ANTI-MYOSTATIN ANTIBODIES, POLYPEPTIDES CONTAINING VARIANT Fc REGIONs, AND METHODS OF USE

Abstract

This disclosure provides anti-myosin antibodies and methods for their formation and use. It also provides nucleic acids encoding anti-myosin antibodies and host cells comprising the aforementioned nucleic acids. This disclosure further provides polypeptides containing a variant Fc region and methods for their formation and use. It also provides nucleic acids encoding the polypeptides and host cells comprising the aforementioned nucleic acids.

Description

[Technical Field]

[0001] This invention relates to anti-myosin antibodies and methods of using them. This invention also relates to polypeptides containing a variant Fc region and methods of using them. [Previous Technology]

[0002] Myostatin, also known as growth differentiation factor 8 (GDF8), is a secreted protein and a member of the transforming growth factor β (TGF-β) superfamily. Members of this superfamily have growth regulation and morphological change characteristics (see, for example, Kingsley et al., Genes Dev. 8(2):133-146 (1994), Hoodless et al., Curr. Top. Microbiol. Immunol. 228:235-272 (1998), and US Patent No. 5,827,733). Myostatin is mainly found in developing and adult skeletal muscle and acts as a negative regulator of muscle growth. Overexpression of the myosin system in adult mice leads to muscle atrophy (see, for example, Zimmers et al., Science 296(5572):1486-1488 (2002)), while myosin knockout mice are characterized by skeletal muscle hypertrophy and proliferation, resulting in two to three times more muscle mass than their wild-type littermates (see, for example, McPherron et al., Nature 387(6628):83-90 (1997)).

[0003] Like other members of the TGF-β family, myosin is synthesized as a large precursor protein containing an N-terminal propeptide domain and a C-terminal domain that is considered the active molecule (see, for example, McPherron and Lee, Proc. Natl. Acad. Sci. USA 94(23):12457-12461(1997); WO 1994 / 021681). The two molecules of the myosin precursor are covalently linked by a single disulfide bond present in the C-terminal growth factor domain. The active, mature myosin (a homodimer composed of disulfide bonds in the C-terminal growth factor domain) is released from the myosin precursor through multiple steps of protein hydrolysis. In the first step of the myosin activation pathway, the peptide bond between the N-terminal propeptide domain and the C-terminal growth factor domain, Arg266-Asp267, is cleaved by furin-type proton convertase in both chains of the homodimer precursor. However, the resulting three peptides (two propeptides and one mature myosin) (i.e., a homodimer with disulfide bonds formed by the growth factor domain) remain bound together, forming a non-covalently bonded, inactive complex known as "latent myosin." The mature myosin is then released from the latent myosin through the breakdown of the propeptide. Members of the bone morphogenetic protein 1 (BMP1) family of metalloproteinases cleave the single peptide bond within the propeptide, Arg98-Asp99, along with the release of the mature, active myosin, a homodimer (see, for example, Szláma et al., FEBS J280(16):3822-3839 (2013)). In addition, latent myostatin can be activated in vitro by dissociating the complex with acid or heat (see, for example, Lee, PloS One 3(2):e1628 (2008)).

[0004] Myosin exerts its action through the transmembrane serine / threonine kinase heterotetrameric receptor family, whose activation enhances receptor transphosphorylation, leading to stimulation of serine / threonine kinase activity. The myosin pathway has been shown to involve the binding of the active myosin dimer to a high-affinity activin receptor type IIB (ActRIIB), which in turn recruits and activates the transphosphorylation of low-affinity receptor activin kinase 4 (ALK4) / activin kinase 5 (ALK5). Proteins Smad 2 and Smad 3 have also been shown to be subsequently activated and form a complex with Smad 4, which then translocates to the nucleus for activation of target gene transcription. ActRIIB has been shown to mediate the effects of myosin in vivo, as the expression of the dominant-negative form of ActRIIB in mice mimics myosin gene knockout (see, for example, Lee, Proc. Natl. Acad. Sci. USA 98(16):9306-9311 (2001)).

[0005] Many diseases and symptoms are associated with muscle atrophy (e.g., reduction or impaired function of muscle tissue), such as muscular dystrophy (MD; including Duchenne's muscular dystrophy), amyotrophic lateral sclerosis (ALS), muscular dystrophy, organ atrophy, asthenia, congestive obstructive pulmonary disease (COPD), sarcopenia, and cachexia caused by cancer or other diseases, as well as kidney disease, heart failure or disease, and liver disease. Increased muscle mass and / or muscle strength would be beneficial to patients; however, currently available treatments for these diseases are limited. Therefore, due to its role as a negative regulator of skeletal muscle growth, myostatin is an ideal target for therapeutic or preventative interventions for such diseases or symptoms, or for monitoring the progression of such diseases or symptoms. In particular, agents that inhibit the activity of myostatin can be beneficial for treatment.

[0006] Inhibition of myosin expression leads to muscle hypertrophy and hyperplasia (McPherron et al., Nature 387(6628):83-90 (1997)). Myosin negatively regulates muscle regeneration after injury, and the lack of myosin in myosin-ineffective (null) mice leads to accelerated muscle regeneration (see, for example, McCroskery et al., J Cell Sci. 118(15):3531-3541 (2005)). Anti-myosin (GDF8) antibodies have been described, for example, in U.S. Patent Nos. 6,096,506, 7,261,893, 7,320,789, 7,807,159, and 7,888,486, and WO 2005 / 094446, WO 2007 / 047112, and WO 2010 / 070094 have been shown to bind myosin in vitro and in vivo and inhibit myosin activity, including myosin activity associated with negative regulation of skeletal muscle mass. In the skeletal muscle of wild-type mice (see, for example, Whittemore et al., Biochem. Biophys. Res. Commun. 300(4):965-971 (2003)) and in mdx mice with muscular dystrophy (see, for example, Bogdanovich et al., Nature 420(6914):418-421 (2002); Wagner., Ann. Neurol. 52(6):832-836 (2002)), myosin-neutralizing antibodies increased body weight, skeletal muscle mass, and muscle size and strength. However, these prior art antibodies are specific to mature myosin rather than latent myosin, and the strategy for inhibiting myosin activity utilizes antibodies that can bind to and neutralize mature myosin.

[0007] Antibodies have attracted attention as pharmaceuticals due to their high stability in the blood and low incidence of side effects (see, for example, Reichert et al., Nat. Biotechnol. 23:1073-1078 (2005) and Pavlou et al., Eur. J. Pharm. Biopharm. 59:389-396 (2005)). Currently, almost all commercially available therapeutic antibodies are human IgG1 subclass antibodies. One of the known functions of IgG subclass antibodies is antibody-dependent cell-mediated cytotoxicity (hereinafter referred to as ADCC activity) (see, for example, Clark et al., Chem. Immunol. 65:88-110 (1997)). For antibodies to exhibit ADCC activity, the antibody Fc region must bind to the Fcγ receptor (hereinafter referred to as FcγR), an antibody-binding receptor present on the surface of effector cells, such as killer cells, natural killer cells, and activated macrophages.

[0008] In humans, isoforms of FcγRIa (CD64A), FcγRIIa (CD32A), FcγRIIb (CD32B), FcγRIIIa (CD16A), and FcγRIIIb (CD16B) have been reported as the FcγR protein family, and their respective allotypes have also been reported (see, for example, Jefferis et al., Immunol. Lett. 82:57-65 (2002)). FcγRIa, FcγRIIa, and FcγRIIIa are referred to as activated FcγRs due to their immunomodulatory functions, while FcγRIIb is referred to as inhibited FcγRs due to their immunosuppressive functions (see, for example, Smith et al., Nat. Rev. Immunol. 10:328-343 (2010)).

[0009] In the binding between the Fc region and FcγR, many amino acid residues in the antibody hinge region and CH2 domain, as well as the sugar chain attached to Asn at position 297 (EU number) bound to the CH2 domain, have been shown to be important (see, for example, Radaev et al., J. Biol. Chem. 276:16478-16483 (2001), Greenwood et al., Eur. J. Immunol. 23:1098-1104 (1993), and Morgan et al., Immunology 86:319-324 (1995)). To date, many variants with FcγR binding properties, mainly antibodies with mutations introduced at these sites, have been studied; and Fc region variants with high binding activity to activated FcγR have been obtained (see, for example, WO 2000 / 042072, WO 2006 / 019447, WO 2004 / 099249 and WO 2004 / 029207).

[0010] When activated FcγR crosslinks with immune complexes, it phosphorylates the immunoreceptor tyrosine-based activating motif (ITAM) contained in the intracellular domain or the γ chain (interacting partner) shared by FcR, activates the signal transducer SYK, and triggers an inflammatory immune response by initiating an activation signal cascade (see, for example, Nimmerjahn et al., Nat. Rev. Immunol. 8:34-47 (2008)).

[0011] FcγRIIb is the only FcγR expressed on B cells (see, for example, Amigorena et al., Eur. J. Immunol. 19:1379-1385 (1989)). The interaction between the antibody Fc region and FcγRIIb has been reported to suppress the primary immune response of B cells (see, for example, Sinclair, J. Exp. Med. 129:1183-1201 (1969)). In addition, it has been reported that when FcγRIIb on B cells and the B cell receptor (BCR) are cross-linked in the blood via immune complexes, the activation of B cells and the antibody production of B cells are suppressed (see, for example, Heyman, Immunol. Lett. 88:157-161 (2003)). In this immunosuppressive signaling mediated by BCR and FcγRIIb, the immunoreceptor tyrosine inhibitory motif (ITIM) contained in the intracellular domain of FcγRIIb is essential (see, for example, Amigorena et al., Science 256:1808-1812 (1992) and Muta et al., Nature 368:70-73 (1994)). When ITIM is phosphorylated by signaling, inositol polyphosphate 5-phosphatase (SHIP) containing SH2 is recruited, the transmission of other activated FcγR signaling cascades is inhibited, and the inflammatory immune response is suppressed (see, for example, Ravetch, Science 290:84-89 (2000)). In addition, the aggregation of FcγRIIb alone has been reported to transiently inhibit calcium ion influx, due to BCR crosslinking and B cell proliferation in a BCR-independent manner without inducing apoptosis in IgM-producing B cells (see, for example, Fournier et al., J. Immunol. 181:5350-5359 (2008)).

[0012] FcγRIIb is also expressed on dendritic cells, macrophages, activated neutrophils, mast cells, and basophils. FcγRIIb inhibits the function of activated FcγR, such as phagocytosis and the release of inflammatory cytokines in these cells, and suppresses inflammatory immune responses (see, for example, Smith et al., Nat. Rev. Immunol. 10:328-343 (2010)).

[0013] To date, the importance of the immunosuppressive function of FcγRIIb has been elucidated through studies using FcγRIIb knockout mice. Reports indicate that in FcγRIIb knockout mice, humoral immunity is not properly regulated (see, for example, J. Immunol. 163:618-622 (1999)), sensitivity to collagen-induced arthritis (CIA) is increased (see, for example, Yuasa et al., J. Exp. Med. 189:187-194 (1999)), symptoms similar to lupus appear, and symptoms similar to Goodpasture's syndrome-like appear (see, for example, Nakamura et al., J. Exp. Med. 191:899-906 (2000)).

[0014] In addition, insufficient control of FcγRIIb has been reported to be associated with human autoimmunity. For example, the relationship between genotypes in transmodal regions and the promoter region of FcγRIIb and the frequency of systemic lupus erythematosus (SLE) (see, for example, Blank, Hum. Genet. 117:220-227 (2005), Olferiev et al., J. Biol. Chem. 282:1738-1746 (2007), Chen et al., Arthritis Rheum. 54:3908-3917 (2006), Floto et al., Nat. Med. 11:1056-1058 (2005), and Li et al., J. Immunol. 176:5321-5328 (2006)), and the reduced expression of FcγRIIb on B cells in SLE patients (see, for example, Mackay et al., J. Exp. Med. 203:2157-2164). (2006) and Yang et al., J. Immunol.178:3272-3280 (2007) have been reported.

[0015] Based on the above mouse models and clinical findings, FcγRIIb is believed to play a role in controlling autoimmune diseases and inflammatory diseases through a specific association with B cells, and it is a potential target molecule for controlling autoimmune diseases and inflammatory diseases.

[0016] IgG1, primarily available as a commercially available therapeutic antibody, is known not only to bind to FcγRIIb but also to strongly activate FcγR (see, for example, Bruhns et al., Blood 113:3716-3725 (2009)). It is possible to develop therapeutic antibodies with stronger immunosuppressive properties than IgG1 by utilizing Fc regions that exhibit enhanced FcγRIIb binding or improved FcγRIIb binding selectivity compared to activated FcγR. For example, it has been suggested that antibodies with variant regions that bind to BCR and Fc with enhanced FcγRIIb binding affinity can inhibit B cell activation (see, for example, Chu et al., Mol. Immunol. 45:3926-3933 (2008)). Previous reports have indicated that cross-linking FcγRIIb to B cells and IgE bound to the B cell receptor inhibits B cell differentiation into plasma cells, resulting in suppression of IgE production; and in human PBMC-transplanted mice, the concentrations of human IgG and IgM are maintained while the concentration of human IgE is reduced (see, for example, Chu et al., J. Allergy Clin. Immunol. 129:1102-1115 (2012)). In addition to IgE, it has been reported that when FcγRIIb and CD79b, a component of the B cell receptor complex, are cross-linked with antibodies, B cell proliferation is inhibited in vitro and arthritis symptoms are relieved in collagen arthritis models (see, for example, Veri et al., Arthritis Rheum. 62:1933-1943 (2010)).

[0017] In addition to B cells, it has been reported that molecules can be used to crosslink FcεRI and FcγRIIb on mast cells. The Fc portion of IgG with enhanced FcγRIIb binding affinity is fused to the Fc portion of IgE, which binds to the IgE receptor FcεRI, causing phosphorylation of FcγRIIb and thus inhibiting FcεRI-dependent calcium ion influx. This shows that it is possible to inhibit FcγRIIb-stimulated degranulation by enhancing FcγRIIb binding affinity (see, for example, Cemerski et al., Immunol. Lett. 143:34-43 (2012)).

[0018] Therefore, antibodies against Fc with improved FcγRIIb binding activity show potential as therapeutic agents for inflammatory diseases (such as autoimmune diseases).

[0019] Furthermore, it has been reported that activation of macrophages and dendritic cells via Toll-like receptor 4 (TLR 4) stimulated by LPS is inhibited in the presence of antibody-antigen immune complexes, and this effect is also shown to be mediated by FcγRIIb immune complexes (see, for example, Wenink et al., J. Immunol. 183:4509-4520 (2009) and Zhang et al., J. Immunol. 182:554-562 (2009)). Therefore, the use of antibodies with enhanced FcγRIIb binding affinity is expected to increase the inhibition of TLR-mediated activation signals; thus, such antibodies have shown potential as therapeutic agents for inflammatory diseases (such as autoimmune diseases).

[0020] Furthermore, mutants with enhanced FcγRIIb binding affinity have shown potential as therapeutic agents for cancer and for inflammatory diseases such as autoimmune diseases. To date, FcγRIIb has been found to play an important role in activating the activity of agonist antibodies against the anti-TNF receptor superfamily. In particular, interaction with FcγRIIb has been shown to be necessary for the catalytic activity of antibodies against CD40, DR4, DR5, CD30, and CD137, which are included in the TNF receptor family (see, for example, Ravetch, Science 333:1030-1034 (2011), Wilson et al., Cancer Cell 19:101-113 (2011), Kohrt et al., J. Clin. Invest. 122:1066-1075 (2012), Xu et al., J. Immunol. 171:562-568 (2003), Zhang et al., Blood 108:705-710 (2006), Chuntharapai et al., J. Immunol. 166:4891-4898 (2001), and Ravetch et al., Proc. Natl. Acad. Sci. USA109:10966-10971 (2012)). Ravetch, Science 333:1030-1034 (2011) showed that the use of antibodies with enhanced FcγRIIb binding affinity increased the antitumor effect of anti-CD40 antibodies. Therefore, antibodies with enhanced FcγRIIb binding affinity are expected to have the effect of increasing the activating activity of agonist antibodies, including antibodies against the anti-TNF receptor superfamily.

[0021] Furthermore, it has been shown that when an antibody recognizing the Kit is used to crosslink FcγRIIb and the Kit onto cells expressing the Kit, which is a receptor tyrosine kinase (RTK), cell proliferation is inhibited. Similar effects have been reported even when this Kit is persistently activated and has mutations that lead to tumorigenesis (see, for example, Cemerski et al., Immunol. Lett. 143:28-33 (2002)). Therefore, the use of antibodies with enhanced FcγRIIb binding affinity is expected to increase the inhibitory effect on cells expressing RTKs with persistently activated mutations.

[0022] Antibodies with improved FcγRIIb binding activity have been reported (see, for example, Chu et al., Mol. Immunol. 45:3926-3933 (2008)). In this literature, FcγRIIb binding activity was improved by adding modifications such as S267E / L328F, G236D / S267E, and S239D / S267E to the Fc region of the antibody. Among them, the antibody with the S267E / L328F mutation bound to FcγRIIb most strongly and maintained the same level of binding to FcγRIa and H-type FcγRIIa, the residue at position 131 of which is His, the same as that of naturally occurring IgG1. However, another report shows that this modification increases the binding to R-type FcγRIIa by hundreds of times to the same extent as the binding to FcγRIIb, where the residue at position 131 of R-type FcγRIIa is Arg, which means that the binding selectivity of FcγRIIb is not improved compared to R-type FcγRIIa (see, for example, US Patent Application No. US2009 / 0136485).

[0023] Only the enhancement of FcγRIIa binding, rather than the increase in FcγRIIb binding, is thought to affect cells that express FcγRIIa but not FcγRIIb (such as platelets) (see, for example, Smith et al., Nat. Rev. Immunol. 10:328-343 (2010)). For example, patients given bevacizumab, an antibody against VEGF, are known to have a higher risk of thromboembolism (see, for example, Scappaticci et al., J. Natl. Cancer. Inst. 99:1232-1239 (2007)). Furthermore, similar thromboembolism has been observed in clinical development testing of antibodies against the CD40 ligand, and clinical testing has been interrupted (see, for example, Arthritis Rheum. 48:719-727 (2003)). In these antibody cases, subsequent studies using animal models and other methods have shown that administered antibodies agglutinate platelets and form thrombi by binding to FcγRIIa on platelets (see, for example, Meyer et al., J. Thromb. Haemost. 7:171-181 (2008) and Robles-Carrillo et al., J. Immunol. 185:1577-1583 (2010)). In systemic lupus erythematosus, an autoimmune disease, platelets are activated through an FcγRIIa-dependent mechanism, and platelet activation has been reported to be correlated with symptom severity (see, for example, Duffau et al., Sci. Transl. Med. 2:47ra63 (2010)). Administration of antibodies with enhanced FcγRIIa binding in these patients, who already have a high risk of thromboembolism, would increase the risk of thromboembolism and is therefore quite dangerous.

