contained purified immunoglobulin aggregates, their administration could cause local painful irritation and adverse systemic reactions due to activation of the immune response via the complement cascade (3,4).

[0005] New purification methods developed in the 1960s and 1970s have enabled the removal of aggregates, making it possible to prepare compositions suitable for intravenous administration at larger doses (3-7). Even though such formulations can also be administered via other routes, such as subcutaneous administration, the acronym "IVIG" remains the commonly used term for these formulations. The primary indication for IVIG formulations remains replacement therapy for immunocompromised patients (8-10).

[0006] In 1981, while treating children with secondary immunodeficiency due to extensive immunosuppressive therapy and refractory immune thrombocytopenic purpura (ITP), Paul Imbach found an unexpected increase in platelet counts after IVIG treatment (11). This effect of IVIG in increasing platelet counts was replicated in ITP patients without immunodeficiency, paving the way for the use of IVIG for its immunomodulatory effects (12-15).

[0007] Currently, IVIG is a treatment option for many different diseases and is recommended as a first-line immunomodulator for many autoimmune disorders. In fact, while the use of IVIG as an alternative immunoglobulin in immunodeficiency syndromes remains an important indication, IVIG is increasingly being used to treat autoimmune disorders.

[0008] Although IVIG formulations are effective in clinical treatment, several issues associated with current practices could significantly impact their sustainability. First, adverse reactions, including allergic reactions, nephropathy, thrombotic complications, and diabetic symptoms, are frequently observed after IVIG administration. Efforts to address these issues include pre-screening patients for IgA deficiency and close monitoring of IgA, factor XI, glucose, and sodium levels. However, each of these steps limits supply capacity and increases commodity and administrative costs. Furthermore, despite these efforts, IVIG use continues to be disadvantaged by adverse effects that have not yet been fully mitigated.

[0009] Furthermore, compared to most biologics, IVIGs are typically administered at very high doses, usually ranging from about 0.5 g to 4 g / kg body weight. Judging from the doses required for efficacy, the one or more therapeutically active components of an IVIG constitute only a very small fraction of the formulation. With the significant challenges posed by the ever-increasing cost of the product and the need to improve the quality of IVIG formulations, there is a strong need to improve alternative compositions and / or methods to address one or more of these problems:

[0010] The problems with IVIG treatment stem partly from the fact that the mechanism of action of IVIG is not yet fully understood and that its effects may vary depending on the indication. Summary of the Invention

[0011] As stated above, there is a great need for improvements in IVIG-based treatments, including one or more alternatives that can eliminate or reduce adverse reactions, can be manufactured with more consistent quality, allow for lower doses while maintaining efficacy, and / or reduce product costs. The inventors hypothesize that recombinant engineering of immunoglobulins will allow for the production of more defined molecules that can be manufactured with consistent quality, allow for lower doses while maintaining efficacy, and reduce product costs.

[0012] Although the mechanism of action of IVIG is not fully understood, the inventors hypothesize that it is possible to associate at least some indications with the antibody structural elements required for the therapeutic efficacy of IVIG in these indications. For example, in the treatment of immunodeficiency, IVIG replenishes serum Ig levels and provides life-saving protection against infectious agents and / or their toxins. Therefore, it can be envisioned that the large diversity of antigen-specificity contained within the variable regions of the aggregated immunoglobulins is responsible for the therapeutic efficacy in these indications. In contrast, research supports the view that the Fc region of immunoglobulins is responsible for the immunomodulatory role of IVIG in the treatment of acute and chronic autoimmune disorders.

[0013] Intact IVIG and its Fc fragments were observed to have equivalent anti-inflammatory activity in ITP treatment and animal models (16). This supports the role of the Fc region in anti-inflammatory function. Furthermore, in a mouse ITP model, the immunomodulatory effect of IVIG was observed to be mediated by the Fc receptor and dependent on dendritic cell (DC)-macrophage cross-interaction, with FcγRIIIa being crucial for the activation phase and FcγRIIb for the effector phase (17). Finally, in a mouse ITP model, treatment with IVIG containing high levels of Ig dimers was observed to reverse platelet depletion more effectively than with normal monomeric immunoglobulins (18). Therefore, the inventors hypothesize that FcγRIIIa and FcγRIIb on the surface of dendritic DCs, which normally have low affinity for the Fc region, can effectively interact with small amounts of oligomeric antibodies present in IVIG formulations via affinity (multiple interactions), with the oligomeric Fc providing said affinity binding, which could further enhance the immunomodulatory effect of IVIG formulations.

[0014] This invention provides methods and materials that completely or partially solve the aforementioned problems. Therefore, in its broad sense, this invention includes a recombinant intravenous immunoglobulin (rIVIG) polypeptide comprising (a) a single-chain Fc peptide containing two or more Fc peptide domains; and (b) an oligomerizing peptide domain. In a specific aspect of this invention, the oligomerizing peptide domain is a trimerizing peptide domain. In a specific embodiment, the rIVIG polypeptide of this invention (also known as a Pan receptor-interacting molecule, or "PRIM") comprises (a) a single-chain Fc peptide containing two Fc peptide domains and (b) an oligomerizing peptide domain, particularly a trimerizing peptide domain. The individual Fc peptide domains in the rIVIG polypeptide of this invention can be linked by a flexible linker. In a specific embodiment of this invention, the flexible linker comprises five repeats of the amino acid sequence GGGGS (Sequence ID 9); namely, GGGGSGGGSGGGGSGGGGSGG-GGS (Sequence ID 10). In other specific embodiments of the invention, the oligopeptide domain comprises amino acids 712 to 768 of sequence ID 4, or amino acids 1 to 79 of sequence ID 6. In some embodiments, the rIVIG polypeptide of the invention comprises an amino acid sequence selected from the group consisting of sequence ID 2, sequence ID 3, sequence ID 4, sequence ID 5, sequence ID 6, sequence ID 7, and sequence ID 8.

[0015] In other embodiments, the present invention includes a nucleotide molecule encoding a recombinant intravenous immunoglobulin (rIVIG) polypeptide, said polypeptide comprising (a) a single-chain Fc peptide containing two or more Fc peptide domains; and (b) an oligomerizing peptide domain. In a specific aspect of the invention, the nucleotide molecule encodes a trimerizing peptide domain. In a specific embodiment, the nucleotide molecule of the present invention encodes an rIVIG polypeptide comprising (a) two Fc peptide domains and (b) one trimerizing peptide domain. In a specific embodiment, the present invention includes a nucleotide molecule encoding an rIVIG polypeptide comprising an amino acid sequence selected from the group consisting of sequence ID 2, sequence ID 3, sequence ID 4, sequence ID 5, sequence ID 6, sequence ID 7, and sequence ID 8.

[0016] On the other hand, the present invention provides a composition for treating immune disorders, the composition comprising a recombinant immunoglobulin (rIVIG) protein, wherein the rIVIG protein comprises an oligomerizing peptide domain that provides a scaffold for aggregating three single-chain Fc domains (scFc). In a specific embodiment, the oligomerizing peptide domain comprises an amino acid sequence selected from the group consisting of amino acids 1 to 79 of sequence ID 6 and amino acids 712 to 768 of sequence ID 4. In a specific aspect of the invention, the composition primarily comprises a single protein species containing three single-chain Fc peptides. The individual Fc domains of the single-chain Fc peptides can interact intramolecularly to form a functional single-chain Fc peptide. In a specific embodiment, the present invention provides a composition primarily comprising rIVIG protein, the rIVIG protein comprising an amino acid sequence selected from the group consisting of sequence ID 2, sequence ID 3, sequence ID 4, sequence ID 5, sequence ID 6, sequence ID 7, and sequence ID 8.

[0017] On the other hand, the present invention provides a method for treating a patient with an autoimmune disorder, the method comprising administering to the patient an effective amount of a composition mainly comprising recombinant immunoglobulin (rIVIG) protein, wherein the rIVIG protein comprises an oligopeptide domain that provides a scaffold for forming a single-chain Fc peptide trimer. In the specific implementation plan, the patient suffers from an immune disorder selected from the following: refractory immune thrombocytopenic purpura (ITP), chronic inflammatory demyelinating polyneuropathy (CIDP), multiple sclerosis (MS), systemic lupus erythematosus (SLE or lupus), Graves' disease, Kawasaki disease, dermatomyositis, myasthenia gravis, Guillain-Barré syndrome, myasthenia gravis, autoimmune hemolytic anemia (IMHA), pernicious anemia, hemolytic anemia, aplastic anemia, paroxysmal nocturnal hemoglobinuria (PNH), Addison's disease, Hashimoto's disease (chronic thyroiditis), Hashimoto's encephalopathy, autoimmune neutropenia, thrombocytopenia, rheumatoid arthritis and reactive arthritis, inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, and Sjögren's syndrome. CREST syndrome, pelvic inflammatory disease (PID), ankylosing spondylitis, Bechtel disease, vasculitis, Lyme disease (chronic or advanced), and type 1 diabetes.

[0018] On the other hand, the present invention provides a method for reducing immune rejection in patients who have received organ transplants, bone marrow transplants, blood transfusions, or stem cell transplants, the method comprising administering to the patient an effective amount of a composition comprising recombinant immunoglobulin (rIVIG) protein, wherein the rIVIG protein comprises an oligomeric peptide domain, the oligomeric peptide domain providing a composition primarily comprising a single-chain Fc peptide trimer.

[0019] On the other hand, the present invention provides a method for treating a non-human mammal suffering from an autoimmune disorder, the method comprising administering to the non-human mammal an effective amount of a composition comprising recombinant intravenous immunoglobulin (rIVIG) protein, wherein the rIVIG protein comprises an oligomeric peptide domain, the oligomeric peptide domain providing a composition primarily comprising a single-chain Fc peptide trimer, and wherein the rIVIG protein comprises an amino acid sequence derived from a non-human mammal of the same species. In a specific embodiment, the non-human mammal suffers from an autoimmune disorder selected from the group consisting of autoimmune hemolytic anemia (AIHA), immune thrombocytopenic purpura (ITP), or rheumatoid arthritis. For example, dogs suffering from AIHA can be treated with a composition primarily comprising a trimer rIVIG protein containing a canine amino acid sequence, such as the amino acid sequences of sequence IDs 7 and 8. Attached Figure Description

[0020] Figure 1 The composition of constructs according to certain embodiments of the present invention is illustrated. P7005H is a prototype for the design of oligomeric functional Fc domains utilizing the inherent trimerizing ability of the extracellular domain of the CD40 ligand. The smallest functional oligomer consists of six polypeptide chains that assemble into three dimer Fc domains at the N-terminus and two trimerized CD40L ECDs at the C-terminus. Based on the complex SEC spectra of P7005H (… Figure 2 This resulted in P8001Z, where a functional Fc domain was generated using the scFc form, and the CD40L ECD was replaced by a collagen trimerization domain. Although the SEC spectrum ( Figure 2 The SEC profile of P8001Z is superior to that of P7005H, but P8001Z still contains a large number of higher oligomers. As a similar construct, P8004Z, with an additional human IgG1 heavy chain hinge region (H), also exhibits a less than ideal SEC profile, showing that simply including the hinge region cannot solve the folding problem. Interestingly, when additional constant regions (CL and CH1) are introduced, both P8003Z and P8020Z proteins fold more efficiently and mainly exhibit correctly folded trimers. Figure 2 ). ( Figure 2The P8020Z construct uses a trimerization scaffold, which allows the oligomeric Fc to be positioned at the C-terminus of the fusion protein. The C-terminal Fc form is expected to be very similar to the orientation of conventional antibodies used for interaction with the Fc receptor. Importantly, unlike P7005H, P8001Z, P8002Z, or P8004Z, homologous compositions of trimer species were successfully obtained from the expression of P8003Z and P8020Z (see [link to documentation]). Figure 5 ).

