Multimeric Hybrid Fc Proteins for Replacement of IVIG

Abstract

The hybrid Fc protein of the present invention comprises IgG and IgM Fc components. The IgG Fc component comprises at least a portion of the hinge region and the CH2 and CH3 regions. The IgM component comprises the Cμ3 and Cμ4 regions of the Cμ constant region. The hybrid Fc protein can form a duplex through interchain disulfide bonds between cysteines in its hinge region. The hybrid Fc protein can be used to treat immune diseases mediated by endogenous IgG, such as diseases previously treated with intravenous immunoglobulins.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. 62 / 767,303, filed Nov. 14, 2018, which is hereby incorporated by reference in its entirety for all purposes.

[0003] Sequence Listing

[0004] This application includes an 85 KB sequence listing written in the txt file 538890WO - ST25, created on Oct. 24, 2019, which is incorporated by reference. BACKGROUND OF THE INVENTION

[0005] Intravenous immunoglobulin (IVIG) is a pooled IgG preparation from thousands of healthy human donors and has been used as a human therapy for immunodeficiency and immune - mediated diseases (Nimmerjahn et al., Annu. Rev. Immunol. 26:513 - 533, 2008; Nagelkerke et al., Front. Immunol. 5:Article 674, 2015; Mitrevski et al., Front. Immunol. 6:Article 4, 2015; Seite et al., Arthritis Rheum. 67:595 - 603, 2015; Afonso et al., Biomolecules 6:15, 2016; Lazarus, Chapter 6 in Imbach (eds), Antibody Therapy, Springer, 2018). IVIG administered at a dosage range of 200 to 500 mg per kilogram of body weight can provide pathogen - specific IgG antibodies from donors to immunodeficient patients to prevent infectious diseases. Since this protective IgG antibody is eventually cleared from the circulation, IVIG requires continuous administration to maintain protection for the patient, typically administered every three to four weeks.

[0006] High-dose IVIG administration (usually 1 to 3 g per kilogram of body weight) has been used as an anti-inflammatory agent for the treatment of acute and chronic immune-mediated diseases, such as idiopathic thrombocytopenic purpura (ITP), Kawasaki disease, Guillain-Barré syndrome, and chronic inflammatory demyelinating polyneuropathy. Off-label uses of IVIG for the treatment of immune-mediated diseases include systemic lupus erythematosus, multiple sclerosis, and autoimmune neutropenia. Two mechanisms of action (MOA) have been proposed and supported by scientific observations: (i) neonatal Fc receptor saturation, and (ii) inhibition of B cell function (Nagelkerke et al., Front. Immunol. 5: Article 674, 2015; Seite et al., Arthritis Rheum. 67:595-603, 2015).

[0007] The neonatal Fc receptor (FcRn) is a heterodimer composed of a transmembrane alpha chain and beta2-microglobulin (β2m). FcRn expressed in endothelial cells mediates the transcytosis of maternal IgG to the fetus and the homeostasis of adult IgG. FcRn captures the pinocytosed IgG antibody in acidified endosomes, rescues the pinocytosed IgG antibody from lysosomal degradation, cycles back to the cell surface, and returns to the circulation. The binding of IgG to FcRn is saturable. When serum IgG concentration rises above normal levels, the excess IgG that cannot bind to FcRn is degraded in lysosomes (Roopenian et al., Nat. Rev. Immunol. 7:715-725, 2007; Kuo et al., J. Clin. Immunol. 30:777-789, 2010; Rath et al., Front. Immunol. 5: Article 664, 2015). High-dose (usually 1 to 3 g / kg) administration of IVIG competes with pathogenic IgG antibodies present in patients with autoimmune diseases for FcRn binding, thus accelerating the clearance of such pathogenic antibodies in the circulation (Nimmerjahn et al., Annu. Rev. Immunol. 26:513-533, 2008; Seite et al., Arthritis Rheum. 67:595-603, 2015).

[0008] CD22 is a member of the Siglec family of type I transmembrane proteins that binds specifically to sialic acid linked to glycans with an affinity of 32 μM (Powell et al., J. Biol. Chem. 13:7523-7532, 1995; Fearon et al., Annu. Rev. Immunol. 18:393-442, 2000; Pillai et al., Annu. Rev. Immunol. 30:357-392, 2012; Nitschke, Glycobiol. 24:807-817, 2014). CD22 plays a crucial regulatory role in establishing the B cell activation threshold. Multivalent cross-linking of CD22 induces intracellular signaling through immunoreceptor tyrosine-based inhibitory motifs (ITIMs) located in its cytoplasmic domain, leading to functional inhibition of B cells. The sialylated moiety of IVIG has been shown to bind to CD22 and negatively regulate the immune response of B cells (Mitrevski et al., Int. Trends Immun. 2:67, 2014; Seite et al., Blood 116:1698-1704, 2010). A positive correlation has been reported between the sialylation level of IVIG (or Fc protein) and its immunosuppressive activity (Schwab et al., Clin. Exp. Immunol. 178:97-99, 2014; Washburn et al., Proc. Natl. Acad. Sci. 112:E1297-E1306, 2015; Bruckner et al., Int. Immunol. 29:499-509, 2017), indicating the importance of CD22 in the therapeutic activity of IVIG in treating inflammatory diseases.

[0009] The involvement of CD32B (also known as Fcγ receptor IIB) is also relevant to the MOA of IVIG; CD32B is a type I transmembrane protein expressed on B cells and myeloid dendritic cells. Unlike CD64 (Fcγ receptor I), CD32A (Fcγ receptor IIA) and CD16 (Fcγ receptor III) which have immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic domains, CD32B contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain and acts as a negative regulator of the immune response. Crosslinking of CD32B induces intracellular signal transduction, leading to downregulation of antibody production in B cells. When CD32B is deficient in the mice used in the studies, IVIG did not show therapeutic effects in mouse models of ITP, rheumatoid arthritis, and nephrotoxis nephritis. However, it is not clear whether IVIG directly interacts with CD32B for immunosuppression. It has been reported that DC-SIGN (dendritic cell-specific ICAM-3-grabbing non-integrin; also known as CD209), rather than CD32B, is the major site of action of CDIG. DC-SIGN is the human interspecies homolog of mouse SIGN-R1 (specific ICAM-3-grabbing non-integrin-related 1), which is expressed on macrophages and dendritic cells. Interaction of sialylated IVIG (or Fc protein) with DC-SIGN on macrophages and dendritic cells can induce the expression of certain cytokines such as IL-33, leading to upregulation of CD32B in antigen-presenting cells and signaling through CD32B, thereby suppressing the immune response. For a literature review, see Samuelsson et al., Science 291:484-486, 2001; Crow et al., Blood 102:558-560, 2003; Bruhns et al., Immunity 18:573-581, 2003; Akilesh et al., J. Clin. Invest. 113:1328-1333, 2004; Zhou et al., Cell. Mol. Immunol. 4:279-283, 2006; Kaneko et al., Science 313:670-673, 2006; Kaneko et al., J. Exp. Med. 203:789-797, 2006; Anthony et al., Proc. Natl. Acad. Sci. 105:19571-19578, 2008; Anthony et al., Nature 475:110-113, 2013; Pagan et al., Cell 172:564-577, 2018.

[0010] Although IVIG has been widely used to treat immunodeficiencies and various immune-mediated diseases in humans, IVIG has an inherent drawback in that it is derived from human blood. Although blood sources have been screened for infectious agents and other diseases unsuitable for blood donation, there is always a minimal possibility that unknown infectious agents will contaminate IVIG products. In addition, batch-to-batch variability of IVIG is inevitable. Moreover, the decline in blood donation has led to a shortage in the supply of IVIG. Therefore, it is crucial to develop a clean, continuously available recombinant product that can functionally replace IVIG in the treatment of immune-mediated diseases.

[0011] There have been several attempts to use recombinant anti-FcRn monoclonal antibodies to block the interaction between IgG and FcRn in order to enhance catabolism and reduce the concentration of IgG molecules in circulation. Nixon et al. (Front. Immunol. 6: Article 176, 2015) generated a human anti-FcRn antibody that resulted in a long-term reduction in IgG levels in cynomolgus monkeys. Kiessling et al. (Sci. Transl. Med. 9: eaan1208, 2017) reported the use of the humanized anti-FcRn monoclonal antibody rozanolixizumab as an alternative to IVIG. In both cynomolgus monkeys and humans, rozanolixizumab was able to reduce the IgG concentration in circulation. Severe treatment-related adverse events were observed in several human subjects who received intravenous injections of 7 mg / kg of rozanolixizumab. Neither the papers by Kiessling nor Nixon showed data on the inhibition of B cell-mediated immune responses by anti-FcRn antibodies.

[0012] As an alternative approach to blocking the interaction between IgG and FcRn, Patel et al. (J. Immunol. 187: 1015 - 1022, 2011) reported an engineered human IgG1 antibody with enhanced FcRn binding; it had Met substituted for Tyr at position 252, Thr substituted for Ser at position 254, Glu substituted for Thr at position 256, Lys substituted for His at position 433, and Phe substituted for Asn at position 434 (MST-HN; positions based on Eu numbering) in the Fc region, which reduced the serum IgG levels in mice. Ulrichts et al. (J. Clin. Invest. JCI97911, 2018) used an Fc fragment derived from human IgG1 with the same five amino acid substitutions (MST-HN) as described above (efgartigimod) as an FcRn antagonist and showed that efgartigimod could reduce the IgG content in humans by up to 50%.

[0013] Czajkowsky et al. (Sci. Reports 5:9526, 2015) reported the generation of a hexameric Fc fragment (Hexa-Fc), in which the leucine residue at position 309 (Eu numbering) was changed to a cysteine residue, and an 18-amino acid-long μ tail-piece was linked to the end of the human IgG1 Fc fragment to form a hexamer as a possible antagonist of FcRn. However, animal data showing the regulation of serum IgG levels using Hexa-Fc were not shown in that paper. The authors also pointed out that the unique three-dimensional structure of Hexa-Fc might hinder its interaction with FcRn.

[0014] Spirig et al. (J. Immunol. 200:2542 - 2553, 2018) also generated a hexameric IgG1 Fc fragment (Fc-μTP-L309C) by introducing a leucine-to-cysteine substitution at position 309 (Eu numbering) and a μ tail-piece at the end of the Fc region. Although Fc-μTP-L309C could effectively inhibit inflammatory arthritis and ITP in mice, it had a short serum half-life in human FcRn transgenic mice (3.1 hours) and rats (2.5 to 3 hours). In contrast, the serum half-life of human IgG in human FcRn transgenic mice was reported to be about 10 days (Tam et al., mAbs 5:397 - 405, 2013). Data on the effect of Fc-μTP-L309C on serum IgG levels or data on the inhibition of immune responses were not reported in the Spirig paper.

[0015] US 9,382,319 reported antibodies or Fc fusion proteins linked to a hybrid heavy chain constant region having IgG or IgA and IgM components. The antibody variable region or a heterologous polypeptide forms a binding site for a target site in a subject, and the hybrid constant region causes the polymerization and activation of cells expressing the target on the surface. SUMMARY OF THE INVENTION

[0016] The present invention provides a hybrid Fc protein that sequentially includes an IgG Fc region, CH2 and CH3 regions, and an IgM Fc region from the N-terminus to the C-terminus; the IgG Fc region includes at least a part of the hinge region, each of the CH2 and CH3 regions is of the IgG isotype, the IgM Fc region includes Cμ3 and Cμ4 regions, wherein at least a part of the hinge region is not linked to (a) an antibody variable region or (b) a heterologous polypeptide that binds to a target, wherein the molecule of the hybrid Fc protein can form a duplex through an interchain disulfide bond between cysteine residues in at least a part of the hinge region, and the duplexes can multimerize with each other through the Cμ3 and Cμ4 regions.

[0017] Optionally, the IgG Fc region is of human IgG1, IgG2, IgG3 or IgG4 isotype, and the Cμ3 and Cμ4 regions are human Cμ3 and Cμ4 regions, respectively. Optionally, at least a portion of the hinge region is not linked to a polypeptide of more than 25 amino acids. Optionally, at least a portion of the hinge region differs from the native human hinge region by substitution of cysteine residues that do not participate in Fc duplex formation in native antibodies. Optionally, the protein consists essentially of at least a portion of the hinge region, the CH2 and CH3 regions, the Cμ3 and Cμ4 regions, and optionally a peptide of up to 25 amino acids linked to at least a portion of the hinge region. Optionally, at least a portion of the hinge region contains the peptide Glu-Pro-Lys-Ser-Ser (SEQ ID NO: 8) at its N-terminus. Optionally, the IgG Fc region and / or the IgM Fc region comprise one or more mutations to reduce ADCC, ADP or CDC. Optionally, the IgG Fc region comprises one or more mutations to increase FcRn binding. Optionally, the IgG Fc region and / or the IgM Fc region comprise one or more mutations to increase sialylation.

[0018] Optionally, positions 234 and 235 (Eu numbering) in the IgG Fc region are alanine residues (e.g., SEQ ID NO: 9). Optionally, positions 433 and 435 (Eu numbering) in the IgM Fc region are alanine and serine residues, respectively (e.g., SEQ ID NO: 10). Optionally, position 428 (Eu numbering) in the IgG Fc region is a leucine residue (e.g., SEQ ID NO: 13). Optionally, position 241 or 243 (Eu numbering) in the IgG Fc region is an alanine residue (e.g., SEQ ID NO: 15 and 16, respectively).

[0019] Optionally, the molecules of the hybrid Fc protein form a duplex through interchain disulfide bonds between cysteine residues in at least a portion of the hinge region, and the duplexes multimerize with each other through the Cμ3 and Cμ4 regions. Optionally, the multimer is a hexamer.

[0020] Optionally, the hybrid Fc protein is at least 99% w / w pure.

[0021] The present invention further provides a pharmaceutical composition comprising any of the above hybrid Fc proteins and a pharmaceutically acceptable carrier.

[0022] The present invention further provides a method for treating immune diseases, the method comprising administering to a subject in need thereof a hybrid Fc protein as described in any one of the foregoing claims in an effective regimen. Optionally, the hybrid Fc protein reduces the half-life of IgG molecules in circulation. Optionally, the hybrid Fc protein reduces the concentration of IgG molecules in circulation. Optionally, the hybrid Fc fusion protein inhibits the immune response of B cells. Optionally, the subject has an immune disease.

[0023] The present invention further provides the use of the hybrid Fc protein as described in any one of the foregoing claims in the preparation of a medicament for treating inflammatory diseases, rejection after organ transplantation, hematological diseases, skin diseases or neuromuscular diseases.

[0024] The present invention further provides the use of the hybrid Fc protein as described in any one of the foregoing claims in the preparation of a medicament for treating autoimmune diseases. Optionally, the disease is idiopathic thrombocytopenic purpura, Kawasaki disease, Guillain-Barré syndrome, or chronic inflammatory demyelinating polyneuropathy. Optionally, the disease is systemic lupus erythematosus, multiple sclerosis, or autoimmune neutropenia. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Schematic structure of the expression vector pVF101.

[0026] Figure 2 : Schematic structure of the hexameric hybrid Fc protein of the present invention.

[0027] Figure 3 : Relative concentration of murine monoclonal IgG antibody ABC2 in murine serum shown with SEM (standard error of the mean) error bars. ABC2 was administered to three mice: without Fc protein (Group A), LS41K-Fc.S (Group B), or LS41K-Fc.SL (Group C).

[0028] Figure 4 : Relative concentration of murine IgG in murine serum from Groups A, B, and C shown with SEM error bars.

[0029] Figure 5 : Concentrations of LS41K-Fc.S and LS41K-Fc.SL in murine serum from Groups B and C respectively shown with SEM error bars.

[0030] Figure 6 : Relative concentration of ABC2 in murine serum shown with SEM error bars. ABC2 was administered to three mice together with 100 μg of LS41K-Fc.SL (Group D) or 400 μg of LS41K-Fc.SL (Group E).

[0031] Figure 7 : The relative concentrations of murine IgG in murine sera from groups D and E are shown as SEM error bars.