[0024] Furthermore, antibodies with enhanced FcγRIIa binding have been reported to enhance macrophage-mediated antibody-dependent phagocytosis (ADCP) (see, for example, Richards et al., Mol. Cancer Ther. 7:2517-2527 (2008)). When an antibody-bound antigen is phagocytosed by macrophages, the antibody itself is also considered to be phagocytosed simultaneously. When antibodies are administered as drugs, peptide fragments from the administered antibody are considered likely to be presented as antigens as well, thus increasing the risk of antibody-anti-therapeutic antibody (anti-therapeutic antibody) formation against the therapeutic antibody. More specifically, enhanced FcγRIIa binding will increase the risk of antibody-anti-therapeutic antibody formation, which will significantly reduce their value as drugs. Furthermore, it has been shown that FcγRIIb on dendritic cells contributes to peripheral tolerance by inhibiting dendritic cell activation resulting from immune complexes formed between antigens and antibodies, or by activating Fcγ receptors to inhibit antigen-presenting cells from T cells (see, for example, Desai et al., J. Immunol. 178:6217-6226 (2007)). Since FcγRIIa is also expressed on dendritic cells, when Fc antibodies with enhanced selective binding to FcγRIIb are used as drugs, the enhanced selective binding to FcγRIIb makes it less likely for antigens to be presented by dendritic cells, thus relatively reducing the risk of anti-drug antibody production. Such antibodies can also be useful in this regard.

[0025] More specifically, when the binding of FcγRIIa is enhanced, it leads to an increased risk of thrombosis via platelet aggregation and an increased risk of the production of anti-therapeutic antibodies due to increased immunogenicity, and its value as a drug will be significantly reduced.

[0026] From this perspective, compared to naturally occurring IgG1, the aforementioned Fc variant with enhanced FcγRIIb binding exhibits significantly enhanced R-type FcγRIIa binding. Therefore, its value as a drug for patients with R-type FcγRIIa is significantly reduced. H-type and R-type FcγRIIa are observed with roughly the same frequency in Caucasians and African Americans (see, for example, Salmon et al., J. Clin. Invest. 97:1348-1354 (1996) and Manger et al., Arthritis Rheum. 41:1181-1189 (1998)). Therefore, when this Fc variant is used to treat autoimmune diseases, the number of patients who can safely use it and enjoy its therapeutic effects will be limited.

[0027] Furthermore, it has been reported that dendritic cells lacking FcγRIIb, or those in which the interaction between FcγRIIb and the Fc portion of the antibody is inhibited by anti-FcγRIIb antibodies, are mature (see, for example, Boruchov et al., J. Clin. Invest. 115:2914-2923 (2005) and Dhodapkar et al., Proc. Natl. Acad. Sci. USA 102:2910-2915 (2005)). This report shows that FcγRIIb actively inhibits dendritic cell maturation in a stable state without inflammation or activation. In addition to FcγRIIb, FcγRIIa is also expressed on the surface of dendritic cells; therefore, dendritic cell maturation can be promoted even with enhanced binding to inhibitory FcγRIIb and even with enhanced binding to activated FcγR (such as FcγRIIa). More specifically, improving not only FcγRIIb binding activity, but also the ratio of FcγRIIb binding activity to FcγRIIa binding activity is considered important in providing antibody immunosuppressive effects.

[0028] Therefore, when considering the production of drugs that utilize FcγRIIb binding-mediated immunosuppression, the Fc variant must not only have enhanced FcγRIIb binding activity but also have the ability to bind to the H and R type FcγRIIa variants to the same degree or at a lower degree compared to naturally occurring IgG1.

[0029] Meanwhile, there have been reports of instances where amino acid alterations were introduced into the Fc region to increase FcγRIIb binding selectivity (see, for example, Armour et al., Mol. Immunol. 40:585-593 (2003)). However, in this report, all variants reported to have improved FcγRIIb selectivity showed reduced FcγRIIb binding compared to naturally occurring IgG1. Therefore, these variants are considered to be practically less likely to induce FcγRIIb-mediated immunosuppression more strongly than IgG1.

[0030] Furthermore, since FcγRIIb plays an important role in the aforementioned catalytic antibodies, enhancing their binding activity is expected to enhance catalytic activity. However, when the binding of FcγRIIa is also enhanced, unintended effects (such as ADCC and ADCP activities) will occur, and this may cause negative effects. Therefore, from the above perspective, selectively enhancing the binding activity of FcγRIIb is preferable.

[0031] These results indicate that when using FcγRIIb to manufacture therapeutic antibodies for the treatment of autoimmune diseases and cancer, it is important that the binding activity to FcγRIIa isoforms is maintained or reduced compared to naturally occurring IgG, and that the binding to FcγRIIb is enhanced. However, in the extracellular region, FcγRIIb shares 93% sequence identity with one of the activated FcγRs, FcγRIIa, and they are structurally very similar. FcγRIIa has isoforms, H-type and R-type, with the amino acid at position 131 being His (H-type) or Arg (R-type), but their interactions with antibodies are different (see, for example, Warmerdam et al., J. Exp. Med. 172:19-25 (1990)). Therefore, generating Fc region variants with enhanced selective binding to FcγRIIb compared to each isoform of FcγRIIa can be a challenging task, as it involves distinguishing highly homologous sequences between FcγRIIa and FcγRIIb. Despite these challenges, many Fc region variants with selective binding activity to FcγRIIb compared to FcγRIIa have been identified to date through comprehensive amino acid modification analysis in the Fc region (see, for example, WO 2012 / 115241, WO 2013 / 047752, WO 2013 / 125667, WO 2014 / 030728 and WO 2014 / 163101).

[0032] There are reports of Fc region variants with selective binding to FcγRIIb related to human FcγR, but there are no reports of Fc region variants with selective binding to FcγRIIb related to monkey FcγR. Because of the lack of such Fc variants, the effect of selective binding of Fc variants to FcγRIIb has not been thoroughly studied in monkeys.

[0033] In addition to the above, there have been reports indicating that by modifying the charge of amino acid residues that may be exposed on the surface of the antibody, it is possible to increase or decrease the isoelectric point (pI) of the antibody, thereby making it possible to adjust the half-life of the antibody in the blood (see, for example, WO 2007 / 114319 and WO 2009 / 041643). This shows that it is possible to prolong the plasma half-life of the antibody by decreasing its pI, and vice versa.

[0034] In addition, it has been reported that modifying the charge of specific amino acid residues, particularly in their CH3 domain, can increase the pI of antibodies, thereby promoting antigen incorporation into cells (see, for example, WO 2014 / 145159). It has also been reported that modifying the charge of amino acid residues in the constant region of antibodies (mainly the CH1 domain) to reduce the pI can prolong the half-life of antibodies in plasma (see, for example, WO 2012 / 016227). [Summary of the Invention]

[0035] This invention provides an anti-myosin antibody and a method of using the same. This invention also provides a protein comprising a mutated Fc region and a method of using the same.

[0036] In some embodiments, the isolated anti-myosin antibody of the present invention binds to latent myosin. In still other embodiments, the antibody binds to the antigenic determinant in the fragment consisting of amino acids 21-100 of the myosin propeptide (Sequence Identification Number: 78). In some embodiments, the isolated anti-myosin antibody of the present invention inhibits the activation of myosin. In still other embodiments, the antibody blocks the release of mature myosin from latent myosin. In still other embodiments, the antibody blocks the release of mature myosin via proteolytic hydrolysis. In still other embodiments, the antibody blocks the spontaneous release of mature myosin. In still other embodiments, the antibody does not bind to mature myosin. In still other embodiments, the antibody binds to the same antigenic determinant as the antibodies described in Table 13. In still other embodiments, the antibody binds to the same antigenic determinant as the antibodies including the VH and VL pairs described in Table 13. In still other embodiments, the antibody binds to the same antigenic determinant as the antibodies described in Table 2a. In some other embodiments, the antibody binds to the same antigenic determinant as antibodies including the VH and VL pairs described in Table 2a. In some other embodiments, the antibody binds to the same antigenic determinant as antibodies described in Table 11a. In some other embodiments, the antibody binds to the same antigenic determinant as antibodies including the VH and VL pairs described in Table 11a. In some other embodiments, the antibody binds to the same antigenic determinant as antibodies described in Tables 2a, 11a, or 13. In some other embodiments, the antibody binds to the same antigenic determinant as antibodies including the VH and VL pairs described in Tables 2a, 11a, or 13.

[0037] In some embodiments, the isolated anti-myosin antibody of the present invention binds to latent myosin with higher affinity at a neutral pH than at an acidic pH. In some embodiments, the anti-myosin antibody binds to latent myosin with higher affinity at pH 7.4 than at pH 5.8. In some embodiments, the isolated anti-myosin antibody of the present invention binds to a polypeptide fragment consisting of amino acids 21-100 of the myosin propeptide (Sequence Identification Number: 78) with higher affinity at a neutral pH than at an acidic pH. In some embodiments, the antibody binds to the myosin antigenic determinant with higher affinity at a neutral pH than at an acidic pH, which is the same as the myosin antigenic determinant bound by the antibodies described in Table 13. In some additional embodiments, the anti-myosin antibody binds to the antigenic determinant with higher affinity at pH 7.4 than at pH 5.8, which is the same as the antigenic determinant bound by the antibodies described in Table 13. In some embodiments, the antibody binds to the antigenic determinant with higher affinity at pH 7.4 than at pH 5.8, and this antigenic determinant is the same as that bound by antibodies including the VH and VL pairs described in Table 13. In some embodiments, the antibody binds to the myostatin antigenic determinant with higher affinity at neutral pH than at acidic pH, and this antigenic determinant is the same as that bound by antibodies described in Table 2a. In some embodiments, the antibody binds to the myostatin antigenic determinant with higher affinity at pH 7.4 than at pH 5.8, and this antigenic determinant is the same as that bound by antibodies including the VH and VL pairs described in Table 2a. In some additional embodiments, the anti-myosin antibody binds to the antigenic determinant with higher affinity at a neutral pH than at an acidic pH, the same antigenic determinant bound by the antibodies described in Table 11a. In still other embodiments, the antibody binds to the myosin antigenic determinant with higher affinity at pH 7.4 than at pH 5.8, the same antigenic determinant bound by the antibodies described in Table 11a. In still other embodiments, the antibody binds to the antigenic determinant with higher affinity at pH 7.4 than at pH 5.8, the same antigenic determinant bound by antibodies including the VH and VL pairs described in Table 11a. In some additional embodiments, the anti-myosin antibody binds to the antigenic determinant with higher affinity at a neutral pH than at an acidic pH, the same antigenic determinant bound by the antibodies described in Tables 2a, 11a, or 13. In some other embodiments, the antibody binds to the myostatin antigenic determinant with higher affinity at pH 7.4 than at pH 5.8, which is the same as the myostatin antigenic determinant bound by the antibodies described in Tables 2a, 11a or 13.In some other embodiments, the antibody binds to the antigenic determinant with higher affinity at pH 7.4 than at pH 5.8, which is the same antigenic determinant bound by antibodies including the VH and VL pairs described in Tables 2a, 11a, or 13.

[0038] In some embodiments, the isolated antimystifier antibody of the present invention competes with the antibodies provided herein for binding to latent myostatin. In some embodiments, the isolated antimystifier antibody of the present invention competes with the antibodies described in Table 13 for binding to latent myostatin. In some embodiments, the isolated antimystifier antibody of the present invention competes with antibodies including the VH and VL pairs described in Table 13 for binding to latent myostatin. In some embodiments, the antibody competes with the antibodies described in Table 2a for binding to latent myostatin. In some embodiments, the isolated antimystifier antibody of the present invention competes with antibodies including the VH and VL pairs described in Table 2a for binding to latent myostatin. In some embodiments, the antibody competes with the antibodies described in Table 11a for binding to latent myostatin. In some additional embodiments, the antimystifier antibody competes with the antibodies described in Tables 2a, 11a, or 13 for binding to latent myostatin. In some additional embodiments, the anti-myosin antibody competitively binds to latent myosin against antibodies including the VH and VL pairs described in Tables 2a, 11a, or 13. In still other embodiments, the anti-myosin antibody binds to latent myosin with higher affinity at neutral pH than at acidic pH. In still other embodiments, the anti-myosin antibody binds to latent myosin with higher affinity at pH 7.4 than at pH 5.8. In still other embodiments, the anti-myosin antibody binds to the polypeptide fragment consisting of amino acids 21-100 of the myosinogen propeptide (Sequence Identification Number: 78) with higher affinity at pH 7.4 than at pH 5.8. Methods for evaluating the ability of an antibody to competitively bind to latent myosin against a reference antibody are described herein and are well known in the art.

[0039] In some embodiments, the isolated anti-myosin antibody of the present invention is a monoclonal antibody. In some embodiments, the isolated anti-myosin antibody of the present invention is a human, humanized, or chimeric antibody. In some embodiments, the isolated anti-myosin antibody of the present invention is an antibody fragment bound to myosin. In some embodiments, the isolated anti-myosin antibody of the present invention is an antibody fragment bound to latent myosin. In some embodiments, the isolated anti-myosin antibody of the present invention is an antibody fragment bound to a polypeptide fragment composed of amino acids 21-100 of the myosin propeptide (Sequence Identification Number: 78). In some embodiments, the isolated anti-myosin antibody of the present invention is a full-length IgG antibody.

[0040] In some embodiments, the anti-myosin antibody of the present invention comprises: (a) (i) HVR-H3, comprising the amino acid sequence GVPAX1SX2GGDX3, wherein X1 is Y or H, X2 is T or H, and X3 is L or K (sequence identification number: 128); (ii) HVR-L3, comprising the amino acid sequence AGGYGGGX1YA, wherein X1 is L or R (sequence identification number: 131); and (iii) HVR-H2, comprising the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, wherein X1 is Y or H, X2 is S or K, X3 is T, M or K, X4 is Y or K, X5 is A, M or E, X6 is S or E, and X7 is G or K (sequence identification number: 127); (b) (i) HVR-H1, comprising the amino acid sequence X1X2DIS, wherein X1 is S or H, and X2 is Y, T, D, or E (sequence identification number: 126); (ii) HVR-H2, comprising the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, wherein X1 is Y or H, X2 is S or K, X3 is T, M, or K, X4 is Y or K, X5 is A, M, or E, X6 is S or E, and X7 is G or K (sequence identification number: 127); and (iii) HVR-H3, comprising the amino acid sequence GVPAX1SX2GGDX3, wherein X1 is Y or H, X2 is T or H, and X3 is L or K (sequence identification number: 128); (c) (i) HVR-H1, comprising the amino acid sequence X1X2DIS, wherein X1 is S or H, and X2 is Y, T, D, or E (sequence identification number: 126); (ii) HVR-H2, comprising the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, wherein X1 is Y or H, X2 is S or K, X3 is T, M, or K, X4 is Y or K, X5 is A, M, or E, X6 is S or E, and X7 is G or K (sequence identification number: 127); (iii) HVR-H3, comprising the amino acid sequence GVPAX1SX2GGDX3, wherein X1 is Y or H, X2 is T or H, and X3 is L or K. (Sequence Identification Number: 128); (iv) HVR-L1, comprising the amino acid sequence X1X2SQX3VX4X5X6NWLS, wherein X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, and X6 is N, D, A, or E (Sequence Identification Number: 129); (v) HVR-L2, comprising the amino acid sequence WAX1TLAX2, wherein X1 is S or E, and X2 is S, Y, F, or W (Sequence Identification Number: 130); and (vi) HVR-L3, comprising the amino acid sequence AGGYGGGX1YA, wherein X1 is L or R (Sequence Identification Number: 131);(d) (i) HVR-L1, comprising the amino acid sequence X1X2SQX3VX4X5X6NWLS, wherein X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, and X6 is N, D, A, or E (sequence identification number: 129); (ii) HVR-L2, comprising the amino acid sequence WAX1TLAX2, wherein X1 is S or E, and X2 is S, Y, F, or W (sequence identification number: 130); and (iii) HVR-L3, comprising the amino acid sequence AGGYGGGX1YA, wherein X1 is L or R (sequence identification number: 131). In some embodiments, the antibody of (b) further comprises a heavy chain variable domain frame FR1, including an amino acid sequence of sequence identification numbers 132-134; FR2, including an amino acid sequence of sequence identification numbers 135-136; FR3, including an amino acid sequence of sequence identification number 137; and FR4, including an amino acid sequence of sequence identification number 138. In some embodiments, the antibody of (d) further comprises a light chain variable domain frame FR1, including an amino acid sequence of sequence identification number 139; FR2, including an amino acid sequence of sequence identification numbers 140-141; FR3, including an amino acid sequence of sequence identification numbers 142-143; and FR4, including an amino acid sequence of sequence identification number 144.

[0041] In some embodiments, the isolated anti-myosin antibody of the present invention comprises: (a) HVR-H3, comprising the amino acid sequence GVPAX1SX2GGDX3, wherein X1 is Y or H, X2 is T or H, and X3 is L or K (sequence identification number: 128); (b) HVR-L3, comprising the amino acid sequence AGGYGGGX1YA, wherein X1 is L or R (sequence identification number: 131); and (c) HVR-H2, comprising the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, wherein X1 is Y or H, X2 is S or K, X3 is T, M or K, X4 is Y or K, X5 is A, M or E, X6 is S or E, and X7 is G or K (sequence identification number: 127).

[0042] In some embodiments, the isolated antimyosin antibody of the present invention comprises: (a) HVR-H1, comprising the amino acid sequence X1X2DIS, wherein X1 is S or H, and X2 is Y, T, D or E (sequence identification number: 126); (b) HVR-H2, comprising the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, wherein X1 is Y or H, X2 is S or K, X3 is T, M or K, X4 is Y or K, X5 is A, M or E, X6 is S or E, and X7 is G or K (sequence identification number: 127); and (c) HVR-H3, comprising the amino acid sequence GVPAX1SX2GGDX3, wherein X1 is Y or H, X2 is T or H, and X3 is L or K (sequence identification number: 128). In some other embodiments, the antibody includes a heavy chain variable domain frame FR1, including an amino acid sequence of sequence identification number 132-134; FR2, including an amino acid sequence of sequence identification number 135-136; FR3, including an amino acid sequence of sequence identification number 137; and FR4, including an amino acid sequence of sequence identification number 138. In some other embodiments, the antibody additionally includes: (a) HVR-L1, comprising the amino acid sequence X1X2SQX3VX4X5X6NWLS, wherein X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, and X6 is N, D, A, or E (sequence identification number: 129); (b) HVR-L2, comprising the amino acid sequence WAX1TLAX2, wherein X1 is S or E, and X2 is S, Y, F, or W (sequence identification number: 130); and (c) HVR-L3, comprising the amino acid sequence AGGYGGGX1YA, wherein X1 is L or R (sequence identification number: 131).

[0043] In some embodiments, the isolated antimyosin antibody of the present invention comprises: (a) HVR-L1, comprising the amino acid sequence X1X2SQX3VX4X5X6NWLS, wherein X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, and X6 is N, D, A or E (sequence identification number: 129); (b) HVR-L2, comprising the amino acid sequence WAX1TLAX2, wherein X1 is S or E, and X2 is S, Y, F or W (sequence identification number: 130); and (c) HVR-L3, comprising the amino acid sequence AGGYGGGX1YA, wherein X1 is L or R (sequence identification number: 131). In some other embodiments, the antibody further comprises: a light chain variable domain frame FR1, including an amino acid sequence with sequence identification number 139; FR2, including an amino acid sequence with sequence identification numbers 140-141; FR3, including an amino acid sequence with sequence identification numbers 142-143; and FR4, including an amino acid sequence with sequence identification number 144.