[0021] Figure 2 The role of the composition of the present invention in a size exclusion chromatography (SEC) analysis model is demonstrated. The rIVIG molecules of the present invention were purified by protein A affinity chromatography and then buffer-exchanged into phosphate-buffered saline at pH 7.2. Each SEC analysis was performed using a Superdex 200 10 / 30 SEC column (GE Healthcare) injected at a rate of 0.5 ml / min at approximately 100 μL of rIVIG sample. Arrows indicate the eluted sites of correctly folded trimer molecules. Figure 2 The results showed that less than about one-third of P7005H, P8001Z, P8002Z, and P8004Z were in correctly folded trimer form. In contrast, more than two-thirds of P8003Z and P8020Z were correctly folded into trimers. These results indicate that the introduction of the CL and CH1 domains can significantly enhance the folding of the trimer form.

[0022] Figure 3 The role of the compositions of the present invention in an FcγR binding model is demonstrated. Individual human Fc receptors fused with GST were coated onto ELISA plates. After blocking unoccupied regions, human IgG1, P8003Z1, and P8003Z3 (non-fucosyl variants of P8003Z, derived from a cell line lacking the α-1,6-fucosyltransferase gene, were administered at sequentially diluted concentrations.) - / - (The product) or P8020Z1 is added to the plate. Human IgG1 and rIVIG variants are quantitatively bound by the fluorescently labeled F(ab)'2 fragment of goat anti-human antibody. Figure 3The figure above shows an example of affinity measurements of human IgG1 and rIVIG with human FcγRIIA (H131). Curve fitting (SoftMax Pro 5.1, Molecular Devices, Sunnyvale, CA) allowed estimation of the KD of rIVIG for recombinant soluble Fc receptors. The table below shows these calculated KDs. Clearly, the trimer rIVIG of the present invention exhibits a significant increase in binding affinity compared to human IgG1; while it has already shown sub-nM affinity for human FcγRI, rIVIG only shows slightly higher affinity. These results confirm that the trimer rIVIG of the present invention, with its affinity advantage, can bind to Fc receptors with higher apparent affinity.

[0023] Figure 4 The therapeutic effects of the composition of this invention in a collagen-induced arthritis (CIA) model were demonstrated. Mice were primed with bovine type II collagen from CFA on day 1, treated with P8020Z (50 mg / kg body weight) on day 18, and boosted with the same collagen from IFA on day 21. Clinical scores for each paw were measured every other day, ranging from 1 to 4, with 4 being the most severe. Clinical scores were added to each group and normalized by the number of mice. Compared to conventional human IVIG formulations that are often used at approximately 2-3 g / kg body weight and administered multiple times during the study, administering P8020Z1 at a single dose of 50 mg / kg represents a dose reduction of 40 to 60 times.

[0024] Figure 5 The therapeutic effects of the compositions of the present invention in autoimmune disorders induced by passive transfer of anti-collagen antibodies were demonstrated. Mice were treated with anti-collagen antibodies, followed by lipopolysaccharide treatment after 3 days, and on day 6, a single injection of plasma-derived IVIG (pd.IVIG) or recombinant IVIG (rIVIG or PRIM) molecules (PM 02, also known as non-fucosylated P8003Z3) at the indicated dose was given. The dosage of pd.IVIG 1K was 1 gm / kg body weight; for pd.IVIG 2K, the dose was 2 gm / kg body weight. PM 02 15 was 15 mg / kg body weight; PM 0250 was 50 mg / kg body weight; and PM 02 150 was 150 mg / kg body weight. Both pd.IVIG 1K and pd.IVIG 2K were slightly more effective between day 9 and day 13. PM 02 15 exhibited therapeutic efficacy comparable to both concentrations of pd.IVIG. Both PM 02 50 and PM 02 150 demonstrated better efficacy than either PD-IVIG administration. Therefore, PM 02 is demonstrated to treat autoimmune disorders induced by passive transfer of anti-collagen antibodies.

[0025] Figure 6 Size exclusion chromatograms of P8003Z1 and P8020Z1 are shown. The peaks representing the trimeric rIVIG protein of this invention demonstrate that the rIVIG peptide of this invention can be prepared in a homogeneous form. Detailed Implementation

[0026] In the following description, numerous specific details are set forth for purposes of explanation in order to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without these specific details, and that various modifications and alterations can be made therein without departing from the broader scope of the invention.

[0027] All publications cited herein are hereby expressly incorporated by reference into this publication in order to teach what they teach.

[0028] As used herein, the term "subject" refers to both mammals and non-mammals. Mammals include any member of the mammal class, including but not limited to humans; non-human primates such as chimpanzees and other ape and monkey species; farm animals such as cattle, horses, sheep, goats, and pigs; livestock such as rabbits, dogs, and cats; laboratory animals including rodents such as rats, mice, and guinea pigs; and so on. Examples of non-mammals include, but are not limited to, birds and fish.

[0029] This invention relates to recombinant intravenous immunoglobulin (rIVIG) protein, compositions containing such rIVIG, and methods for producing, purifying, and using rIVIG compositions for treating various immune disorders and conditions.

[0030] In this invention, the design of recombinant immunoglobulin (rIVIG) focuses on modifying nucleic acid and protein molecules containing multiple copies of the human IgG1 Fc domain and a domain that enhances the formation of oligomeric rIVIG molecules. While not wishing to be bound by any theory, it is anticipated that the rIVIG molecules of this invention will be able to bind not only high-affinity FcγRI but also low-affinity Fc receptors, namely FcγRII and FcγRIII receptors. The enhanced binding to low-affinity receptors is likely due to the affinity interaction between oligomeric Fc and Fc receptors present on the cell surface.

[0031] Biochemically, this invention provides methods and materials designed to aggregate Fc domains and oligomeric protein scaffolds to produce correctly folded fusion proteins exhibiting desired characteristics for use as therapeutic products. Therapeutically, the rIVIG protein of this invention can be used to treat many immune disorders and can be used as an immunomodulator for many autoimmune disorders. Furthermore, considering the involvement of various complement proteins in many autoimmune disorders, this invention may optionally include additional structural elements, such as elements capable of activating cascade clearance components along the complement pathway.

[0032] The inventors have designed and expressed numerous rIVIG molecules using various protein scaffolds to oligomerize variants of the Fc construct. In some preferred embodiments, the rIVIG molecules of the present invention comprise oligomeric scaffold domains that preferentially aggregate three single-chain Fc peptides or three Fc dimers together to form three functional Fc domains.

[0033] The methods and materials of this invention can be used to treat immune disorders, including but not limited to autoimmune diseases, or any disorder, disease or syndrome that requires immune regulation. Indications for use of this invention include, but are not limited to, immune thrombocytopenic purpura (ITP), chronic inflammatory demyelinating polyneuropathy (CIDP), multiple sclerosis (MS), systemic lupus erythematosus (SLE or lupus), Graves' disease, Kawasaki disease, Addison's disease, celiac-ostomy, dermatomyositis, myasthenia gravis, dermatitis, Hashimoto's disease (chronic thyroiditis), Hashimoto's encephalopathy, Guillain-Barré syndrome, myasthenia gravis, autoimmune hemolytic anemia (IMHA), pernicious anemia, hemolytic anemia, aplastic anemia, paroxysmal nocturnal hemoglobinuria (PNH), autoimmune neutropenia, thrombocytopenia, rheumatoid arthritis and reactive arthritis, inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, Sjögren's syndrome, CREST syndrome, pelvic inflammatory disease (PID), ankylosing spondylitis, Bechtel's disease, vasculitis, Lyme disease (chronic or advanced), and type I diabetes.

[0034] The methods and materials of this invention can also be used to treat disorders caused by autoantibodies, as well as organ-specific autoimmune disorders, including myocarditis, post-myocardial infarction syndrome nephritis, Goodpasture syndrome, interstitial cystitis, autoimmune hepatitis, primary biliary cirrhosis and primary sclerosing cholangitis (PSC), antisynthetic enzyme syndrome; alopecia areata, autoimmune angioedema, dermatitis, psoriasis, systemic scleroderma, lymphoproliferative syndrome, antiphospholipid syndrome, autoimmune retinopathy, uveitis, and Meniere's disease.

[0035] The methods and materials of this invention can also be used to prevent, reduce and / or treat immune or antibody-mediated responses to procedures, including organ transplantation, bone marrow transplantation, blood transfusion, or stem cell transplantation.

[0036] The methods and materials of this invention can also be used for any autoimmune indication in which any commercially available intravenous immunoglobulin (IVIG) has already been used. Commercially available IVIGs include: Gammaked TM Gammaplex -C、 and The specific uses and autoimmune indications for commercially available IVIG that have been approved include the following: chronic inflammatory demyelinating polyneuropathy (CIDP); chronic immune thrombocytopenic purpura (ITP); multifocal motor neuropathy (MMN); control of bleeding in ITP; and prevention of coronary aneurysms associated with Kawasaki syndrome in pediatric patients.

[0037] This invention comprises a recombinant IVIG (rIVIG) protein, which can be expressed and purified as a homogeneous substance containing three functional scFc domains. This trimeric Fc oligomer can bind to both high-affinity and low-affinity Fc receptors due to affinity (multivalent) interactions. These enhanced affinities for various Fc receptors are reminiscent of the immunomodulatory effects attributed to human IVIG, which contains small amounts of oligomeric antibodies in human IVIG formulations. Due to its mimicry of enhanced interactions with Fc receptors, the rIVIG of this invention is expected to replace conventional IVIG for its immunomodulatory applications rather than its passive immunoprotective applications.