[0032] Figure 8 : The concentrations of LS41K-Fc.SL in murine sera from groups D and E are shown as SEM error bars.

[0033] Figures 9A, B : Schematic diagrams of the expression vectors for (A) ST6GAL1 and (B) B4GALT1.

[0034] Figure 10A A, B, C: The sequences of (A) γ-1 (SEQ ID NO: 29-32), γ-2 (SEQ ID NO: 33-36), γ-3 (SEQ ID NO: 37-40), (B) γ-4 (SEQ ID NO: 41-44), α-1 (SEQ ID NO: 45-47), α-2 (SEQ ID NO: 48-50), and (C) the μ heavy chain constant region (SEQ ID NO: 51-54) and the J chain (SEQ ID NO: 55). The underlined part in the Cμ sequence is an 18-amino acid long μ tail fragment. The first 22 amino acids shown in the J chain are the cleaved signal peptide.

[0035] Figures 11A - D : FACS analysis of binding to human and murine FcRn: (A) Binding of Erbitux (a murine-human chimeric IgG1 antibody) to human FcRn at pH 6.0 and pH 7.5, (B) Binding of LS41K-Fc.SL to human FcRn at pH 6.0 and pH 7.5, (C) Binding of Erbitux to murine FcRn at pH 6.0 and pH 7.5, and (D) Binding of LS41K-Fc.SL to murine FcRn at pH 6.0 and pH 7.5. Detailed Description of the Invention

[0036] Definitions

[0037] The present hybrid Fc protein is typically provided in isolated form. This means that the hybrid Fc protein is typically 50% w / w pure of interfering proteins and other contaminants resulting from its production or purification, but does not exclude the possibility that the hybrid Fc protein is combined with an excess of pharmaceutically acceptable carriers or other vehicles for its use. Sometimes the hybrid Fc protein is at least 60%, 70%, 80%, 90%, 95% or 99% w / w pure of interfering proteins and other contaminants resulting from production or purification. Generally, the hybrid Fc protein is the major macromolecular component remaining after purification.

[0038] The hybrid Fc protein binds specifically to FcRn. The specific binding is at a detectable magnitude and is higher, distinguishing it from non-specific binding to at least one unrelated target. Specific binding can be the result of bond formation between specific functional groups or specific spatial fits (e.g., lock and key type), while non-specific binding is usually the result of van der Waals forces. However, specific binding does not necessarily mean that the hybrid Fc protein binds to one and only one target. For example, they can also specifically bind to CD22 through sialylation.

[0039] The basic antibody structural unit is a tetramer of subunits. Each tetramer includes two pairs of identical polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50 - 70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids, which is mainly responsible for antigen recognition. When initially expressed, the variable region is linked to a cleavable signal peptide. The variable region without the signal peptide is sometimes referred to as the mature variable region. Thus, for example, the light chain mature variable region refers to the light chain variable region without the light chain signal peptide. However, referring to the variable region does not mean that a signal sequence must be present; in fact, once the antibody or fusion protein of the present invention is expressed and secreted, the signal sequence is cleaved. A pair of heavy and light chain variable regions defines the binding region of the antibody. The carboxyl-terminal portions of the light and heavy chains define the light and heavy chain constant regions, respectively. The heavy chain constant region is mainly responsible for effector functions. In IgG antibodies, the heavy chain constant region is divided into CH1, hinge, CH2, and CH3 regions. In IgA, the heavy constant region is divided into CH1, CH2, and CH3. The CH1 region binds to the light chain constant region through disulfide bonds and non-covalent bonds. The hinge region provides flexibility between the binding region and the effector region of the antibody and also provides a site for intermolecular disulfide bond binding between the two heavy chain constant regions in the tetrameric subunit. The CH2 and CH3 regions are the main sites for effector functions and FcRn binding. In IgM antibodies, the μ heavy chain constant region (Cμ) is divided into four regions Cμ1, Cμ2, Cμ3, and Cμ4. The Cμ3 and Cμ4 regions, sometimes in combination with one or more J chains, provide a multimerization function in native IgM antibodies and the hybrid Fc proteins of the present invention. The μ tail piece is an 18-amino acid-long polypeptide located at the C-terminus of the IgM heavy chain constant region. IgM polymerizes to form a pentameric structure in the presence of J chain and a hexameric structure in the absence of J chain.

[0040] The light chains are divided into κ or λ. The heavy chains are divided into γ, μ, α, δ or ε, and the isotypes of antibodies are defined as IgG, IgM, IgA, IgD and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined together by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. (See generally Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7), which is incorporated herein by reference in its entirety for all purposes).

[0041] The mature variable regions of each light / heavy chain pair form the antibody binding site. Thus, a complete antibody has two binding sites and is thus bivalent. In native antibodies, the binding sites are identical. However, bispecific antibodies in which the two binding sites are different can be prepared (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)). These variable regions all exhibit the same general structure, namely relatively conserved framework regions (FRs) connected by three hypervariable regions, which are also called complementarity determining regions or CDRs. The CDRs of the two chains of each pair are aligned by the framework so that they can bind to a specific epitope. From the N-terminus to the C-terminus, the light and heavy chains both contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. The assignment of amino acids in each domain is defined according to Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989). Kabat also provides a widely used numbering rule (Kabat numbering), in which corresponding residues between different heavy chains or different light chains are given the same number. Although Kabat numbering can be used for the antibody constant region, the EU index numbering (EU index) is more commonly used, as is the case in this application.

[0042] The multimerization unit of the hybrid Fc protein is typically a duplex of two such proteins, which are linked by interchain disulfide bonds between one or more cysteines in their respective hinge regions.

[0043] Polymerization refers to the association of at least two polymerization units (more typically five or six such units) through the binding of the Cμ portion of the hybridizing constant region. Polymerization of hybrid Fc protein units can sometimes form structures of higher or lower order than the pentameric or hexameric structure of normal IgM. This is sometimes indicated by characterizing the complex formed by polymerization, which has at least about five or six units.

[0044] The heterologous polypeptide in the fusion protein is a polypeptide that is not naturally linked to an immunoglobulin constant region. Such a polypeptide can be a full-length protein or a fragment thereof that is long enough to retain the specific binding of the antigen that binds to the full-length protein. For example, the heterologous polypeptide can be a receptor extracellular domain or its ligand.

[0045] "Subject" includes a human or other mammal receiving prophylactic or therapeutic treatment. Other non-human animals include animal models of human conditions (e.g., rodents, non-human primates) and veterinary subjects.

[0046] The term "target" refers to a target molecule (e.g., a protein, nucleic acid, or carbohydrate) present in a subject to which a drug (e.g., an antibody or Fc fusion protein) can specifically bind to effectively treat or prevent a medical condition in the subject.

[0047] To classify an amino acid substitution as conservative or non-conservative, the amino acids are grouped as follows: Group I (hydrophobic side chains): methionine, alanine, valine, leucine, isoleucine; Group II (neutral hydrophilic side chains): cysteine, serine, threonine; Group III (acidic side chains): aspartic acid, glutamic acid; Group IV (basic side chains): asparagine, glutamine, histidine, lysine, arginine; Group V (residues affecting chain orientation): glycine, proline; and Group VI (aromatic side chains): tryptophan, tyrosine, phenylalanine. Conservative substitutions include substitutions between members of the same class of amino acids. Non-conservative substitutions are replacements of a member of one of these classes with a member of another of these classes.

[0048] The percent sequence identity is determined by maximizing the match of the antibody sequence by the Kabat numbering convention (for variable regions) or EU numbering (for constant regions). After alignment, if the subject antibody region (e.g., the entire mature variable region of the heavy or light chain) is compared to the same region of a reference antibody, then the percent sequence identity between the subject antibody region and the reference antibody region is the number of positions with identical amino acids in the subject antibody region and the reference antibody region divided by the total number of positions aligned for the two regions, disregarding gaps, and multiplied by 100 to convert to a percentage.

[0049] A composition or method "comprising" one or more recited elements may include other elements not specifically recited. For example, a composition containing an antibody may contain only the antibody or a combination of the antibody with other components.

[0050] "Consisting essentially of" is used, according to convention, to denote the basic and novel features of a composition or method and does not exclude other components or steps that may be present that do not materially affect the basic and novel features.

[0051] The pH-dependent binding of an antibody to the FcRn receptor means that the antibody binds this receptor more tightly at pH 6.0 than at pH 7.5. After internalization by endocytosis, FcRn binding at the low pH in endosomes rescues IgG antibodies from catabolic degradation in lysosomes. The rescued IgG antibodies are then released from FcRn at neutral pH and recycled into the circulation. This pH-dependent FcRn binding is the basis of the molecular mechanism for the long serum half-life of IgG antibodies (Ghetie et al., Annu. Rev. Immunol. 18:739-766, 2000). For example, human IgG antibodies bind to the human neonatal Fc receptor (FcRn) at pH 6.0, while binding to FcRn only weakly at pH 7.5. The FcRn binding site in IgG antibodies is located at the junction of the CH2 and CH3 domains. Since the μ heavy chain does not bind to FcRn at pH 6.0 or 7.5, native IgM cannot utilize the FcRn-mediated pathway to rescue antibodies from lysosomal degradation and thus generally has a shorter half-life than native IgG antibodies. Some of the hybrid Fc proteins of the present invention show little significant difference in binding to FcRn at pH 6.0 and 7.5, which contributes to their ability to compete with IgG for binding to FcRn.

[0052] Detailed Description

[0053] I. General Overview

[0054] The hybrid Fc proteins of the invention comprise IgG and IgM Fc moieties. The IgG Fc moiety comprises at least a portion of the hinge region and the CH2 and CH3 regions. The IgM moiety comprises the Cμ3 and Cμ4 regions of the Cμ constant region. The hybrid Fc proteins can form dimers through interchain disulfide bonds between cysteines in their hinge regions. The dimers can in turn be multiplexed through disulfide bonds of the IgM Fc portion. The IgG portion of the hybrid Fc protein, like other IgG molecules, has a specific affinity for the FcRn receptor. However, due to IgM Fc-mediated multimerization, the binding affinity is increased. Although not necessary to understand the mechanism to practice the invention, it is believed that the binding of the hybrid Fc protein to FcRn competes with the binding of endogenous IgG to FcRn, thereby reducing the half-life of endogenous IgG. Shortening the half-life of endogenous IgG can be used to treat immune diseases mediated by endogenous IgG, such as those previously treated with intravenous immunoglobulins. In contrast to the antibodies or fusion proteins previously described in US 9,382,319, this mechanism of action does not require treatment in a subject through target binding of a binding region provided by an antibody variable region or a heterologous polypeptide linked to the hybrid Fc region. The above advantages can be achieved without in vitro manipulation, except for those involved in preparing nucleic acid constructs for expressing the hybrid Fc proteins.

[0055] II. Components of the Hybrid Fc Region

[0056] The hybrid Fc protein comprises an IgG Fc portion and an IgM Fc portion. The IgG Fc portion comprises at least a part of the hinge region and the CH2 and CH3 regions. The CH2 and CH3 regions are responsible for or at least partly responsible for FcRn binding, protein A and G binding, ADCC (antibody-dependent cell cytotoxicity), CDC (complement-dependent cell cytotoxicity), and opsonization. At least a part of the hinge region serves to provide cysteine residues to form interchain disulfide bonds of the double-stranded IgG Fc region. At least a part of the hinge region comprises at least one (usually 2 or more) cysteine of the native hinge region and flanking residues sufficient to support the desired interchain disulfide bonds for forming the duplex. However, not all cysteine residues in the native hinge region contribute to duplex formation between Fc regions, and any or all such other cysteine residues can be removed or replaced by another residue (such as serine or alanine or glycine) to avoid cysteine participating in non-natural disulfide bonding. Thus, a part of the hinge region, usually the N-terminal part, can be replaced by a synthetic peptide, which is usually no more than 25, 20, 15, 10, or 5 residues. In addition to providing cysteine residues, the hinge region including any synthetic peptide also provides flexibility for the formation of duplexes and multimers. Glycine, alanine, and serine are exemplary residues for this purpose. The synthetic peptide is synthetic in the sense that it does not occur in nature as a discrete peptide and has a sequence non-naturally linked to the hinge or a part thereof to which it is attached, although the synthetic peptide can be a mutant form of a part (especially the N-terminal part) of the hinge region as described in this example. The synthetic peptide is usually overexpressed in alanine, glycine, and / or serine (i.e., at least 25%, 35%, or 50% of all residues in the synthetic peptide are alanine, glycine, and / or serine).

[0057] The Cμ portion comprises Cμ3 and Cμ4 of the Cμ constant region. The Cμ portion is responsible for polymerizing multiple monovalent or divalent binding units into a multivalent complex. Although it is not necessary to understand the mechanism to practice the present invention, it is believed that the polymerization of the hybrid Fc fusion protein is similar to that in native IgM antibodies and is carried out through interchain disulfide bonds between the Cμ3 regions of different monomers and between μ tail pieces. Some multimers of IgM also contain one or more J chains that bind to the μ tail piece. In the presence of one or more J chains, IgM can form a pentameric structure, and in the absence of J chains, it can form a hexameric structure. It has been reported that hexameric IgM has stronger CDC than pentameric IgM. Although it is believed that the hybrid Fc protein of the present invention forms pentameric or hexameric complexes in the same manner as IgM, other multiplicities larger or smaller than or in place of the pentameric and hexameric forms can also be formed.

[0058] The above components are arranged in the following order from the N-terminus to the C-terminus: synthetic peptide (if present), at least a portion of the IgG hinge region, IgG CH2 region, IgG CH3 region, Cμ3 region, and Cμ4 region.

[0059] Typically, all IgG regions have the same isotype and subtype. That is, all IgG regions are from IgG1, IgG2, IgG3, or IgG4. Optionally, the IgG CH2 and CH3 regions of the hybrid Fc protein comprise different isotypes and subtypes.

[0060] Preferably, the IgG region is human IgG. Similarly, the Cμ3 and Cμ4 regions are preferably human. Figure 10A Exemplary sequences of the human IgG1, IgG2, IgG3, IgG4, IgM heavy chain constant regions are shown in A, B, C, in which the components (CH1, hinge, CH2, CH3, Cμ1, Cμ2, Cμ3, and Cμ4, and J chain) are differentiated. However, regions from other species can also be used, including non-human primates, camelids, cartilaginous fish, mice, or rats. Exemplary sequences of human IgG1 hybrid Fc proteins are SEQ ID NOs. 7, 11, 13, 15, and 16. Exemplary sequences of IgG2, IgG3, and IgG4 hybrid Fc proteins are SEQ ID NOs. 26 - 28, respectively.

[0061] Other components that are typically found in therapeutic proteins or fusion proteins may or may not be present, but are not required. For example, the hybrid Fc protein of the present invention does not need to include the IgG CH1 constant region (since there is no light chain to pair), a heavy or light chain variable region that forms a binding site that specifically binds to a target present in a human or other subject, or a heterologous polypeptide (such as a receptor ECD or ligand) that specifically binds to a target present in a human or other subject and that is typically found in Fc fusion proteins.

[0062] References to human IgG, IgA or IgM regions (i.e., CH1, hinge, CH2, CH3, Cμ3 and Cμ4) or the J chain, including references to the hybrid Fc proteins of the invention, are to exemplary sequences or their allotypes or their isoallotypes or other variant sequences that have at least 90, 95, 98 or 99% sequence identity with the exemplary sequence and / or differ from the exemplary sequence by at most 1, 2, 3, 4, 5, 10 or 15 amino acid deletions, substitutions or internal insertions in the case of CH1, CH2, CH3, Cμ3 and Cμ4 and the J chain and 1, 2 or 3 deletions, substitutions or internal insertions in the case of the IgG1, 2, 4 hinge regions and at most 1, 2, 3, 4, 5 or 6 deletions in the case of the IgG3 hinge. If there are substitutions, they are preferably conservative. Human constant regions exhibit allotypic and isoallotypic variation between different individuals, i.e., the constant regions of different individuals may differ at one or more polymorphic positions. The difference between allotypes and isoallotypes is that sera that recognize an isoallotype bind to non-polymorphic regions of one or more other isotypes. References to human constant regions include constant regions having any natural allotype (including isoallotype) or any permutation of residues that occupy polymorphic positions in natural allotypes. Sequences of non-human constant regions are provided by, for example, the Swiss-Prot or Genbank databases. References to non-human constant regions likewise include allotypic or isoallotypic variants, and their permutations, or other variant sequences that are the same as or different from the native sequence. The range of variants is defined by sequence identity and / or the number of substitutions relative to the native sequence of the non-human constant region, in a manner similar to the description of variants of human constant regions above. The Eu numbering convention is used to define corresponding positions between isotypes or different species, or to define mutant positions.