[0044] In some embodiments, the isolated anti-myosin antibody of the present invention comprises: a heavy chain variable domain frame FR1, comprising an amino acid sequence of sequence identification number 132-134; FR2, comprising an amino acid sequence of sequence identification number 135-136; FR3, comprising an amino acid sequence of sequence identification number 137; and FR4, comprising an amino acid sequence of sequence identification number 138. In some embodiments, the isolated anti-myosin antibody of the present invention comprises a light chain variable domain frame FR1, comprising an amino acid sequence of sequence identification number 139; FR2, comprising an amino acid sequence of sequence identification number 140-141; FR3, comprising an amino acid sequence of sequence identification number 142-143; and FR4, comprising an amino acid sequence of sequence identification number 144.

[0045] In some embodiments, the isolated anti-myosin antibody of the present invention comprises: (a) a VH sequence having at least 95% sequence identity with an amino acid sequence of any one of sequence identification numbers: 13, 16-30, 32-34, and 86-95; (b) a VL sequence having at least 95% sequence identity with an amino acid sequence of any one of sequence identification numbers: 15, 31, 35-38, and 96-99; or (c) the VH sequence as described in (a) and the VL sequence as described in (b). In still some embodiments, the antibody comprises a VH sequence of any one of sequence identification numbers: 13, 16-30, 32-34, and 86-95. In still some embodiments, the antibody comprises a VL sequence of any one of sequence identification numbers: 15, 31, 35-38, and 96-99. In some embodiments, the antibody includes a VH sequence of any one of sequence identification numbers: 13, 16-30, 32-34, and 86-95. In still other embodiments, the antibody includes a VH sequence of any one of sequence identification numbers: 13, 16-30, 32-34, and 86-95; and a VL sequence of any one of sequence identification numbers: 15, 31, 35-38, and 96-99.

[0046] The present invention also provides isolated nucleic acids encoding the anti-myosin antibody of the present invention. The present invention also provides host cells comprising the nucleic acids of the present invention. The present invention also provides a method for manufacturing antibodies, comprising culturing the host cells of the present invention to produce antibodies.

[0047] In some embodiments, the present invention provides a method for manufacturing an anti-myosin antibody, comprising: (a) culturing the host cells of the present invention to produce the antibody; or (b) immunizing an animal against a polypeptide, wherein the polypeptide comprises a region corresponding to an amino acid at positions 21-100 of a myosinogen peptide (Sequence Identification Number: 78).

[0048] The present invention further provides a method for manufacturing anti-myosin antibodies. In some embodiments, the method includes immunizing an animal with a polypeptide, wherein the polypeptide includes a region corresponding to an amino acid at positions 21-100 of a myosinogen peptide (Sequence Identification Number: 78).

[0049] The present invention also provides a pharmaceutical formulation comprising the antimyosin antibody of the present invention and a pharmaceutically acceptable carrier.

[0050] The present invention provides a polypeptide including a mutated Fc region and a method for manufacturing and using the polypeptide.

[0051] In one embodiment, the present invention provides an FcγRIIB binding polypeptide comprising a mutated Fc region and a method of using the same. In some embodiments, the mutated Fc region of the present invention having enhanced FcγRIIB binding activity comprises at least one amino acid alteration in the parent Fc region. In still other embodiments, the ratio of [KD value of the parent Fc region to monkey FcγRIIb] to [KD value of the mutated Fc region to monkey FcγRIIb] is 2 or greater. In still other embodiments, the ratio of [KD value of the parent Fc region to monkey FcγRIIb] to [KD value of the mutated Fc region to monkey FcγRIIb] is 0.5 or less. In still other embodiments, the ratio of [KD value of the parent Fc region to human FcγRIIb] to [KD value of the mutated Fc region to human FcγRIIb] is 2 or greater. In some embodiments, the ratio of [KD value of the parental Fc region to human FcγRIIIa] to [KD value of the variant Fc region to human FcγRIIIa] is 0.5 or less. In some other embodiments, the ratio of [KD value of the parental Fc region to human FcγRIIa (H type)] to [KD value of the variant Fc region to human FcγRIIa (H type)] is 5.0 or less. In some other embodiments, the ratio of [KD value of the parental Fc region to human FcγRIIa (R type)] to [KD value of the variant Fc region to human FcγRIIa (R type)] is 5.0 or less. In another embodiment, the KD value of the variant Fc region to monkey FcγRIIb is 1.0 × 10⁻⁶ M or less. In another embodiment, the KD value of the variant Fc region to monkey FcγRIIIa is 5.0 × 10⁻⁷ M or greater. In another example, the KD value of the mutated Fc region for human FcγRIIb is 2.0 × 10⁻⁶ M or less. In another embodiment, the KD value of the mutated Fc region for human FcγRIIIa is 1.0 × 10⁻⁶ M or greater. In another embodiment, the KD value of the mutated Fc region for human FcγRIIa (H type) is 1.0 × 10⁻⁷ M or greater. In another embodiment, the KD value of the mutated Fc region for human FcγRIIa (R type) is 2.0 × 10⁻⁷ M or greater.

[0052] In some embodiments, the variant Fc region of the present invention having enhanced FcγRIIb binding activity includes at least one amino acid modification at a position selected from at least one group consisting of: 231, 232, 233, 234, 235, 236, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334 and 396 (according to EU numbering).

[0053] In some other embodiments, the variant Fc region having enhanced FcγRIIb binding activity includes at least two amino acid modifications, including: (a) an amino acid modification at position 236; and (b) at least one amino acid modification selected from at least: (i) positions 231, 232, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334 and 396; (ii) positions 231, 232, 235, 239, 268, 295, 298, 326, 330 and 396; or (iii) positions of the group consisting of positions 268, 295, 326 and 330 (according to EU numbering).

[0054] In some other embodiments, the variant Fc region having enhanced FcγRIIb binding activity includes at least two amino acid modifications, including: (a) an amino acid modification at position 236; and (b) at least one amino acid modification at a position selected from the group consisting of: 231, 232, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334 and 396 (according to EU numbering).

[0055] In some other embodiments, the variant Fc region having enhanced FcγRIIb binding activity includes at least two amino acid modifications, including: (a) an amino acid modification at position 236; and (b) at least one amino acid modification at a position selected from the group consisting of 231, 232, 235, 239, 268, 295, 298, 326, 330 and 396 (according to EU numbering).

[0056] In some other embodiments, the variant Fc region having enhanced FcγRIIb binding activity includes at least two amino acid modifications, including: (a) an amino acid modification at position 236; and (b) at least one amino acid modification at a position selected from the group consisting of 268, 295, 326 and 330 (according to EU numbering).

[0057] In some embodiments, the variant Fc region of the present invention having enhanced FcγRIIb binding activity comprises at least one amino acid selected from: (a) Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 231; (b) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, G ln, Arg, Ser, Thr, Val, Trp, Tyr are at position 232; (c) Asp is at position 233; (d) Trp, Tyr are at position 234; (e) Trp is at position 235; (f) Ala, Asp, Glu, His, Ile, Leu, Met, Asn, Gln, Ser, Thr, Val are at position 236; (g) Asp, Tyr are at position 237; (h) Glu, Ile, Met, Gln, Tyr are at position 238; (i) Ile Leu, Asn, Pro, Val are at position 239; (j) Ile is at position 264; (k) Phe is at position 266; (l) Ala, His, Leu are at position 267; (m) Asp, Glu are at position 268; (n) Asp, Glu, Gly are at position 271; (o) Leu is at position 295; (p) Leu is at position 298; (q) Glu, Phe, Ile, Leu are at position 325; (r) Thr is at position 326; (s) Ile, Asn are at position 239. The group consisting of 327; (t) Thr at position 328; (u) Lys, Arg at position 330; (v) Glu at position 331; (w) Asp at position 332; (x) Asp, Ile, Met, Val, Tyr at position 334; and (y) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 396 (according to EU number).

[0058] In some other embodiments, the variant Fc region having enhanced FcγRIIb binding activity includes at least one amino acid selected from the group consisting of: (a) Gly, Thr at position 231; (b) Asp at position 232; (c) Trp at position 235; (d) Asn, Thr at position 236; (e) Val at position 239; (f) Asp, Glu at position 268; (g) Leu at position 295; (h) Leu at position 298; (i) Thr at position 326; (j) Lys, Arg at position 330; and (k) Lys, Met at position 396 (according to EU numbering).

[0059] In another embodiment, the present invention provides a polypeptide comprising a variant Fc region with an increased isoelectric point (pI) and a method of using the same. In some embodiments, the polypeptide comprising a variant Fc region with an increased pI includes at least two amino acids modified in the parental Fc region. In still other embodiments, each amino acid modification increases the isoelectric point of the variant Fc region relative to the isoelectric point (pI) of the parental Fc region. In still other embodiments, the amino acids may be exposed on the surface of the variant Fc region. In still other embodiments, the polypeptide comprises a variant Fc region and an antigen-binding domain. In still other embodiments, the antigen-binding activity of the antigen-binding domain varies depending on ion concentration conditions. In yet other embodiments, the Fc region of the invention with increased pI variation comprises at least two amino acid modifications at positions selected from at least two groups consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431 (according to EU designations). In yet other embodiments, the Fc region with increased pI variation comprises Arg or Lys at each selected position.

[0060] In some embodiments, the modified Fc region of the present invention includes amino acid modifications as described in Tables 14-30.

[0061] In some embodiments, the polypeptide includes the variant Fc region of the present invention. In still other embodiments, the parental Fc region is derived from human IgG1. In still other embodiments, the polypeptide is an antibody. In still other embodiments, the polypeptide is an Fc fusion protein.

[0062] The present invention provides a polypeptide comprising any amino acid sequence in sequence identification numbers 229-381.

[0063] The present invention also provides isolated nucleic acids encoding polypeptides comprising the variant Fc region of the present invention. The present invention also provides host cells comprising the nucleic acids of the present invention. The present invention also provides a method for manufacturing polypeptides comprising the variant Fc region, comprising culturing the host cells of the present invention to manufacture the polypeptides.

[0064] The present invention further provides a pharmaceutical formulation comprising a polypeptide comprising a variant of the Fc region of the present invention and a pharmaceutically acceptable carrier.

Implementation Method

[0065] The techniques or procedures described or cited herein are conventional methods well-known and commonly used by those skilled in the art, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (edited by FM Ausubel et al. (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (edited by MJ MacPherson, BD Hames, and GR Taylor (1995)); Harlow and Lane (edited by Harlow and Lane (1988)); Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (edited by MJ Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis (ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, ed., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and CC Blackwell, ed.); Gene Transfer Vectors for Mammalian Cells (JMMiller and MP Calos (eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (JE Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty, ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD. The widely used methods described in Capra (edited, Harwood Academic Publishers, 1995) and Cancer: Principles and Practice of Oncology (edited by VT DeVita et al., JB Lippincott Company, 1993) are as follows. I. Definitions.

[0066] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Singleton et al., *Dictionary of Microbiology and Molecular Biology*, 2nd ed., J. Wiley & Sons (New York, NY 1994), and *March, Advanced Organic Chemistry Reactions, Mechanisms and Structure*, 4th ed., John Wiley & Sons (New York, NY 1992) provide general guidance to those skilled in the art regarding many of the terms used in this application. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0067] For the purpose of interpreting this specification, the following definitions will be used, and terms used in the singular may also include plural forms, and vice versa, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. In the event of any conflict between any definition set forth below and any document incorporated herein by reference, the following definition shall prevail.

[0068] For the purposes of this document, a “receptor human framework” is a framework comprising an amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from the human immunoglobulin framework or the human common framework as defined below. A receptor human framework “derived from” the human immunoglobulin framework or the human common framework may include its same amino acid sequence, or it may include amino acid sequence alterations. In some embodiments, the number of amino acid alterations is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, the VL receptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or the human common framework sequence.

[0069] "Affinity" refers to the total strength of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, "binding affinity" as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by methods known in the art, including those described herein. Specific descriptions and exemplary embodiments for measuring binding affinity are described below.

[0070] "Affinity-matured" antibodies refer to antibodies that have one or more alterations in one or more hypervariable regions (HVRs) compared to parent antibodies that do not have such alterations, resulting in improved antibody affinity for antigens.

[0071] The terms "anti-myosin antibody" and "antibody bound to myosin" refer to an antibody that binds to myosin with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent targeting myosin. In one embodiment, for example by radioimmunoassay (RIA), the degree to which the anti-myosin antibody binds to unrelated non-myosin proteins is less than about 10% of the antibody's binding to myosin. In a particular embodiment, the antibody bound to myosin has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸ M or less, e.g., from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In a particular embodiment, the anti-myosin antibody binds to the antigenic determinant of myosin, which is conserved with myosin from different species.

[0072] The term “antibody” is used in the broadest sense herein and covers a variety of antibody structures, including but not limited to monoclonal antibodies, multiclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0073] "Antibody fragment" refers to a molecule other than a complete antibody that includes a portion of a complete antibody, which binds to the antigen bound by that complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabody; linear antibody; single-chain antibody molecule (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0074] "Antibody that binds to the same antigenic determinant" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen in a competition assay, and / or conversely, the reference antibody blocks the binding of the antibody to its antigen in a competition assay. This article provides an exemplary competition assay.

[0075] The term “chimeric” antibody refers to an antibody in which part of the heavy chain and / or light chain comes from a specific source or species, while the rest of the heavy chain and / or light chain comes from different sources or species.

[0076] The "type" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main types of antibodies: IgA, IgD, IgE, IgG, and IgM. Several of these types can be further divided into subgroups (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different types of immunoglobulins are respectively called α, δ, ε, γ, and μ.

[0077] As used herein, the term “cytotoxic agent” refers to a substance that inhibits or prevents cell function and / or causes cell death or damage. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioisotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vincristine, vinblastine, etoposide, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleolysins; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof; and a variety of antitumor or anticancer agents disclosed below.

[0078] "Effective function" refers to those biological activities attributable to the Fc region of an antibody, which vary with antibody isotype. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; negative regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0079] The “effective amount” of a reagent, such as a pharmaceutical formulation, refers to the amount that, at the dosage and within the required time, is effective in achieving the desired therapeutic or preventive outcome.

[0080] The term "antigen determinant" includes any determinant that can be bound by an antibody. An antigen determinant is a region of the antigen that is bound by an antibody targeting that antigen, and contains a specific amino acid that directly contacts the antibody. Antigen determinant sites may comprise chemically active surface clusters of molecules (e.g., amino acids, sugar branches, phosphate groups, or sulfonyl groups) and may have specific three-dimensional structural characteristics and / or specific charge characteristics. Generally, antibodies specific to a particular target antigen will preferentially recognize antigen determinants on the target antigen in a complex mixture of proteins and / or macromolecules.

[0081] “Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a natural human FcR. In some embodiments, the FcR is a receptor (γ receptor) that binds to an IgG antibody and comprises the FcγRI, FcγRII, and FcγRIII subclasses, which include allelic variants and alternatively spliced ​​forms of these receptors. The FcγRII receptor comprises FcγRIIA (“activated receptor”) and FcγRIIB (“inhibitory receptor”), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activated receptor FcγRIIA contains the immunoreceptor tyrosine activation motif (ITAM) in its cytoplasmic domain. The inhibited receptor FcγRIIB contains the immunoreceptor tyrosine inhibition motif (ITIM) in its cytoplasmic domain (see, for example, Daëron, Annu. Rev. Immunol.15:203-234 (1997)). FcRs have been reviewed in, for example, Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are covered by the term “FcR” in this paper.

[0082] The term “Fc receptor” or “FcR” also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and the constant regulation of immunoglobulins. Methods for measuring FcRn binding are known (see, for example, Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.). In vivo binding to human FcRn and the plasma half-life of peptides that bind to human FcRn with high affinity can be analyzed, for example, by administering peptides with a variant Fc region to genetically modified mice or transfected human cell lines expressing human FcRn, or in primates. WO 2000 / 42072 (Presta) describes antibody variants that exhibit improved or reduced binding to FcR. See also, for example, Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0083] The term “Fc region” as used herein is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. This term includes both natural sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0084] The term "Fc region-comprising antibody" refers to an antibody that includes an Fc region. The C-terminal lysine residue (477, according to the EU numbering system) of the Fc region may be removed, for example, during antibody purification or by recombinant engineering of the nucleic acid encoding the antibody. Therefore, compositions including the antibodies of the present invention having an Fc region may include antibodies having K447, antibodies with all K447 removed, or mixtures of antibodies having and not having K447 residues.

[0085] “Frame” or “FR” refers to the variable domain residues outside the highly variable region (HVR) residues. The variable domain FR is generally composed of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences generally appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0086] The terms “full-length antibody”, “intact antibody” and “all antibody” are used interchangeably in this document to refer to an antibody that has a structure substantially similar to that of a natural antibody or has a heavy chain containing an Fc region as defined herein.

[0087] A “functional Fc region” possesses an “effective function” of a natural sequence Fc region. Exemplary “effective functions” include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; negative regulation of cell surface receptors (e.g., B cell receptors), etc. These effector functions typically require an Fc region to bind to a binding domain (e.g., antibody variable domain) and can be evaluated using a variety of revealed analyses, such as those defined herein.

[0088] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably to refer to cells in which exogenous nucleic acids have been introduced, including progeny of such cells. Host cells include “transformers” and “transformed cells,” which include the initially transformed cells and their derived progeny, regardless of passage number. The nucleic acid content of the progeny may not be exactly the same as that of the parent cells and may contain mutations. This document includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells.

[0089] A “human antibody” is one having an amino acid sequence corresponding to an antibody produced by humans or human cells or derived from a non-human source, wherein the non-human source amino acid sequence is encoded by a human antibody library or other human antibody sequences. This definition of a human antibody specifically excludes humanized antibodies that include non-human antigen-binding residues.

[0090] The “human common framework” is a framework representing the most frequently occurring amino acid residues in the selection of the human immunoglobulin VL or VH framework sequence. Generally, the selection of the human immunoglobulin VL or VH sequence is based on a isotype of the variable domain sequence. Typically, the isotype is the one described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the isotype is kappa I, as described in Kabat et al., supra. In one embodiment, for VH, the isotype is isotype III, as described in Kabat et al., supra.

[0091] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In a particular embodiment, a humanized antibody will include at least one, and typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of the non-human antibody, and all or substantially all of the FRs correspond to those of the human antibody. A humanized antibody may optionally include at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

[0092] As used herein, the term “hypervariant region” or “HVR” refers to each region of an antibody variable domain that is highly variable in sequence (“complementarity-determining region” or “CDR”), and / or forms a structurally defined loop (“hypervariant loop”), and / or contains antigen contact residues (“antigen contact”). Generally, an antibody comprises six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs in this paper include: (a) highly variable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, MD (1991)); (c) Antigen contacts present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and (d) combinations of (a), (b), and / or (c) containing HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0093] Unless otherwise stated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered according to Kabat et al., supra.

[0094] An "immunoconjugate" is an antibody conjugated to one or more heterogeneous molecules (including but not limited to cytotoxic agents).

[0095] "Individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In a particular embodiment, the individual or object is a human.

[0096] An “isolated” antibody is an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, which is determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). A review of methods for assessing antibody purity can be found, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0097] "Isolated" nucleic acid refers to a nucleic acid molecule that has been isolated from components of its natural environment. Isolated nucleic acid includes nucleic acid molecules contained in cells that normally contain such nucleic acid molecules, but which are located outside chromosomes or at chromosomal locations other than their natural chromosomal locations.