[0038] Immunoglobulins

[0039] Fc fragment

[0040] This invention utilizes a CH2-CH3 domain comprising the constant region 2 (CH2) and constant region 3 (CH3) of the human heavy chain of IgG (preferably IgG1) (CH2-CH3). When using two or more Fc fragments, such as the CH2-CH3 domain, they are typically synthesized or expressed as single-chain Fc peptides, wherein the CH2-CH3 domains are linked using a flexible peptide linker, such as (GGGGS)5 (=GGGGSGGGGSGGGGSGGGGSGGGGS (Sequence ID 10)). This flexible peptide linker facilitates intramolecular interactions between the separate CH2-CH3 domains of the single-chain Fc peptide, allowing the single-chain Fc peptide to present a three-dimensional conformation optimized for biological function. A hinge region (H) from human IgG (preferably IgG1) may also be present at the N-terminus of each CH2 region to promote appropriate conformation for optimized biological activity.

[0041] In some embodiments, the rIVIG protein of the present invention includes other regions of the Fc molecule. For example, rIVIG may include one or more constant region 1 (CH1) domains of IgG (preferably IgG1) and one or more Igκ or light chain constant region (CL) domains. A short linker sequence (e.g., (GGGGS)2) can be used to link the C-terminus of the CL domain to the N-terminus of the CH1 domain. In this construct, the CH1 domain can interact with the CL domain via intramolecular disulfide bonds, which are thermodynamically more favorable than intermolecular disulfide bonds. Furthermore, the CL / CH1 domain plays a role in clearing complement components, which can further improve complement immune responses present in many autoimmune disorders.

[0042] Human antibody isotype

[0043] Several different antibody isotypes are known to exist, each with different binding modes, resulting in different functional effects in vivo. The binding affinity of each isotype is generally known (Gillis et al. (2014) Frontier Immunology 5:1-13) and is shown in Table 1 below. The Fc fragments of each antibody isotype bind differently to the Fc receptor. For example, human IgG3 has at least 50-fold higher binding affinity to FcγRIIIIA (0.1 μM KD) than human IgG1 to the same receptor (5-10 μM KD). Similarly, human IgG1 binds to FcγRIIA 15-fold stronger than human IgG4. Therefore, although the examples herein use Fc fragments derived from IgG1, Fc fragments from each isotype can be used in this invention. For example, variants of P8003Z or P8020Z carrying the isotype constant region of IgG2, IgG3, or IgG4 are expected to exhibit different affinities than their parental counterparts, P8003Z or P8020Z, which carry the isotype constant region of IgG1. Since many autoimmune disorders are associated with differentially combined expression of Fc receptors, the rIVIG of the present invention, derived from each isotype variant, can provide different therapeutic benefits.

[0044] Table 1: Affinity of human antibody isotypes to Fc receptors

[0045]

[0046] Non-human mammalian antibody subclasses

[0047] It is known that certain non-human mammalian species possess antibody subclasses similar to human antibody isotypes. For example, canine immunoglobulins have four known subclasses: subclass A, subclass B, subclass C, and subclass D. These subclasses share functional characteristics with four human IgG isotypes. Canine subclasses A and D have been reported to be effector-negative, while subclasses B and C bind to the canine Fcγ receptor and are ADCC-positive. Furthermore, all canine subclasses except subclass C have been reported to bind to the neonatal Fc receptor (22).

[0048] Glycosylation of the Fc domain of immunoglobulins and enhanced interactions between non-fucosylated antibodies and FcγRIII (human) and FcγRIV.

[0049] In addition to isoform differences, differential glycosylation at a single glycosylation site (Asn-297) is known to play a crucial role in Fc-Fc receptor interactions. Indeed, it is evident that glycoform alterations at Asn-297 residues occur under both physiological and pathological conditions (23). Furthermore, differential sialylation has been reported to affect the inflammatory properties of IgG, and it has been proposed as a mechanism for inducing anti-inflammatory symptoms through molecular switches (24). Moreover, removal of the glycan completely impairs the ability of Fc to interact with all Fc receptors except for the neonatal Fc receptor (FcRn) (25). Most interestingly, it has been found that the removal of the core fucose in the N-glycan complex leads to a selectivity enhancement of up to 100-fold for Fc-FcγRIII interactions (26). Antibodies in non-fucosylated forms can be used in host cell lines lacking the α-1,6-fucosyltransferase gene (FUT8). - / - The antibodies expressed in both P8003Z1 and P8003Z3 are very similar. The core fucosylation of P8003Z1 and P8003Z3 differs in that P8003Z1 is produced in FUT8 competent cells, while P8003Z3 is produced in FUT8 deficient cells. As expected, the unfucosylated P8003Z3 exhibits enhanced binding to human FcγRIII and mouse FcγRIV (see [link to relevant documentation]). Figure 3 (KD table in the table).

[0050] Therefore, it will be clear to those skilled in the art that the modified glycosylated rIVIG protein, the cell lines and culture media that produce the modified glycosylated rIVIG protein can be used in this invention, and the use of the cell lines and culture media for the production of rIVIG and the use of the modified glycosylated rIVIG protein in the treatment of immune disorders form part of this invention.

[0051] Oligomeric scaffold structural domain

[0052] As used herein, the terms “oligomery domain,” “oligomery scaffold domain,” and “oligomery protein scaffold” are used interchangeably to indicate that the specified sequence plays a role in forming an oligomeric structure. Oligomeric scaffold domains that can be used in this invention include those that induce the trimerization of their fusion partner (e.g., a single-chain Fc peptide) to form a trimer rIVIG molecule, wherein each rIVIG molecule contains two H-CH2-CH3 Fc domains (hinge region-heavy chain constant region 2-heavy chain constant region 3). In some embodiments, such as illustrated by P8020Z (Sequence ID 6), the oligomery scaffold domain may be located at the N-terminus of the construct, in which case the C-terminus of the oligomery scaffold domain may be directly or indirectly connected to the N-terminus of the first hinge region (H) or CH2 region or CL domain via a short linker sequence such as GGGGS. In other embodiments, such as P8003Z (Sequence ID 4), the oligomerized scaffold domain can be located at the C-terminus of the construct, in which case the N-terminus of the oligomerized scaffold domain can be directly or indirectly connected to the C-terminus of the final CH3 domain via a short connector sequence such as GGGGS (Sequence ID 9).

[0053] Connectors and flexible connectors

[0054] The linkers and flexible linkers that can be used in this invention include peptide linkers rich in glycine and / or serine, having a plurality of glycine or serine residues and defining a polypeptide of sufficient length to span the distance between the C-terminus of a first domain and the N-terminus of a second domain. The term "flexible linker" is used to define a polypeptide sequence of sufficient length to allow the formation of a flexible, unstructured polypeptide conformation that is inherently free of secondary structure in aqueous solution, and the linker provides a manner for connecting two protein domains such that chimeric or fusion proteins can be generated as a single polypeptide molecule from a single nucleic acid construct.

[0055] The length of the linker can vary, thereby allowing the formation of a three-dimensional conformation optimized for biological function in a manner that allows intramolecular interactions between separate regions. As used herein, the term "flexible linker" generally applies to linkers with a length of ten or more amino acids. Suitable flexible linkers typically have a length of at least 10 amino acid residues and comprise linker polypeptides having about 10 to 36 amino acid residues. Preferred flexible linkers are those having more than at least about 50% glycine residues and a length of about 10 to about 30 amino acids, more preferably about 12 to 25 amino acids, or about 15 to 25 amino acids. Flexible linkers that can be used in this invention include, for example, (GGGGS). n The amino acid sequence; where n is 2 to 7. The term "G4S" is used interchangeably to refer to the sequence GGGGS (sequence ID 9). Preferred flexible linkers include (GGGGS).n The amino acid sequence is specified; where n is 2 to 6; and more preferably n is 3 to 5. Such glycine-rich and / or serine-rich peptide linkers are well known and have been used to link antibody domains to form single-chain Fv(sFv) proteins, which integrate the complete antibody binding site into a single polypeptide chain. Peptide linkers of fewer than twelve amino acid residues, rich in serine and / or rich in glycine, can also be used as linkers for peptide domains, but generally do not provide sufficient flexibility to allow for conformations in which adjacent fused peptide domains can interact intramolecularly. A specific flexible linker usable in this invention comprises the amino acid sequence (GGGGS)5 (Sequence ID 9). Shorter linkers usable in this invention (where a flexible linker is not required) include linkers containing the amino acids GGGGS and (GGGGS)2. Typically, linkers may contain other amino acid residues with non-reactive side chains, such as alanine and threonine. However, linkers should generally not contain charged amino acid residues and cysteine ​​residues that can form disulfide bonds. Suitable flexible peptide linkers and DNA constructs that can be used to prepare them are described in U.S. Patents 5,258,498, 5,482,858 and 5,525,491.

[0056] purification:

[0057] This invention also relates to compositions comprising primarily one or more rIVIG proteins of the present invention. As used herein, when referring to the weight of a composition, the term "primarily comprising" one or more rIVIG proteins means, by weight of the total weight of the composition, that the composition comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the specified rIVIG protein. When used relative to the amount of protein in a composition, the term "primarily comprising" one or more rIVIG proteins means, by mole percentage (in mole percentage) of the total protein present in the composition, that the composition comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the specified rIVIG protein.

[0058] Compositions primarily comprising one or more rIVIG proteins of the present invention can be obtained using conventional IgG purification methods that utilize protein A-agarose that binds to the Fc region of IgG or protein G-agarose that preferentially binds to the Fc region of IgG but may also bind to the Fab region of IgG (making it usable for purifying F(ab')2). According to the present invention, other purification methods known in the art can be used to further purify compositions of rIVIG proteins according to the present invention, including size exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC). See http: / / www.kpl.com / docs / techdocs / purifigg.pdf (accessed March 23, 2016) and the references cited therein; Surolia et al. (1982) Trends Biochem. Sci. 7:74-76; Harlow and Lane (eds.) Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratory, NY), pp. 617-618; Langone (1982) J. Immunological Methods 55:277-296; Lindmark et al. (1983) J. Immunological Methods 62:1-13; and Thruston and Henley (1988) Walker (ed.) Methods in Molecular Biology, Vol. 3 – New Protein Techniques (Humana Press: Clifton, NJ), pp. 149-158.

[0059] Composition

[0060] This invention also relates to compositions of rIVIG proteins already combined with pharmaceutically acceptable adjuvants or carriers. As used, the term "pharmaceutically acceptable" means acceptable for use in the pharmaceutical field, i.e., not having unacceptable toxicity, or otherwise not unsuitable for administration to mammals. Examples of pharmaceutically acceptable adjuvants include, but are not limited to, diluents, excipients, etc. See Remington's: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, 2005, generally used for guidance on pharmaceutical formulations.

[0061] The pharmaceutical composition may further include other ingredients, such as preservatives, buffers, tension agents, antioxidants and stabilizers, nonionic wetting agents or clarifying agents, thickeners, etc.