[0063] For various purposes, various substitutions can be made in the IgG or IgM Fc region or both. For example, there are many known mutations in IgG Fc that increase the binding to FcRn. Exemplary substitutions include Gln at position 250 and / or Leu at position 428, Ser or Asn at position 434, Tyr at position 252, Thr at position 254, and Glu at position 256 (EU numbering). The increased binding to FcRn is beneficial for the hybrid Fc protein of the present invention to more strongly compete with endogenous IgG for binding to FcRn. There are also many mutations known in IgG and IgM Fc that reduce any one of ADCC, ADP (antibody-dependent phagocytosis), or CDC. (See, for example, Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006). For example, substitution at any one of positions 234, 235, 236, and / or 237 reduces the affinity for Fcγ receptors (especially the FcγRI receptor) (see, for example, US 6,624,821). Optionally, positions 234, 236, and / or 237 in human IgG2 are substituted with alanine, and position 235 is substituted with glutamine or glutamate (see, for example, US 5,624,821). Other substitutions that reduce effector function include: Ala at position 268, Gly or Ala at position 297, Leu at position 309, Ala at position 322, Gly at position 327, Ser at position 330, Ser at position 331, Ser at position 238, Ala at position 268, Leu at position 309 (Eu numbering). Other substitutions in IgG or IgM Fc are advantageous in stimulating sialylation, which is useful for increasing the binding to CD22. For example, it is known that in the IgG Fc region, substitution of Phe at position 241 with Ala, substitution of Phe at position 243 with Ala, substitution of Val at position 262 with Glu, and substitution of Val at position 264 with Glu (EU numbering) all enhance the sialylation of IgG molecules (Yu et al, J. Am. Chem. Soc. 2013 135:9723-9732). Other IgG Fc mutants that enhance the sialylation of IgG molecules are reported in U.S. Patents 9187552, 9328170, and 9663581.

[0064] As further discussed below, with the possible exception of synthetic linkers that replace some or all of the hinge region and one or more amino acid substitutions that enhance or inhibit effector function or FcRn binding, the hybrid Fc proteins preferably do not contain other sequences than the hinge, CH2, CH3, Cμ3, and Cμ4 regions described above. As previously mentioned, the CH1 region or the heavy or light chain variable regions are not required. Nevertheless, other sequences, such as a hexahistidine tag, can be added, but this is not essential. Thus, preferred hybrid Fc proteins consist of, or consist essentially of: the full or partial hinge, CH2, CH3, Cμ3, and Cμ4 regions as described above, optionally, an additional peptide of up to 5, 10, 15, 20, or 25 residues, such as the synthetic peptides described above, and optionally a J chain. Some hybrid Fc proteins consist of, or consist essentially of: the full or partial hinge (optionally modified to remove one or more cysteine residues involved in light chain pairing), CH2, CH3, Cμ3, and Cμ4 regions as described above. Some hybrid Fc proteins consist of or consist essentially of: the fully human IgG hinge, CH2, CH3, and Cμ3 and Cμ4 regions, and optionally a J chain, with the possible exception of one or more mutated cysteine residues in the hinge region. Hybrid Fc proteins formed entirely or substantially of human sequences are poorly immunogenic in humans. Any other sequences present preferably do not increase the immunogenicity of the hybrid Fc protein in humans.

[0065] A standard immunoglobulin structure, which includes two heavy chains, has a maximum of four sialic acid residues (two per chain). Because of their multiplicity (e.g., hexamer) and because the Cμ3 and Cμ4 regions provide additional sialic acid attachment sites, the hybrid Fc proteins of the present invention can have more than two. For example, each protein molecule of the present hybrid Fc fusion can have an average of 2.1 or more sialic acids (e.g., 2.1 - 5). Thus, for a hexamer of the present hybrid Fc molecule, each hexamer can have an average of more than 12, 15, or 20 sialic acid molecules (e.g., 12.1 - 30). Higher sialylation favors immunosuppression by binding to CD22.

[0066] III. Genetic Engineering and Expression

[0067] Hybrid Fc proteins are produced by recombinant expression. A DNA fragment encoding the IgG Fc portion is fused in-frame with a DNA fragment encoding the Cμ portion to obtain a hybrid Fc protein. Preferably, the last amino acid of the CH3 exon of IgG is fused in-frame to the first amino acid of the Cμ3 exon.

[0068] The order of genetic element fusion in constructing constructs encoding several components is not important. The fragments can also be ligated simultaneously by ligating overlapping oligonucleotides encoding the individual fragments in an overlapping PCR-type reaction. In practice, once an expression vector encoding the hybrid constant region has been generated, the same vector can be used for inserting any heavy chain variable region or other binding region, with respect to the fusion protein (and sometimes the light chain variable region), without having to recreate the DNA fragment encoding the hybrid constant region.

[0069] Mammalian cells are a host for expressing nucleotide fragments encoding the hybrid Fc proteins of the invention (see Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987)). Many suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas, including Sp2 / 0 and NS0. Preferably, the cells are non-human. The cells used for antibody production may or may not endogenously express the J chain. If the endogenous J chain is not expressed or is expressed at insufficient levels, the host cells can be genetically modified to express the J chain (i.e., by introducing a construct encoding it). However, host cells that do not express the J chain can also be used. The choice of cells with or without the J chain affects the valency of the antibody or fusion protein produced (e.g., pentamers with the J chain and hexamers without the J chain). Preferably, the hybrid Fc protein is expressed from a monoclonal cell line.

[0070] The expression vectors for these cells can include expression control sequences such as origins of replication, promoters, enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription termination sequences. Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0071] The cells are transfected with a vector encoding the hybrid Fc protein to be expressed. The hybrid Fc protein is expressed, processed to remove the signal peptide, assembled, and secreted from the host cell. It is believed that multimerization and binding to the J chain occur at least mainly intracellularly, such that the hybrid Fc protein is secreted mainly as a multimer, particularly a multimer in which five or six units are bound through the Cμ portion of the hybrid constant region.

[0072] The hybrid Fc protein can be purified from cell culture supernatants by conventional antibody purification methods. The purification can include chromatographic steps using Protein A or Protein G as affinity reagents. Conventional antibody purification processes such as ion exchange, hydroxyapatite chromatography, or HPLC can also be used (see generally Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0073] IV. Methods of Treatment and Pharmaceutical Compositions

[0074] The hybrid Fc proteins of the invention can be used to treat a variety of conditions mediated by antibody or B cell function, particularly those previously treated with IVIG as shown in the background art. Such conditions include immune diseases, inflammatory diseases, rejection after organ transplantation, hematological diseases, dermatological diseases, or neuromuscular diseases. The designations of the conditions are not mutually exclusive. Thus, for example, an immune disease can also be an inflammatory disease. The hybrid Fc proteins can treat these conditions by reducing the half-life of endogenous IgG molecules in circulation, inhibiting the immune response of endogenous B cells, and decreasing the concentration of endogenous IgG molecules in circulation.

[0075] One class of immune diseases treatable by the hybrid Fc proteins of the invention is transplant rejection. When allogeneic cells or organs (e.g., skin, kidney, liver, heart, lung, pancreas, and bone marrow) are transplanted into a host (i.e., the donor and recipient are different individuals of the same species), the host's immune system is likely to mount an immune response against foreign antigens in the graft (graft-versus-host disease), leading to the destruction of the transplanted tissue. The hybrid Fc proteins of the invention can be particularly useful for blocking the immune response induced by alloantigens in the recipient.

[0076] A related use of the hybrid Fc proteins of the invention is to modulate the immune response associated with "graft-versus-host" disease (GVHD). GVHD is a potentially fatal disease that occurs when immunocompetent cells are transferred to an allogeneic recipient. In this situation, the donor's immunocompetent cells may attack tissues in the recipient. Skin, intestinal epithelium, and liver tissues are common targets and may be destroyed during GVHD. The disease causes particularly severe problems when immunological tissues are transplanted, such as in bone marrow transplantation. However, milder cases of GVHD have also been reported in other situations (including heart and liver transplantation).

[0077] Further situations requiring immunosuppression are the treatment of autoimmune diseases, such as idiopathic thrombocytopenic purpura, Kawasaki disease, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, systemic lupus erythematosus, multiple sclerosis, autoimmune neutropenia type 1 diabetes, Crohn's disease, ulcerative colitis, multiple sclerosis, stiff-person syndrome, rheumatoid arthritis, myasthenia gravis, and lupus erythematosus. Other treatable diseases include acute disseminated encephalomyelitis, acute motor axonal neuropathy, Addison's disease, painful adiposity, adult Still's disease, alopecia areata, ankylosing spondylitis, anti-glomerular basement membrane nephritis, anti-neutrophil cytoplasmic antibody-associated vasculitis, anti-N-methyl-D-aspartic acid receptor encephalitis, antiphospholipid syndrome, anti-synthetase syndrome, aplastic anemia, autoimmune angioedema, autoimmune encephalitis, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune polyendocrine syndrome type 2, autoimmune polyendocrine syndrome type 3, autoimmune progesterone dermatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura, autoimmune thyroiditis, autoimmune urticaria, autoimmune uveitis, Balo concentric sclerosis, Behçet's disease, Bickerstaff encephalitis, bullous pemphigoid, celiac disease, chronic fatigue syndrome, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, complex regional pain syndrome, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes, discoid lupus erythematosus, endometriosis, enteritis, enthesitis-related arthritis, eosinophilic esophagitis, eosinophilic fasciitis, epidermolysis bullosa, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, Felty syndrome, fibromyalgia, gastritis, pemphigoid gestationis, giant cell arteritis, Goodpasture syndrome, Graves' disease, Graves' ophthalmopathy, Hashimoto encephalopathy, Hashimoto thyroiditis, Henoch-Schönlein purpura, lymphadenitis suppurativa, idiopathic dilated cardiomyopathy, idiopathic inflammatory demyelinating diseases, IgA nephropathy, IgG4-related systemic diseases, inflammatory bowel disease (IBD), intermediate uveitis, interstitial cystitis, juvenile arthritis, Kawasaki disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneousconjunctivitis), linear IgA disease, lupus nephritis, Meniere's disease, microscopic colitis, microscopic polyangiitis, mixed connective tissue disease, Mooren's corneal ulcer, morphea, Mucha-Habermann disease, myasthenia gravis, myocarditis, myositis, neuromyelitis optica, neuromyotonia, opsoclonus-myoclonus syndrome, optic neuritis, Ord's thyroiditis, relapsing polychondritis, paraneoplastic cerebellar degeneration, Parry-Romberg syndrome, Parsonage-Turner syndrome, pediatric autoimmune neuropsychiatric disorder associated with Streptococcus (PANDAS), pemphigus vulgaris, pernicious anemia, pityriasis lichenoides et varioliformis acuta, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary immunodeficiency, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, rheumatoid vasculitis, sarcoidosis, Schnitzler syndrome, scleroderma, Sjögren's syndrome, stiff-person syndrome, subacute bacterial endocarditis, Susac syndrome, Sydenham's chorea, sympathetic ophthalmia, systemic scleroderma, thrombocytopenia, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, urticaria, urticarial vasculitis, vasculitis, and vitiligo.

[0078] In any of these diseases, the body mounts a humoral immune response against one of its own antigens, resulting in the destruction of cells expressing that antigen and potentially causing disability and / or death. Autoimmune diseases can be treated by administering a hybrid Fc protein.

[0079] Other immune diseases treatable by the hybrid Fc proteins of the invention include asthma, allergy, celiac disease, psoriasis, and uveitis. Celiac disease, psoriasis, and uveitis are autoimmune diseases.

[0080] The hybrid Fc protein is administered according to an effective regime, meaning a dose, route of administration, and frequency of administration that delays onset, reduces severity, inhibits further deterioration, and / or improves at least one symptom or condition of a disease. If the subject already has a disease, the regime may be referred to as a therapeutically effective regime. If the subject is at high risk for the disease relative to the general population but has not yet manifested symptoms, the regime may be referred to as a prophylactically effective regime. In some cases, therapeutic or prophylactic efficacy may be observed in an individual subject relative to that subject's historical control or past experience. In other cases, therapeutic or prophylactic efficacy may be demonstrated in a group of treated subjects relative to a control group of untreated subjects in preclinical or clinical trials.

[0081] Exemplary doses of the hybrid Fc protein are 0.01 - 20 or 0.5 - 5, or 0.01 - 1, or 0.01 - 0.5 or 0.05 - 0.5 mg / kg body weight (e.g., 0.1, 0.5, 1, 2, 3, 4, or 5 mg / kg) or as a fixed dose of 10 - 1500 mg. The dose depends on the patient's condition and response to previous treatment (if any), whether the treatment is prophylactic or therapeutic, and whether the disease is acute or chronic, among other factors.

[0082] Administration may be parenteral, intravenous, oral, subcutaneous, intra - arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Administration into the systemic circulation is preferably by intravenous or subcutaneous routes. Intravenous administration may be by infusion, for example, over a duration of 30 - 90 minutes.

[0083] The frequency of administration depends on the half - life of the hybrid Fc protein in the circulation, the subject's condition, the route of administration, and other factors. The frequency may be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the subject's condition or the progression of the disease being treated. In a continuous course of treatment, an exemplary frequency of intravenous administration is between weekly and quarterly, although higher or lower frequencies may also be possible. For subcutaneous administration, exemplary frequencies are from daily to monthly, although higher or lower frequencies may also be possible.

[0084] The number of doses administered depends on whether the disease is acute or chronic and also on the response of the disease to treatment. For an acute disease or an acute exacerbation of a chronic disease, between 1 and 10 doses are usually sufficient. Sometimes, a single bolus dose (optionally in divided form) is sufficient to address an acute disease or an acute exacerbation of a chronic disease. For a recurrence of an acute disease or acute exacerbation, treatment may be repeated. For a chronic disease, the hybrid Fc protein of the invention may be administered regularly, e.g., weekly, bi - weekly, monthly, quarterly, every six months for at least 1, 5, or 10 years, or for the lifetime of the subject.

[0085] The pharmaceutical composition is preferably suitable for parenteral administration to humans. Such a composition is preferably sterile, substantially isotonic and manufactured under GMP conditions. The pharmaceutical composition can be provided in unit dosage form (i.e., the dose for a single administration). One or more pharmaceutically acceptable carriers, diluents, excipients or adjuvants can be used to formulate the pharmaceutical composition. Pharmaceutically acceptable means suitable for parenteral administration to humans are, for example, approved by the FDA. The formulation depends on the chosen route of administration. For injection, the hybrid Fc protein of the present invention can be formulated in an aqueous solution, preferably in a physiologically compatible buffer (such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer) (to reduce discomfort at the injection site). The solution can contain formulatory agents such as suspending agents, stabilizers and / or dispersing agents. Alternatively, the hybrid Fc protein of the present invention can be in lyophilized form for reconstitution prior to use with a suitable carrier (such as sterile pyrogen-free water).

[0086] Treatment with the hybrid Fc protein of the present invention can be combined with other treatments effective for the disease being treated. For the treatment of immune diseases, conventional treatment methods include mast cell degranulation inhibitors, corticosteroids, non-steroidal anti-inflammatory drugs, and stronger anti-inflammatory drugs such as azathioprine, cyclophosphamide, leukeran, FK506 and cyclosporine. Biological anti-inflammatory drugs can also be used, such as (natalizumab) or (adalimumab).