[0098] “isolated nucleic acid encoding antimyosin antibody” refers to one or more nucleic acid molecules encoding the heavy chain and light chain (or fragments thereof) of the antibody, such nucleic acid molecules contained in a single carrier or separate carriers, and such nucleic acid molecules present at one or more locations in the host cell.

[0099] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody family, i.e., a single antibody comprising the same family and / or binding to the same antigenic determinant, except, for example, antibodies containing naturally occurring mutations or possible variants that may arise during the preparation of monoclonal antibodies (such variants are typically present in small amounts). Unlike the preparation of polyclonal antibodies, which typically comprises different antibodies targeting different determinants (antigenic determinants), each monoclonal antibody prepared targets a single determinant on the antigen. Therefore, the modifier "monoclonal" refers to the antibody characteristics obtained from a substantially homogeneous antibody family and is not construed as requiring any specific method to produce the antibody. For example, monoclonal antibodies to be used according to the present invention can be prepared by a variety of techniques, including but not limited to fusion tumor technology, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin locus (LOCI), such methods and other exemplary methods for producing monoclonal antibodies will be described herein.

[0100] “Naked antibody” refers to an antibody that is not conjugated to a heterologous portion (e.g., a cytotoxic portion) or a radiolabel. Naked antibodies may be present in pharmaceutical formulations.

[0101] “Natural antibodies” refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains bound by disulfide bonds. From the N to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of their constant domains, the light chains of antibodies can be classified into one of two types, called κ and λ.

[0102] “Natural sequence Fc region” includes amino acid sequences that are identical to the amino acid sequences of Fc regions found in nature. Natural sequence human Fc regions include natural sequence human IgG1 Fc region (non-A and A variants); natural sequence human IgG2 Fc region; natural sequence human IgG3 Fc region; and natural sequence human IgG4 Fc region, as well as their naturally occurring variants.

[0103] The term “package insert” refers to the instructions typically included in the commercial packaging of a therapeutic product, which contain information about indications, usage, dosage, administration, combination therapy, contraindications and / or warnings about the use of such therapeutic products.

[0104] The "percentage (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after sequence alignment and, where necessary, the introduction of gaps to achieve maximum percentage sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved in various ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. However, for the purposes of this document, the % amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted with the user file to the U.S. Copyright Office, Washington DC, 20559, and is registered with the U.S. Copyright Office under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available at Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

[0105] When using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A compared to and / or relative to a given amino acid sequence B (or it can be expressed as a given amino acid sequence A having or including a specific % amino acid sequence identity compared to and / or relative to a given amino acid sequence B) is calculated as follows: 100 multiplied by a fraction X / Y, where X is the number of amino acid residues scored as a match by the sequence alignment program ALIGN-2 in the A and B alignments of this program, and where Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A compared to B will not be equal to the % amino acid sequence identity of B compared to A. Unless explicitly stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraphs.

[0106] The term “medical formulation” refers to a preparation which is in a form in which the bioactivity of the active ingredient contained therein is permitted and which does not contain any additional ingredients that would have unacceptable toxicity to the subject to which the formulation will be applied.

[0107] "Pharmaceutical acceptable carrier" refers to a component in a pharmaceutical formulation other than the active ingredient that is non-toxic to the target organism. Pharmaceutical acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0108] As used herein, the term "myosin" can refer to any natural myosin from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Unless otherwise stated, the term "myosin" refers to a human myosin protein having the amino acid sequence shown in Sequence Identification No. 1 and containing the terminal promyostatin domain of human myosin shown in Sequence Identification No. 75 or 78. This term covers "full-length," untreated myosin, and any form of myosin produced by intracellular processing. This term also covers naturally occurring variants of myosin, such as splice variants or allelic variants. An exemplary amino acid sequence of human myosin is shown in Sequence Identification No. 1 (promyostatin). An exemplary amino acid sequence of the C-terminal growth factor domain of human myosin is shown in Sequence Identification No. 2. The amino acid sequences of the N-terminal propeptide domain of exemplary human myosin are shown in sequence identification numbers 75 or 78. Active, mature myosin is a homodimer composed of two C-terminal growth factor domains linked by disulfide bonds. Inactive, latent myosin is a complex of two propeptides and mature myosin linked non-covalently. As disclosed herein, the antibodies of the present invention bind to inactive latent myosin but not to the mature, active myosin homodimer. In some embodiments, the antibodies of the present invention bind to the antigenic determinant within the fragment consisting of amino acids 21-100 of the myosin propeptide (sequence identification number: 78) but not to the mature, active myosin homodimer. The amino acid sequences of exemplary rhesus monkey and murine myosin (protomyosin) are shown in sequence identification numbers 3 and 5, respectively. The amino acid sequences of the C-terminal growth factor domain of exemplary rhesus monkey and murine myosin are shown in sequence identification numbers 4 and 6, respectively. The amino acid sequences of the N-terminal propeptide domain of exemplary rhesus monkey and mouse myostatin are shown in sequence identification numbers 76 or 79, and 77 or 80, respectively. GDF-11 (BMP-11) is a molecule closely related to myostatin, and both are members of the TGF-β superfamily. Similar to myostatin, GDF11 is first synthesized as a precursor propeptide, which is then cleaved into an N-terminal prodomain and a C-terminal mature GDF11. The amino acid sequence of human GDF11 (precursor) is shown in sequence identification number 81. The amino acid sequence of C-terminal mature human GDF11 is shown in sequence identification number 82. The amino acid sequence of the N-terminal prodomain of human GDF11 is shown in sequence identification number 83 or 84. The amino acid sequences of sequence identification numbers 1, 3, 5, 78, 79, 80, 81 and 84 contain message sequences corresponding to their amino acids 1-24, and the message sequences are removed during cell processing.

[0109] As used herein, “treatment” (and its grammatical variations) refers to a clinical intervention that attempts to alter the natural course of disease in an individual being treated, and may be performed for prevention or during clinicopathological processes. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological outcome of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and reducing or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the development of disease or slow its progression.

[0110] The term “variable region” or “variable domain” refers to a domain of the heavy or light chain of an antibody that binds to an antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, each comprising four conserved frame regions (FRs) and three highly variable regions (HVRs) (see, for example, Kindt et al., Kuby Immunology, 6thed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer specificity for antigen binding. Furthermore, antibodies binding to a specific antigen can be isolated using the VH or VL domains of the antibody binding that antigen, respectively, by screening complementary VL or VH domain databases. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0111] Due to at least one amino acid modification (alteration), preferably one or more amino acid substitutions, the "mutated Fc region" includes an amino acid sequence different from the natural sequence Fc region. Preferably, the modified Fc region has at least one amino acid substitution compared to the natural sequence Fc region or the Fc region of the parent polypeptide, for example, about 1 to 10 amino acid substitutions, preferably about 1 to 5 amino acid substitutions in the natural sequence Fc region or the Fc region of the parent polypeptide. The modified Fc region herein preferably has at least about 80% homology with the natural sequence Fc region and / or the Fc region of the parent polypeptide, and most preferably at least about 90% homology, more preferably at least about 95% homology.

[0112] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors as autonomously replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell to which they have been introduced. Specific vectors can indicate the expression of the nucleic acids to which they are effectively linked. Such vectors are referred to herein as "expression vectors." II. Composition and Methods

[0113] In one embodiment, the invention is, in part, based on anti-myosin antibodies and their use. In a particular embodiment, an antibody that binds to myosin is provided. The antibodies of the present invention are beneficial, for example, in the diagnosis and treatment of diseases.

[0114] In one embodiment, the present invention is, in part, based on polypeptides including a variant Fc region and their use. In one embodiment, a polypeptide including a variant Fc region having enhanced FcγRIIb binding activity is provided. In another embodiment, a polypeptide including a variant Fc region having an increased pI is provided. In some specific embodiments, the polypeptides of the present invention are antibodies. The polypeptides of the present invention including a variant Fc region are beneficial, for example, in the diagnosis and treatment of diseases. A. Exemplary antimyosin antibody and polypeptide including a variant Fc region

[0115] In one embodiment, the present invention provides an isolated antibody that binds to myosin. In some specific embodiments, the anti-myosin antibody of the present invention binds to latent myosin. In still other embodiments, the anti-myosin antibody of the present invention binds to the myosin native peptide (human: sequence number: 75 or 78; rhesus monkey: sequence number: 76 or 79; mouse: sequence number: 77 or 80). In still other embodiments, the antibody binds to an antigenic determinant within a fragment consisting of amino acids 21-100 of the myosin native peptide (sequence number: 78). The native peptide is contained in latent myosin as a component, as described above. In some specific embodiments, the anti-myosin antibody of the present invention inhibits the activation of myosin. In some specific embodiments, the anti-myosin antibody prevents the release of mature myosin from latent myosin. It has been reported that mature myosin is released from latent myosin via proteolytic and non-proteolytic steps. The anti-myosin antibody of the present invention can inhibit the proteolytic and / or non-proteolytic release of mature myosin from latent myosin. In some specific embodiments, the anti-myosin antibody inhibits the proteolytic cleavage of latent myosin. In some specific embodiments, the anti-myosin antibody prevents a protease from approaching latent myosin (particularly to the cleavage site of latent myosin proteolysis (Arg98-Asp99)). In still other embodiments, the protease may be a metalloproteinase of the BMP1 / TLD family, such as BMP1, TED, tolloid-like protein-1 (TLL-1), or tolloid-like protein-2 (TLL-2). In another embodiment, the anti-myosin antibody inhibits the non-proteolytic release of mature myosin from latent myosin. As used herein, non-proteolytic release refers to the spontaneous release of mature myosin from latent myosin without accompanying proteolytic cleavage of latent myosin. Non-protein hydrolysis release includes, for example, releasing mature myostatin by culturing latent myostatin under conditions such as 37°C in the absence of proteases that cleave latent myostatin. In some specific embodiments, the anti-myostatin antibody of the present invention does not bind to mature myostatin. In some embodiments, the anti-myostatin antibody binds to the same antigenic determinant as the antibodies described in Table 2a. In some embodiments, the anti-myostatin antibody competitively binds to latent myostatin as the antibodies described in Table 2a. In some additional embodiments, the anti-myostatin antibody competitively binds to latent myostatin as the antibody includes the VH and VL pairs described in Table 2a. In some embodiments, the anti-myostatin antibody competitively binds to the fragment consisting of amino acids 21-100 of the myostatin native peptide (Sequence Identification Number: 78) as the antibody described in Table 2a. In still other embodiments, the anti-myostatin antibody binds to the same antigenic determinant as the antibodies described in Table 11a or 13.In some embodiments, the anti-myosin antibody competes with the antibodies described in Table 11a or 13 for binding to latent myosin. In some embodiments, the anti-myosin antibody competes with the antibodies described in Table 11a or 13 for binding to the fragment consisting of amino acids 21-100 of the myosin native peptide (Sequence Identification Number: 78).

[0116] In some embodiments, the anti-myosin antibody of the present invention binds to latent myosin and inhibits myosin activation. In still other embodiments, the antibody: (a) inhibits the release of mature myosin from latent myosin; (b) inhibits the protein hydrolysis release of mature myosin; (c) inhibits the spontaneous release of mature myosin; or (d) does not bind to mature myosin; or binds to the antigenic determinant within the fragment composed of amino acids 21-100 of the myosin native peptide (Sequence Identification Number: 78). In still other embodiments, the antibody competes with antibodies including the VH and VL pairs described in Tables 2a, 11a, or 13 for binding to latent myosin, or binds to the same antigenic determinant with antibodies including the VH and VL pairs described in Tables 2a, 11a, or 13. In yet other embodiments, the antibody binds to latent myostatin with higher affinity at neutral pH (e.g., pH 7.4) than at acidic pH (e.g., pH 5.8). In yet other embodiments, the antibody is (a) a monoclonal antibody; (b) a human, humanized, or chimeric antibody; (c) a full-length IgG antibody; or (d) an antibody fragment that binds to latent myostatin or myostatin native peptide.

[0117] In another embodiment, the antimystin antibody of the present invention does not bind to GDF11. In some specific embodiments, the antimystin antibody of the present invention does not inhibit the activation of GDF11. In some specific embodiments, the antimystin antibody does not prevent the release of mature GDF11 from latent GDF11. The antimystin antibody of the present invention does not prevent the release of mature GDF11 from latent GDF11 via proteolysis and non-proteolysis. In some specific embodiments, the antimystin antibody does not prevent the proteolytic cleavage of latent GDF11. In some specific embodiments, the antimystin antibody does not prevent the protease from approaching latent GDF11 (particularly to the proteolytic cleavage site of latent GDF11). In still other embodiments, the protease may be a metalloproteinase of the BMP1 / TLD family, such as BMP1, TED, tolloid-like protein 1 (TLL-1), or tolloid-like protein 2 (TLL-2). The non-protein hydrolysis release used in this article refers to the spontaneous release of mature GDF11 from latent GDF11 without the accompanying proteolytic cleavage of latent GDF11. Non-protein hydrolysis release includes, for example, the release of mature GDF11 by culturing latent GDF11 under conditions such as 37°C in the absence of proteases that cleave latent GDF11. Most known anti-myosin antibodies are not specific to myosin. These antibodies have high affinity for other members of the TGF-β superfamily (such as GDF11) and neutralize their biological activity. GDF11 plays an important role during embryogenesis and is responsible for homologous transformation of the axial skeleton. Homozygous GDF11 knockout mice are perinatal lethal; mice with a copy of the wild-type GDF11 gene can survive but have skeletal defects. Because GDF11 plays a crucial role during embryonic development, GDF11 antagonists pose a theoretical safety risk, potentially causing toxicity in treated patients or reproductive toxicity in women of reproductive age, for example. Therefore, specific inhibition of myosin activity is necessary for the treatment of myosin-related conditions, particularly in women of reproductive age.

[0118] In another embodiment, the invention provides an anti-myosin antibody exhibiting pH-dependent binding properties. As used herein, the term "pH-dependent" means that the antibody exhibits "reduced binding to myosin at acidic pH compared to neutral pH" (the two terms may be used interchangeably for the purposes of this disclosure). For example, an antibody "having pH-dependent binding properties" comprises an antibody that binds to myosin with a higher affinity at neutral pH compared to acidic pH. In a particular embodiment, the antibody of the present invention binds to myosin at neutral pH with an affinity of at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times compared to acidic pH. In some embodiments, the antibody binds to myosin (e.g., latent myosin or protopeptide myosin) with a higher affinity at pH 7.4 than at pH 5.8. In still other embodiments, the antibody of the present invention binds to myosin with an affinity at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times at pH 7.4 than at pH 5.8.

[0119] When the antigen is a soluble protein, the binding of the antibody to the antigen can lead to a prolonged half-life of the antigen in the plasma (i.e., reduced clearance of the antigen from the plasma), because the antibody has a longer half-life in the plasma than the antigen itself and can act as a carrier of the antigen. This is due to the recycling of the antigen-antibody complex via the endosome pathway in the cell through FcRn (Roopenian, Nat. Rev. Immunol.7(9): 715-725 (2007)). However, antibodies with pH-dependent binding properties bind to their antigens in a neutral extracellular environment, and release the antigens into the acidic intracellular compartments upon entering the cell. These pH-dependent binding antibodies are expected to have superior performance in antigen neutralization and clearance, which is related to their corresponding pH-dependent binding (Igawa et al., Nature Biotechnol. 28(11):1203-1207 (2010); Devanaboyina et al., mAbs5(6):851-859 (2013); WO 2009 / 125825).

[0120] The “affinity” of an antibody to myostatin, for the purposes of this disclosure, is expressed in the terminology of the antibody’s KD. The KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The higher the KD value of an antigen binding to its antigen, the weaker its affinity for that particular antigen. Therefore, as used herein, “higher affinity at neutral pH than at acidic pH” (or equivalently, “pH-dependent binding”) means that the KD of an antibody binding to myostatin at acidic pH is greater than the KD of an antibody binding to myostatin at neutral pH. For example, in the context of this invention, if the KD of an antibody binding to myostatin at acidic pH is at least twice greater than the KD of an antibody binding to myostatin at neutral pH, the antibody is considered to bind to myostatin with higher affinity at neutral pH than at acidic pH. Therefore, the present invention includes antibodies whose KD (Knowledge Difference) to myosin at acidic pH is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times greater than the KD of the antibody to myosin at neutral pH. In another embodiment, the KD value of the antibody at neutral pH may be 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M, or less. In another embodiment, the KD value of the antibody at acidic pH may be 10⁻⁹ M, 10⁻⁸ M, 10⁻⁷ M, 10⁻⁶ M, or greater.

[0121] In some further embodiments, if the KD of the antibody binding to myosin (e.g., latent myosin or protopeptide myosin) at pH 5.8 is at least twice greater than the KD of the antibody binding to myosin at pH 7.4, the antibody is considered to bind to myosin with higher affinity at neutral pH compared to acidic pH. In some embodiments, the provided antibody has a KD of at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more than the KD of the antibody binding to myosin at pH 7.4. In another embodiment, the KD value of the antibody at pH 7.4 may be 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M or less. In another embodiment, the KD value of the antibody at pH 5.8 may be 10⁻⁹ M, 10⁻⁸ M, 10⁻⁷ M, 10⁻⁶ M or greater.

[0122] The binding characteristics of an antibody to a specific antigen can also be represented by the antibody's kd. The antibody's kd refers to the dissociation rate constant of the antibody to a specific antigen, expressed in the reciprocal of seconds (i.e., sec⁻¹). An increased kd value indicates that the antibody binds weaker to its antigen. The present invention therefore includes antibodies that bind to myosin at acidic pH with a higher kd value compared to neutral pH. The present invention includes antibodies whose kd for binding to myosin at acidic pH is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times greater than the kd for binding to myosin at neutral pH. In another embodiment, the KD value of the antibody may be 10⁻²¹ / s, 10⁻³¹ / s, 10⁻⁴¹ / s, 10⁻⁵¹ / s, 10⁻⁶¹ / s, or less at neutral pH. In another embodiment, the KD value of the antibody may be 10⁻³¹ / s, 10⁻²¹ / s, 10⁻¹¹ / s, or greater at acidic pH. The present invention also includes antibodies that bind to myosin (e.g., latent myosin or protopeptide myosin) at pH 5.8 with a larger KD value than at pH 7.4. The present invention comprises antibodies whose kd (kD) to myosin at pH 5.8 is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times greater than the kd to myosin at pH 7.4. In another embodiment, the kd value of the antibody at pH 7.4 may be 10⁻² lb / s, 10⁻³ lb / s, 10⁻⁴ lb / s, 10⁻⁵ lb / s, 10⁻⁶ lb / s, or less. In another embodiment, the kd value of the antibody at pH 5.8 may be 10⁻³ lb / s, 10⁻² lb / s, 10⁻¹¹ lb / s, or greater.