[0062] Suitable preservatives for use in solution may include polyquaternium-1, benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenethyl alcohol, disodium EDTA, sorbic acid, benzyl chloride, etc. Typically (but not always), such preservatives are used in amounts of 0.001%–1.0% (by weight).

[0063] Buffer solutions are typically (but not always) used to maintain preparations at or near physiological pH. Suitable buffers include boric acid, sodium bicarbonate and potassium bicarbonate, sodium borate and potassium borate, sodium carbonate and potassium carbonate, sodium acetate, sodium diphosphate, etc., in amounts sufficient to maintain the pH between approximately pH 6 and pH 8, preferably between approximately pH 7 and pH 7.5.

[0064] Suitable tensioning agents include dextran 40, dextran 70, dextrose, glycerol, potassium chloride, propylene glycol, sodium chloride, etc., so that the sodium chloride equivalent of the injectable solution is in the range of 0.9% ± 0.2%.

[0065] Suitable antioxidants and stabilizers include sodium bisulfite, sodium metabisulfite, sodium thiosulfate, and thiourea. Suitable wetting agents and clarifying agents include polysorbate 80, polysorbate 20, poloxamer 282, and tyloxapine. Suitable thickeners include dextran 40, dextran 70, gelatin, glycerin, hydroxyethyl cellulose, hydroxymethylpropyl cellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and carboxymethyl cellulose.

[0066] The choice of adjuvant depends on the intended route of administration of the composition, and may also take into account the intended indication and the patient. In one embodiment of the invention, the compound is formulated for administration by infusion, or by subcutaneous or intravenous injection, and thus can be used as an aqueous solution in a sterile and pyrogen-free form, optionally buffered or prepared as an isotonic solution. Therefore, the compound can be administered in distilled water, or more ideally in saline, phosphate-buffered saline, or a 5% dextran solution. In addition to the foregoing, the formulations of the present invention may also contain other active and / or inactive ingredients, including solvents, diluents, suspending agents, thickeners or emulsifiers, binders, stabilizers, lubricants, etc., to suit a particular dosage and route of administration. Unless any conventional carrier medium is incompatible with the components of the present invention, for example by producing any undesirable effects or otherwise interacting in a harmful manner with any other one or more components of the formulation, its use is considered to be within the scope of the invention.

[0067] Administration method:

[0068] The pharmaceutical composition is suitable for various routes of administration as described herein, including, for example, systemic or local administration. The pharmaceutical composition may be in the form of an injectable solution or in a form suitable for oral administration. The pharmaceutical compositions described herein may be packaged in a single unit dose or in multiple doses. In some embodiments, the pharmaceutical composition is suitable for administration to an individual, vertebrate, mammal, or human via any of the routes of administration described herein, including oral or intravenous administration.

[0069] The compositions described herein can be administered to an individual via any route, including but not limited to intravenous (e.g., via an infusion pump), intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, inhalation, intracapsular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, percutaneous, transthoracic, intra-arterial, local, inhalation (e.g., as a mist in a spray), mucosal (e.g., via nasal mucosa), subcutaneous, percutaneous, gastrointestinal, intra-articular, intracisional, intraventricular, rectal (i.e., via suppository), vaginal (i.e., via pessary), intracranial, intraurethral, ​​intrahepatic, and intratumoral. In some embodiments, the compositions are administered systemically (e.g., via intravenous injection). In some embodiments, the compositions are administered locally (e.g., via intra-arterial or intraocular injection).

[0070] In some embodiments, the composition is administered intravascularly, such as intravenously or intra-arterially. In some embodiments (e.g., for treating kidney disease), the composition is administered directly into an artery (e.g., the renal artery). In a preferred embodiment, the composition is administered subcutaneously.

[0071] In some embodiments, the composition may be administered directly to the eye or ocular tissue. In some embodiments, the composition may be administered topically to the eye, for example, as eye drops. In some embodiments, the composition may be administered by injection into the eye (intraocular injection) or to tissues associated with the eye. The composition may be administered, for example, by intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intra-anterior chamber injection, subconjectval injection, subconjunctival injection, subfascial injection, retrobulbar injection, peribulbar injection, or posterior scleral delivery. These methods are known in the art. For example, for a description of exemplary peribulbar routes for retinal drug delivery, see Periodic routes for retinal drug delivery, Raghava et al. (2004), Expert Opin. Drug Deliv. 1(1):99-114. The composition may be administered, for example, to the vitreous body, aqueous humor, sclera, conjunctiva, area between the sclera and conjunctiva, retinal choroidal tissue, macula, or other areas in or near the individual eye.

[0072] The composition can also be administered to an individual as an implant. Preferred implants are biocompatible and / or biodegradable sustained-release formulations that gradually release the compound over a period of time. Ocular implants for drug delivery are well known in the art. See, for example, US 5,501,856, 5,476,511 and 6,331,313. The composition can also be administered to an individual using iontophoresis, including but not limited to the iontophoresis methods described in US 4,454,151 and US 2003 / 0181531 and 2004 / 0058313.

[0073] dose:

[0074] The optimal effective amount of the composition can be determined empirically and depends on the type and severity of the disease, route of administration, disease progression, and the individual's health, weight, and body surface area. Such determinations are within the skill of those skilled in the art. The effective amount can also be determined based on in vitro assays. Examples of dosages of the composition that can be used in the methods described herein include, but are not limited to, effective amounts within any dosage range of about 0.01 μg / kg to about 300 mg / kg, or about 0.1 μg / kg to about 40 mg / kg, or about 1 μg / kg to about 20 mg / kg, or about 1 μg / kg to about 10 mg / kg. For example, when administered subcutaneously, the composition can be given in low microgram ranges, including, for example, about 0.1 μg / kg or less, about 0.05 μg / kg or less, or 0.01 μg / kg or less. In some embodiments, the amount of composition administered to an individual is from about 10ug to about 500mg per dose, including, for example, any one of about 10ug to about 50ug per dose, about 50ug to about 100ug, about 100ug to about 200ug, about 200ug to about 300ug, about 300ug to about 500ug, about 500ug to about 1mg, about 1mg to about 10mg, about 10mg to about 50mg, about 50mg to about 100mg, about 100mg to about 200mg, about 200mg to about 300mg, about 300mg to about 400mg, or about 400mg to about 500mg.

[0075] The composition can be administered as a single daily dose, or the total daily dose can be administered in divided doses twice, three, or four times daily. The composition can also be administered at less frequent frequencies than daily, such as six, five, four, three, twice, once, every two weeks, every three weeks, once a month, once every two months, once every three months, or once every six months. The composition can also be administered as a sustained-release formulation, for example, in an implant, which gradually releases the composition for use over a period of time, and can reduce the frequency of administration, such as once a month, once every 2-6 months, once a year, or even a single dose. Sustained-release devices (e.g., pellets, nanoparticles, microparticles, nanospheres, microspheres, etc.) can be administered by injection or surgically implanted at different sites within the body.

[0076] Co-administration

[0077] This invention provides a method for improving the treatment of immune disorders or diseases, comprising co-administering the rIVIG composition of the invention with one or more other active agents having preventive or therapeutic activity, or which have been approved for use in treating such immune disorders or diseases. In these methods, the rIVIG composition may be administered before, simultaneously with, or after the administration of the additional active agent. For example, in the treatment of rheumatoid arthritis (RA), the rIVIG composition of the invention may be co-administered with... The rIVIG composition is co-administered with adilimumab (AbbVie Inc.) (a therapeutic antibody approved for RA). The rIVIG composition is expected to provide additional relief for patients with RA, and its effects may be related to... The synergistic effect.

[0078] This invention provides a method for improving the treatment of patients receiving organ transplants or other procedures such as stem cell transplants or blood transfusions, comprising administering the rIVIG composition of this invention before, during, or after such transplantation or other procedures. This treatment according to the invention provides a method for preventing or reducing antibody-mediated immune responses (i.e., immune rejection) against the transplanted organ. The rIVIG composition may be administered co-administered with one or more additional active agents having preventive or therapeutic activity against such antibody-mediated immune responses or transplant rejection.

[0079] For example, in the treatment of kidney transplant recipients, the rIVIG composition of the present invention can be co-administered with compositions containing immunosuppressive drugs such as cyclosporine. Other immunosuppressive drugs that can be co-administered with the compositions of the present invention include calcineurin inhibitors such as tacrolimus; mTOR inhibitors such as sirolimus; antiproliferative agents such as mycophenolate mofetil and azathioprine; and steroids such as prednisone. The rIVIG composition is expected to provide additional relief for patients with immune rejection, and the effect of the rIVIG composition can synergize with the effect of immunosuppressants. Furthermore, this treatment according to the present invention may allow for a reduction in the dosage of such immunosuppressants.

[0080] Encoding nucleotide molecules, recombinant vectors, and recombinant cell lines

[0081] Methods for synthesizing nucleotide molecules encoding the rIVIG protein of the present invention are known in the art. Using the genetic code, the amino acid sequence of the rIVIG protein of the present invention can be easily back-translated and codon optimized using online tools (27); and the encoding nucleotide molecule can be synthesized using strategies such as the hierarchical approach to gene synthesis described by Kim et al. (28).

[0082] For the expression of the rIVIG protein of the present invention, it is known that the encoding nucleotide sequence can be expressed in host cells using a recombinant vector, wherein the nucleic acid sequence encoding the rIVIG protein is controlled by a suitable promoter that drives the expression of the rIVIG protein in the host cell. Suitable host cells include, for example, mammalian CHO cells and 293T cells (29).

[0083] Gene therapy

[0084] Molecules can also be delivered via the expression of fusion proteins in vivo, a practice commonly referred to as "gene therapy." For example, cells can be engineered in vitro with polynucleotides (DNA or RNA) encoding the fusion protein, and then the engineered cells can be provided to an individual to be treated with the fusion protein. These methods are well known in the art. For instance, cells can be engineered using retroviral particles containing RNA encoding the fusion protein of the present invention, according to procedures known in the art. Local delivery of the rIVIG protein of the present invention using gene therapy can deliver the therapeutic agent to a local target region.

[0085] Gene delivery methods are known in the art. These methods include, but are not limited to, direct DNA transfer, see, for example, Wolff et al. (1990) Science 247:1465-1468; 2) liposome-mediated DNA transfer, see, for example, Caplen et al. (1995) Nature Med. 3:39-46; Crystal (1995) Nature Med. 1:15-17; Gao and Huang (1991) Biochem. Biophys. Res. Comm. 179:280-285; 3) retrovirus-mediated DNA transfer, see, for example, Kay et al. (1993) Science 262:117-119; Anderson (1992) Science 256:808-813; 4) DNA virus-mediated DNA transfer. Such DNA viruses include adenoviruses (preferably Ad2 or Ad5-based vectors), herpesviruses (preferably herpes simplex virus-based vectors), and parvoviruses (preferably “defective” or non-autonomous parvovirus-based vectors, more preferably adeno-associated virus-based vectors, and most preferably AAV-2-based vectors). See, for example, Ali et al. (1994) GeneTherapy 1:367-384; U.S. Patent No. 4,797,368 (incorporated herein by reference) and U.S. Patent No. 5,139,941.