[0087] For all purposes, all patent documents, websites, other publications, accession numbers, etc., cited above or below are hereby incorporated by reference in their entirety to the same extent as if each individual item was specifically and individually indicated to be incorporated by reference. If different versions of a sequence are associated with an accession number at different times, it means the version associated with the accession number on the effective filing date of this application. The effective filing date means the earlier of the actual filing date or the filing date of the priority application (if any) that mentions the accession number. Similarly, if different versions of a publication, website, etc. are published at different times, unless otherwise stated, it means the version published most recently on the effective filing date of this application. Unless otherwise expressly stated, any feature, step, element, embodiment, or aspect of the present invention can be used in combination with any other. Although the present invention has been described in detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be made within the scope of the appended claims.

[0088] Examples

[0089] Example 1: General Methods and Materials

[0090] Recombinant DNA manipulations, as well as the expression, purification, and characterization of recombinant proteins, are performed using standard laboratory techniques (such as those described in Green and Sambrook (Molecular Cloning, A Laboratory Manual, Fourth Edition, 2012, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), Greenfield (Antibodies, A Laboratory Manual, Second Edition, 2014, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), Kostelny et al. (Int. J. Cancer 93:556 - 565, 2001), Cole et al. (J. Immunol. 159:3613 - 3621, 1997), and Tsurushita et al. (Methods 36:69 - 83, 2005)).

[0091] The mammalian expression vector pVF101( Figure 1 ) is designed to produce a multimeric hybrid Fc protein that contains, from the N - terminus to the C - terminus, an artificial signal peptide (sp), a hinge, the CH2 and CH3 regions of the human IgG1 isotype, and then the human Cμ3 and Cμ4 regions, and contains the following genetic components. From Figure 1Starting from the SalI site of pVF101 in the clockwise direction, the plasmid contains the human cytomegalovirus (CMV) major immediate early promoter and enhancer (CMV-P in the figure) to initiate the transcription of the region encoding LS41A-Fc (defined below). After the CMV promoter is an exon (SEQ ID NO: 1) encoding a signal peptide fused to a hinge region (hinge; SEQ ID NO: 2), an exon encoding CH2 (SEQ ID NO: 3), an exon encoding CH3 fused to Cμ3 (SEQ ID NO: 5) and Cμ4 (SEQ ID NO: 6) (SEQ ID NO: 4), and a polyadenylation site with intervening introns. The hinge (H in the figure), CH2 and CH3 regions, and the polyadenylation site are all from the human γ-1 heavy chain gene. After the transcription unit of the hybrid Fc protein is the SV40 early promoter (SV40-P), the puromycin N-acetyltransferase gene (puro) for resistance to puromycin, and a fragment containing the SV40 polyadenylation site (SV40-A). Finally, pVF101 contains a portion of plasmid pUC19, including the bacterial origin of replication (pUCori) and the β-lactamase gene (β-lactamase). The arrows in the figure indicate the direction of transcription. The amino acid sequence (LS41A-Fc) of the mature hybrid Fc protein encoded in pVF101, which consists of the hinge, CH2, CH3, Cμ3, and Cμ4 regions, is as follows:

[0092] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 7). The schematic structure of the hexameric form of the disulfide-linked LS41A-Fc dimer is shown as Figure 2 shown.

[0093] As described below, the mammalian expression vector pVF101 was modified in the coding region of LS41A-Fc to generate a new expression vector pVF102. The first five amino acid residues in the hinge region of the mature LS41A-Fc sequence were replaced with the artificial pentapeptide EPKSS (SEQ ID NO: 8) in pVF102. The leucine residues at positions 234 and 235 in CH2 (Eu numbering of Kabat et al., Sequences of Proteins of Immunological Interests, 5th edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services, 1991) were changed to alanine residues (L234A / L235A) (SEQ ID NO: 9) to eliminate the potential effector functions associated with IgG molecules (Xu et al. 2000 Cell. Immunol. 200:16-26; Hezareh et al. 2001 J. Virol. 75:12161-12168). The proline residue at position 433 and another proline residue at position 435 (Eu numbering) in Cμ3 were changed to alanine and serine residues, respectively (P433A / P435S) (SEQ ID NO: 10) to eliminate the potential CDC activity associated with IgM molecules (Arya et al., 1994 J. Immunol. 152:1206-1212). No other changes were introduced into pVF101 to generate pVF102. The amino acid sequence of the mature hybrid Fc protein (LS41K-Fc.S) encoded in pVF102, which consists of the artificial pentapeptide, a part of the hinge, CH2, CH3, Cμ3, and Cμ4, is as follows:

[0094] EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY(SEQ ID NO:11).

[0095] The mammalian expression vector pVF102 was modified by replacing the methionine residue at position 428 in CH3 (Eu numbering) (SEQ ID NO: 12) with a leucine residue to produce a new expression vector pVF103. No other changes were introduced into pVF102 to produce pVF103. The amino acid sequence of the mature hybrid Fc protein (LS41K-Fc.SL) encoded in pVF103, which consists of an artificial pentapeptide, a part of the hinge, CH2, CH3, Cμ3 and Cμ4, is as follows:

[0096] EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY(SEQ ID NO:13).

[0097] The concentrations of LS41A-Fc, LS41K-Fc.S and LS41K-Fc.SL in the culture supernatants or mouse sera were measured by sandwich ELISA. In a typical experiment, an ELISA plate was coated overnight at 4 °C with 100 μl / well of a 1 / 2,000 dilution of goat anti-human IgG Fcγ chain-specific antibody (Jackson ImmunoResearch, West Grove, PA) in PBS (phosphate-buffered saline, pH 7.4), washed with Wash Buffer (PBS containing 0.05% Tween 20), and blocked for 1 h at room temperature with 200 μl / well of ELISA buffer (PBS containing 2% non-fat dry milk and 0.05% Tween 20). After washing with the Wash Buffer, the test samples appropriately diluted in the ELISA buffer were loaded onto the ELISA plate (100 μl / well). Purified LS41A-Fc, LS41K-Fc.S or LS41K-Fc.SL was used as a standard. After incubating the ELISA plate for 1 h at room temperature and washing with the Wash Buffer, the bound Fc proteins were detected using 100 μl / well of a 1 / 2,000 dilution of HRP-conjugated goat anti-human γ chain antibody (SouthernBiotech, Birmingham, AL) in the ELISA buffer. After incubating for 30 min at room temperature and washing with the Wash Buffer, color development was initiated with 100 μl / well of ABTS substrate and terminated with 100 μl / well of 2% oxalic acid. Absorbance was read at 405 nm.

[0098] As described above, the concentration of the mouse monoclonal anti-human CD122 IgG1 / κ antibody ABC2 (U.S. Patent 9,028,830) in mouse sera was measured by sandwich ELISA, except that (1) the ELISA plate was coated with the extracellular region of human CD122 fused with six histidine residues (CD122-His; SEQ ID NO: 14) produced in JN Biosciences; (2) HRP-conjugated goat anti-mouse κ chain antibody (Bethyl Laboratories, Montgomery, TX) was used to detect the bound ABC2, and (3) ABC2 was used as a standard.

[0099] As described above, the concentration of mouse IgG in mouse sera was measured by sandwich ELISA, except that (1) it was coated with goat anti-mouse IgG Fcγ chain-specific antibody (Jackson ImmunoResearch), (2) HRP-conjugated goat anti-mouse κ chain antibody (Bethyl Laboratories) was used for detection, and (3) ABC2 was used as a standard.

[0100] Example 2: Expression and Purification of Multimeric Hybrid Fc Proteins

[0101] The expression vectors pVF102 and pVF103 were introduced into the chromosomes of the Chinese hamster ovary cell line CHO-K1, respectively, to obtain cell lines stably producing LS41K-Fc.S and LS41K-Fc.SL. The CHO-K1 cells were grown in SFM4CHO medium (GE Healthcare, Chicago, IL) at 37 °C in a 7.5% CO2 incubator. They were stably transfected into CHO-K1 by electroporation. Before transfection, each expression vector was linearized using FspI. In a typical experiment, approximately 10 7 cells were transfected with 20 μg of linearized plasmid, suspended in SFM4CHO medium, and seeded into several 96-well plates after appropriate dilution of the cells. After 48 hours, puromycin was added to isolate stable transfectants. Approximately twelve days after the selection began, the culture supernatants of the transfectants were assayed to detect antibody production.

[0102] As described above, the expression of LS41K-Fc.S and LS41K-Fc.SL was measured by sandwich ELISA. The previously purified LS41K-Fc.S or LS41K-Fc.SL was used as a standard. The CHO-K1 stable transfectants producing each of LS41K-Fc.S and LS41K-Fc.SL were amplified in SFM4CHO until the cell viability became less than 50%. After centrifugation and filtration, the culture supernatant was loaded onto a Protein A column (HiTrap MABSelect SuRe, GE Healthcare). The column was washed with PBS and then the hybrid Fc protein was eluted with 0.1 M glycine-HCl (pH 3.0) containing 0.1 M NaCl. The buffer of the eluted hybrid Fc protein was neutralized with 1 M Tris-HCl (pH 8.0) and then changed to PBS by dialysis. The concentration of the hybrid Fc protein was determined by measuring the absorbance at 280 nm (1 mg / ml = 1 OD).

[0103] The molecular sizes of native - form purified LS41K - Fc.S and LS41K - Fc.SL were analyzed by gel filtration using an AKTA Basic FPLC system with a Superose 6 10 / 300GL column (GE Healthcare). PBS was used as the running buffer. A single dominant peak was observed for each of the purified LS41K - Fc.S and LS41K - Fc.SL. By comparing the elution profile with molecular size markers, the sizes of native - form LS41K - Fc.S and LS41K - Fc.SL were estimated to be approximately 600 kDa, which is consistent with the size of the hexamer of the disulfide - linked hybrid Fc dimer of the present invention, containing the hinge, CH2, CH3, Cμ3, and Cμ4 regions.

[0104] Example 3: Analysis of the Pharmacokinetics (PK) and Pharmacodynamics (PD) of LS41K - Fc.S in Mice

[0105] In the presence and absence of 400 μg of LS41K - Fc.S in 50 μL of PBS (Groups A and B, respectively), fifty (50) μg of the murine monoclonal anti - human CD122 IgG1 antibody ABC2 was administered intracardially to three Balb / c mice in each group. Serum samples were collected from these mice one day before dosing (Day - 1), two hours after dosing (2HR), one day (Day 1), three days (Day 3), five days (Day 5), and eight days (Day 8) after dosing.

[0106] As described above, the concentration of ABC2 in the serum samples was measured by ELISA. The concentration of ABC2 at each time point (Day 1, Day 3, Day 5, and Day 8) was normalized to the concentration in the 2HR sample for each mouse. The data were plotted in Figure 3 . The average relative concentration of ABC2 in Group A (ABC2 only) was 100% (2HR), 52.0% (Day 1), 36.7% (Day 3), 29.9% (Day 5), and 19.3% (Day 8). In contrast, the average percentage concentration of ABC2 in Group B (ABC2 and LS41K - Fc.S) was 100% (2HR), 39.1% (Day 1), 20.1% (Day 3), 14.9% (Day 5), and 8.8% (Day 8). Compared with the administration of ABC2 alone, the administration of ABC2 and LS41K - Fc.S could reduce the concentration of ABC2 in the serum of mice more rapidly. This may be due to the high affinity of LS41K - Fc.S for binding to FcRn, resulting in enhanced catabolism of ABC2 in lysosomes.

[0107] As described above, the concentration of murine IgG in serum samples was measured by ELISA. The murine IgG concentration at each time point (2HR, day 1, day 3, day 5, and day 8) was normalized to the concentration in the sample from each mouse on day -1. The data were plotted in Figure 4 . In group A (ABC2 only), the mean relative concentration of murine IgG remained essentially unchanged. The mean concentrations of murine IgG were 100% (day -1), 92.8% (2HR), 86.2% (day 1), 90.7% (day 3), 96.6% (day 5), and 96.8% (day 8). In contrast, the mean relative IgG concentration in group B (ABC2 and LS41K-Fc.S) dropped to two-thirds of that on day -1 on day 3 and recovered to 80% level on day 8. The mean relative IgG concentrations were 100% (day -1), 88.4% (2HR), 72.1% (day 1), 67.6% (day 3), 74.8% (day 5), and 80.4% (day 8). These results indicate that LS41K-Fc.S effectively competes with murine IgG for binding to FcRn and prevents murine IgG from recycling into the circulation.

[0108] As described above, the concentration of LS41K-Fc.S in serum samples was measured by ELISA. The data were plotted in Figure 5 . The mean concentration of LS41K-Fc.S was 230 μg / ml (2HR), 144 μg / ml (day 1), 76.4 μg / ml (day 3), 50.0 μg / ml (day 5), and 5.1 μg / ml (day 8). The sudden drop in concentration on day 8 may be due to the human-derived immune response against LS41K-Fc.S in mice. The half-life of LS41K-Fc.S in the murine circulation calculated using samples from day 1, day 3, and day 5 was 62 hours.

[0109] Example 4: PK and PD Analysis of LS41K-Fc.SL in Mice

[0110] It is known that a leucine residue (M428L) in the γ heavy chain of human IgG replaces the methionine residue at position 428 (Eu numbering), thereby increasing the serum half-life of this modified IgG antibody (Hinton et al., J. Biol. Chem. 279:6213 - 6219, 2004; Hinton et al., J. Immunol. 176:346 - 356, 2006). To increase the half-life in circulation, a variant of LS41K-Fc.S called LS41K-Fc.SL was generated, in which the methionine residue at position 428 in CH3 was replaced by a leucine residue.

[0111] A mixture of 50 μg of ABC2 and 400 μg of LS41K-Fc.SL in 50 μg of PBS was administered intracardially to three Balb / c mice (Group C). Serum samples were collected from these mice on the day before administration (Day -1), two hours after administration (2HR), one day (Day 1), three days (Day 3), five days (Day 5), and eight days (Day 8). This experiment was conducted together with Group A (ABC2 only) and Group B (ABC2 and LS41K-Fc.S) described above.

[0112] As described above, the concentration of LS41K-Fc.SL in the serum samples was measured by ELISA. The average concentration of LS41K-Fc.SL was 279 μg / ml (2HR), 86.0 μg / ml (Day 1), 20.1 μg / ml (Day 3), and 7.2 μg / ml (Day 5). The data were plotted in Figure 5 . The concentration in the Day 8 sample was below the detection limit. The LS41K-Fc.SL concentration on Day 5 in Group C was 2.6% of the 2HR concentration, while the LS41K-Fc.SL concentration on Day 5 in Group B was 21.7% of the 2HR concentration. The half-life of LS41K-Fc.SL in the circulation calculated using Day 1, Day 3, and Day 5 was 27 hours. Despite the M428L mutation in the Fc region, LS41K-Fc.SL was cleared from the circulation faster than LS41K-Fc.S.

[0113] As described above, the concentration of ABC2 in the serum samples was measured by ELISA. The ABC2 concentration at each time point (Day 1, Day 3, Day 5, and Day 8) was normalized to the concentration in the 2HR sample for each mouse. The data were plotted in Figure 3 . The average relative concentration of ABC2 in Group C (ABC2 and LS41K-Fc.SL) was 37.3% (Day 1), 12.7% (Day 3), 10.2% (Day 5), and 8.0% (Day 8). On Day 3 and Day 5, LS41K-Fc.SL decreased the concentration of ABC2 in the serum samples more drastically than LS41K-Fc.S.

[0114] As described above, the concentration of mouse IgG in the serum samples was measured by ELISA. The mouse IgG concentration at each time point (2HR, Day 1, Day 3, Day 5, and Day 8) was normalized to the concentration in the Day -1 sample for each mouse. The data were plotted in Figure 4 . The average relative concentration of mouse IgG was 87.2% (2HR), 68.6% (Day 1), 55.3% (Day 3), 67.1% (Day 5), and 118.8% (Day 8). The mouse IgG concentration on Day 3 of LS41K-Fc.SL in Group C reached nearly half of the pre-dose concentration (Day -1).