[0123] In a specific example, "reduced binding to myostatin at acidic pH compared to neutral pH" is expressed as the ratio of the KD value of antibody binding to myostatin at acidic pH to the KD value of antibody binding to myostatin at neutral pH (or vice versa). For example, for the purposes of this invention, if an antibody has an acidic / neutral KD ratio of 2 or greater, then the antibody can be considered to have "reduced binding to myostatin at acidic pH compared to neutral pH." In some specific embodiments, the pH 5.8 / pH 7.4 KD ratio of the anti-myostatin antibody of this invention may be 2 or greater. In some specific exemplary embodiments, the acid / neutral KD ratio of the antibody of the present invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or greater. In another embodiment, the KD value of the antibody at neutral pH may be 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M or less. In another embodiment, the KD value of the antibody at acidic pH may be 10⁻⁹ M, 10⁻⁸ M, 10⁻⁷ M, 10⁻⁶ M or greater. In a further example, if the antibody has a pH 5.8 / pH 7.4 KD ratio of 2 or greater, the antibody can be considered to have "reduced binding to myosin (e.g., latent myosin) at acidic pH compared to at neutral pH." In some specific exemplary embodiments, the antibody's pH 5.8 / pH 7.4 KD ratio may be 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or greater. In another embodiment, the antibody's KD value at pH 7.4 may be 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M or less. In another embodiment, the KD value of the antibody at pH 5.8 may be 10⁻⁹ M, 10⁻⁸ M, 10⁻⁷ M, 10⁻⁶ M or greater.

[0124] In a specific example, "reduced binding to myostatin at acidic pH compared to neutral pH" is expressed as the ratio of the kd value of antibody binding to myostatin at acidic pH to the kd value of antibody binding to myostatin at neutral pH (or vice versa). For example, for the purposes of this invention, if an antibody has an acidic / neutral kd ratio of 2 or greater, then the antibody may be considered to have "reduced binding to myostatin at acidic pH compared to neutral pH." In some specific exemplary embodiments, the pH 5.8 / pH 7.4 kd ratio of the antibody of this invention may be 2 or greater. In some specific exemplary embodiments, the acid / neutral kd ratio of the antibody of the present invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or greater. In another embodiment, the kd value of the antibody at neutral pH may be 10⁻²¹ / s, 10⁻³¹ / s, 10⁻⁴¹ / s, 10⁻⁵¹ / s, 10⁻⁶¹ / s or less. In another embodiment, the kd value of the antibody at acidic pH may be 10⁻³¹ / s, 10⁻²¹ / s, 10⁻¹¹ / s or greater. In some specific exemplary embodiments, the pH 5.8 / pH 7.4 kd ratio of the antibody may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or greater. In another embodiment, the kd value of the antibody at pH 7.4 may be 10⁻²¹ / s, 10⁻³¹ / s, 10⁻⁴¹ / s, 10⁻⁵¹ / s, 10⁻⁶¹ / s, or less. In another embodiment, the kd value of the antibody at pH 5.8 may be 10⁻³¹ / s, 10⁻²¹ / s, 10⁻¹¹ / s, or greater.

[0125] As used herein, the term "acidic pH" refers to a pH between 4.0 and 6.5. The term "acidic pH" includes any pH value among 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. In a particular sample, "acidic pH" is 5.8.

[0126] As used herein, the term "neutral pH" refers to a pH between 6.7 and 10.0. The term "neutral pH" includes any pH value among 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0. In a particular sample, "neutral pH" is 7.4.

[0127] KD and kd values, as indicated herein, can be determined using a biosensor based on surface plasma resonance to characterize antibody-antigen interactions (see, for example, Example 7 herein). KD and kd values ​​can be determined at 25°C or 37°C.

[0128] In some specific embodiments, the anti-myosin antibody of the present invention binds to myosin from more than one species. In still other embodiments, the anti-myosin antibody binds to myosin from humans and non-humans. In still other embodiments, the anti-myosin antibody binds to myosin from humans, mice, and monkeys (rhesus monkeys, rhesus macaques, marmosets, chimpanzees, or baboons).

[0129] In some specific embodiments, the antimystifier antibody of the present invention binds to latent myostatin from more than one species. In still other embodiments, the antimystifier antibody binds to latent myostatin from humans and non-humans. In still other embodiments, the antimystifier antibody binds to latent myostatin from humans, mice, and monkeys.

[0130] In some specific embodiments, the antimyosin antibody of the present invention binds to protopeptide myosin from more than one species. In still other embodiments, the antimyosin antibody binds to protopeptide myosin from humans and non-humans. In still other embodiments, the antimyosin antibody binds to protopeptide myosin from humans, mice, and monkeys.

[0131] In yet another embodiment, the invention provides an anti-myosin antibody that forms an immune complex (i.e., an antigen-antibody complex) with myosin. In some specific embodiments, two or more anti-myosin antibodies bind to two or more myosin molecules to form an immune complex. This is possible because myosin exists as a homodimer comprising two myosin molecules, and an antibody has two antigen-binding sites. The anti-myosin antibody may bind to the same antigenic determinant on the myosin molecule, or it may bind to different antigenic determinants on the myosin molecule, much like a bispecific antibody. In general, when two or more antibodies form immune complexes with two or more antigens, the resulting immune complexes can strongly bind to Fc receptors present on the cell surface due to the affinity effect of the Fc region of the antibody in the complex, and can then be taken up into the cell with high efficiency. Therefore, the aforementioned anti-myosin antibodies capable of forming immune complexes containing two or more anti-myosin antibodies and two or more myosin molecules can lead to rapid clearance of myosin from the plasma in vivo through strong binding to Fc receptors attributable to the affinity effect.

[0132] In addition, antibodies with pH-dependent binding properties are considered to have superior performance in antigen neutralization and clearance, which is related to their corresponding pH-dependent binding (Igawa et al., Nature Biotech. 28(11): 1203-1207 (2010); Devanaboyina et al., mAbs 5(6): 851-859 (2013); WO 2009 / 125825). Therefore, antibodies with the above two properties, namely, antibodies with pH-dependent binding properties and antibodies that form immune complexes containing two or more antibodies and two or more antigens, are expected to have even superior performance in greatly accelerating the elimination of antigens from the plasma (WO 2013 / 081143).

[0133] In another embodiment, the invention provides an anti-myosin antibody comprising at least one, two, three, four, five, or six highly variable regions (HVRs), selected from (a) HVR-H1 comprising an amino acid sequence of any one of sequence identification numbers: 55-57, 114-115, 126; (b) HVR-H2 comprising an amino acid sequence of any one of sequence identification numbers: 58-60, 116-120, 127; (c) HVR-H3 comprising an amino acid sequence of any one of sequence identification numbers: 61-64, 121, 128; (d) HVR-L1 comprising an amino acid sequence of any one of sequence identification numbers: 65-69, 122-124, 129; (e) HVR-L2 comprising an amino acid sequence of any one of sequence identification numbers: 70-72, 125, 130; and (f) HVR-L3 includes any of the amino acid sequences in sequence identification numbers 73-74 and 131.

[0134] In another embodiment, the invention provides an anti-myosin antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising an amino acid sequence of any one of sequence identification numbers: 55-57; (b) HVR-H2 comprising an amino acid sequence of any one of sequence identification numbers: 58-60; (c) HVR-H3 comprising an amino acid sequence of any one of sequence identification numbers: 61-64; (d) HVR-L1 comprising an amino acid sequence of any one of sequence identification numbers: 65-69; (e) HVR-L2 comprising an amino acid sequence of any one of sequence identification numbers: 70-72; and (f) HVR-L3 comprising an amino acid sequence of any one of sequence identification numbers: 73-74.

[0135] In another embodiment, the invention provides an anti-myosin antibody comprising at least one, two, three, four, five, or six highly variable regions (HVRs), selected from (a) HVR-H1 comprising an amino acid sequence of sequence identification number 114-115; (b) HVR-H2 comprising an amino acid sequence of sequence identification number 116-120; (c) HVR-H3 comprising an amino acid sequence of sequence identification number 121; (d) HVR-L1 comprising an amino acid sequence of sequence identification number 122-124; (e) HVR-L2 comprising an amino acid sequence of sequence identification number 125; and (f) HVR-L3 comprising an amino acid sequence of sequence identification number 73-74.

[0136] In another embodiment, the invention provides an anti-myosin antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising an amino acid sequence of sequence identification number: 114; (b) HVR-H2 comprising an amino acid sequence of sequence identification number: 58; (c) HVR-H3 comprising an amino acid sequence of sequence identification number: 63; (d) HVR-L1 comprising an amino acid sequence of sequence identification number: 122; (e) HVR-L2 comprising an amino acid sequence of sequence identification number: 71; and (f) HVR-L3 comprising an amino acid sequence of sequence identification number: 74. In another embodiment, the invention provides an anti-myosin antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising an amino acid sequence of sequence identification number 114; (b) HVR-H2 comprising an amino acid sequence of sequence identification number 58; (c) HVR-H3 comprising an amino acid sequence of sequence identification number 63; (d) HVR-L1 comprising an amino acid sequence of sequence identification number 123; (e) HVR-L2 comprising an amino acid sequence of sequence identification number 71; and (f) HVR-L3 comprising an amino acid sequence of sequence identification number 74.

[0137] In one embodiment, the invention provides an anti-myosin antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of sequence identification number 126; (b) HVR-H2 comprising the amino acid sequence of sequence identification number 127; (c) HVR-H3 comprising the amino acid sequence of sequence identification number 128; (d) HVR-L1 comprising the amino acid sequence of sequence identification number 129; (e) HVR-L2 comprising the amino acid sequence of sequence identification number 130; and (f) HVR-L3 comprising the amino acid sequence of sequence identification number 131.

[0138] In one embodiment, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) an amino acid sequence comprising any one of sequence identification numbers: 55-57, 114-115, 126; (b) an amino acid sequence comprising any one of sequence identification numbers: 58-60, 116-120, 127; and (c) an amino acid sequence comprising any one of sequence identification numbers: 61-64, 121, 128. In one embodiment, the antibody comprises HVR-H3, which comprises an amino acid sequence comprising any one of sequence identification numbers: 61-64, 121, 128. In another embodiment, the antibody comprises: HVR-H3, which includes an amino acid sequence of any one of sequence identification numbers: 61-64, 121, and 128; and HVR-L3, which includes an amino acid sequence of any one of sequence identification numbers: 73-74 and 131. In yet another embodiment, the antibody comprises: HVR-H3, which includes an amino acid sequence of any one of sequence identification numbers: 61-64, 121, and 128; HVR-L3, which includes an amino acid sequence of any one of sequence identification numbers: 73-74 and 131; and HVR-H2, which includes an amino acid sequence of any one of sequence identification numbers: 58-60, 116-120, and 127. In another embodiment, the antibody includes (a) HVR-H1 comprising an amino acid sequence of any one of sequence identification numbers: 55-57, 114-115, 126; (b) HVR-H2 comprising an amino acid sequence of any one of sequence identification numbers: 58-60, 116-120, 127; and (c) HVR-H3 comprising an amino acid sequence of any one of sequence identification numbers: 61-64, 121, 128.

[0139] In one embodiment, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising an amino acid sequence of any one of sequence identification numbers: 55-57; (b) HVR-H2 comprising an amino acid sequence of any one of sequence identification numbers: 58-60; and (c) HVR-H3 comprising an amino acid sequence of any one of sequence identification numbers: 61-64. In one embodiment, the antibody comprises HVR-H3 comprising an amino acid sequence of any one of sequence identification numbers: 61-64. In another embodiment, the antibody comprises HVR-H3 comprising an amino acid sequence of any one of sequence identification numbers: 61-64, and HVR-L3 comprising an amino acid sequence of any one of sequence identification numbers: 73-74. In another embodiment, the antibody includes: HVR-H3, which includes an amino acid sequence of any one of sequence identification numbers 61-64; HVR-L3, which includes an amino acid sequence of any one of sequence identification numbers 73-74; and HVR-H2, which includes an amino acid sequence of any one of sequence identification numbers 58-60. In yet another embodiment, the antibody includes (a) HVR-H1, which includes an amino acid sequence of any one of sequence identification numbers 55-57; (b) HVR-H2, which includes an amino acid sequence of any one of sequence identification numbers 58-60; and (c) HVR-H3, which includes an amino acid sequence of any one of sequence identification numbers 61-64.

[0140] In one embodiment, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) an amino acid sequence comprising any one of sequence identification numbers: 114-115; (b) an amino acid sequence comprising any one of sequence identification numbers: 116-120; and (c) an amino acid sequence comprising an amino acid sequence comprising sequence identification number: 121. In one embodiment, the antibody comprises HVR-H3, which comprises an amino acid sequence comprising sequence identification number: 121. In another embodiment, the antibody comprises: HVR-H3, which comprises an amino acid sequence comprising sequence identification number: 121, and HVR-L3, which comprises an amino acid sequence comprising any one of sequence identification numbers: 73-74. In another embodiment, the antibody includes: HVR-H3, which includes an amino acid sequence of sequence identification number 121; HVR-L3, which includes an amino acid sequence of any one of sequence identification numbers 73-74; and HVR-H2, which includes an amino acid sequence of any one of sequence identification numbers 116-120. In yet another embodiment, the antibody includes (a) HVR-H1, which includes an amino acid sequence of any one of sequence identification numbers 114-115; (b) HVR-H2, which includes an amino acid sequence of any one of sequence identification numbers 116-120; and (c) HVR-H3, which includes an amino acid sequence of sequence identification number 121.

[0141] In another embodiment, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising an amino acid sequence of sequence identification number: 114; (b) HVR-H2 comprising an amino acid sequence of sequence identification number: 58; and (c) HVR-H3 comprising an amino acid sequence of sequence identification number: 63. In one embodiment, the antibody comprises HVR-H3 comprising an amino acid sequence of sequence identification number: 63. In another embodiment, the antibody comprises HVR-H3 comprising an amino acid sequence of sequence identification number: 63, and HVR-L3 comprising an amino acid sequence of sequence identification number: 74. In another embodiment, the antibody includes: HVR-H3, which includes the amino acid sequence at sequence identification number 63; HVR-L3, which includes the amino acid sequence at sequence identification number 74; and HVR-H2, which includes the amino acid sequence at sequence identification number 58. In yet another embodiment, the antibody includes (a) HVR-H1, which includes the amino acid sequence at sequence identification number 114; (b) HVR-H2, which includes the amino acid sequence at sequence identification number 58; and (c) HVR-H3, which includes the amino acid sequence at sequence identification number 63.

[0142] In another embodiment, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising an amino acid sequence of sequence identification number: 126; (b) HVR-H2 comprising an amino acid sequence of sequence identification number: 127; and (c) HVR-H3 comprising an amino acid sequence of sequence identification number: 128. In one embodiment, the antibody comprises HVR-H3 comprising an amino acid sequence of sequence identification number: 128. In another embodiment, the antibody comprises HVR-H3 comprising an amino acid sequence of sequence identification number: 128, and HVR-L3 comprising an amino acid sequence of sequence identification number: 131. In another embodiment, the antibody includes: HVR-H3, which includes the amino acid sequence at sequence identification number 128; HVR-L3, which includes the amino acid sequence at sequence identification number 131; and HVR-H2, which includes the amino acid sequence at sequence identification number 127. In yet another embodiment, the antibody includes (a) HVR-H1, which includes the amino acid sequence at sequence identification number 126; (b) HVR-H2, which includes the amino acid sequence at sequence identification number 127; and (c) HVR-H3, which includes the amino acid sequence at sequence identification number 128.

[0143] In another embodiment, the present invention provides an antibody comprising at least one, at least two, or all three selected from (a) an HVR-L1 comprising an amino acid sequence of any one of sequence identification numbers: 65-69, 122-124, 129; (b) an HVR-L2 comprising an amino acid sequence of any one of sequence identification numbers: 70-72, 125, 130; and (c) an HVR-L3 comprising an amino acid sequence of any one of sequence identification numbers: 73-74, 131. In one embodiment, the antibody includes (a) HVR-L1 comprising an amino acid sequence of any one of sequence identification numbers: 65-69, 122-124, 129; (b) HVR-L2 comprising an amino acid sequence of any one of sequence identification numbers: 70-72, 125, 130; and (c) HVR-L3 comprising an amino acid sequence of any one of sequence identification numbers: 73-74, 131.

[0144] In another embodiment, the present invention provides an antibody comprising at least one, at least two, or all three selected from (a) an HVR-L1 comprising an amino acid sequence of any one of sequence identification numbers: 65-69; (b) an HVR-L2 comprising an amino acid sequence of any one of sequence identification numbers: 70-72; and (c) an HVR-L3 comprising an amino acid sequence of any one of sequence identification numbers: 73-74. In one embodiment, the antibody comprises (a) an HVR-L1 comprising an amino acid sequence of any one of sequence identification numbers: 65-69; (b) an HVR-L2 comprising an amino acid sequence of any one of sequence identification numbers: 70-72; and (c) an HVR-L3 comprising an amino acid sequence of any one of sequence identification numbers: 73-74.

[0145] In another embodiment, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) an HVR-L1 comprising an amino acid sequence of any one of sequence identification numbers: 122-124; (b) an HVR-L2 comprising an amino acid sequence of sequence identification number: 125; and (c) an HVR-L3 comprising an amino acid sequence of any one of sequence identification numbers: 73-74. In one embodiment, the antibody comprises (a) an HVR-L1 comprising an amino acid sequence of any one of sequence identification numbers: 122-124; (b) an HVR-L2 comprising an amino acid sequence of sequence identification number: 125; and (c) an HVR-L3 comprising an amino acid sequence of any one of sequence identification numbers: 73-74. In another embodiment, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) an amino acid sequence comprising sequence identification number 122; (b) an amino acid sequence comprising sequence identification number 71; and (c) an amino acid sequence comprising sequence identification number 74. In one embodiment, the antibody comprises (a) an amino acid sequence comprising sequence identification number 122; (b) an amino acid sequence comprising sequence identification number 71; and (c) an amino acid sequence comprising any one of sequence identification number 74. In another embodiment, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) an amino acid sequence comprising sequence identification number 123; (b) an amino acid sequence comprising sequence identification number 71; and (c) an amino acid sequence comprising sequence identification number 74. In one embodiment, the antibody comprises (a) an amino acid sequence comprising sequence identification number 123; (b) an amino acid sequence comprising sequence identification number 71; and (c) an amino acid sequence comprising any one of sequence identification number 74.

[0146] In another embodiment, the present invention provides an antibody comprising at least one, at least two, or all three selected from (a) an HVR-L1 comprising an amino acid sequence of sequence identification number 129; (b) an HVR-L2 comprising an amino acid sequence of sequence identification number 130; and (c) an HVR-L3 comprising an amino acid sequence of sequence identification number 131. In one embodiment, the antibody comprises (a) an HVR-L1 comprising an amino acid sequence of sequence identification number 129; (b) an HVR-L2 comprising an amino acid sequence of sequence identification number 130; and (c) an HVR-L3 comprising any of the amino acid sequences of sequence identification number 131.

[0147] In another embodiment, the antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) an amino acid sequence comprising any one of sequence identification numbers 55-57, 114, 115, 126; (ii) an amino acid sequence comprising any one of sequence identification numbers 58-60, 116-120, 127; and (iii) an amino acid sequence comprising any one of sequence identification numbers 61-64, 121, 128; and (b) a VL domain comprising at least one, at least two, or all three VVR-L1 sequences selected from (i) an amino acid sequence comprising any one of sequence identification numbers 65-69, 122-124, 129; and (ii) an amino acid sequence comprising any one of sequence identification numbers 65-69, 122-124, 129. HVR-L2 including any of the amino acid sequences in sequence identification numbers 70-72, 125, and 130, and (c) VL HVR sequences of HVR-L3 including any of the amino acid sequences in sequence identification numbers 73-74 and 131.