[0086] Retroviruses that can derive the retroviral plasmid vectors described above include, but are not limited to, Moloney's mouse leukemia virus, spleen necrosis virus, retroviruses such as Raoul's sarcoma virus, Harvey's sarcoma virus, avian leukemia virus, gibberish leukemia virus, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moloney's mouse leukemia virus.

[0087] Adenoviruses have the advantage of a broad host range, capable of infecting quiescent or terminally differentiated cells, such as neurons or hepatocytes, and exhibiting largely non-oncogenic behavior. See, for example, Ali et al. (1994), ibid., p. 367. Adenoviruses do not integrate into the host genome. Because they exist extrachromosomally, the risk of insertional mutations is greatly reduced. Ali et al. (1994), ibid., p. 373.

[0088] Adeno-associated viruses (AAVs) exhibit similar advantages to adenovirus-based vectors. However, AAVs show site-specific integration on human chromosome 19 (Ali et al. (1994), ibid., p. 377).

[0089] Gene therapy vectors may include one or more promoters. In some embodiments, the vector has a promoter that drives expression in multiple cell types. In some embodiments, the vector has a promoter that drives expression in a specific cell type (e.g., retinal cells or kidney cells). Suitable promoters that may be used include, but are not limited to, retroviral LTRs; SV40 promoters; and human cytomegalovirus (CVM) promoters described by Miller et al. (Miller et al. (1989) Biotechniques 7(9):980-990), or any other promoters (e.g., cellular promoters such as eukaryotic cell promoters, including but not limited to histone, pol III, and β-actin promoters). Other viral promoters that may be used include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. Based on the teaching contained herein, the selection of a suitable promoter will be apparent to those skilled in the art.

[0090] The nucleic acid sequence encoding the rIVIG protein is preferably controlled by a suitable promoter. Suitable promoters that can be used include, but are not limited to, adenovirus promoters, such as the adenovirus major late promoter; or heterologous promoters, such as cytomegalovirus (CMV) promoters; respiratory syncytial virus (RSV) promoters; inducible promoters, such as MMT promoters, metallothionein promoters; heat shock promoters; albumin promoters; ApoA1 promoters; human globin promoters; viral thymidine kinase promoters, such as the herpes simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs described above); β-actin promoters; and human growth hormone promoters.

[0091] Retroviral plasmid vectors can be used to transduce packaging cell lines to form production cell lines. Examples of packaging cells that can be transfected are described in Miller (1990) Human Gene Therapy 1:5-14. The vector can transduce packaging cells by any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 precipitation. In one alternative, the retroviral plasmid vector can be encapsulated in or conjugated with lipids and then administered to the host. Production cell lines produce infectious retroviral vector particles that include one or more nucleic acid sequences encoding a polypeptide. Such retroviral vector particles can then be used to transduce eukaryotic cells in vitro or in vivo. The transduced eukaryotic cells will express one or more nucleic acid sequences encoding a polypeptide. Transducible eukaryotic cells include, but are not limited to, embryonic stem cells, embryonic cancer cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.

[0092] In vitro drug delivery

[0093] In some embodiments, the immunomodulatory effects of rIVIG proteins can be achieved by ex vivo contact of a composition containing said molecules with bodily fluids under conditions that allow the molecules to function in regulating immune responses. Suitable bodily fluids include those that can be returned to the individual, such as blood, plasma, or lymph. Affinity adsorption apheresis is generally described in Nilsson et al. (1988) Blood 58(1):38-44; Christie et al. (1993) Transfusion 33:234-242; Richter et al. (1997) ASAIO J.43(1):53-59; Suzuki et al. (1994) Autoimmunity 19:105-112; US Patent No. 5,733,254; Richter et al. (1993) Metabol. Clin. Exp. 42:888-894; and Wallukat et al. (1996) Int'l J. Card. 54:1910-195.

[0094] Therefore, the present invention includes a method for treating one or more diseases described herein in an individual, the method comprising treating the individual’s blood in vitro (i.e., in vitro or ex vivo) with a composition containing the molecules, under conditions that allow the molecules to function to modulate the immune response, and returning the blood to the individual.

[0095] Unit dose, products and reagent kits

[0096] Unit dosage forms of the composition are also provided, each dose containing about 0.01 mg to about 50 mg, including, for example, any one of about 0.1 mg to about 50 mg, about 1 mg to about 50 mg, about 5 mg to about 40 mg, about 10 mg to about 20 mg, or about 15 mg molecules. In some embodiments, the unit dosage form of the molecular composition comprises any one of about 0.01 mg-0.1 mg, 0.1 mg-0.2 mg, 0.2 mg-0.25 mg, 0.25 mg-0.3 mg, 0.3 mg-0.35 mg, 0.35 mg-0.4 mg, 0.4 mg-0.5 mg, 0.5 mg-1.0 mg, 10 mg-20 mg, 20 mg-50 mg, 50 mg-80 mg, 80 mg-100 mg, 100 mg-150 mg, 150 mg-200 mg, 200 mg-250 mg, 250 mg-300 mg, 300 mg-400 mg, or 400 mg-500 mg molecules. In some embodiments, the unit dosage form comprises about 0.25 mg molecules. The term "unit dosage form" refers to a physically discrete unit suitable as an individual unit dose, each unit containing a predetermined amount of active substance, calculated to produce the desired therapeutic effect, and used in combination with a suitable drug carrier, diluent, or excipient. These unit dosage forms can be stored in suitable packaging as single or multiple unit doses and can be further sterilized and sealed.

[0097] Articles comprising the compositions described herein in suitable packaging are also provided. Suitable packaging for compositions (e.g., ophthalmic compositions) described herein is known in the art and includes, for example, vials (e.g., sealed vials), containers, ampoules, bottles, wide-mouth bottles, flexible packaging (e.g., sealed polyester film or plastic bags), etc. These articles may be further sterilized and / or sealed.

[0098] The present invention also provides kits comprising the compositions described herein (or unit dose forms and / or articles), and may further include one or more descriptions of methods of using the compositions (e.g., the uses described herein). The kits described herein may further include other materials required from a commercial and user perspective, including additional buffers, diluents, filters, needles, syringes, and packaging instructions with instructions for carrying out any of the methods described herein.

[0099] The compositions and formulations of the present invention can be used to treat conditions associated with the regulation of immune responses.

[0100] Veterinary uses

[0101] In addition to the above, the present invention also provides methods and materials that can be used for veterinary indications, including the treatment of immune disorders and diseases in non-human mammals. In specific embodiments, the methods and materials of the present invention for veterinary use include peptide domains derived from the same species as the veterinary host / patient. Non-human mammals may suffer from any immune disorder or disease, including autoimmune hemolytic anemia (AIHA), immune thrombocytopenic purpura (ITP), rheumatoid arthritis, or reactive arthritis. While non-human mammals can be of any species, certain breeds of dogs are known to be particularly susceptible to autoimmune disorders. For example, for the treatment of dogs, one or more Fc peptide domains and an oligomeric peptide domain, each of which is canine in origin, can be used. Specifically, dogs are known to be susceptible to immune disorders such as autoimmune hemolytic anemia (AIHA), in which the dog's own immune system binds to and destroys its red blood cells. Human-derived IVIGs have been used to treat dogs with AIHA or immune thrombocytopenic purpura (ITP) that are unresponsive to conventional therapies, or in severe ITP where the risk of fatal bleeding is considered significant. See, for example, Kellerman et al. (1997) J Vet Int Med, 11:327-332. However, dogs treated with human IVIG consistently develop canine anti-human antibodies (DAHA), which can trigger anaphylactic reactions upon repeated administration of human IVIG. Therefore, the treatment of veterinary patients with currently available IVIG compositions is severely limited. Therefore, the methods and materials of the present invention provide canine-derived rIVIG compositions and methods for treating dogs exhibiting canine immune disorders such as AIHA and ITP.

[0102] In veterinary indications, the present invention includes rIVIG peptides comprising peptide domains derived from the same species as the veterinary host / patient. Therefore, for the treatment of dogs, the present invention includes rIVIG peptides comprising one or more canine Fc peptide domains and canine oligomeric peptide domains. As in human treatment, a preferred embodiment of the invention comprises two or more Fc moieties linked by flexible linkers to allow intramolecular interactions, and a trimerizing peptide domain. rIVIG peptides comprising canine-derived oligomeric peptide domains can be used to treat dogs exhibiting canine immunodeficiency.

[0103] Recombinant immunoglobulin fusion protein

[0104] P7005H is a fusion protein composed of the human Fc moiety containing the CH2 and CH3 regions of the human IgG1 heavy chain and the extracellular domain (ECD) of human CD40L. The human Fc moiety can dimerize, and the CD40L ECD can trimerize. Therefore, the fusion protein is expected to form a hexamer containing three dimer Fc and two trimer CD40L. Mature P7005H contains three dimer Fc moieties containing the CH2 and CH3 regions of the human IgG1 heavy chain and is expected to exhibit excellent IVIG-mimicking activity. However, because each functional Fc domain is located on a separate peptide chain, the formation of dimer Fc is not homogeneous. Furthermore, the disulfide bonds between the expressed peptide chains can vary significantly, and intermolecular interactions can also occur intramolecularly, resulting in “zipper” oligomers that are much larger than hexamers. Therefore, the compositions formed from P7005H are significantly less homogeneous than desired and include improperly folded and therefore inactive aggregated proteins. Therefore, to make protein compositions containing P7005H more acceptable, further purification steps are required to isolate the most active hexamer. This need for purification makes P7005H less commercially viable, as the preparation of a homogeneous composition necessitates further purification.

[0105] To address the uniformity issue of the rIVIG compositions of this invention, the inventors have developed a series of single-chain human Fc fusion peptides.

[0106] P8001Z is a fusion protein comprising a single-chain human Fc, a GXY triplet repeat derived from human collagen 21, and an NC1 domain. The single-chain human Fc contains two tandem human CH2-CH3 Fc domains, each containing the CH2 and CH3 regions of the human IgG1 heavy chain. The single-chain Fc peptide includes a flexible linker (GGGGS)5 between the two CH2-CH3 Fc domains, which allows thermodynamically favorable intramolecular interactions and promotes the formation of functional Fc peptides within the single chain. This intramolecular interaction is expected to minimize intermolecular disulfide bond formation and maximize the formation of a single type of functional single-chain Fc peptide. The GXY triplet repeat sequence is responsible for collagen trimerization and is expected to aggregate the three Fc regions together, within which the two tandem CH2-CH3 Fc domains connected by the flexible linker can interact. Therefore, the product of P8001Z is expected to be more homogeneous than that of the P7005H construct.