[0115] Compared with the parental LS41K-Fc.S, the presence of the M428L mutation in LS41K-Fc.SL resulted in a decrease in serum half-life rather than the expected increase. In addition, LS41K-Fc.SL was more effective than LS41K-Fc.S in reducing the concentrations of ABC2 and murine IgG in circulation.

[0116] Example 5: Dose-dependence of LS41K-Fc.SL

[0117] Fifty (50) μg of the murine monoclonal IgG1 antibody ABC2 was administered intracardially to three Balb / c mice per group together with 100 μg (Group D) or 400 μg (Group E) of LS41K-Fc.SL. Serum samples were collected from these mice at two hours (2HR), one day (Day 1), three days (Day 3), five days (Day 5), and eight days (Day 8) after administration. The concentrations of ABC2, murine IgG, and LS41K-Fc.SL in the serum samples were measured by ELISA as described above.

[0118] The ABC2 concentration at each time point (Day 1, Day 3, Day 5, and Day 8) was normalized to the concentration in the 2HR sample for each mouse. The data was plotted in Figure 6 . The mean relative concentration of ABC2 with 100 μg LS41K-Fc.SL (Group D) was 100% at 2HR, 34.0% at Day 1, 19.6% at Day 3, 16.4% at Day 5, and 12.3% at Day 8. The mean concentration of ABC2 with 400 μg LS41K-Fc.SL (Group E) was 100% at 2HR, 27.2% at Day 1, 10.7% at Day 3, 8.0% at Day 5, and 5.5% at Day 8. The decrease in the ABC2 concentration in the serum samples was dependent on the dose of LS41K-Fc.SL. Administration of 400 μg of LS41K-Fc.SL (Group E) reduced the concentration of ABC2 in circulation compared to the administration of 100 μg of LS41K-Fc.SL (Group D).

[0119] As described above, the concentration of murine IgG in the serum samples was measured by ELISA. The murine IgG concentration at each time point (Day 1, Day 3, Day 5, and Day 8) was normalized to the concentration in the 2HR sample for each mouse. The data was plotted in Figure 7In the D group, the average relative mouse IgG concentration was 100% at 2HR, 83.6% on day 1, 73.6% on day 3, 76.5% on day 5, and 92.8% on day 8. In the E group, the average relative mouse IgG concentration was 100% at 2HR, 74.8% on day 1, 61.4% on day 3, 63.6% on day 5, and 88.3% on day 8. In both the D and E groups, the mouse IgG levels decreased on days 1 and 3 and gradually increased starting on day 5. Compared with the D group (100 μg of LS41K-Fc.SL), the mouse IgG concentration in the E group (400 μg of LS41K-Fc.SL) decreased significantly on days 1, 3, and 5.

[0120] Figure 8 The concentration of LS41K-Fc.SL in the serum samples is shown. The average LS41K-Fc.SL in the D group was 72.8 μg / ml at 2HR, 21.9 μg / ml on day 1, 5.3 μg / ml on day 3, and 2.1 μg / ml on day 5. The average LS41K-Fc.SL concentration in the E group was 311.3 μg / ml at 2HR, 97.4 μg / ml on day 1, 23.8 μg / ml on day 3, and 9.2 μg / ml on day 5. In both dose groups, the LS41K-Fc.SL concentration on day 8 was below the detection limit. For the 100 μg and 400 μg dosing groups, the average serum half-life of LS41K-Fc.SL calculated using the data on days 1, 3, and 5 was 28 hours.

[0121] Example 6: Fc Mutations to Enhance Sialylation

[0122] It has been shown that substitution of the phenylalanine residue at each of positions 241 and 243 (Eu numbering) in the IgG Fc region (F241A and F243A, respectively) with an alanine residue enhanced the sialylation of N-linked glycans in IgG molecules and Fc proteins (Yu et al., J. Am. Chem. Soc. 135:9723-9732, 2013; Ahmed et al., J. Mol. Biol. 426:3166–3179; Fiebiger et al. Proc. Natl. Acad. Sci. 112:E2385–E2394, 2015; Mimura et al., J. Immunol. Methods 428:30-36, 2016).

[0123] A variant of LS41K-Fc.SL was generated by replacing the phenylalanine residue at position 241 (Eu numbering) in the CH2 of the human γ-1 chain in pVF103 with an alanine residue to generate pVF104. The amino acid sequence of the mature hybrid Fc protein encoded in pVF104 (LS41K-Fc.SL.F241A), which consists of an artificial pentapeptide, a part of the hinge, CH2, CH3, Cμ3, and Cμ4, is as follows:

[0124] EPKSSDKTHTCPPCPAPEAAGGPSVALFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY(SEQ ID NO:15).

[0125] Another variant of LS41K-Fc.SL was generated by replacing the phenylalanine residue at position 243 in the CH2 of the human γ-1 chain in pVF103 with an alanine residue. The amino acid sequence of the mature hybrid Fc protein encoded in pVF103 (LS41K-Fc.SL), which consists of an artificial pentapeptide, a part of the hinge, CH2, CH3, Cμ3, and Cμ4, is as follows: The amino acid sequence of the mature hybrid Fc protein encoded in pVF105 (LS41K-Fc.SL.F243A), which consists of an artificial pentapeptide, a part of the hinge, CH2, CH3, Cμ3, and Cμ4, is as follows:

[0126] EPKSSDKTHTCPPCPAPEAAGGPSVFLAPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY(SEQ ID NO:16).

[0127] Example 7: Expression of β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) and β-1,4-galactosyltransferase 1 (B4GALT1)

[0128] Although IgG molecules expressed in mammalian cells are weakly sialylated (Wang et al., Biotech. Bioeng. 2018 115:1378-1393; Friedman et al., 1988 Cancer Lett. 43:79), sialylated IVIG has been reported to be associated with anti-inflammatory activity (Anthony et al., J. Clin. Immunol. 30:9-14, 2010; Seite et al., Arthritis Rheum. 67:595-603, 2015). Terminal sialic acid can be linked to galactose via α-2,3-, α-2,6-, or α-2,8-bonds. CD22 and DC-SIGN bind only to α-2,6-linked sialic acid (Powell et al., J. Biol. Chem. 13:7523-7532, 1995; Anthony et al., Proc. Natl. Acad. Sci. 105:9571-19578, 2008). Two β-galactoside α-2,6-sialyltransferases (ST6GAL1 and ST6GAL2) are responsible for attaching α-2,6-linked sialic acid to the terminal galactose of N-linked carbohydrates in mammalian cells.

[0129] Raymond et al. (mAbs 7:571-583, 2015) reported that the expression of recombinant ST6GAL1 in CHO-K1 cells increased the level of α-2,6-sialylated recombinant human IgG1 antibody.

[0130] Co-expression of recombinant B4GALT1 (β-1,4-galactosyltransferase 1) and recombinant ST6GAL1 in CHO-K1 cells further increased the α-2,6 sialylation level of IgG molecules. This co-expression catalyzes the addition of galactose molecules to the terminal N-acetylglucosamine of N-linked glycans, and recombinant ST6GAL1 in CHO-K1 cells further increases the α-2,6 sialylation level of IgG molecules.

[0131] Genes encoding human ST6GAL1 and B4GALT1 were synthesized as SpeI-EagI fragments at Synbio Technologies (Monmouth Junction, NJ). The expression vector of human ST6GAL1 (SEQ ID NO: 17) (pFCm512; Figure 9A) has the same as pVF101 ( Figure 1)The same structure, except that (i) the SpeI-EagI fragment encoding LS41A-Fc was replaced with a synthetic gene encoding human ST6GAL1 and (ii) the puromycin N-acetyltransferase gene (puro) was replaced with the blasticidin S deaminase gene (Bsr in the figure). Another expression vector for human B4GALT1 (SEQ ID NO: 18) (pFCm513; Figure 9B) has the same structure as pVF101, except that (i) the SpeI-EagI fragment encoding LS41A-Fc was replaced with a synthetic gene encoding human B4GALT1, and (ii) the puromycin N-acetyltransferase gene (puro) was replaced with the bleomycin resistance gene from Streptoalloteichus hindustanus (Zeo in the figure).

[0132] Example 8: Involvement of CD22

[0133] Each of the expression vectors pVF103, pVF104, and pVF105 was transiently transfected into HEK293 cells by the polyethyleneimine method (Durocher et al. Nucl. Acids Res. 30: e9, 2002) to express LS41K-Fc.SL, LS41K-Fc.SL.F241A, and LS41K-Fc.SL.F243A, respectively. Each of these three expression vectors was also co-transfected with (i) pFCm512 or (ii) pFCm512 and pFCm513 into HEK293 cells.

[0134] The binding of the thus-expressed LS41K-Fc.SL, LS41K-Fc.SL.F241A, and LS41K-Fc.SL.F243A to CD22 was analyzed in human Burkitt lymphoma cell lines Ramos and Raji by flow cytometry. The activity of LS41K-Fc.SL, LS41K-Fc.SL.F241A, and LS41K-Fc.SL.F243A in reducing the viability of Ramos cells through CD22 crosslinking was analyzed by the method described by Seite et al. (Blood 116: 1698-1704, 2010).

[0135] Example 9: PK and PD Analysis of LS41K-Fc.SL, LS41K-Fc.SL.F241A, and LS41K-Fc.SL.F243A in Non-Human Primates

[0136] A group of three cynomolgus monkeys was intravenously administered the humanized anti-CD122 IgG1 antibody HuABC2 (U.S. Patent 9,028,830) at a dose of 5 mg / kg. Another group of three cynomolgus monkeys was also intravenously administered HuABC2 and 20 mg / kg of LS41K-Fc.SL, LS41K-Fc.SL.F241A, or LS41K-Fc.SL.F243A. Serum samples were collected from these monkeys on the day before administration (Day -1), two hours after administration (2HR), one day (Day 1), four days (Day 4), seven days (Day 7), ten days (Day 10), and fourteen days (Day 14).

[0137] As described above, the concentration of HuABC2 in the serum samples was determined by ELISA, except that (1) the fusion of the extracellular region of human CD122 and six histidine residues produced by JNBiosciences (CD122-His; SEQ ID NO: 14) was used for coating the ELISA plate, (2) an HRP-conjugated goat anti-human κ chain antibody was used to detect the bound antibody, and (3) HuABC2 was used as a standard to demonstrate the ability of LS41K-Fc.SL, LS41K-Fc.SL.F241A, and LS41K-Fc.SL.F243A to rapidly clear HuABC2 from the circulation of macaques.

[0138] As described above, the concentration of total cynomolgus monkey IgG in the serum samples was measured by ELISA, except that (1) a goat anti-cynomolgus monkey IgG Fcγ chain-specific antibody was used for coating, (2) an HPR-conjugated goat anti-cynomolgus monkey κ chain antibody was used to detect the bound antibody, and (3) cynomolgus monkey IgG was used as a standard to demonstrate the ability of LS41K-Fc.SL, LS41K-Fc.SL.F241A, and LS41K-Fc.SL.F243A to reduce the IgG concentration in the circulation of cynomolgus monkeys.

[0139] B cells were isolated from cynomolgus monkeys administered LS41K-Fc.SL, LS41K-Fc.SL.F241A, or LS41K-Fc.SL.F243A. To demonstrate the ability of LS41K-Fc.SL, LS41K-Fc.SL.F241A, and LS41K-Fc.SL.F243A to inhibit the immune response, the activity of B cells in response to an antigen (such as endotoxin) was monitored by analyzing the production of anti-endotoxin antibodies. Additionally, the B cell immune response generated by analyzing surface CD40 binding was analyzed by flow cytometry to measure the expression level of CD95 on the surface.

[0140] Example 10: Sialylation level of LS41K-Fc.SL.F243A

[0141] As described above, CHO-K1 cells were stably transfected with pFCm512 expressing human ST6GAL1 by electroporation. Stable transfectants of CHO-K1 expressing ST6GAL1 (CHO-K1 / ST6GAL1) were isolated by selection in the presence of blasticidin and then used for stable transfection with pVF105 expressing LS41K-Fc.SL.F243A. As described above, puromycin-resistant CHO-K1 / ST6GAL1 cells expressing LS41K-Fc.SL.F243A were amplified in SFM4CHO medium. As described above, LS41K-Fc.SL.F243A was purified using a protein A affinity column. The purified LS41K-Fc.SL.F243A showed a single major peak of the expected size (about 600 kDa) in a gel filtration analysis performed using a Superose 6 column.

[0142] The sialylation of LS41K-Fc.SL.F243A purified from CHO-K1 / ST6GAL1 cells was analyzed using an EnzyChrom Sialic Acid Assay Kit (BioAssay Systems, Hayward, CA). Humanized IgG1 monoclonal antibody (trastuzumab) was used as a reference for this assay. The average number of sialic acids attached to each LS41K-Fc.SL.F243A molecule was determined to be 21.3. The average number of sialic acids attached to each Herceptin was 0.14.

[0143] Example 11: Binding to FcRn

[0144] To express human FcRn on the cell surface, a new vector pFCm239 was constructed. Vector pFCm239 has the same structure as pVF101 ( Figure 1 ), except that (1) the Spe-EagI fragment was replaced with a DNA fragment encoding, from the N-terminus to the C-terminus: the signal peptide and extracellular region of human FcRn (SEQ ID NO: 19), the polypeptide linker Thr-Gly-Gly-Gly, the FLAG polypeptide (SEQ ID NO: 20), the polypeptide linker Gly-Gly-Gly, and the GPI-anchoring signal of human CD55 (SEQ ID NO: 21) (hFcRn-FLAG-GPI; SEQ ID NO: 22) and (2) the puromycin N-acetyltransferase gene (puro) was replaced with the Escherichia coli xanthine-guanine phosphoribosyltransferase to select transfectants in the presence of mycophenolic acid.

[0145] Expression vector pFCm240 has the same structure as pVF101, except that the Spe-EagI fragment is replaced by a DNA fragment (SEQ ID NO: 23) encoding the entire human β2-microglobulin.

[0146] The murine myeloma cell line NS0 was maintained in DME medium containing 10% fetal bovine serum (FBS). NS0 cells were stably transfected with pFCm239 by electroporation (Bebbington et al. Bio / Technology 10:169-175, 1992) and selected in DME medium containing 10% FBS, 1 μg / ml mycophenolic acid, HT medium supplement (Sigma-Aldrich, St. Louis, MO), and 0.25 mg / ml xanthine. Then, the surface expression of hFcRn-FLAG-GPI was detected by flow cytometry using the rat anti-FLAG peptide antibody L5 (BioLegend, San Diego, CA) and the phycoerythrin-labeled goat anti-rat IgG antibody (SouthernBiotech, Birmingham, AL). NS0 cells expressing FcRn-FLAG-GPI were further stably transfected with pFCm240 by electroporation. The puromycin-resistant NS0 transfectants were tested for the expression of human β2-microglobulin by flow cytometry using the mouse anti-human β2-microglobulin antibody 2M2 (BioLegend) and the phycoerythrin-labeled goat anti-mouse IgG antibody (SouthernBiotech). The NS0 transfected cell line expressing hFcRn-FLAG-GPI and human β2-microglobulin was designated NS0 / hFcRn. Human IgG antibody binds to NS0 / hFcRn at pH 6.0.

[0147] Expression vector pFCm380 has the same structure as pVF101( Figure 1)Identical structure, except that the Spe-EagI fragment is replaced by a DNA fragment encoding, from the N-terminus to the C-terminus: the signal peptide and extracellular region of murine FcRn (SEQ ID NO: 24), the polypeptide linker Thr-Gly-Gly-Gly, the FLAG polypeptide (SEQ ID NO: 20), the polypeptide linker Gly-Gly-Gly, and the GPI-anchoring signal of human CD55 (SEQ ID NO: 21) (mFcRn-FLAG-GPI; SEQ ID NO: 25). NS0 cells were stably transfected with pFCm380 by electroporation. Expression of mFcRn-FLAG-GPI on the surface of puromycin-resistant NS0 cells was detected by flow cytometry using the rat anti-FLAG peptide antibody L5 and the phycoerythrin-conjugated goat anti-rat IgG antibody. The NS0 transfected cell line expressing mFcRn-FLAG-GPI associated with endogenous murine β2-microglobulin on the surface was designated NS0 / mFcRn. Murine IgG antibodies bind to NS0 / mFcRn cells at pH 6.0.