[0148] In another embodiment, the antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising any amino acid sequence of sequence identification numbers 55-57, (ii) HVR-H2 comprising any amino acid sequence of sequence identification numbers 58-60, and (iii) HVR-H3 comprising any amino acid sequence of sequence identification numbers 61-64; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising any amino acid sequence of sequence identification numbers 65-69, (ii) HVR-L2 comprising any amino acid sequence of sequence identification numbers 70-72, and (c) HVR-L3 comprising any amino acid sequence of sequence identification numbers 73-74.

[0149] In another embodiment, the antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) an amino acid sequence comprising any one of sequence identification numbers 114-115, (ii) an amino acid sequence comprising any one of sequence identification numbers 116-120, and (iii) an amino acid sequence comprising an amino acid sequence comprising an amino acid sequence comprising an amino acid sequence comprising a ...

[0150] In another embodiment, the antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three selected from (i) HVR-H1 comprising an amino acid sequence of sequence identification number 114, (ii) HVR-H2 comprising an amino acid sequence of sequence identification number 58, and (iii) HVR-H3 comprising an amino acid sequence of sequence identification number 63; and (b) a VL domain comprising at least one, at least two, or all three selected from (i) HVR-L1 comprising an amino acid sequence of sequence identification number 122, (ii) HVR-L2 comprising an amino acid sequence of sequence identification number 71, and (c) HVR-L3 comprising an amino acid sequence of sequence identification number 74. In another embodiment, the antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising an amino acid sequence of sequence identification number 114, (ii) HVR-H2 comprising an amino acid sequence of sequence identification number 58, and (iii) HVR-H3 comprising an amino acid sequence of sequence identification number 63; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising an amino acid sequence of sequence identification number 123, (ii) HVR-L2 comprising an amino acid sequence of sequence identification number 71, and (c) HVR-L3 comprising an amino acid sequence of sequence identification number 74.

[0151] In another embodiment, the antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three selected from (i) HVR-H1 comprising an amino acid sequence of sequence identification number 126, (ii) HVR-H2 comprising an amino acid sequence of sequence identification number 127, and (iii) HVR-H3 comprising an amino acid sequence of sequence identification number 128; and (b) a VL domain comprising at least one, at least two, or all three selected from (i) HVR-L1 comprising an amino acid sequence of sequence identification number 129, (ii) HVR-L2 comprising an amino acid sequence of sequence identification number 130, and (c) HVR-L3 comprising an amino acid sequence of sequence identification number 131.

[0152] In another embodiment, the invention provides an antibody comprising: (a) HVR-H1 comprising an amino acid sequence comprising any one of sequence identification numbers: 55-57, 114-115, 126; (b) HVR-H2 comprising an amino acid sequence comprising any one of sequence identification numbers: 58-60, 116-120, 127; (c) HVR-H3 comprising an amino acid sequence comprising any one of sequence identification numbers: 61-64, 121, 128; (d) HVR-L1 comprising an amino acid sequence comprising any one of sequence identification numbers: 65-69, 122-124, 129; (e) HVR-L2 comprising an amino acid sequence comprising any one of sequence identification numbers: 70-72, 125, 130; and (f) HVR-L3 includes any of the amino acid sequences in sequence identification numbers 73-74 and 131.

[0153] In another embodiment, the invention provides an antibody comprising: (a) an HVR-H1 comprising an amino acid sequence of any one of sequence identification numbers 55-57 and 114-115; (b) an HVR-H2 comprising an amino acid sequence of any one of sequence identification numbers 58-60 and 116-120; (c) an HVR-H3 comprising an amino acid sequence of any one of sequence identification numbers 61-64 and 121; (d) an HVR-L1 comprising an amino acid sequence of any one of sequence identification numbers 65-69 and 122-124; (e) an HVR-L2 comprising an amino acid sequence of any one of sequence identification numbers 70-72 and 125; and (f) an HVR-L3 comprising an amino acid sequence of any one of sequence identification numbers 73-74.

[0154] In another embodiment, the invention provides an antibody comprising: (a) an HVR-H1 comprising an amino acid sequence of any one of sequence identification numbers: 114-115; (b) an HVR-H2 comprising an amino acid sequence of any one of sequence identification numbers: 116-120; (c) an HVR-H3 comprising an amino acid sequence of sequence identification number: 121; (d) an HVR-L1 comprising an amino acid sequence of any one of sequence identification numbers: 122-124; (e) an HVR-L2 comprising an amino acid sequence of sequence identification number: 125; and (f) an HVR-L3 comprising an amino acid sequence of any one of sequence identification numbers: 73-74. In another embodiment, the invention provides an antibody comprising: (a) HVR-H1 comprising an amino acid sequence of sequence identification number 114; (b) HVR-H2 comprising an amino acid sequence of sequence identification number 58; (c) HVR-H3 comprising an amino acid sequence of sequence identification number 63; (d) HVR-L1 comprising an amino acid sequence of sequence identification number 122; (e) HVR-L2 comprising an amino acid sequence of sequence identification number 71; and (f) HVR-L3 comprising an amino acid sequence of sequence identification number 74. In another embodiment, the invention provides an antibody comprising: (a) HVR-H1 comprising an amino acid sequence of sequence identification number 114; (b) HVR-H2 comprising an amino acid sequence of sequence identification number 58; (c) HVR-H3 comprising an amino acid sequence of sequence identification number 63; (d) HVR-L1 comprising an amino acid sequence of sequence identification number 123; (e) HVR-L2 comprising an amino acid sequence of sequence identification number 71; and (f) HVR-L3 comprising an amino acid sequence of sequence identification number 74.

[0155] In another embodiment, the invention provides an antibody comprising: (a) an HVR-H1 comprising an amino acid sequence of sequence identification number 126; (b) an HVR-H2 comprising an amino acid sequence of sequence identification number 127; (c) an HVR-H3 comprising an amino acid sequence of sequence identification number 128; (d) an HVR-L1 comprising an amino acid sequence of sequence identification number 129; (e) an HVR-L2 comprising an amino acid sequence of sequence identification number 130; and (f) an HVR-L3 comprising an amino acid sequence of sequence identification number 131.

[0156] In some specific embodiments, any one or more amino acids of the anti-myosin antibody provided above are substituted at the following HVR positions: (a) at positions 1 and 2 in HVR-H1 (sequence identification number: 55); (b) at positions 4, 7, 8, 10, 11, 12 and 16 in HVR-H2 (sequence identification number: 58); (c) at positions 5, 7 and 11 in HVR-H3 (sequence identification number: 61); (d) at positions 1, 2, 5, 7, 8 and 9 in HVR-L1 (sequence identification number: 65); (e) at positions 3 and 7 in HVR-L2 (sequence identification number: 70); and (f) at position 8 in HVR-L3 (sequence identification number: 73).

[0157] In some specific embodiments, one or more amino acid substitutions of the antimyosin antibody are conservative substitutions, as provided herein. In some specific embodiments, any one or more of the following substitutions may be performed in any combination: (a) in HVR-H1 (sequence identification number: 55), S1H; Y2T, D or E; (b) in HVR-H2 (sequence identification number: 58), Y4H; S7K; T8M or K; Y10K; A11M or E; S12E; G16K; (c) in HVR-H3 (sequence identification number: 61), Y5H; T7H; L11K; (d) in HVR-L1 (sequence identification number: 65), Q1T, S2T; S5E; Y7F; D8H; N9D or A or E; (e) in HVR-L2 (sequence identification number: 70), S3E; S7Y or F or W; and (f) in HVR-L3 (sequence identification number: 73), L8R.

[0158] All possible combinations of the above substitutions are covered by the sequence identification numbers 126, 127, 128, 129, 130 and 131 of the common sequences of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 respectively.

[0159] In any of the above embodiments, the anti-myosin antibody may be humanized. In one embodiment, the anti-myosin antibody includes the HVR as in any of the above embodiments, and further includes a receptor human framework, such as a human immunoglobulin framework or a human common framework. In another embodiment, the anti-myosin antibody includes the HVR as in any of the above embodiments, and further includes VH or VL, which includes an FR sequence. In yet another embodiment, the anti-myosin antibody includes the following heavy chain and / or light chain variable domain FR sequences. For the heavy chain variable domain, FR1 includes any amino acid sequence of sequence identification numbers 132-134, FR2 includes any amino acid sequence of sequence identification numbers 135-136, FR3 includes an amino acid sequence of sequence identification number 137, and FR4 includes an amino acid sequence of sequence identification number 138. For the light chain variable domain, FR1 includes the amino acid sequence with sequence identification number 139, FR2 includes any amino acid sequence with sequence identification numbers 140-141, FR3 includes any amino acid sequence with sequence identification numbers 142-143, and FR4 includes the amino acid sequence with sequence identification number 144.

[0160] In one embodiment, the invention provides an anti-myosin antibody comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising an amino acid sequence of any one of sequence identification numbers: 157-162; (b) HVR-H2 comprising an amino acid sequence of any one of sequence identification numbers: 163-168; (c) HVR-H3 comprising an amino acid sequence of any one of sequence identification numbers: 169-174; (d) HVR-L1 comprising an amino acid sequence of any one of sequence identification numbers: 175-180; (e) HVR-L2 comprising an amino acid sequence of any one of sequence identification numbers: 181-186; and (f) HVR-L3 comprising an amino acid sequence of any one of sequence identification numbers: 187-192.

[0161] In one embodiment, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising an amino acid sequence of any one of sequence identification numbers: 157-162; (b) HVR-H2 comprising an amino acid sequence of any one of sequence identification numbers: 163-168; and (c) HVR-H3 comprising an amino acid sequence of any one of sequence identification numbers: 169-174. In one embodiment, the antibody comprises HVR-H3 comprising an amino acid sequence of any one of sequence identification numbers: 169-174. In another embodiment, the antibody comprises: HVR-H3 comprising an amino acid sequence of any one of sequence identification numbers: 169-174, and HVR-L3 comprising an amino acid sequence of any one of sequence identification numbers: 187-192. In another embodiment, the antibody includes: HVR-H3, which includes an amino acid sequence of any one of sequence identification numbers 169-174; HVR-L3, which includes an amino acid sequence of any one of sequence identification numbers 187-192; and HVR-H2, which includes an amino acid sequence of any one of sequence identification numbers 163-168. In yet another embodiment, the antibody includes (a) HVR-H1, which includes an amino acid sequence of any one of sequence identification numbers 157-162; (b) HVR-H2, which includes an amino acid sequence of any one of sequence identification numbers 163-168; and (c) HVR-H3, which includes an amino acid sequence of any one of sequence identification numbers 169-174.

[0162] In another embodiment, the antibody of the invention comprises at least one, at least two, or all three selected from (a) an HVR-L1 comprising an amino acid sequence of any one of sequence identification numbers: 175-180; (b) an HVR-L2 comprising an amino acid sequence of any one of sequence identification numbers: 181-186; and (c) an HVR-L3 comprising an amino acid sequence of any one of sequence identification numbers: 187-192. In one embodiment, the antibody comprises (a) an HVR-L1 comprising an amino acid sequence of any one of sequence identification numbers: 175-180; (b) an HVR-L2 comprising an amino acid sequence of any one of sequence identification numbers: 181-186; and (c) an HVR-L3 comprising an amino acid sequence of any one of sequence identification numbers: 187-192.

[0163] In another embodiment, the antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising any amino acid sequence of sequence identification numbers 157-162, (ii) HVR-H2 comprising any amino acid sequence of sequence identification numbers 163-168, and (iii) HVR-H3 comprising any amino acid sequence of sequence identification numbers 169-174; and (b) a VL domain comprising at least one, at least two, or all three VVR-L1 comprising any amino acid sequence of sequence identification numbers 175-180, (ii) HVR-L2 comprising any amino acid sequence of sequence identification numbers 181-186, and (c) The VL HVR sequence of HVR-L3 includes any amino acid sequence in sequence identification numbers 187-192.

[0164] In another embodiment, the invention provides an antibody comprising (a) an HVR-H1 comprising an amino acid sequence of any one of sequence identification numbers 157-162; (b) an HVR-H2 comprising an amino acid sequence of any one of sequence identification numbers 163-168; (c) an HVR-H3 comprising an amino acid sequence of any one of sequence identification numbers 169-174; (d) an HVR-L1 comprising an amino acid sequence of any one of sequence identification numbers 175-180; (e) an HVR-L2 comprising an amino acid sequence of any one of sequence identification numbers 181-186; and (f) an HVR-L3 comprising an amino acid sequence of any one of sequence identification numbers 187-192.

[0165] In another embodiment, the anti-myosin antibody includes a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to any of the amino acid sequences in sequence identification numbers 13, 16-30, 32-34, and 86-95. In some specific embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity comprises substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-myosin antibody including that sequence retains the ability to bind to myosin. In some specific embodiments, a total of 1 to 10 amino acids in any of sequence identification numbers 13, 16-30, and 32-34 are substituted, inserted, and / or deleted. In some specific embodiments, substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the antimyosin antibody includes a VH sequence of any one of sequence identification numbers 13, 16-30, and 32-34, which contains a post-translational modification of that sequence. In one specific embodiment, the VH includes one, two, or three HVRs selected from: (a) HVR-H1 comprising an amino acid sequence of any one of sequence identification numbers 55-57, (b) HVR-H2 comprising an amino acid sequence of any one of sequence identification numbers 58-60, and (c) HVR-H3 comprising an amino acid sequence of any one of sequence identification numbers 61-64.

[0166] In another embodiment, the anti-myosin antibody includes a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to any of the amino acid sequences in sequence identification numbers 13, 16-30, 32-34, and 86-95. In some specific embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity comprises substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-myosin antibody including that sequence retains the ability to bind to myosin. In some specific embodiments, a total of 1 to 10 amino acids in any of sequence identification numbers 13, 16-30, 32-34, and 86-95 are substituted, inserted, and / or deleted. In some specific embodiments, substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the antimyosin antibody includes a VH sequence of any one of sequence identification numbers: 13, 16-30, 32-34, and 86-95, which contains a post-translational modification of that sequence. In one specific embodiment, the VH includes one, two, or three HVRs selected from: (a) HVR-H1 including an amino acid sequence of any one of sequence identification numbers: 55-57, 114-115, and 126; (b) HVR-H2 including an amino acid sequence of any one of sequence identification numbers: 58-60, 116-120, and 127; and (c) HVR-H3 including an amino acid sequence of any one of sequence identification numbers: 61-64, 121, and 128.

[0167] In another embodiment, the anti-myosin antibody includes a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to any of the amino acid sequences in sequence identification numbers 13, 16-30, 32, 33, and 34. In some specific embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity comprises substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-myosin antibody including that sequence retains the ability to bind to myosin. In some specific embodiments, a total of 1 to 10 amino acids in any of sequence identification numbers 13, 16-30, 32, 33, and 34 are substituted, inserted, and / or deleted. In some specific embodiments, substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the antimyosin antibody includes a VH sequence of any one of sequence identification numbers 13, 16-30, 32, 33, and 34, which contains a post-translational modification of that sequence. In one specific embodiment, the VH includes one, two, or three HVRs selected from: (a) HVR-H1 comprising an amino acid sequence of any one of sequence identification numbers 55-57, (b) HVR-H2 comprising an amino acid sequence of any one of sequence identification numbers 58-60, and (c) HVR-H3 comprising an amino acid sequence of any one of sequence identification numbers 61-64.

[0168] In another embodiment, the anti-myosin antibody includes a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to any of the amino acid sequences in sequence identification numbers 86-95. In some specific embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity comprises substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-myosin antibody including that sequence retains the ability to bind to myosin. In some specific embodiments, in any of sequence identification numbers 86-95, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. In some specific embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-myosin antibody includes a VH sequence of any one of sequence identification numbers 86-95, which contains a post-translational modification of that sequence. In a particular embodiment, the VH includes one, two, or three HVRs selected from: (a) HVR-H1 including an amino acid sequence of any one of sequence identification numbers 114-115, 126; (b) HVR-H2 including an amino acid sequence of any one of sequence identification numbers 116-120, 127; and (c) HVR-H3 including an amino acid sequence of any one of sequence identification numbers 121, 128.

[0169] In another embodiment, the anti-myosin antibody includes a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of sequence identification number 86. In some specific embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity comprises substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the anti-myosin antibody including that sequence retains the ability to bind to myosin. In some specific embodiments, a total of 1 to 10 amino acids in sequence identification number 86 are substituted, inserted, and / or deleted. In some specific embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-myosin antibody includes a VH sequence of sequence identification number 86, which contains a post-translational modification of that sequence. In a particular embodiment, the VH includes one, two, or three HVRs, selected from: (a) HVR-H1 including the amino acid sequence of sequence identification number 114, (b) HVR-H2 including the amino acid sequence of sequence identification number 58, and (c) HVR-H3 including the amino acid sequence of sequence identification number 63.

[0170] In another embodiment, the anti-myosin antibody includes a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of sequence identification number: 92. In some specific embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity comprises substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the anti-myosin antibody including that sequence retains the ability to bind to myosin. In some specific embodiments, a total of 1 to 10 amino acids in sequence identification number: 92 are substituted, inserted, and / or deleted. In some specific embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-myosin antibody includes the VH sequence of sequence identification number 92, which contains a post-translational modification of that sequence. In a particular embodiment, the VH includes one, two, or three HVRs, selected from: (a) HVR-H1 including the amino acid sequence of sequence identification number 114, (b) HVR-H2 including the amino acid sequence of sequence identification number 58, and (c) HVR-H3 including the amino acid sequence of sequence identification number 63.

[0171] In another embodiment, an anti-myosin antibody is provided, wherein the antibody comprises a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the amino acid sequences of sequence identification numbers 15, 31, 35-38, and 96-99. In some specific embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity comprises substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-myosin antibody including that sequence retains the ability to bind to myosin. In some specific embodiments, a total of 1 to 10 amino acids in any of sequence identification numbers 15, 31, 35-38, and 96-99 are substituted, inserted, and / or deleted. In some specific embodiments, substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the antimyosin antibody includes a VL sequence of any one of sequence identification numbers: 15, 31, 35-38, and 96-99, which contains a post-translational modification of that sequence. In one specific embodiment, the VL includes one, two, or three HVRs selected from: (a) HVR-L1 including an amino acid sequence of any one of sequence identification numbers: 65-69, 122-124, and 129; (b) HVR-L2 including an amino acid sequence of any one of sequence identification numbers: 70-72, 125, and 130; and (c) HVR-L3 including an amino acid sequence of any one of sequence identification numbers: 73-74 and 131.

[0172] In another embodiment, an anti-myosin antibody is provided, wherein the antibody comprises a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the amino acid sequences of sequence identification numbers 15, 31, 35, 36, 37, and 38. In some specific embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity comprises substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-myosin antibody including that sequence retains the ability to bind to myosin. In some specific embodiments, a total of 1 to 10 amino acids in any of sequence identification numbers 15, 31, 35, 36, 37, and 38 are substituted, inserted, and / or deleted. In some specific embodiments, substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the antimyosin antibody includes a VL sequence of any one of sequence identification numbers 15, 31, 35, 36, 37, and 38, which contains a post-translational modification of that sequence. In one specific embodiment, the VL includes one, two, or three HVRs selected from: (a) HVR-L1 comprising an amino acid sequence of any one of sequence identification numbers 65-69, (b) HVR-L2 comprising an amino acid sequence of any one of sequence identification numbers 70-72, and (c) HVR-L3 comprising an amino acid sequence of any one of sequence identification numbers 73-74.