[0107] P8003Z is a fusion protein comprising a single-chain human IgG κ or light chain constant region (CL), a first Fc domain containing the complete IgG constant region (CH1, CH2, and CH3), and a second Fc domain (containing CH2 and CH3). The second Fc domain is tandemly linked to the C-terminus of the first Fc region via a flexible linker, preferably a (G4S)5 linker. The flexible linker allows the construct to adopt a conformation in which the first and second Fc domains can interact intramolecularly. The C-terminus of the second Fc domain is tandemly linked to a collagen GXY triplet repeat and an NC1 domain (trimerizing domain). Similar to P8001Z, the collagen GXY repeat and NC1 domain exhibit intrinsic trimerizing activity to aggregate three single-chain Fc peptides, each containing a first CH2-CH3 Fc domain linked to the second CH2-CH3 Fc domain via a flexible linker, in which the first and second CH2-CH3 Fc domains can interact. It should also be mentioned that the CL and CH1 domains undergo heterodimerization. The CL / CH1 domain plays a role in clearing complement components, which can further alleviate the complement immune response present in many autoimmune disorders.

[0108] P8020Z is a fusion protein composed of an N-terminal portion of human mannose-binding protein (MBP) and a single-chain Fc peptide (similar to P8003Z), arranged in the following order from N-terminus to C-terminus: CL-CH1-CH2-CH3-flexible linker-CH2-CH3. The N-terminal portion of human MBP possesses intrinsic trimerizing capability and is responsible for the oligomerization of the fusion protein. In contrast to the design of P8003Z, the oligomerizing domain of P8020Z is located at the N-terminus of the fusion protein, while the Ig Fc region is located at the C-terminus, as found in innate immunoglobulin molecules. The structure of P8020Z, with its single-chain Fc peptide located at the C-terminus of the fusion protein, was expected to closely resemble the orientation of conventional antibody-Fc receptor interactions.

[0109] The recombinant rIVIG construct described above has been prepared and expressed in 293T cells, and the resulting protein can be purified using purification techniques known in the art.

[0110] To determine whether rIVIG proteins were correctly folded so that they primarily contained hexameric Fc structures, size exclusion chromatography (SEC) was used to analyze the purified proteins. Figure 2 The SEC spectra of each protein product are shown.

[0111] The FcγR binding activity of these rIVIG proteins was analyzed and compared with that of purified monomeric human IgG1 antibody. Figure 3 ).

[0112] The therapeutic effects of the P8003Z and P8020Z constructs were also tested using a mouse collagen-induced arthritis model.

[0113] Mice were primed with bovine type II collagen with complete Freund's adjuvant (CFA) and boosted with the same collagen with incomplete Freund's adjuvant (IFA) on day 21. P8020Z was administered intraperitoneally on day 18. Inflamed paws were scored starting on day 26. Figure 4 The results showed that mice treated with P8020 exhibited significantly reduced inflammation compared to control mice treated with PBS.

[0114] While the following examples illustrate the practice of the invention in various embodiments, these examples should not be construed as limiting the scope of the invention. Other embodiments will be apparent to those skilled in the art in light of the specification and examples.

[0115] Example

[0116] Example 1:

[0117] Construction of P7005H

[0118] The P7005H protein is expressed by the mammalian expression plasmid pMEhFcN1-7005, which encodes a 395-amino acid protein under the control of the cytomegalovirus (CMV) instantaneous early gene promoter. Starting from the N-terminus, the encoded product consists of a human IgG1 hinge, CH2 and CH3 regions, and an extracellular domain linked to human CD40L. The following is the coding sequence (Sequence ID 1) of the mature protein product (375 amino acids) produced from the production system.

[0119] The protein sequence of P7005H (375 amino acids) (Sequence ID 1):

[0120]

[0121] Table 2

[0122]

[0123] Example 2:

[0124] Construction of P8001Z

[0125] The P8001Z protein is expressed by the mammalian expression plasmid pHCM-rIVIG V1, which encodes a 539-amino acid protein under the control of the cytomegalovirus (CMV) instantaneous early gene promoter. Starting from the N-terminus, the encoded product consists of: a first CH2-CH3 Fc domain containing the CH2 and CH3 regions of the human IgG1 heavy chain; followed by a flexible linker containing five repeats of the G4S linker (GGGGS)5; followed by a second CH2-CH3 Fc domain containing the CH2 and CH3 regions of the human IgG1 heavy chain; and then eleven copies of the GXY triplet and NC1 domain ((GXY)11-NC1) from human collagen 21 A1. The following is the coding sequence (Sequence ID 2) of the mature protein product (519 amino acids) produced from the production system.

[0126] The protein sequence of P8001Z (519 amino acids) (Sequence ID 2):

[0127]

[0128] Table 3

[0129]

[0130] Example 3:

[0131] Construction of P8002Z

[0132] The P8002Z protein is expressed by the mammalian expression plasmid pHCM-rIVIG V2, which encodes a 529-amino acid protein under the control of the cytomegalovirus (CMV) instantaneous early gene promoter. Starting from the N-terminus, the encoded product consists of: a first CH2-CH3 Fc domain containing the CH2 and CH3 regions of the human IgG1 heavy chain; followed by three repeated G4S linkers (GGGGS)3; followed by a second CH2-CH3 Fc domain composed of the CH2 and CH3 regions of the human IgG1 heavy chain; followed by the GGGGS linker; and then eleven copies of the GXY triplet from human collagen 21 A1 and the NC1 domain ((GXY)11-NC1). The following is the coding sequence (Sequence ID 3) of the mature protein product (509 amino acids) produced from the production system.

[0133] The protein sequence of P8002Z (509 amino acids) (Sequence ID 3):

[0134]

[0135] Table 4

[0136]

[0137] Example 4:

[0138] Construction of P8003Z

[0139] The P8003Z protein was expressed by the mammalian expression plasmid pHCM-rIVIG V3, which encodes a 788-amino acid protein under the control of the cytomegalovirus (CMV) instantaneous early gene promoter. Starting from the N-terminus, the encoded product consists of: a human κ light chain constant region (CL); followed by two repeated G4S linkers (G4S)2; then a CH1-hinge-CH2-CH3 Fc domain containing the human IgG1 heavy chain constant region (CH1-hinge-CH2-CH3); followed by a flexible linker with five repeated G4S linkers (GGGGS)5; then a hinge-CH2-CH3 Fc domain containing the human IgG1 heavy chain hinge, CH2, and CH3 regions; followed by eleven copies of the GXY triplet and NC1 domain from human collagen 21 A1 ((GXY)11-NC1). The following is the sequence (Sequence ID 4) of the mature protein product (768 amino acids) produced from the production system.

[0140] The protein sequence of P8003Z (768 amino acids) (Sequence ID 4):

[0141]

[0142] Table 5

[0143]

[0144] Example 5:

[0145] Construction of P8004Z

[0146] The P8004Z protein is expressed by the mammalian expression plasmid pHCM-rIVIG V4, which encodes a 569-amino acid protein under the control of the cytomegalovirus (CMV) instantaneous early gene promoter. Starting from the N-terminus, the encoded product consists of: a first hinge-CH2-CH3 Fc domain (hinge-CH2-CH3) containing the human IgG1 heavy chain hinge, CH2, and CH3 regions; followed by a flexible linker (GGGGS)5; then a second hinge-CH2-CH3 Fc domain containing the human IgG1 heavy chain hinge, CH2, and CH3 regions; followed by eleven copies of the GXY triplet and NC1 domain from human collagen 21 A1 (GXY11-NC1). The following is the coding sequence (Sequence ID 5) of the mature protein product (549 amino acids) produced from the production system.

[0147] The protein sequence of P8004Z (549 amino acids) (Sequence ID 5):

[0148]

[0149] Table 6

[0150]

[0151] Example 6:

[0152] Construction of P8020Z

[0153] The P8020Z protein was expressed by the mammalian expression plasmid pHCM-rIVIG V20, which encodes an 816-amino acid protein under the control of the cytomegalovirus (CMV) instant early gene promoter. Starting from the N-terminus, the encoded product consists of: a human mannose-binding protein (hMBP) N-terminal peptide-hMBP collagen triple helix domain; followed by three repeated G4S linkers (GGGGS)3; followed by the human κ light chain constant region (CL); followed by two repeated G4S linkers (G4S)2; followed by a first CH1-hinge-CH2-CH3 Fc domain (CH1-hinge-CH2-CH3) containing the human IgG1 heavy chain constant region; followed by a flexible linker containing five repeated GGGGS linkers (GGGGS)5; followed by a hinge-CH2-CH3 Fc domain containing the human IgG1 heavy chain hinge, CH2, and CH3 regions. The following is the sequence of the mature protein product (796 amino acids) produced from the production system:

[0154] The protein sequence of P8020Z (796 amino acids) (Sequence ID 6):

[0155]

[0156] Table 7

[0157]

[0158]

[0159] Example 7:

[0160] K8020Z Construction

[0161] The K8020Z protein is expressed by the mammalian expression plasmid pHCM-rIVIG V40, which encodes an 822-amino acid protein under the control of the cytomegalovirus (CMV) instantaneous early gene promoter. Starting from the N-terminus, the encoded product consists of: a canine mannose-binding protein (MBP) N-terminal peptide-canine MBP collagen triple helix domain; followed by three repeated G4S linkers (GGGGS)3; followed by the canine κ light chain constant region (CL); followed by two repeated G4S linkers (GGGGS)2; followed by a canine CH1-hinge-CH2-CH3 Fc domain (CH1-hinge-CH2-CH3) containing the IgG subclass B heavy chain constant region; followed by a flexible linker containing five repeated G4S (GGGGS)5; followed by a canine hinge-CH2-CH3 Fc domain containing the canine IgG subclass B heavy chain hinge, CH2, and CH3 regions. The following is the coding sequence (Sequence ID 7) of the mature protein product (802 amino acids) produced from the production system.

[0162] The protein sequence of K8020Z (802 amino acids) (Sequence ID 7):

[0163]

[0164] Table 8

[0165]

[0166] Example 8:

[0167] Construction of K8003Z

[0168] The K8003Z protein was expressed by the mammalian expression plasmid pHCM-rIVIG V42, which encodes a 779-amino acid protein under the control of the cytomegalovirus (CMV) instantaneous early gene promoter. Starting from the N-terminus, the encoded product consists of: a canine κ light chain constant region (CL); followed by two repeated G4S linkers (GGGGS)2; followed by a canine CH1-hinge-CH2-CH3 Fc domain (CH1-hinge-CH2-CH3) containing the IgG subclass B heavy chain constant region; followed by a flexible linker containing five repeated G4S (GGGGS)5; followed by a canine hinge-CH2-CH3 Fc domain containing the canine IgG subclass B heavy chain hinge, CH2, and CH3 regions; followed by eleven copies of the GXY triplet and NC1 domain ((GXY)11-NC1) from canine collagen 21 A1. The following is the sequence (Sequence ID 8) of the mature protein product (779 amino acids) produced from the production system.