[0148] The binding of Erbitux (cetuximab; murine-human chimeric anti-EGFR IgG1 antibody) and LS41K-Fc.SL to NS0 / hFcRn cells was tested in PBS containing 0.5% BSA and 0.05% sodium azide at pH 7.5 (FACS Buffer (pH 7.5)) and 6.0 (FACS Buffer (pH 6.0)). For FcRn binding, Erbitux and LS41K-SL were incubated with approximately 100,000 NS0 / hFcRn at 2,000 ng / ml, 400 ng / ml, and 80 ng / ml in 200 μl of FACS buffer (pH 7.5) or FACS buffer (pH 6.0) for 30 minutes at room temperature. After washing, the NS0 / hFcRn cells were incubated with 1 μg / ml of phycoerythrin-conjugated donkey anti-human IgG F(ab’)2 antibody (Bethyl Laboratories, Montgomery, TX) for 30 minutes at room temperature in FACS buffer at the same pH as in the initial binding step. The cells were then washed and resuspended in FACS buffer at the same pH as in the initial binding step, and then flow cytometry was performed.

[0149] Cetuximab showed binding to human FcRn in a dose-dependent manner at pH 6.0. Compared with the binding to FcRn at pH 6.0, the binding of cetuximab to human FcRn was severely reduced at pH 7.5 (Figure 11A). This is consistent with the reported observation that the binding of human IgG antibodies to human FcRn is pH-dependent; there is strong binding at pH 6.0 and little binding at pH 7.5 (Hinton et al. 2006 J. Immunol. 176:346–356). In contrast, the hybrid Fc fusion protein (LS41K-Fc.SL) of the present invention bound to human FcRn in a dose-dependent manner at both pH 6.0 and 7.5 (Figure 11B). No major difference in the binding of LS41K-Fc.SL to human FcRn was observed between pH 6.0 and pH 7.5.

[0150] As described above, the binding of cetuximab and LS41K-Fc.SL to murine FcRn was also tested using NS0 / mFcRn cells at pH 6.0 and pH 7.5. Cetuximab showed dose-dependent binding to murine FcRn at pH 6.0, while its binding to murine FcRn was hardly detectable at pH 7.5 (Figure 11C). LS41K-Fc.SL bound to murine FcRn in a dose-dependent manner at both pH 6.0 and pH 7.5 (Figure 11D). No major difference in the binding of LS41K-Fc.SL to murine FcRn was observed between pH 6.0 and pH 7.5.

[0151] Example 12: Hybrid Fc Proteins Comprising Human IgG2, IgG3, and IgG4 Fc Regions

[0152] The coding sequences of the hinge, CH2, and CH3 regions encoding LS41K-Fc.SL in pVF103 were respectively replaced with the coding sequences of the hinge, CH2, and CH3 regions of human IgG2 to construct pVF103-G2. The amino acid sequence of the human IgG2-based hybrid Fc protein encoded in pVF103-G2 is:

[0153] ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTWVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFCVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY(SEQ ID NO: 26).

[0154] The coding sequences of the hinge, CH2, and CH3 regions in pVF103 were replaced with the coding sequences of the last repeated hinge, CH2, and CH3 regions of human IgG3, respectively, to construct pVF103-G3. The amino acid sequence of the human IgG3-based hybrid Fc protein encoded in pVF103-G3 is:

[0155] EPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCPVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY(SEQ ID NO: 27).

[0156] The coding sequences of the hinge, CH2, and CH3 regions in pVF103 were replaced with the coding sequences of the hinge, CH2, and CH3 regions of human IgG4, respectively, to construct pVF103-G4. The amino acid sequence of the human IgG4-based hybrid Fc protein encoded by pVF103-G4 is as follows:

[0157] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVRVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEDNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGKDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY(SEQ ID NO: 28).

[0158] Sequence Listing

[0159] SEQ ID NO: 1

[0160] Amino acid sequence of the signal peptide for LS41A-Fc encoded in pVF101

[0161] MGWSWIFFFLLSGTASVLS

[0162] SEQ ID NO: 2

[0163] Amino acid sequence of the human γ-1 heavy chain hinge region encoded in pVF101

[0164] EPKSCDKTHTCPPCP

[0165] SEQ ID NO: 3

[0166] Amino acid sequence of the human γ-1 heavy chain CH2 region encoded in pVF101

[0167] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0168] SEQ ID NO:4

[0169] Amino acid sequence of the human γ-1 heavy chain CH3 region encoded in pVF101

[0170] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0171] SEQ ID NO:5

[0172] Amino acid sequence of the human μ heavy chain Cμ3 region encoded in pVF101

[0173] DQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPK

[0174] SEQ ID NO:6

[0175] Amino acid sequence of the human μ heavy chain Cμ4 region encoded in pVF101

[0176] GVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0177] SEQ ID NO:7

[0178] Amino acid sequence of the mature LS41A-Fc protein encoded in pVF101

[0179] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0180] SEQ ID NO:8

[0181] Pentapeptide amino acid sequence fused to a portion of the hinge region encoded in pVF102

[0182] EPKSS

[0183] SEQ ID NO:9

[0184] Amino acid sequence of the modified CH2 region of the human γ-1 heavy chain encoded in pVF102

[0185] APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0186] SEQ ID NO:10

[0187] Amino acid sequence of the human μ heavy chain Cμ3 region encoded in pVF102

[0188] DQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTISRPK

[0189] SEQ ID NO:11

[0190] Amino acid sequence of the mature LS41K-Fc.S protein encoded in pVF102

[0191] EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0192] SEQ ID NO:12

[0193] Amino acid sequence of the modified CH3 region of the human γ-1 heavy chain encoded in VF103

[0194] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0195] SEQ ID NO:13

[0196] Amino acid sequence of the mature LS41K-Fc.SL protein encoded in pVF103

[0197] EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0198] SEQ ID NO:14

[0199] Amino acid sequence of the mature form of the extracellular region of human CD122 fused with six histidine residues at the C-terminus (CD122-His)

[0200] SAAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTTGGGAHHHHHH

[0201] SEQ ID NO:15

[0202] Amino acid sequence of the mature LS41K-Fc.SL.F241A protein encoded in pVF104

[0203] EPKSSDKTHTCPPCPAPEAAGGPSVALFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0204] SEQ ID NO:16

[0205] The amino acid sequence of the mature LS41K-Fc.SL.F243A protein encoded in pVF105

[0206] EPKSSDKTHTCPPCPAPEAAGGPSVFLAPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0207] SEQ ID NO:17

[0208] Amino acid sequence of human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) encoded in pFCm512

[0209] MIHTNLKKKFSCCVLVFLLFAVICVWKEKKKGSYYDSFKLQTKEFQVLKSLGKLAMGSDSQSVSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDSSSKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC

[0210] SEQ ID NO:18

[0211] Amino acid sequence of human β-1,4-galactosyltransferase 1 (B4GALT1) encoded in pFCm513

[0212] MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS

[0213] SEQ ID NO:19

[0214] Amino acid sequence of human FcRn signal peptide and extracellular region

[0215] MGVPRPQPWALGLLLFLLPGSLGAESHLSLLYHLTAVSSPAPGTPAFWVSGWLGPQQYLSYNSLRGEAEPCGAWVWENQVSWYWEKETTDLRIKEKLFLEAFKALGGKGPYTLQGLLGCELGPDNTSVPTAKFALNGEEFMNFDLKQGTWGGDWPEALAISQRWQQQDKAANKELTFLLFSCPHRLREHLERGRGNLEWKEPPSMRLKARPSSPGFSVLTCSAFSFYPPELQLRFLRNGLAAGTGQGDFGPNSDGSFHASSSLTVKSGDEHHYCCIVQHAGLAQPLRVELESPAKSS

[0216] SEQ ID NO:20

[0217] Amino acid sequence of FLAG peptide

[0218] DYKDDDDK

[0219] SEQ ID NO:21

[0220] Amino acid sequence of GPI-anchoring signal of human CD55

[0221] PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

[0222] SEQ ID NO:22

[0223] Amino acid sequence of hFcRn-FLAG-GPI encoded in pFCm239

[0224] MGVPRPQPWALGLLLFLLPGSLGAESHLSLLYHLTAVSSPAPGTPAFWVSGWLGPQQYLSYNSLRGEAEPCGAWVWENQVSWYWEKETTDLRIKEKLFLEAFKALGGKGPYTLQGLLGCELGPDNTSVPTAKFALNGEEFMNFDLKQGTWGGDWPEALAISQRWQQQDKAANKELTFLLFSCPHRLREHLERGRGNLEWKEPPSMRLKARPSSPGFSVLTCSAFSFYPPELQLRFLRNGLAAGTGQGDFGPNSDGSFHASSSLTVKSGDEHHYCCIVQHAGLAQPLRVELESPAKSSTGGGDYKDDDDKGGGPNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

[0225] SEQ ID NO:23

[0226] Amino acid sequence of human β2-microglobulin

[0227] MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

[0228] SEQ ID NO:24

[0229] Amino acid sequence of mouse FcRn signal peptide and extracellular region

[0230] MGMPLPWALSLLLVLLPQTWGSETRPPLMYHLTAVSNPSTGLPSFWATGWLGPQQYLTYNSLRQEADPCGAWMWENQVSWYWEKETTDLKSKEQLFLEALKTLEKILNGTYTLQGLLGCELASDNSSVPTAVFALNGEEFMKFNPRIGNWTGEWPETEIVANLWMKQPDAARKESEFLLNSCPERLLGHLERGRRNLEWKEPPSMRLKARPGNSGSSVLTCAAFSFYPPELKFRFLRNGLASGSGNCSTGPNGDGSFHAWSLLEVKRGDEHHYQCQVEHEGLAQPLTVDLDSSARSS

[0231] SEQ ID NO:25

[0232] Amino acid sequence of mFcRn-FLAG-GPI encoded in pFCm380

[0233] MGMPLPWALSLLLVLLPQTWGSETRPPLMYHLTAVSNPSTGLPSFWATGWLGPQQYLTYNSLRQEADPCGAWMWENQVSWYWEKETTDLKSKEQLFLEALKTLEKILNGTYTLQGLLGCELASDNSSVPTAVFALNGEEFMKFNPRIGNWTGEWPETEIVANLWMKQPDAARKESEFLLNSCPERLLGHLERGRRNLEWKEPPSMRLKARPGNSGSSVLTCAAFSFYPPELKFRFLRNGLASGSGNCSTGPNGDGSFHAWSLLEVKRGDEHHYQCQVEHEGLAQPLTVDLDSSARSSTGGGDYKDDDDKGGGPNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

[0234] SEQ ID NO:26

[0235] Amino acid sequence of hybrid Fc protein based on human IgG2

[0236] ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTWVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFCVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0237] SEQ ID NO:27

[0238] Amino acid sequence of a human IgG3-based hybrid Fc protein

[0239] EPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCPVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0240] Amino acid sequence of the hybrid Fc protein based on human IgG4, SEQ ID NO:28