[0173] In another embodiment, an anti-myosin antibody is provided, wherein the antibody comprises a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the amino acid sequences in sequence identification numbers 96-99. In some specific embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity comprises substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-myosin antibody including that sequence retains the ability to bind to myosin. In some specific embodiments, a total of 1 to 10 amino acids in any of sequence identification numbers 96-99 are substituted, inserted, and / or deleted. In some specific embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR. Optionally, the anti-myosin antibody includes a VL sequence of any one of sequence identification numbers 96-99, which contains a post-translational modification of that sequence. In a particular embodiment, the VL includes one, two, or three HVRs selected from: (a) HVR-L1 including an amino acid sequence of any one of sequence identification numbers 122-124, 129; (b) HVR-L2 including an amino acid sequence of any one of sequence identification numbers 125, 130; and (c) HVR-L3 including an amino acid sequence of sequence identification number 131.

[0174] In another embodiment, an anti-myosin antibody is provided, wherein the antibody comprises a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of Sequence Identification Number: 96. In some specific embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity comprises substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-myosin antibody comprising that sequence retains the ability to bind to myosin. In some specific embodiments, a total of 1 to 10 amino acids in Sequence Identification Number: 96 are substituted, inserted, and / or deleted. In some specific embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR. Optionally, the anti-myosin antibody comprises the VL sequence of Sequence Identification Number: 96, which includes post-translational modifications of that sequence. In one particular embodiment, VL includes one, two, or three HVRs, selected from: (a) HVR-L1 comprising the amino acid sequence of sequence identification number 122, (b) HVR-L2 comprising the amino acid sequence of sequence identification number 71, and (c) HVR-L3 comprising the amino acid sequence of sequence identification number 74. In another embodiment, an anti-myosin antibody is provided, wherein the antibody comprises VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of sequence identification number 97. In some specific embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, relative to the reference sequence, includes substitutions (e.g., conserved substitutions), insertions, or deletions, but the anti-myosin antibody including that sequence retains the ability to bind to myosin. In some specific embodiments, in sequence identification number 97, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. In some specific embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR. Optionally, the anti-myosin antibody includes the VL sequence of sequence identification number 97, which includes post-translational modifications of that sequence. In a particular embodiment, VL includes one, two, or three HVRs selected from: (a) HVR-L1 including the amino acid sequence of sequence identification number 123, (b) HVR-L2 including the amino acid sequence of sequence identification number 71, and (c) HVR-L3 including the amino acid sequence of sequence identification number 74.

[0175] In another embodiment, an anti-myosin antibody is provided, wherein the antibody comprises the VH sequence as described in any of the embodiments provided above, and the VL sequence as described in any of the embodiments provided above. In one embodiment, the antibody comprises a VH sequence of any one of sequence identification numbers 13, 16-30, 32-34, and 86-95, and a VL sequence of any one of sequence identification numbers 15, 31, 35-38, and 96-99, comprising post-translational modifications of those sequences. In one embodiment, the antibody comprises a VH sequence of any one of sequence identification numbers 13, 16-30, 32-34, and 86-95, and a VL sequence of any one of sequence identification numbers 15, 31, 35-38, and 96-99, comprising post-translational modifications of those sequences. In one embodiment, the antibody comprises VH sequences of sequence identification numbers 86-59 and VL sequences of sequence identification numbers 96-90, which contain post-translational modifications of those sequences.

[0176] In another embodiment, an anti-myosin antibody is provided, wherein the antibody comprises the VH sequence as provided in any of the embodiments described above, and the VL sequence as provided in any of the embodiments described above. In one embodiment, the antibody comprises a VH sequence of any one of sequence identification numbers 13, 16-30, and 32-34, and a VL sequence of any one of sequence identification numbers 15, 3, and 35-38, comprising post-translational modifications of those sequences. In one embodiment, the antibody comprises a VH sequence of any one of sequence identification numbers 13, 16-30, and 32-34, and a VL sequence of any one of sequence identification numbers 15, 3, and 35-38, comprising post-translational modifications of those sequences.

[0177] In another embodiment, an anti-myosin antibody is provided, wherein the antibody comprises the VH as described in any of the embodiments provided above, and the VL as described in any of the embodiments provided above. In one embodiment, the antibody comprises a VH sequence of any one of sequence identification numbers 86-95 and a VL sequence of any one of sequence identification numbers 96-99, respectively, and includes post-translational modifications of those sequences. In one embodiment, the antibody comprises a VH sequence of any one of sequence identification numbers 86-95 and a VL sequence of any one of sequence identification numbers 96-99, respectively, and includes post-translational modifications of those sequences. In one embodiment, the antibody comprises a VH sequence of any one of sequence identification numbers 86-95 and a VL sequence of any one of sequence identification numbers 96-99, respectively, and includes post-translational modifications of those sequences. In another embodiment, an anti-myosin antibody is provided, wherein the antibody comprises the VH as described in any of the embodiments provided above, and the VL as described in any of the embodiments provided above. In one embodiment, the antibody comprises a VH sequence of sequence identification number 86 and a VL sequence of sequence identification number 96, wherein the VH sequence comprises a VH sequence of sequence identification number 92 and a VL sequence of sequence identification number 97, wherein the VL ...

[0178] In another embodiment, an anti-myosin antibody is provided, wherein the antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to any of the amino acid sequences in sequence identification numbers 12, 145-150. In some specific embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity comprises substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-myosin antibody including that sequence retains the ability to bind to myosin. In some specific embodiments, a total of 1 to 10 amino acids in any of sequence identification numbers 12, 145-150 are substituted, inserted, and / or deleted. In some specific embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-myosin antibody includes a VH sequence of any one of sequence identification numbers 12, 145-150, which contains a post-translational modification of that sequence. In a particular embodiment, the VH includes one, two, or three HVRs selected from: (a) HVR-H1 including an amino acid sequence of any one of sequence identification numbers 55, 157-162; (b) HVR-H2 including an amino acid sequence of any one of sequence identification numbers 58, 163-168; and (c) HVR-H3 including an amino acid sequence of any one of sequence identification numbers 61, 169-174.

[0179] In another embodiment, an anti-myosin antibody is provided, wherein the antibody includes a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the amino acid sequences in sequence identification numbers 14, 151-156. In some specific embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity comprises substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-myosin antibody including that sequence retains the ability to bind to myosin. In some specific embodiments, a total of 1 to 10 amino acids in any of sequence identification numbers 14, 151-156 are substituted, inserted, and / or deleted. In some specific embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-myosin antibody includes a VL sequence of any one of sequence identification numbers 14 and 151-156, which contains a post-translational modification of that sequence. In a particular embodiment, the VL includes one, two, or three HVRs selected from: (a) HVR-L1 including an amino acid sequence of any one of sequence identification numbers 65 and 175-180, (b) HVR-L2 including an amino acid sequence of any one of sequence identification numbers 70 and 181-186, and (c) HVR-L3 including an amino acid sequence of any one of sequence identification numbers 73 and 187-192.

[0180] In another embodiment, an anti-myosin antibody is provided, wherein the antibody comprises VH as in any of the embodiments provided above, and VL as in any of the embodiments provided above. In one embodiment, the antibody comprises a VH sequence of any one of sequence identification numbers 12, 145-150 and a VL sequence of any one of sequence identification numbers 14, 151-156, respectively, containing post-translational modifications of those sequences.

[0181] In some specific embodiments, the antimyosin antibody of the present invention comprises VH as provided in any of the embodiments above, and a heavy chain constant region comprising an amino acid sequence comprising any of sequence identification numbers 7, 9, 11, 193, 195-198, 227, 228, 229-381. In some specific embodiments, the antimyosin antibody of the present invention comprises VL as provided in any of the embodiments above, and a light chain constant region comprising an amino acid sequence comprising any of sequence identification numbers 8 and 10.

[0182] In yet another embodiment, the invention provides an antibody that binds to the same antigenic determinant as the anti-myosin antibody provided herein. In yet another embodiment, the invention provides an antibody that binds to the same antigenic determinant as the antibody described in Table 2a. In yet another embodiment, the invention provides an antibody that binds to the same antigenic determinant as the antibody described in Table 11a or 13. In some specific embodiments, the provided antibody binds to the antigenic determinant of a fragment of myosinogen peptide composed of amino acids 21-100 of sequence identification number 78. Alternatively, the antibody binds to a fragment of myosinogen peptide composed of amino acids 21-80, 41-100, 21-60, 41-80, 61-100, 21-40, 41-60, 61-80, or 81-100 of sequence identification number 78.

[0183] In yet another embodiment, the anti-myosin antibody according to any of the foregoing embodiments is a monoclonal antibody comprising chimeric, humanized, or human antibodies. In one embodiment, the anti-myosin antibody is an antibody fragment, such as Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length IgG antibody, such as a complete IgG1 or IgG4 antibody, or other antibody species or isotypes as defined herein.

[0184] In another sample, the anti-myosin antibody according to any of the above embodiments may incorporate any feature, either alone or in combination, as described in sections 1-7 below. 1. Antibody Affinity

[0185] In some specific embodiments, the antibodies provided herein have a dissociation constant (Kd) of ≤1 µm, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸ M or less, e.g., from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M).

[0186] In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed in the form of Fab of the antibody of interest and its antigen. For example, the solution binding affinity of Fab to the antigen is measured by equilibrating Fab with the minimum concentration of (125I)-labeled antigen in the presence of a titration series of unlabeled antigen, and then capturing the bound antigen with a disc coated with anti-Fab antibody (see, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the assay conditions, 5 μg / ml capture with anti-Fab antibody (Cappel Labs) coated MICROTITER® discs (Thermo Scientific) in 50 mM sodium carbonate (pH 9.6) overnight is followed by blocking with 2% (w / v) bovine serum albumin in PBS at room temperature (approximately 23°C) for 2 to 5 hours. In a non-adsorbent plate (Nunc#269620), 100 pM or 26 pM [125I]-antigen was mixed with serially diluted Fabs of interest (e.g., consistent with the evaluation of anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fabs of interest were then incubated overnight; however, incubation could be extended (e.g., approximately 65 hours) to ensure equilibration. The mixture was then transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution was then removed and the plate was washed eight times with PBS containing 0.1% polysorbate 20 (TWEEN-20®). Once the plate was dry, 150 μl / well of scintillation buffer (MICROSCINT-20™; Packard) was added, and the plate was counted for 10 minutes using a TOPCOUNT™ gamma counter (Packard). Each Fab was selected at a concentration less than or equal to 20% of its maximum binding for competitive binding assays.

[0187] According to another embodiment, Kd is measured using the BIACORE® surface plasma resonance assay. For example, the assay is performed at 25°C with an immobilized antigen CM5 wafer at approximately 10 response units (RU) using the BIACORE®-2000 or BIACORE®-3000 (BIACORE®, Inc., Piscataway, NJ) assay. In one embodiment, the carboxymethylated polydextrose biosensor wafer (CM5, BIACORE®, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Before injecting the conjugated protein at a flow rate of 5 μl / min to obtain approximately 10 response units (RU), the antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, serially diluted Fab (0.78 nM to 500 nM) was injected at approximately 25 μl / min into PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20™) surfactant at 25 °C. Binding rate (kon) and dissociation rate (koff) were calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting binding and dissociation sensor maps. The equilibrium dissociation constant (Kd) was calculated as a ratio of koff / kon. Please refer, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If, according to the above-mentioned surface plasma resonance assay, the binding rate exceeds 10⁶ M⁻¹ s⁻¹, then the binding rate is determined using fluorescence quenching technology. This is measured at 25°C at pH 7.2, by the increase or decrease in the fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS. The measurement is performed in the presence of an increase in antigen concentration using a spectrometer, such as an Aviv Instruments spectrophotometer equipped with a stop-flow device or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirring colorimetric tube. 2. Antibody fragments

[0188] In some specific embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, and other fragments as described below. For a review of specific antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of specific scFv fragments, see, for example, Pluckthün, The Pharmacology of Monoclonal Antibodies, vol. 113, edited by Rosenburg and Moore, (Springer-Verlag, New York), pp. 269-315 (1994); also see WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that include salvage receptor-binding antigenic determinant residues and have an increased in vivo half-life, please refer to U.S. Patent No. 5,869,046.

[0189] A diobody is an antibody fragment with two antigen-binding sites. It can be bivalent or bispecific, see, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Tri- and tetra-antibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0190] A single-domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In some specific embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516).

[0191] Antibody fragments can be formed using a variety of techniques, including but not limited to the protein hydrolysis of intact antibodies, and production via recombinant host cells (e.g., E. coli or bacteriophages), as described herein. 3. Chimeric and humanized antibodies

[0192] In some specific embodiments, the antibodies provided herein are chimeric antibodies. Specific chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). In one example, the chimeric antibody includes a non-human variable region (e.g., a variable region derived from a mouse, rat, rodent, rabbit, or non-human primate, such as a monkey) and a human constant region. In yet another example, the chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been changed from that of the parent antibody. The chimeric antibody contains its antigen-binding fragment.

[0193] In some specific embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce their immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies include one or more variable domains, wherein the HVR, for example, the CDR (or a portion thereof) is derived from the non-human antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Humanized antibodies may also optionally include at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are replaced by residues corresponding to those in the non-human antibody (e.g., antibodies from which the HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0194] Humanized antibodies and methods for their preparation are reviewed in, for example, Almagro, Front. Biosci. 13:1619-1633 (2008), and further described in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (description of specificity-determining region (SDR) shift); Padlan, Mol. Immunol. 28:489-498 (1991). (Description of “Surface Remodeling”); Dall'Acqua et al., Methods36:43-60 (2005) (Description of “FR Remodeling”); and Osbourn et al., Methods36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (Description of “Directed Selection” Approach to FR Remodeling).

[0195] Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the “optimal” method (see, for example, Sims et al., J. Immunol. 151:2296 (1993); framework regions of the common sequence of human antibodies derived from the light or heavy chain variable regions of a specific subtype (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); and Presta et al., J. Immunol. 151:2623 (1993); human mature (somatic mutant) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR databases (see, for example, Baca et al., J. Biol. Chem. 272:10678-10684). (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)). 4. Human antibodies

[0196] In some specific embodiments, the antibodies provided herein are human antibodies. Human antibodies can be manufactured using various known techniques. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-374 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0197] Human antibodies can be prepared by administering an immunogen to genetically modified transgenic animals that produce complete human antibodies in response to antigen attack or complete antibodies with human variable regions. These animals typically contain all or part of the human immunoglobulin loci, which replace endogenous immunoglobulin loci, or are located extrachromosomally or randomly integrated into the animal's chromosome. In these transgenic mice, endogenous immunoglobulin loci are generally inactive. For a review of methods for obtaining human antibodies from transgenic animals, please refer to Lonberg, Nat. Biotech. 23:1117-1125 (2005). Please also refer to, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HuMab® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Publication No. US 2007 / 0061900, which describes VelociMouse® technology. The human variable region of the intact antibody produced by such animals can be further modified, for example, by combining it with different human constant regions.

[0198] Human antibodies can also be generated using fusion tumor-based methods. Human myeloma and mouse-human fusion myeloma cell lines used to generate human monoclonal antibodies have been described. (See, for example, Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol. 147:86 (1991). Human antibodies produced by human B-cell fusion tumor technology are also described in Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006). Other methods include those described, for example, U.S. Patent No. 7,189,826 (description of monoclonal human IgM antibody production from fusion tumor cell lines) and Ni, Xiandai Mianyixue 26(4):265-268 (2006).) (Methods describing human-human fusion tumors). Human fusion tumor technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology 27(3):185-191 (2005).

[0199] Human antibodies can also be generated by isolating variable domain sequences from Fv strains derived from human phage display databases. These variable domain sequences can then be combined with desired human constant domains. The technique for selecting human antibodies from antibody databases is described below. 5. Antibodies derived from databases

[0200] The antibodies of the present invention can be isolated by screening for antibodies with the desired activity in a combinatorial database. For example, various methods for generating phage display databases and screening for antibodies with the desired binding characteristics in such databases are known in the art. Such methods are reviewed in, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (2000); edited by O'Brien et al., Human Press, Totowa, NJ, 2001, and further in, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0201] In certain phage display methods, repertoires of the VH and VL genes are selected and randomly recombined into a phage database by polymerase chain reaction (PCR), and antigen-binding phages can then be screened from them, as described by Winter et al., Ann. Rev. Immunol. 12:433-455 (1994). Phages typically present antibody fragments in the form of single-stranded Fv (ScFv) fragments or Fab fragments. Databases derived from immune sources can provide high-affinity antibodies against immunogens without the need for fusion tumor construction. Alternatively, natural libraries can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-autoantigens and autoantigens without any immunization, as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, a natural database can also be synthetically generated by transfecting an unrearranged V gene fragment from stem cells and using a PCR primer containing a random sequence to encode the highly variable CDR3 region, and by achieving in vitro rearrangement, as described in Hoogenboom and Winter, J. Mol. Biol. 227:381-388 (1992). Patent publications describing human antibody phage databases include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publications Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0202] Antibodies or antibody fragments isolated from human antibody databases are considered human antibodies or human antibody fragments in this paper. 6. Multispecific antibodies

[0203] In some specific embodiments, the antibody provided herein is a multispecific antibody, such as a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different sites. In some specific embodiments, one of the binding specificities is against myosin and the other is against any other antigen. In some specific embodiments, a bispecific antibody may bind to two different antigenic determinants of myosin. Bispecific antibodies can also be used to target cytotoxic agents to cells expressing myosin. Bispecific antibodies may be formulated as full-length antibodies or antibody fragments.

[0204] Techniques for manufacturing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 1993 / 08829, and Traunecker et al., EMBO J.10:3655 (1991)), and “knob-in-hole” engineering (see, for example, US Patent No. 5,731,168). Multispecific antibodies can also be prepared by: engineering the electrostatic attraction of the Fc-heterodimer molecules used to prepare antibodies (WO 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, for example, US Patent No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); using “dual antibody” technology to generate bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, for example, Gruber et al., J. Immunol. 152:5368). (1994)); and the preparation of trispecific antibodies, such as those described in Tutt et al., J. Immunol. 147:60 (1991).

[0205] This article also includes engineered antibodies having three or more functional antigen-binding sites, including “octopus antibodies” (see, for example, US 2006 / 0025576A1).