[0169] Protein sequence of K8003Z (779 amino acids) (Sequence ID No. 8):

[0170]

[0171] Table 9

[0172]

[0173] References

[0174] 1. Behring and Kitasato (1890) uber das Zustandekommen der Diphtherie-Immunidat und der Tetanus-Immunitat bei Thieren. Dtsch med Wochenschr 16: 1113-1114

[0175] 2. Bruton (1952) Agaqmmaglobulinemia. Pediatrics 9: 722-728.

[0176] 3. Barandun et al. (1962) Intravenous administration of human gamma-globulin. Vox Sang. 7: 157-174.

[0177] 4. Schultze and Schwick (1962) On new possibilities of intravenous gammaglobulin administration. Dtsch Med Wochenschr. 87: 1643-1644

[0178] 5. Kornhuber (1971) Intravenous g-Globulin-Therapie. Erfahrungen miteiner neuartigen Praparation. Mschr Kinderheilk 119: 528-530.

[0179] 6.Morell and Skvaril(1980)Structure and biological properties ofimmunoglobulins and gamma-globulin preparations.II.Properties of gamma-globulin preparations.Schweiz Mfed Wochenschr.110(3):80-85.

[0180] 7.Stephan(1975)Undegraded human immunoglobulin for intravenoususe.Vox Sang.28:422-437.

[0181] 8.Hansi et al.(1980)Clinical results with a new intravenousimmunoglobulin preparation.Dtsch Med Wochenschr.105:1675-1680.

[0182] 9.Luthardt(1980)Intravenous immunoglobulin administration forantibody deficiency.Dtsch Med Wochenschr.105:993-997.

[0183] 10.Nolte et al.(1979)Intravenous immunoglobulin therapy for antibodydeficiency.Clin Exp Immunol.36:237-243.

[0184] 11.Imbach et al.(1981)Igh-dose intravenous gammaglobulin foridiopathic thrombocytopenic purpura in childhood.Lancet 317:1228-1231.

[0185] 12.Noseworthy et al.(2000)IV immunoglobulin does not reverseestablished weakness in MS.Neurology.55:1135-1143.

[0186] 13.Fehr et al.(1982)Transient reversal of thrombocytopenia inidiopathic thrombocytopenic purpura by high-dose intravenous gamma globulin.NEngl J Med.306:1254-1258.

[0187] 14.Newland et al.(1983)High-dose intravenous IgG in adults withautoimmune thrombocytopenia.Lancet.1:84-87.

[0188] 15.Bussel and Hilgartner(1984)The use and mechanism of action ofintravenous immunoglobulin in the treatment of immune haematologic disease.BrJ Haematol.56:1-7.

[0189] 16.Debré et al.(1993)Infusion of Fc gamma fragments for treatment ofchildren with acute immune thrombocytopenic purpura.Lancet.342:945-949.

[0190] 17.Samuelsson et al.(2001)Anti-inflammatory activity of IVIG mediatedthrough the inhibitory Fc receptor.Science 291:484-486.

[0191] 18.Teeling et al.(2001)Therapeutic efficacy of intravenousimmunoglobulin preparations depeods on the immunoglobulin G dimers:studies inexperimental immune thrombocytopenia.Blood.98:1095-1099.

[0192] 19.Jain et al.(2012)Fully recombinant IgG2a Fc multimers(stradomers)effectiveIy treat collagen-induced arthritis and prevent idiopathicthrombocytopenc purpura in mice.Arthritis Res Ther.14:R192.

[0193] 20.Huang et al.(2010)Dendritic cells modulate platelet activity inIVIg-mediated amelioration of ITP in mice.Blood.116:5002-5009.

[0194] 21.Gillis et a1.(2014)Frontier Immunology 5:1-13

[0195] 22.Bergeron et al.(2014)Comparative functional characterization ofcanine IgG subclasses.Vet.Immunol.Immunopathol.157:31-41.

[0196] 23.Anthony et al.(2012)Novel roles for the IgG Fc glycan.Ann N Y AcadSci.1253:170-80

[0197] 24. Kaneko et al. (2006) Anti-inflammatory activity of immunoglobulin Gresulting from Fc sialylation. Science 313: 670-673

[0198] 25. Amold et al. (2007) Tbe impact of glycosylation on the biological function and structure of human immunoglobulins. Annu Rev Immunol. 25: 21-50

[0199] 26. Yamane-Ohnuki et al. (2004) Establishment of FUT8 knockout Chinesehamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicityBiotechnol Bioeng. 87: 614-622).

[0200] 27. Fugslang (2003) Protein Expression and Purification 31: 247-249.

[0201] 28. Kim et al. (2011) J. Biotechnology 151: 319-324.