[0241] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVRVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEDNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGKDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY Sequence Listing <110> JN Biosciences Co., Ltd. Naoya Tsurashita J. Yun Cao <120> Multimeric Hybrid Fc Proteins for Replacement of IVIG <130> 057761-538890 <150> US 62 / 767,303 <151> 2018-11-14 <160> 55 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 1 Met Gly Trp Ser Trp Ile Phe Phe Phe Leu Leu Ser Gly Thr Ala Ser 1 5 10 15 Val Leu Ser <210> 2 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 2 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 3 <211> 110 <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 100 105 110 <210> 4 <211> 107 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic <400> 4 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 5 <211> 106 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic <400> 5 Asp Gln Asp Thr Ala Ile Arg Val Phe Ala Ile Pro Pro Ser Phe Ala 1 5 10 15 Ser Ile Phe Leu Thr Lys Ser Thr Lys Leu Thr Cys Leu Val Thr Asp 20 25 30 Leu Thr Thr Tyr Asp Ser Val Thr Ile Ser Trp Thr Arg Gln Asn Gly 35 40 45 Glu Ala Val Lys Thr His Thr Asn Ile Ser Glu Ser His Pro Asn Ala 50 55 60 Thr Phe Ser Ala Val Gly Glu Ala Ser Ile Cys Glu Asp Asp Trp Asn 65 70 75 80 Ser Gly Glu Arg Phe Thr Cys Thr Val Thr His Thr Asp Leu Pro Ser 85 90 95 Pro Leu Lys Gln Thr Ile Ser Arg Pro Lys 100 105 <210> 6 <211> 131 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 6 Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro Ala Arg 1 5 10 15 Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu Val Thr 20 25 30 Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg Gly Gln 35 40 45 Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro Glu Pro 50 55 60 Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val Ser Glu 65 70 75 80 Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala His Glu 85 90 95 Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly 100 105 110 Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr Ala Gly 115 120 125 Thr Cys Tyr 130 <210> 7 <211> 469 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 7 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 Lys Asp Gln Asp Thr Ala Ile Arg Val 225 230 235 240 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 245 250 255 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 260 265 270 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 275 280 285 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 290 295 300 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 305 310 315 320 Val Thr His Thr Asp Leu Pro Ser Pro Leu Lys Gln Thr Ile Ser Arg 325 330 335 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 340 345 350 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 355 360 365 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 370 375 380 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 385 390 395 400 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 405 410 415 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 420 425 430 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 435 440 445 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 450 455 460 Ala Gly Thr Cys Tyr 465 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 8 Glu Pro Lys Ser Ser 1 5 <210> 9 <211> 110 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 9 Ala Pro Glu Ala Ala 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 100 105 110 <210> 10 <211> 106 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 10 Asp Gln Asp Thr Ala Ile Arg Val Phe Ala Ile Pro Pro Ser Phe Ala 1 5 10 15 Ser Ile Phe Leu Thr Lys Ser Thr Lys Leu Thr Cys Leu Val Thr Asp 20 25 30 Leu Thr Thr Tyr Asp Ser Val Thr Ile Ser Trp Thr Arg Gln Asn Gly 35 40 45 Glu Ala Val Lys Thr His Thr Asn Ile Ser Glu Ser His Pro Asn Ala 50 55 60 Thr Phe Ser Ala Val Gly Glu Ala Ser Ile Cys Glu Asp Asp Trp Asn 65 70 75 80 Ser Gly Glu Arg Phe Thr Cys Thr Val Thr His Thr Asp Leu Ala Ser 85 90 95 Ser Leu Lys Gln Thr Ile Ser Arg Pro Lys 100 105 <210> 11 <211> 469 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 11 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Ala Ala 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 Lys Asp Gln Asp Thr Ala Ile Arg Val 225 230 235 240 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 245 250 255 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 260 265 270 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 275 280 285 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 290 295 300 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 305 310 315 320 Val Thr His Thr Asp Leu Ala Ser Ser Leu Lys Gln Thr Ile Ser Arg 325 330 335 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 340 345 350 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 355 360 365 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 370 375 380 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 385 390 395 400 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 405 410 415 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 420 425 430 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 435 440 445 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 450 455 460 Ala Gly Thr Cys Tyr 465 <210> 12 <211> 107 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic <400> 12 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 13 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 13 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Ala Ala 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 Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys Asp Gln Asp Thr Ala Ile Arg Val 225 230 235 240 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 245 250 255 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 260 265 270 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 275 280 285 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 290 295 300 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 305 310 315 320 Val Thr His Thr Asp Leu Ala Ser Ser Leu Lys Gln Thr Ile Ser Arg 325 330 335 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 340 345 350 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 355 360 365 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 370 375 380 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 385 390 395 400 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 405 410 415 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 420 425 430 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 435 440 445 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 450 455 460 Ala Gly Thr Cys Tyr 465 <210> 14 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 14 Ser Ala Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser 1 5 10 15 Arg Ala Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp 20 25 30 Thr Ser Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln 35 40 45 Thr Cys Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu 50 55 60 Ile Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val 65 70 75 80 Thr Leu Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala 85 90 95 Ile Gln Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile 100 105 110 Ser Leu Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp 115 120 125 Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu 130 135 140 Ala Arg Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu 145 150 155 160 Thr Leu Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro 165 170 175 Asp Thr Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu 180 185 190 Phe Thr Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys 195 200 205 Pro Ala Ala Leu Gly Lys Asp Thr Thr Gly Gly Gly Ala His His His 210 215 220 His His His 225 <210> 15 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 15 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Ala Ala Gly Gly Pro Ser Val Ala 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 Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys Asp Gln Asp Thr Ala Ile Arg Val 225 230 235 240 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 245 250 255 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 260 265 270 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 275 280 285 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 290 295 300 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 305 310 315 320 Val Thr His Thr Asp Leu Ala Ser Ser Leu Lys Gln Thr Ile Ser Arg 325 330 335 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 340 345 350 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 355 360 365 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 370 375 380 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 385 390 395 400 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 405 410 415 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 420 425 430 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 435 440 445 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 450 455 460 Ala Gly Thr Cys Tyr 465 <210> 16 <211> 469 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 16 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Ala 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 Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys Asp Gln Asp Thr Ala Ile Arg Val 225 230 235 240 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 245 250 255 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 260 265 270 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 275 280 285 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 290 295 300 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 305 310 315 320 Val Thr His Thr Asp Leu Ala Ser Ser Leu Lys Gln Thr Ile Ser Arg 325 330 335 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 340 345 350 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 355 360 365 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 370 375 380 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 385 390 395 400 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 405 410 415 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 420 425 430 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 435 440 445 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 450 455 460 Ala Gly Thr Cys Tyr 465 <210> 17 <211> 406 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic <400> 17 Met Ile His Thr Asn Leu Lys Lys Lys Phe Ser Cys Cys Val Leu Val 1 5 10 15 Phe Leu Leu Phe Ala Val Ile Cys Val Trp Lys Glu Lys Lys Lys Gly 20 25 30 Ser Tyr Tyr Asp Ser Phe Lys Leu Gln Thr Lys Glu Phe Gln Val Leu 35 40 45 Lys Ser Leu Gly Lys Leu Ala Met Gly Ser Asp Ser Gln Ser Val Ser 50 55 60 Ser Ser Ser Thr Gln Asp Pro His Arg Gly Arg Gln Thr Leu Gly Ser 65 70 75 80 Leu Arg Gly Leu Ala Lys Ala Lys Pro Glu Ala Ser Phe Gln Val Trp 85 90 95 Asn Lys Asp Ser Ser Ser Lys Asn Leu Ile Pro Arg Leu Gln Lys Ile 100 105 110 Trp Lys Asn Tyr Leu Ser Met Asn Lys Tyr Lys Val Ser Tyr Lys Gly 115 120 125 Pro Gly Pro Gly Ile Lys Phe Ser Ala Glu Ala Leu Arg Cys His Leu 130 135 140 Arg Asp His Val Asn Val Ser Met Val Glu Val Thr Asp Phe Pro Phe 145 150 155 160 Asn Thr Ser Glu Trp Glu Gly Tyr Leu Pro Lys Glu Ser Ile Arg Thr 165 170 175 Lys Ala Gly Pro Trp Gly Arg Cys Ala Val Val Ser Ser Ala Gly Ser 180 185 190 Leu Lys Ser Ser Gln Leu Gly Arg Glu Ile Asp Asp His Asp Ala Val 195 200 205 Leu Arg Phe Asn Gly Ala Pro Thr Ala Asn Phe Gln Gln Asp Val Gly 210 215 220 Thr Lys Thr Thr Ile Arg Leu Met Asn Ser Gln Leu Val Thr Thr Glu 225 230 235 240 Lys Arg Phe Leu Lys Asp Ser Leu Tyr Asn Glu Gly Ile Leu Ile Val 245 250 255 Trp Asp Pro Ser Val Tyr His Ser Asp Ile Pro Lys Trp Tyr Gln Asn 260 265 270 Pro Asp Tyr Asn Phe Phe Asn Asn Tyr Lys Thr Tyr Arg Lys Leu His 275 280 285 Pro Asn Gln Pro Phe Tyr Ile Leu Lys Pro Gln Met Pro Trp Glu Leu 290 295 300 Trp Asp Ile Leu Gln Glu Ile Ser Pro Glu Glu Ile Gln Pro Asn Pro 305 310 315 320 Pro Ser Ser Gly Met Leu Gly Ile Ile Ile Met Met Thr Leu Cys Asp 325 330 335 Gln Val Asp Ile Tyr Glu Phe Leu Pro Ser Lys Arg Lys Thr Asp Val 340 345 350 Cys Tyr Tyr Tyr Gln Lys Phe Phe Asp Ser Ala Cys Thr Met Gly Ala 355 360 365 Tyr His Pro Leu Leu Tyr Glu Lys Asn Leu Val Lys His Leu Asn Gln 370 375 380 Gly Thr Asp Glu Asp Ile Tyr Leu Leu Gly Lys Ala Thr Leu Pro Gly 385 390 395 400 Phe Arg Thr Ile His Cys 405 <210> 18 <211> 398 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 18 Met Arg Leu Arg Glu Pro Leu Leu Ser Gly Ser Ala Ala Met Pro Gly 1 5 10 15 Ala Ser Leu Gln Arg Ala Cys Arg Leu Leu Val Ala Val Cys Ala Leu 20 25 30 His Leu Gly Val Thr Leu Val Tyr Tyr Leu Ala Gly Arg Asp Leu Ser 35 40 45 Arg Leu Pro Gln Leu Val Gly Val Ser Thr Pro Leu Gln Gly Gly Ser 50 55 60 Asn Ser Ala Ala Ala Ile Gly Gln Ser Ser Gly Glu Leu Arg Thr Gly 65 70 75 80 Gly Ala Arg Pro Pro Pro Pro Leu Gly Ala Ser Ser Gln Pro Arg Pro 85 90 95 Gly Gly Asp Ser Ser Pro Val Val Asp Ser Gly Pro Gly Pro Ala Ser 100 105 110 Asn Leu Thr Ser Val Pro Val Pro His Thr Thr Ala Leu Ser Leu Pro 115 120 125 Ala Cys Pro Glu Glu Ser Pro Leu Leu Val Gly Pro Met Leu Ile Glu 130 135 140 Phe Asn Met Pro Val Asp Leu Glu Leu Val Ala Lys Gln Asn Pro Asn 145 150 155 160 Val Lys Met Gly Gly Arg Tyr Ala Pro Arg Asp Cys Val Ser Pro His 165 170 175 Lys Val Ala Ile Ile Ile Pro Phe Arg Asn Arg Gln Glu His Leu Lys 180 185 190 Tyr Trp Leu Tyr Tyr Leu His Pro Val Leu Gln Arg Gln Gln Leu Asp 195 200 205 Tyr Gly Ile Tyr Val Ile Asn Gln Ala Gly Asp Thr Ile Phe Asn Arg 210 215 220 Ala Lys Leu Leu Asn Val Gly Phe Gln Glu Ala Leu Lys Asp Tyr Asp 225 230 235 240 Tyr Thr Cys Phe Val Phe Ser Asp Val Asp Leu Ile Pro Met Asn Asp 245 250 255 His Asn Ala Tyr Arg Cys Phe Ser Gln Pro Arg His Ile Ser Val Ala 260 265 270 Met Asp Lys Phe Gly Phe Ser Leu Pro Tyr Val Gln Tyr Phe Gly Gly 275 280 285 Val Ser Ala Leu Ser Lys Gln Gln Phe Leu Thr Ile Asn Gly Phe Pro 290 295 300 Asn Asn Tyr Trp Gly Trp Gly Gly Glu Asp Asp Asp Ile Phe Asn Arg 305 310 315 320 Leu Val Phe Arg Gly Met Ser Ile Ser Arg Pro Asn Ala Val Val Gly 325 330 335 Arg Cys Arg Met Ile Arg His Ser Arg Asp Lys Lys Asn Glu Pro Asn 340 345 350 Pro Gln Arg Phe Asp Arg Ile Ala His Thr Lys Glu Thr Met Leu Ser 355 360 365 Asp Gly Leu Asn Ser Leu Thr Tyr Gln Val Leu Asp Val Gln Arg Tyr 370 375 380 Pro Leu Tyr Thr Gln Ile Thr Val Asp Ile Gly Thr Pro Ser 385 390 395 <210> 19 <211> 297 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 19 Met Gly Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe 1 5 10 15 Leu Leu Pro Gly Ser Leu Gly Ala Glu Ser His Leu Ser Leu Leu Tyr 20 25 30 His Leu Thr Ala Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp 35 40 45 Val Ser Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asn Ser Leu 50 55 60 Arg Gly Glu Ala Glu Pro Cys Gly Ala Trp Val Trp Glu Asn Gln Val 65 70 75 80 Ser Trp Tyr Trp Glu Lys Glu Thr Thr Asp Leu Arg Ile Lys Glu Lys 85 90 95 Leu Phe Leu Glu Ala Phe Lys Ala Leu Gly Gly Lys Gly Pro Tyr Thr 100 105 110 Leu Gln Gly Leu Leu Gly Cys Glu Leu Gly Pro Asp Asn Thr Ser Val 115 120 125 Pro Thr Ala Lys Phe Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp 130 135 140 Leu Lys Gln Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile 145 150 155 160 Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr 165 170 175 Phe Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu His Leu Glu Arg 180 185 190 Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys 195 200 205 Ala Arg Pro Ser Ser Pro Gly Phe Ser Val Leu Thr Cys Ser Ala Phe 210 215 220 Ser Phe Tyr Pro Pro Glu Leu Gln Leu Arg Phe Leu Arg Asn Gly Leu 225 230 235 240 Ala Ala Gly Thr Gly Gln Gly Asp Phe Gly Pro Asn Ser Asp Gly Ser 245 250 255 Phe His Ala Ser Ser Ser Leu Thr Val Lys Ser Gly Asp Glu His His 260 265 270 Tyr Cys Cys Ile Val Gln His Ala Gly Leu Ala Gln Pro Leu Arg Val 275 280 285 Glu Leu Glu Ser Pro Ala Lys Ser Ser 290 295 <210> 20 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 20 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 21 <211> 37 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 21 Pro Asn Lys Gly Ser Gly Thr Thr Ser Gly Thr Thr Arg Leu Leu Ser 1 5 10 15 Gly His Thr Cys Phe Thr Leu Thr Gly Leu Leu Gly Thr Leu Val Thr 20 25 30 Met Gly Leu Leu Thr 35 <210> 22 <211> 349 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 22 Met Gly Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe 1 5 10 15 Leu Leu Pro Gly Ser Leu Gly Ala Glu Ser His Leu Ser Leu Leu Tyr 20 25 30 His Leu Thr Ala Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp 35 40 45 Val Ser Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asn Ser Leu 50 55 60 Arg Gly Glu Ala Glu Pro Cys Gly Ala Trp Val Trp Glu Asn Gln Val 65 70 75 80 Ser Trp Tyr Trp Glu Lys Glu Thr Thr Asp Leu Arg Ile Lys Glu Lys 85 90 95 Leu Phe Leu Glu Ala Phe Lys Ala Leu Gly Gly Lys Gly Pro Tyr Thr 100 105 110 Leu Gln Gly Leu Leu Gly Cys Glu Leu Gly Pro Asp Asn Thr Ser Val 115 120 125 Pro Thr Ala Lys Phe Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp 130 135 140 Leu Lys Gln Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile 145 150 155 160 Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr 165 170 175 Phe Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu His Leu Glu Arg 180 185 190 Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys 195 200 205 Ala Arg Pro Ser Ser Pro Gly Phe Ser Val Leu Thr Cys Ser Ala Phe 210 215 220 Ser Phe Tyr Pro Pro Glu Leu Gln Leu Arg Phe Leu Arg Asn Gly Leu 225 230 235 240 Ala Ala Gly Thr Gly Gln Gly Asp Phe Gly Pro Asn Ser Asp Gly Ser 245 250 255 Phe His Ala Ser Ser Ser Leu Thr Val Lys Ser Gly Asp Glu His His 260 265 270 Tyr Cys Cys Ile Val Gln His Ala Gly Leu Ala Gln Pro Leu Arg Val 275 280 285 Glu Leu Glu Ser Pro Ala Lys Ser Ser Thr Gly Gly Gly Asp Tyr Lys 290 295 300 Asp Asp Asp Asp Lys Gly Gly Gly Pro Asn Lys Gly Ser Gly Thr Thr 305 310 315 320 Ser Gly Thr Thr Arg Leu Leu Ser Gly His Thr Cys Phe Thr Leu Thr 325 330 335 Gly Leu Leu Gly Thr Leu Val Thr Met Gly Leu Leu Thr 340 345 <210> 23 <211> 119 <212> PRT <213> Homo sapiens <400> 23 Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser 1 5 10 15 Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg 20 25 30 His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser 35 40 45 Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu 50 55 60 Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp 65 70 75 80 Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp 85 90 95 Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile 100 105 110 Val Lys Trp Asp Arg Asp Met 115 <210> 24 <211> 297 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 24 Met Gly Met Pro Leu Pro Trp Ala Leu Ser Leu Leu Leu Val Leu Leu 1 5 10 15 Pro Gln Thr Trp Gly Ser Glu Thr Arg Pro Pro Leu Met Tyr His Leu 20 25 30 Thr Ala Val Ser Asn Pro Ser Thr Gly Leu Pro Ser Phe Trp Ala Thr 35 40 45 Gly Trp Leu Gly Pro Gln Gln Tyr Leu Thr Tyr Asn Ser Leu Arg Gln 50 55 60 Glu Ala Asp Pro Cys Gly Ala Trp Met Trp Glu Asn Gln Val Ser Trp 65 70 75 80 Tyr Trp Glu Lys Glu Thr Thr Asp Leu Lys Ser Lys Glu Gln Leu Phe 85 90 95 Leu Glu Ala Leu Lys Thr Leu Glu Lys Ile Leu Asn Gly Thr Tyr Thr 100 105 110 Leu Gln Gly Leu Leu Gly Cys Glu Leu Ala Ser Asp Asn Ser Ser Val 115 120 125 Pro Thr Ala Val Phe Ala Leu Asn Gly Glu Glu Phe Met Lys Phe Asn 130 135 140 Pro Arg Ile Gly Asn Trp Thr Gly Glu Trp Pro Glu Thr Glu Ile Val 145 150 155 160 Ala Asn Leu Trp Met Lys Gln Pro Asp Ala Ala Arg Lys Glu Ser Glu 165 170 175 Phe Leu Leu Asn Ser Cys Pro Glu Arg Leu Leu Gly His Leu Glu Arg 180 185 190 Gly Arg Arg Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys 195 200 205 Ala Arg Pro Gly Asn Ser Gly Ser Ser Val Leu Thr Cys Ala Ala Phe 210 215 220 Ser Phe Tyr Pro Pro Glu Leu Lys Phe Arg Phe Leu Arg Asn Gly Leu 225 230 235 240 Ala Ser Gly Ser Gly Asn Cys Ser Thr Gly Pro Asn Gly Asp Gly Ser 245 250 255 Phe His Ala Trp Ser Leu Leu Glu Val Lys Arg Gly Asp Glu His His 260 265 270 Tyr Gln Cys Gln Val Glu His Glu Gly Leu Ala Gln Pro Leu Thr Val 275 280 285 Asp Leu Asp Ser Ser Ala Arg Ser Ser 290 295 <210> 25 <211> 349 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 25 Met Gly Met Pro Leu Pro Trp Ala Leu Ser Leu Leu Leu Val Leu Leu 1 5 10 15 Pro Gln Thr Trp Gly Ser Glu Thr Arg Pro Pro Leu Met Tyr His Leu 20 25 30 Thr Ala Val Ser Asn Pro Ser Thr Gly Leu Pro Ser Phe Trp Ala Thr 35 40 45 Gly Trp Leu Gly Pro Gln Gln Tyr Leu Thr Tyr Asn Ser Leu Arg Gln 50 55 60 Glu Ala Asp Pro Cys Gly Ala Trp Met Trp Glu Asn Gln Val Ser Trp 65 70 75 80 Tyr Trp Glu Lys Glu Thr Thr Asp Leu Lys Ser Lys Glu Gln Leu Phe 85 90 95 Leu Glu Ala Leu Lys Thr Leu Glu Lys Ile Leu Asn Gly Thr Tyr Thr 100 105 110 Leu Gln Gly Leu Leu Gly Cys Glu Leu Ala Ser Asp Asn Ser Ser Val 115 120 125 Pro Thr Ala Val Phe Ala Leu Asn Gly Glu Glu Phe Met Lys Phe Asn 130 135 140 Pro Arg Ile Gly Asn Trp Thr Gly Glu Trp Pro Glu Thr Glu Ile Val 145 150 155 160 Ala Asn Leu Trp Met Lys Gln Pro Asp Ala Ala Arg Lys Glu Ser Glu 165 170 175 Phe Leu Leu Asn Ser Cys Pro Glu Arg Leu Leu Gly His Leu Glu Arg 180 185 190 Gly Arg Arg Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys 195 200 205 Ala Arg Pro Gly Asn Ser Gly Ser Ser Val Leu Thr Cys Ala Ala Phe 210 215 220 Ser Phe Tyr Pro Pro Glu Leu Lys Phe Arg Phe Leu Arg Asn Gly Leu 225 230 235 240 Ala Ser Gly Ser Gly Asn Cys Ser Thr Gly Pro Asn Gly Asp Gly Ser 245 250 255 Phe His Ala Trp Ser Leu Leu Glu Val Lys Arg Gly Asp Glu His His 260 265 270 Tyr Gln Cys Gln Val Glu His Glu Gly Leu Ala Gln Pro Leu Thr Val 275 280 285 Asp Leu Asp Ser Ser Ala Arg Ser Ser Thr Gly Gly Gly Asp Tyr Lys 290 295 300 Asp Asp Asp Asp Lys Gly Gly Gly Pro Asn Lys Gly Ser Gly Thr Thr 305 310 315 320 Ser Gly Thr Thr Arg Leu Leu Ser Gly His Thr Cys Phe Thr Leu Thr 325 330 335 Gly Leu Leu Gly Thr Leu Val Thr Met Gly Leu Leu Thr 340 345 <210> 26 <211> 465 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 26 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val 1 5 10 15 Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu Val Thr Trp Val Val Val Asp Val Ser 35 40 45 His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 65 70 75 80 Phe Cys Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro 100 105 110 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220 Ser Pro Gly Lys Asp Gln Asp Thr Ala Ile Arg Val Phe Ala Ile Pro 225 230 235 240 Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr Lys Leu Thr Cys 245 250 255 Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr Ile Ser Trp Thr 260 265 270 Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn Ile Ser Glu Ser 275 280 285 His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala Ser Ile Cys Glu 290 295 300 Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr Val Thr His Thr 305 310 315 320 Asp Leu Ala Ser Ser Leu Lys Gln Thr Ile Ser Arg Pro Lys Gly Val 325 330 335 Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln 340 345 350 Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu Val Thr Gly Phe 355 360 365 Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg Gly Gln Pro Leu 370 375 380 Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gln Ala 385 390 395 400 Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val Ser Glu Glu Glu 405 410 415 Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala His Glu Ala Leu 420 425 430 Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro 435 440 445 Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys 450 455 460 Tyr 465 <210> 27 <211> 469 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 27 Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 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 Gln Phe Lys 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 Phe 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 Thr Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Pro Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Asn Thr Thr Pro Pro Met 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 Ile Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys Asp Gln Asp Thr Ala Ile Arg Val 225 230 235 240 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 245 250 255 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 260 265 270 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 275 280 285 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 290 295 300 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 305 310 315 320 Val Thr His Thr Asp Leu Ala Ser Ser Leu Lys Gln Thr Ile Ser Arg 325 330 335 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 340 345 350 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 355 360 365 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 370 375 380 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 385 390 395 400 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 405 410 415 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 420 425 430 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 435 440 445 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 450 455 460 Ala Gly Thr Cys Tyr 465 <210> 28 <211> 466 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 28 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Arg Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asp Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Pro Gly Lys Asp Gln Asp Thr Ala Ile Arg Val Phe Ala Ile 225 230 235 240 Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr Lys Leu Thr 245 250 255 Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr Ile Ser Trp 260 265 270 Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn Ile Ser Glu 275 280 285 Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala Ser Ile Cys 290 295 300 Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr Val Thr His 305 310 315 320 Thr Asp Leu Ala Ser Ser Leu Lys Gln Thr Ile Ser Arg Pro Lys Gly 325 330 335 Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro Ala Arg Glu 340 345 350 Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu Val Thr Gly 355 360 365 Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg Gly Gln Pro 370 375 380 Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gln 385 390 395 400 Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val Ser Glu Glu 405 410 415 Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala His Glu Ala 420 425 430 Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly Lys 435 440 445 Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr Ala Gly Thr 450 455 460 Cys Tyr 465 <210> 29 <211> 98 <212> PRT <213> Homo sapiens <400> 29 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val <210> 30 <211> 15 <212> PRT <213> Homo sapiens <400> 30 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 31 <211> 110 <212> PRT <213> Homo sapiens <400> 31 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 100 105 110 <210> 32 <211> 107 <212> PRT <213> Homo sapiens <400> 32 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 33 <211> 98 <212> PRT <213> Homo sapiens <400> 33 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val <210> 34 <211> 12 <212> PRT <213> Homo sapiens <400> 34 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro 1 5 10 <210> 35 <211> 109 <212> PRT <213> Homo sapiens <400> 35 Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 20 25 30 Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 35 40 45 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55 60 Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln 65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 85 90 95 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 100 105 <210> 36 <211> 107 <212> PRT <213> Homo sapiens <400> 36 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 1 5 10 15 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 37 <211> 98 <212> PRT <213> Homo sapiens <400> 37 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val <210> 38 <211> 62 <212> PRT <213> Homo sapiens <400> 38 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys 1 5 10 15 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 20 25 30 Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu 35 40 45 Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 50 55 60 <210> 39 <211> 110 <212> PRT <213> Homo sapiens <400> 39 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 Gln Phe Lys 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 Phe 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 Thr Lys 100 105 110 <210> 40 <211> 107 <212> PRT <213> Homo sapiens <400> 40 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 1 5 10 15 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 41 <211> 98 <212> PRT <213> Homo sapiens <400> 41 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val <210> 42 <211> 12 <212> PRT <213> Homo sapiens <400> 42 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro 1 5 10 <210> 43 <211> 110 <212> PRT <213> Homo sapiens <400> 43 Ala Pro Glu Phe 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 Gln Glu Asp Pro Glu Val Gln 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 Phe 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 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 44 <211> 107 <212> PRT <213> Homo sapiens <400> 44 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 1 5 10 15 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 100 105 <210> 45 <211> 102 <212> PRT <213> Homo sapiens <400> 45 Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Cys Ser Thr 1 5 10 15 Gln Pro Asp Gly Asn Val Val Ile Ala Cys Leu Val Gln Gly Phe Phe 20 25 30 Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln Gly Val 35 40 45 Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp Leu Tyr 50 55 60 Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Leu Ala Gly 65 70 75 80 Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Pro Ser Gln Asp 85 90 95 Val Thr Val Pro Cys Pro 100 <210> 46 <211> 120 <212> PRT <213> Homo sapiens <400> 46 Val Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro 1 5 10 15 Ser Pro Ser Cys Cys His Pro Arg Leu Ser Leu His Arg Pro Ala Leu 20 25 30 Glu Asp Leu Leu Leu Gly Ser Glu Ala Asn Leu Thr Cys Thr Leu Thr 35 40 45 Gly Leu Arg Asp Ala Ser Gly Val Thr Phe Thr Trp Thr Pro Ser Ser 50 55 60 Gly Lys Ser Ala Val Gln Gly Pro Pro Glu Arg Asp Leu Cys Gly Cys 65 70 75 80 Tyr Ser Val Ser Ser Val Leu Pro Gly Cys Ala Glu Pro Trp Asn His 85 90 95 Gly Lys Thr Phe Thr Cys Thr Ala Ala Tyr Pro Glu Ser Lys Thr Pro 100 105 110 Leu Thr Ala Thr Leu Ser Lys Ser 115 120 <210> 47 <211> 131 <212> PRT <213> Homo sapiens <400> 47 Gly Asn Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser Glu 1 5 10 15 Glu Leu Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg Gly 20 25 30 Phe Ser Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln Glu 35 40 45 Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro Ser 50 55 60 Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala Ala 65 70 75 80 Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val Gly His Glu 85 90 95 Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Leu Ala Gly 100 105 110 Lys Pro Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp Gly 115 120 125 Thr Cys Tyr 130 <210> 48 <211> 102 <212> PRT <213> Homo sapiens <400> 48 Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Asp Ser Thr 1 5 10 15 Pro Gln Asp Gly Asn Val Val Val Ala Cys Leu Val Gln Gly Phe Phe 20 25 30 Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln Asn Val 35 40 45 Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp Leu Tyr 50 55 60 Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Pro Asp Gly 65 70 75 80 Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Pro Ser Gln Asp 85 90 95 Val Thr Val Pro Cys Pro 100 <210> 49 <211> 107 <212> PRT <213> Homo sapiens <400> 49 Val Pro Pro Pro Pro Pro Cys Cys His Pro Arg Leu Ser Leu His Arg 1 5 10 15 Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser Glu Ala Asn Leu Thr Cys 20 25 30 Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly Ala Thr Phe Thr Trp Thr 35 40 45 Pro Ser Ser Gly Lys Ser Ala Val Gln Gly Pro Pro Glu Arg Asp Leu 50 55 60 Cys Gly Cys Tyr Ser Val Ser Ser Val Leu Pro Gly Cys Ala Gln Pro 65 70 75 80 Trp Asn His Gly Glu Thr Phe Thr Cys Thr Ala Ala His Pro Glu Leu 85 90 95 Lys Thr Pro Leu Thr Ala Asn Ile Thr Lys Ser 100 105 <210> 50 <211> 131 <212> PRT <213> Homo sapiens <400> 50 Gly Asn Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser Glu 1 5 10 15 Glu Leu Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg Gly 20 25 30 Phe Ser Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln Glu 35 40 45 Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro Ser 50 55 60 Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala Ala 65 70 75 80 Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val Gly His Glu 85 90 95 Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Leu Ala Gly 100 105 110 Lys Pro Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp Gly 115 120 125 Thr Cys Tyr 130 <210> 51 <211> 104 <212> PRT <213> Homo sapiens <400> 51 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro 100 <210> 52 <211> 112 <212> PRT <213> Homo sapiens <400> 52 Val Ile Ala Glu Leu Pro Pro Lys Val Ser Val Phe Val Pro Pro Arg 1 5 10 15 Asp Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu Ile Cys Gln Ala 20 25 30 Thr Gly Phe Ser Pro Arg Gln Ile Gln Val Ser Trp Leu Arg Glu Gly 35 40 45 Lys Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val Gln Ala Glu Ala 50 55 60 Lys Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser Thr Leu Thr Ile 65 70 75 80 Lys Glu Ser Asp Trp Leu Ser Gln Ser Met Phe Thr Cys Arg Val Asp 85 90 95 His Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser Met Cys Val Pro 100 105 110 <210> 53 <211> 106 <212> PRT <213> Homo sapiens <400> 53 Asp Gln Asp Thr Ala Ile Arg Val Phe Ala Ile Pro Pro Ser Phe Ala 1 5 10 15 Ser Ile Phe Leu Thr Lys Ser Thr Lys Leu Thr Cys Leu Val Thr Asp 20 25 30 Leu Thr Thr Tyr Asp Ser Val Thr Ile Ser Trp Thr Arg Gln Asn Gly 35 40 45 Glu Ala Val Lys Thr His Thr Asn Ile Ser Glu Ser His Pro Asn Ala 50 55 60 Thr Phe Ser Ala Val Gly Glu Ala Ser Ile Cys Glu Asp Asp Trp Asn 65 70 75 80 Ser Gly Glu Arg Phe Thr Cys Thr Val Thr His Thr Asp Leu Pro Ser 85 90 95 Pro Leu Lys Gln Thr Ile Ser Arg Pro Lys 100 105 <210> 54 <211> 131 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (114)..(131) <223> μ tail fragment <400> 54 Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro Ala Arg 1 5 10 15 Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu Val Thr 20 25 30 Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg Gly Gln 35 40 45 Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro Glu Pro 50 55 60 Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val Ser Glu 65 70 75 80 Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala His Glu 85 90 95 Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly 100 105 110 Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr Ala Gly 115 120 125 Thr Cys Tyr 130 <210> 55 <211> 159 <212> PRT <213> Homo sapiens <220> <221> Signal <222> (1)..(22) <400> 55 Met Lys Asn His Leu Leu Phe Trp Gly Val Leu Ala Val Phe Ile Lys 1 5 10 15 Ala Val His Val Lys Ala Gln Glu Asp Glu Arg Ile Val Leu Val Asp 20 25 30 Asn Lys Cys Lys Cys Ala Arg Ile Thr Ser Arg Ile Ile Arg Ser Ser 35 40 45 Glu Asp Pro Asn Glu Asp Ile Val Glu Arg Asn Ile Arg Ile Ile Val 50 55 60 Pro Leu Asn Asn Arg Glu Asn Ile Ser Asp Pro Thr Ser Pro Leu Arg 65 70 75 80 Thr Arg Phe Val Tyr His Leu Ser Asp Leu Cys Lys Lys Cys Asp Pro 85 90 95 Thr Glu Val Glu Leu Asp Asn Gln Ile Val Thr Ala Thr Gln Ser Asn 100 105 110 Ile Cys Asp Glu Asp Ser Ala Thr Glu Thr Cys Tyr Thr Tyr Asp Arg 115 120 125 Asn Lys Cys Tyr Thr Ala Val Val Pro Leu Val Tyr Gly Gly Glu Thr 130 135 140 Lys Met Val Glu Thr Ala Leu Thr Pro Asp Ala Cys Tyr Pro Asp 145 150 155