[0202] 29. Lai et al. (2013) Pharmaceuticals 6: 579-603. sequence list <110> AB Biosciences, Inc. Hsu Yen-Ming <120> Recombinant intravenous immunoglobulin (rIVIG) composition and its production and use methods <130> ABB-USPTO-201-Prov <140> 62 / 315,483 <141> 2016 - 03 - 30 <160> 10 <170> PatentIn Version 3.5 <210> 1 <211> 375 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 1 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Ile Leu Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val 225 230 235 240 Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu 245 250 255 Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly 260 265 270 Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln 275 280 285 Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile 290 295 300 Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu 305 310 315 320 Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser 325 330 335 Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe 340 345 350 Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr 355 360 365 Ser Phe Gly Leu Leu Lys Leu 370 375 <210> 2 <211> 519 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 2 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly 210 215 220 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 225 230 235 240 Ser Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 245 250 255 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 260 265 270 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 275 280 285 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 290 295 300 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 305 310 315 320 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 325 330 335 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 340 345 350 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 355 360 365 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 370 375 380 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 385 390 395 400 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 405 410 415 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 420 425 430 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 435 440 445 Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Pro 450 455 460 Pro Gly Ile Ser Gly Pro Pro Gly Asp Pro Gly Leu Pro Gly Lys Asp 465 470 475 480 Gly Asp His Gly Lys Pro Gly Ile Gln Gly Gln Pro Gly Pro Pro Gly 485 490 495 Ile Cys Asp Pro Ser Leu Cys Phe Ser Val Ile Ala Arg Arg Asp Pro 500 505 510 Phe Arg Lys Gly Pro Asn Tyr 515 <210> 3 <211> 509 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 3 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly 210 215 220 Gly Ser Gly Gly Gly Gly Ser Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly 435 440 445 Gly Gly Gly Ser Gly Pro Pro Gly Ile Ser Gly Pro Pro Gly Asp Pro 450 455 460 Gly Leu Pro Gly Lys Asp Gly Asp His Gly Lys Pro Gly Ile Gln Gly 465 470 475 480 Gln Pro Gly Pro Pro Gly Ile Cys Asp Pro Ser Leu Cys Phe Ser Val 485 490 495 Ile Ala Arg Arg Asp Pro Phe Arg Lys Gly Pro Asn Tyr 500 505 <210> 4 <211> 768 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 4 Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 1 5 10 15 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 20 25 30 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 35 40 45 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 50 55 60 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 65 70 75 80 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 85 90 95 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys 465 470 475 480 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 485 490 495 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 500 505 510 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 515 520 525 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 530 535 540 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 545 550 555 560 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 565 570 575 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 580 585 590 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 595 600 605 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 610 615 620 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 625 630 635 640 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 645 650 655 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 660 665 670 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 675 680 685 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 690 695 700 Pro Gly Gly Gly Gly Gly Ser Gly Pro Pro Gly Ile Ser Gly Pro Pro 705 710 715 720 Gly Asp Pro Gly Leu Pro Gly Lys Asp Gly Asp His Gly Lys Pro Gly 725 730 735 Ile Gln Gly Gln Pro Gly Pro Pro Gly Ile Cys Asp Pro Ser Leu Cys 740 745 750 Phe Ser Val Ile Ala Arg Arg Asp Pro Phe Arg Lys Gly Pro Asn Tyr 755 760 765 <210> 5 <211> 549 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 5 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 225 230 235 240 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 245 250 255 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 260 265 270 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 275 280 285 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 290 295 300 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 305 310 315 320 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 325 330 335 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 340 345 350 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 355 360 365 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 370 375 380 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 385 390 395 400 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 405 410 415 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 420 425 430 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 435 440 445 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 450 455 460 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 465 470 475 480 Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Pro Pro Gly 485 490 495 Ile Ser Gly Pro Pro Gly Asp Pro Gly Leu Pro Gly Lys Asp Gly Asp 500 505 510 His Gly Lys Pro Gly Ile Gln Gly Gln Pro Gly Pro Pro Gly Ile Cys 515 520 525 Asp Pro Ser Leu Cys Phe Ser Val Ile Ala Arg Arg Asp Pro Phe Arg 530 535 540 Lys Gly Pro Asn Tyr 545 <210> 6 <211> 796 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 6 Glu Thr Val Thr Cys Glu Asp Ala Gln Lys Thr Cys Pro Ala Val Ile 1 5 10 15 Ala Cys Ser Ser Pro Gly Ile Asn Gly Phe Pro Gly Lys Asp Gly Arg 20 25 30 Asp Gly Thr Lys Gly Glu Lys Gly Glu Pro Gly Gln Gly Leu Arg Gly 35 40 45 Leu Gln Gly Pro Pro Gly Lys Leu Gly Pro Pro Gly Asn Pro Gly Pro 50 55 60 Ser Gly Ser Pro Gly Pro Lys Gly Gln Lys Gly Asp Pro Gly Lys Gly 65 70 75 80 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Thr 85 90 95 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 100 105 110 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 115 120 125 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 130 135 140 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 145 150 155 160 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 165 170 175 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 180 185 190 Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly 195 200 205 Gly Gly Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 210 215 220 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 225 230 235 240 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 245 250 255 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 260 265 270 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 275 280 285 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 290 295 300 Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 305 310 315 320 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 325 330 335 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 340 345 350 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 355 360 365 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 370 375 380 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 385 390 395 400 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 405 410 415 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 420 425 430 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 435 440 445 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 450 455 460 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 465 470 475 480 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 485 490 495 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 500 505 510 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 515 520 525 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly 530 535 540 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 545 550 555 560 Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 565 570 575 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 580 585 590 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 595 600 605 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 610 615 620 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 625 630 635 640 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 645 650 655 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 660 665 670 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 675 680 685 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 690 695 700 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 705 710 715 720 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 725 730 735 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 740 745 750 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 755 760 765 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 770 775 780 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 785 790 795 <210> 7 <211> 802 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 7 Asp Lys Glu Ala Leu Ser Glu Ala Gln Arg Thr Cys Pro Val Val Thr 1 5 10 15 Cys Ala Leu Pro Gly Arg Asp Gly Arg Asp Gly Leu Lys Gly Glu Lys 20 25 30 Gly Glu Pro Gly Gln Gly Leu Arg Gly Leu Gln Gly Pro Pro Gly Lys 35 40 45 Val Gly Pro Pro Gly Asn Thr Gly Ala Pro Gly Ala Pro Gly Leu Lys 50 55 60 Gly His Lys Gly Asp Arg Gly Asp Gly Gly Gly Gly Ser Gly Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Arg Asn Asp Ala Gln Pro Ala Val Tyr 85 90 95 Leu Phe Gln Pro Ser Pro Asp Gln Leu His Thr Gly Ser Ala Ser Val 100 105 110 Val Cys Leu Leu Asn Ser Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp 115 120 125 Lys Val Asp Gly Val Ile Gln Asp Thr Gly Ile Gln Glu Ser Val Thr 130 135 140 Glu Gln Asp Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Met 145 150 155 160 Ser Ser Thr Glu Tyr Leu Ser His Glu Leu Tyr Ser Cys Glu Ile Thr 165 170 175 His Lys Ser Leu Pro Ser Thr Leu Ile Lys Ser Phe Gln Arg Ser Glu 180 185 190 Cys Gln Arg Val Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala 195 200 205 Ser Thr Thr Ala Pro Ser Val Phe Pro Leu Ala Pro Ser Cys Gly Ser 210 215 220 Thr Ser Gly Ser Thr Val Ala Leu Ala Cys Leu Val Ser Gly Tyr Phe 225 230 235 240 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ser Leu Thr Ser Gly 245 250 255 Val His Thr Phe Pro Ser Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 260 265 270 Ser Ser Met Val Thr Val Pro Ser Ser Arg Trp Pro Ser Glu Thr Phe 275 280 285 Thr Cys Asn Val Ala His Pro Ala Ser Lys Thr Lys Val Asp Lys Pro 290 295 300 Val Pro Lys Arg Glu Asn Gly Arg Val Pro Arg Pro Pro Asp Cys Pro 305 310 315 320 Lys Cys Pro Ala Pro Glu Met Leu Gly Gly Pro Ser Val Phe Ile Phe 325 330 335 Pro Pro Lys Pro Lys Asp Thr Leu Leu Ile Ala Arg Thr Pro Glu Val 340 345 350 Thr Cys Val Val Val Asp Leu Asp Pro Glu Asp Pro Glu Val Gln Ile 355 360 365 Ser Trp Phe Val Asp Gly Lys Gln Met Gln Thr Ala Lys Thr Gln Pro 370 375 380 Arg Glu Glu Gln Phe Asn Gly Thr Tyr Arg Val Val Ser Val Leu Pro 385 390 395 400 Ile Gly His Gln Asp Trp Leu Lys Gly Lys Gln Phe Thr Cys Lys Val 405 410 415 Asn Asn Lys Ala Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala 420 425 430 Arg Gly Gln Ala His Gln Pro Ser Val Tyr Val Leu Pro Pro Ser Arg 435 440 445 Glu Glu Leu Ser Lys Asn Thr Val Ser Leu Thr Cys Leu Ile Lys Asp 450 455 460 Phe Phe Pro Pro Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln 465 470 475 480 Glu Pro Glu Ser Lys Tyr Arg Thr Thr Pro Pro Gln Leu Asp Glu Asp 485 490 495 Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp 500 505 510 Gln Arg Gly Asp Thr Phe Ile Cys Ala Val Met His Glu Ala Leu His 515 520 525 Asn His Tyr Thr Gln Lys Ser Leu Ser His Ser Pro Gly Gly Gly Gly 530 535 540 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Pro Lys Arg Glu Asn Gly Arg Val Pro Arg 565 570 575 Pro Pro Asp Cys Pro Lys Cys Pro Ala Pro Glu Met Leu Gly Gly Pro 580 585 590 Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Leu Ile Ala 595 600 605 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Leu Asp Pro Glu Asp 610 615 620 Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Gln Met Gln Thr 625 630 635 640 Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn Gly Thr Tyr Arg Val 645 650 655 Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu Lys Gly Lys Gln 660 665 670 Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser Pro Ile Glu Arg 675 680 685 Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro Ser Val Tyr Val 690 695 700 Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr Val Ser Leu Thr 705 710 715 720 Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp Val Glu Trp Gln 725 730 735 Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg Thr Thr Pro Pro 740 745 750 Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val 755 760 765 Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile Cys Ala Val Met 770 775 780 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser His Ser 785 790 795 800 Pro Gly <210> 8 <211> 779 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 8 Arg Asn Asp Ala Gln Pro Ala Val Tyr Leu Phe Gln Pro Ser Pro Asp 1 5 10 15 Gln Leu His Thr Gly Ser Ala Ser Val Val Cys Leu Leu Asn Ser Phe 20 25 30 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Val Asp Gly Val Ile Gln 35 40 45 Asp Thr Gly Ile Gln Glu Ser Val Thr Glu Gln Asp Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Met Ser Ser Thr Glu Tyr Leu Ser 65 70 75 80 His Glu Leu Tyr Ser Cys Glu Ile Thr His Lys Ser Leu Pro Ser Thr 85 90 95 Leu Ile Lys Ser Phe Gln Arg Ser Glu Cys Gln Arg Val Asp Gly Gly 100 105 110 Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Thr Thr Ala Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Cys Gly Ser Thr Ser Gly Ser Thr Val Ala 130 135 140 Leu Ala Cys Leu Val Ser Gly Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ser Leu Thr Ser Gly Val His Thr Phe Pro Ser Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr Val Pro 180 185 190 Ser Ser Arg Trp Pro Ser Glu Thr Phe Thr Cys Asn Val Ala His Pro 195 200 205 Ala Ser Lys Thr Lys Val Asp Lys Pro Val Pro Lys Arg Glu Asn Gly 210 215 220 Arg Val Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala Pro Glu Met 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Leu 260 265 270 Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys 275 280 285 Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn Gly 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu 305 310 315 320 Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser 325 330 335 Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro 340 345 350 Ser Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr 355 360 365 Val Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp 370 375 380 Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg 385 390 395 400 Thr Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser 405 410 415 Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile 420 425 430 Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser His Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 450 455 460 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro 465 470 475 480 Lys Arg Glu Asn Gly Arg Val Pro Arg Pro Pro Asp Cys Pro Lys Cys 485 490 495 Pro Ala Pro Glu Met Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro 500 505 510 Lys Pro Lys Asp Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys 515 520 525 Val Val Val Asp Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp 530 535 540 Phe Val Asp Gly Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu 545 550 555 560 Glu Gln Phe Asn Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly 565 570 575 His Gln Asp Trp Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn 580 585 590 Lys Ala Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly 595 600 605 Gln Ala His Gln Pro Ser Val Tyr Val Leu Pro Pro Ser Arg Glu Glu 610 615 620 Leu Ser Lys Asn Thr Val Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe 625 630 635 640 Pro Pro Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro 645 650 655 Glu Ser Lys Tyr Arg Thr Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser 660 665 670 Tyr Phe Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg 675 680 685 Gly Asp Thr Phe Ile Cys Ala Val Met His Glu Ala Leu His Asn His 690 695 700 Tyr Thr Gln Lys Ser Leu Ser His Ser Pro Gly Gly Gly Gly Gly Ser 705 710 715 720 Gly Pro Pro Gly Ile Ser Lys Glu Gly Pro Pro Gly Asp Pro Gly Leu 725 730 735 Pro Gly Lys Asp Gly Asp His Gly Lys Pro Gly Ile Gln Gly Gln Pro 740 745 750 Gly Pro Pro Gly Ile Cys Asp Pro Ser Leu Cys Phe Ser Val Ile Val 755 760 765 Gly Arg Asp Pro Phe Arg Lys Gly Pro Asn Tyr 770 775 <210> 9 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 9 Gly Gly Gly Gly Ser 1 5 <210> 10 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Synthesized in La <400> 10 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25

Claims

1. A recombinant intravenous immunoglobulin (rIVIG) polypeptide comprising: (a) a CL domain, a CH1 domain and a single-chain Fc peptide containing two CH2-CH3 Fc domains; and (b) an oligomerized peptide domain, wherein the rIVIG polypeptide comprises an amino acid sequence of sequence ID 4 or sequence ID 6.

2. A nucleic acid encoding the rIVIG polypeptide as described in claim 1.

3. A recombinant vector comprising the nucleic acid as described in claim 2.

4. A recombinant cell comprising the recombinant vector as described in claim 3.

5. The recombinant cell of claim 4, wherein the cell lacks the α-1,6-fucosyltransferase gene (FUT8). - / - ).

6. A composition for treating immune disorders, comprising recombinant immunoglobulin (rIVIG) protein, said rIVIG protein being composed of: The rIVIG protein comprises a CL domain, a CH1 domain, a single-chain Fc peptide containing two CH2-CH3 Fc domains, and an oligomeric peptide domain; and the rIVIG protein consists of an amino acid sequence of sequence ID 4 or sequence ID 6.

7. The composition of claim 6, wherein the composition comprises a homotrimeric Fc dimer.

8. The composition of claim 6, wherein the rIVIG protein comprises the amino acid sequence of sequence ID 6.

9. The composition of claim 6, wherein the rIVIG protein comprises the amino acid sequence of sequence ID 4.

10. The composition of any one of claims 6, 7, 8 and 9, wherein the rIVIG protein is unfucosylated.

11. Use of recombinant intravenous immunoglobulin (rIVIG) protein in the preparation of a medicament for treating patients with autoimmune disorders, wherein said rIVIG protein comprises the following: The rIVIG protein comprises a CL domain, a CH1 domain, a single-chain Fc peptide containing two CH2-CH3 Fc domains, and an oligomeric peptide domain; and the rIVIG protein consists of an amino acid sequence of sequence ID 4 or sequence ID 6.

12. The use as described in claim 11, wherein the patient suffers from refractory immune thrombocytopenic purpura.

13. Use of recombinant intravenous immunoglobulin (rIVIG) protein in the preparation of a medicament for reducing immune rejection in patients who have received organ transplants, wherein the rIVIG protein comprises: a CL domain, a CH1 domain, a single-chain Fc peptide containing two CH2-CH3 Fc domains, and an oligomerized peptide domain; and wherein the rIVIG protein comprises an amino acid sequence of sequence ID 4 or sequence ID 6.

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