Claims

1. A hybrid Fc protein that sequentially comprises an IgG Fc region, CH2 and CH3 regions, and an IgM Fc region from the N-terminus to the C-terminus; the IgG Fc region comprises at least a portion of the hinge region, each of the CH2 and CH3 regions is of IgG isotype, the IgM Fc region comprises Cμ3 and Cμ4 regions, wherein the at least a portion of the hinge region is not linked to (a) an antibody variable region or (b) a heterologous polypeptide that specifically binds to a target, wherein the molecule of the hybrid Fc protein is capable of forming a duplex through an interchain disulfide bond between cysteine residues in the at least a portion of the hinge region, and the duplexes are capable of polymerizing with each other through the Cμ3 and Cμ4 regions; Wherein, The hybrid Fc protein presents a hexameric form of a duplex; wherein the amino acid sequence of the hybrid Fc protein is as shown in any one of SEQ ID NO: 11, 13, 15 and 16, or the hybrid Fc protein consists of any one of SEQ ID NO: 11, 13, 15 and 16 and an additional sequence, and the additional sequence is added to at least one end of any one of SEQ ID NO: 11, 13, 15 and 16; and wherein the additional sequence is selected from at least one of the following: a hexahistidine tag, and a signal peptide.

2. The hybrid Fc protein according to claim 1, wherein, Each molecule of the hybrid Fc protein has more than two sialic acid residues.

3. The hybrid Fc protein according to claim 1, each hexamer having more than 12 sialic acid molecules.

4. The hybrid Fc protein according to any one of claims 1-3, wherein the hybrid Fc protein is at least 99% pure by weight.

5. A pharmaceutical composition comprising the hybrid Fc protein according to any one of claims 1-4 and a pharmaceutically acceptable carrier.

Images