[0206] The antibodies or fragments described herein also contain “dual-acting FAbs” or “DAFs,” which include antigen-binding sites that bind to both myosin and another distinct antigen (see, for example, US 2008 / 0069820). 7. Antibody Variants

[0207] In some specific embodiments, the amino acid sequence variants of the antibodies provided herein are contemplated. For example, enhancing the binding affinity and / or other biological properties of the antibody may be desirable. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be achieved in the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding. a. Substitution, Insertion, and Deletion Variants

[0208] In some specific embodiments, antibody variants with one or more amino acid substitutions are provided. The sites of interest for substitution mutagenesis include HVR and FR. Conserved substitutions are shown under the heading "Preferred Substitutions" in Table 1. More substantial changes are provided under the heading "Exemplary Substitutions" in Table 1, and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into antibodies of interest and screened into products with desired activities, such as retained / improved antibody binding, reduced immunogenicity, or improved ADCC or CDC. Table 1 Original residues Exemplary replacement Preferred replacement Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Leucine Leu Leu (L) Leucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Leucine Leu

[0209] Amino acids can be classified according to their common side-chain characteristics as: (1) hydrophobic: leucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will result in the exchange of members of one of these groups with members of another group.

[0210] Substitution variants are a class of variants involving the substitution of one or more highly variable residues in a parent antibody (e.g., a humanized or human antibody). Generally, the variants selected for further research will have modifications (e.g., improvements) in specific biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and / or will have substantially retained the specific biological properties of the parent antibody. Exemplary substitution variants are affinity-matured antibodies that can be readily generated, for example, using phage display-based affinity maturation techniques as described herein. In short, one or more HVR residues are mutated and a variant antibody is displayed on a phage, followed by screening for specific biological activities (e.g., binding affinity).

[0211] Modifications (e.g., substitutions) can be formed in HVRs to improve antibody affinity. Such modifications can be formed in HVR “hotspots,” which are residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008), and / or residues that contact the antigen, and the binding affinity of the resulting variants VH or VL can be tested. Affinity maturation through construction and reselection from a second database has been, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Press, Totowa, NJ). As described in (2001). In some embodiments of affinity maturation, diversity is introduced into the variant gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis). A second database is then generated. The database is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves HVR directed methods, in which several HVR residues (e.g., 4 to 6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, by using alanine scan mutagenesis or by establishing a model. CDR-H3 and CDR-L3 are particularly commonly targeted.

[0212] In some specific embodiments, substitution, insertion, or deletion may occur within one or more HVRs, provided that such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conserved modifications that do not substantially reduce binding affinity (e.g., conserved substitutions as provided herein) may be formed in the HVR. Such modifications may, for example, be outside the antigen-contacting residues in the HVR. In the specific embodiments of the variant VH and VL sequences provided above, each HVR is either unmodified or contains no more than one, two, or three amino acid substitutions.

[0213] A method for identifying residues or regions of antibodies that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells, Science 244:1081-1085 (1989). In this method, a residue or group of residues (e.g., charged residues such as arg, asp, his, lys, and glu) can be identified and substituted with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Further substitutions can be introduced at amino acid sites that show functional sensitivity to the initial substitution. Alternatively, or additionally, the crystal structure of the antigen-antibody complex identifies contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

[0214] Amino acid sequence inserts include amino- and / or carboxyl-terminal fusions of polypeptides ranging in length from one residue to 100 or more residues, and intra-sequence inserts of single or multiple amino acid residues. Examples of terminal inserts include antibodies having an N-terminal methionine residue. Other insertion variants of antibody molecules include fusions of the N- or C-terminus of the antibody with an enzyme (e.g., ADEPT) or polypeptide, which increase the serum half-life of the antibody. b. Glycation variants

[0215] In some specific embodiments, the antibodies provided herein are modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites on the antibody can be conveniently accomplished by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0216] When an antibody includes an Fc region, the sugars attached thereto can be modified. Naturally occurring antibodies produced by mammalian cells typically include branched, bitennary oligosaccharides, generally with an N-linked Asn297 attached to the CH2 domain of the Fc region, see, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can contain various sugars, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the “backbone” of the bitennary oligosaccharide structure. In some embodiments, oligosaccharide formation modifications can be made in the antibodies of the present invention to produce antibody variants with specific improved properties.

[0217] In one embodiment, the provided antibody variant has a glycostructure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all glycosyl structures attached to Asn297 (e.g., complexes, hybrids, and high-mannose structures) as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to the aspartic acid residue located approximately at position 297 (Eu number of the Fc region residue) in the Fc region; however, due to minor sequence variations in the antibody, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucoidan variants may have improved ADCC function, see, for example, U.S. Patent Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publicly available publications involving “defucosylated” or “fucose-deficient” antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Publication No. 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312, Adams et al., particularly in Example 11), and knockout cell lines such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda et al., Biotechnol. Bioeng. 94(4):680-688 (2006); and WO2003 / 085107).

[0218] Further, antibody variants having bisected oligosaccharides are provided, for example, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GICNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, WO 2003 / 011878 (Jean-Mairet et al.), US Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S); and WO 1999 / 22764 (Raju, S). c. Fc region variants

[0219] In some specific embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to generate Fc region variants. Fc region variants may include human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc regions) that include amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0220] In some specific embodiments, the present invention envisions antibody variants having some, but not all, effector functions, making them ideal candidates for applications where the in vivo half-life of the antibody is important, but specific effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. The main cells regulating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of molecules of interest are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see, Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA); and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood monocytes (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example in animal models, as disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Clq binding assays can also be performed to determine if the antibody cannot bind Clq and therefore lacks CDC activity. See, for example, Clq and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402.To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg et al., Blood 101:1045-1052 (2003); and Cragg, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, for example, Petkova et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0221] Antibodies with reduced effector function include those antibodies with substitutions in one or more of the residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions in two or more of the amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with residues 265 and 297 substituted with alanine (US Patent No. 7,332,581).

[0222] Specific antibody variants that have improved or weakened binding to FcR have been described (see, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0223] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that enhance ADCC, for example, substitutions at positions 298, 333 and / or 334 (EU numbers of residues) of the Fc region.

[0224] In some embodiments, alterations are formed in the Fc region that result in altered (i.e., improved or reduced) Clq binding and / or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent Nos. 6,194,551, WO 1999 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0225] Antibodies that exhibit increased half-life and improved binding to the neonatal Fc receptor (FcRn) are described in US2005 / 0014934 A1 (Hinton et al.), the neonatal Fc receptor being responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). These antibodies include one or more Fc regions substituted therein that improve the binding of the Fc regions to FcRn. Such Fc variants contain one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, with substitutions, such as substitution of Fc region residue 434 (US Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan, Nature 322:738-40 (1988); US Patent Nos. 5,648,260 and 5,624,821; and WO 1994 / 29351. d) Cysteine-modified antibody variants

[0226] In certain embodiments, it is desirable to generate cysteine-modified antibodies, such as "thioMAb," in which one or more residues of the antibody are replaced by cysteine ​​residues. In particular embodiments, the substituted residues are located in easily accessible positions on the antibody. By replacing those residues with cysteine, a reactive thiol group is placed in an easily accessible position on the antibody and can be used to conjugate the antibody to other parts, such as a pharmaceutical part or to link a pharmaceutical part, to create an immunoconjugate, as further described herein. In certain embodiments, any or more of the following residues may be replaced by cysteine: V205 (Kabat number) of the light chain; A118 (EU number) of the heavy chain; and S400 (EU number) of the Fc region of the heavy chain. Cysteine-modified antibodies can be generated, for example, as described in U.S. Patent No. 7,521,541. e) Antibody Derivatives

[0227] In some specific embodiments, the antibodies provided herein may be further modified to include additional non-protein moiety known in the art and readily available. Suitable moiety for antibody derivatization includes, but is not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, polydextrose, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homomers or random copolymers), and polydextrose or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. Polymers can have any molecular weight and can be branched or unbranched. The number of polymers attached to an antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on, but not limited to, considerations of the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used in therapy under defined conditions.

[0228] In another embodiment, an antibody and a conjugate of a non-protein portion that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-protein portion is carbon nanotubes (Kam et al., Proc. Natl. Acad. Sci. USA102:11600-11605 (2005)). The radiation may have any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat the non-protein portion to near the temperature at which the antibody-non-protein portion kills cells. 8. Mutant Fc region

[0229] In one state, the invention provides an isolated polypeptide comprising a variant Fc region having enhanced FcγRIIb binding activity. In some states, the polypeptide is an antibody. In some states, the polypeptide is an Fc fusion protein. In some specific embodiments, the variant Fc region comprises at least one amino acid residue modification (e.g., substitution) compared to the corresponding sequence in the Fc region of a natural or reference variant sequence (generally referred to herein collectively as the “parental” Fc region). In some specific embodiments, the variant Fc region of the invention has increased binding activity to monkey FcγRIIb compared to the parental Fc region. In some specific embodiments, the monkey FcγRIIb is rhesus monkey FcγRIIb (Sequence Identification Number: 223).

[0230] In some specific embodiments, the ratio of [KD value of the parental Fc region to monkey FcγRIIb] to [KD value of the variant Fc region to monkey FcγRIIb] can be 2.0 or greater, 3.0 or greater, 4.0 or greater, 5.0 or greater, 6.0 or greater, 7.0 or greater, 8.0 or greater, 9.0 or greater, 10 or greater, 15 or greater, 20 or greater, 25 or greater, 30 or greater, 40 or greater, or 50 or greater. In still other embodiments, the variant Fc region has reduced binding activity to monkey FcγRIIIa. In some specific embodiments, the ratio of [KD value of the parental Fc region to monkey FcγRIIIa] to [KD value of the variant Fc region to monkey FcγRIIIa] can be 0.50 or less, 0.40 or less, 0.30 or less, 0.20 or less, 0.10 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, or 0.01 or less. In some specific embodiments, monkey FcγRIIb has the sequence identification number 223 (rhesus monkey). In some specific embodiments, monkey FcγRIIIa has the sequence identification number 224 (rhesus monkey).

[0231] In some other embodiments, the variant Fc region exhibits enhanced binding activity to human FcγRIIb. In some specific embodiments, the ratio of [KD value of parental Fc region to human FcγRIIb] to [KD value of variant Fc region to human FcγRIIb] may be 2.0 or greater, 3.0 or greater, 4.0 or greater, 5.0 or greater, 6.0 or greater, 7.0 or greater, 8.0 or greater, 9.0 or greater, 10 or greater, 15 or greater, 20 or greater, 25 or greater, 30 or greater, 40 or greater, or 50 or greater. In some other embodiments, the variant Fc region exhibits reduced binding activity to human FcγRIIIa. In some specific embodiments, the ratio of [KD value of parental Fc region to human FcγRIIIa] to [KD value of variant Fc region to human FcγRIIIa] may be 0.50 or less, 0.40 or less, 0.30 or less, 0.20 or less, 0.10 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, or 0.01 or less. In some specific embodiments, human FcγRIIb has a sequence identification number of 212, 213, or 214. In some specific embodiments, human FcγRIIIa has a sequence identification number of 215, 216, 217, or 218.

[0232] In some other embodiments, the variant Fc region exhibits reduced binding activity to human FcγRIIa (H type). In some specific embodiments, the ratio of [KD value of parental Fc region to human FcγRIIa (H type)] to [KD value of variant Fc region to human FcγRIIa (H type)] may be 5.0 or less, 4.0 or less, 3.0 or less, 2.0 or less, 1.0 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In some other embodiments, the variant Fc region exhibits reduced binding activity to human FcγRIIa (R type). In some specific embodiments, the ratio of [KD value of parental Fc region to human FcγRIIa (R type)] to [KD value of variant Fc region to human FcγRIIa (R type)] can be 5.0 or less, 4.0 or less, 3.0 or less, 2.0 or less, 1.0 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In some specific embodiments, human FcγRIIa (H type) has a sequence with sequence identification number 211. In some specific embodiments, human FcγRIIa (R type) has a sequence with sequence identification number 210.

[0233] In some specific embodiments, the ratio of [KD value of parental Fc region to monkey FcγRIIa] / [KD value of variant Fc region to monkey FcγRIIa] can be 2.0 or greater, 3.0 or greater, 4.0 or greater, 5.0 or greater, 6.0 or greater, 7.0 or greater, 8.0 or greater, 9.0 or greater, 10 or greater, 15 or greater, 20 or greater, 25 or greater, 30 or greater, 40 or greater, or 50 or greater. In some specific embodiments, monkey FcγRIIa is selected from: monkey FcγRIIa1 (e.g., rhesus monkey FcγRIIa1 (sequence identification number: 220)), monkey FcγRIIa2 (e.g., rhesus monkey FcγRIIa2 (sequence identification number: 221)) and monkey FcγRIIa3 (e.g., rhesus monkey FcγRIIa3 (sequence identification number: 222)).

[0234] In another embodiment, the KD value of the variant Fc region for monkey FcγRIIb can be 1.0×10-6M or less, 9.0×10-7M or less, 8.0×10-7M or less, 7.0×10-7M or less, 6.0×10-7M or less, 5.0×10-7M or less, 4.0×10-7M or less, 3.0×10-7M or less, 2.0×10-7M or less, or 1.0×10-7M or less. In another embodiment, the KD value of the variant Fc region for monkey FcγRIIIa can be 5.0×10⁻⁷ M or greater, 6.0×10⁻⁷ M or greater, 7.0×10⁻⁷ M or greater, 8.0×10⁻⁷ M or greater, 9.0×10⁻⁷ M or greater, 1.0×10⁻⁶ M or greater, 2.0×10⁻⁶ M or greater, 3.0×10⁻⁶ M or greater, 4.0×10⁻⁶ M or greater, 5.0×10⁻⁶ M or greater, 6.0×10⁻⁶ M or greater, 7.0×10⁻⁶ M or greater, 8.0×10⁻⁶ M or greater, 9.0×10⁻⁶ M or greater, or 1.0×10⁻⁵ M or greater. In another embodiment, the KD value of the variant Fc region for human FcγRIIb can be 2.0×10⁻⁶ M or less, 1.0×10⁻⁶ M or less, 9.0×10⁻⁷ M or less, 8.0×10⁻⁷ M or less, 7.0×10⁻⁷ M or less, 6.0×10⁻⁷ M or less, 5.0×10⁻⁷ M or less, 4.0×10⁻⁷ M or less, 3.0×10⁻⁷ M or less, 2.0×10⁻⁷ M or less, or 1.0×10⁻⁷ M or less. In another embodiment, the KD value of the variant Fc region for human FcγRIIIa can be 1.0×10⁻⁶ M or greater, 2.0×10⁻⁶ M or greater, 3.0×10⁻⁶ M or greater, 4.0×10⁻⁶ M or greater, 5.0×10⁻⁶ M or greater, 6.0×10⁻⁶ M or greater, 7.0×10⁻⁶ M or greater, 8.0×10⁻⁶ M or greater, 9.0×10⁻⁶ M or greater, 1.0×10⁻⁵ M or greater, 2.0×10⁻⁵ M or greater, 3.0×10⁻⁵ M or greater, 4.0×10⁻⁵ M or greater, or 5.0×10⁻⁵ M or greater. In another embodiment, the KD value of the variant Fc region for human FcγRIIa (H type) can be 1.0×10⁻⁷ M or greater, 2.0×10⁻⁷ M or greater, 3.0×10⁻⁷ M or greater, 4.0×10⁻⁷ M or greater, 5.0×10⁻⁷ M or greater, 6.0×10⁻⁷ M or greater, 7.0×10⁻⁷ M or greater, 8.0×10⁻⁷ M or greater, 9.0×10⁻⁷ M or greater, 1.0×10⁻⁶ M or greater, 2.0×10⁻⁶ M or greater, 3.0×10⁻⁶ M or greater, 4.0×10⁻⁶ M or greater, or 5.0×10⁻⁶ M or greater.In another embodiment, the KD value of the variant Fc region for human FcγRIIa (R type) can be 2.0×10⁻⁷ M or greater, 3.0×10⁻⁷ M or greater, 4.0×10⁻⁷ M or greater, 5.0×10⁻⁷ M or greater, 6.0×10⁻⁷ M or greater, 7.0×10⁻⁷ M or greater, 8.0×10⁻⁷ M or greater, 9.0×10⁻⁷ M or greater, 1.0×10⁻⁶ M or greater, 2.0×10⁻⁶ M or greater, 3.0×10⁻⁶ M or greater, 4.0×10⁻⁶ M or greater, or 5.0×10⁻⁶ M or greater.

[0235] In another embodiment, the KD value of the variant Fc region for monkey FcγRIIa can be 1.0 × 10⁻⁶ M or less, 9.0 × 10⁻⁷ M or less, 8.0 × 10⁻⁷ M or less, 7.0 × 10⁻⁷ M or less, 6.0 × 10⁻⁷ M or less, 5.0 × 10⁻⁷ M or less, 4.0 × 10⁻⁷ M or less, 3.0 × 10⁻⁷ M or less, 2.0 × 10⁻⁷ M or less, or 1.0 × 10⁻⁷ M or less. In some specific embodiments, monkey FcγRIIa can be selected from any one of monkey FcγRIIa1, monkey FcγRIIa2, and monkey FcγRIIa3.

[0236] When developing pharmaceutical products for treating human diseases, it is important to evaluate their efficacy and safety in monkeys due to the biological similarity between monkeys and humans. Therefore, it is preferable that the pharmaceutical product to be developed exhibits cross-responsiveness in both humans and monkeys in its target binding activity.

[0237] “Fcγ receptor” (hereinafter referred to as Fcγ receptor, FcγR or FcgR) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3 and IgG4 monoclonal antibodies, and actually represents any member of the protein family encoded by the Fcγ receptor gene. In humans, this family includes FcγRI (CD64), which includes isoforms FcγRIa, FcγRIb and FcγRIc; FcγRII (CD32), which includes isoforms FcγRIIa (including isoforms H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16), which includes isoforms FcγRIIIa (including isoforms V158 and F158) and FcγRIIIb (including isoforms FcγRIIIb-NA1 and FcγRIIIb-NA2), and any undiscovered FcγRs, FcγR isoforms or isoforms, but is not limited thereto. FcγRIIb1 and FcγRIIb2 have been reported as splice variants of human FcγRIIb. Furthermore, a splice variant named FcγRIIb3 has been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splice variants, human FcγRIIb includes all splice variants registered with the NCBI, namely NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3. In addition, human FcγRIIb includes each previously reported genetic polymorphism as well as FcγRIIb (Arthritis Rheum. 48:3242-3252 (2003); Kono et al., Hum. Mol. Genet. 14:2881-2892 (2005); and Kyogoju et al., Arthritis Rheum. 46:1242-1254 (2002)) and each genetic polymorphism to be reported in the future.

[0238] In FcγRIIa, there are two isoforms, one of which is histidine at amino acid position 131 (H type), and the other is substituted with arginine at amino acid position 131 (R type) (Warrmerdam, J. Exp. Med. 172:19-25 (1990)).

[0239] FcγR includes, but is not limited to, human, mouse, rat, rabbit, and monkey-derived FcγRs, and may be derived from any of these organisms. Mouse FcγR includes, but is not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2) and any mouse FcγR or FcγR isoform. Unless otherwise stated, the term “monkey FcγR” or its variants refer to rhesus monkey FcγRIIa1 (sequence number: 220), FcγRIIa2 (sequence number: 221), FcγRIIa3 (sequence number: 222), FcγRIIb (sequence number: 223), or FcγRIIIaS (sequence number: 224).

[0240] The polynucleotide sequence of human FcγRI is shown in sequence identification number: 199 (NM_000566.3); the polynucleotide sequence of human FcγRIIa is shown in sequence identification number: 200 (BC020823.1) or sequence identification number: 201 (NM_001136219.1); the polynucleotide sequence of human FcγRIIb is shown in sequence identification number: 202 (BC146678.1) or sequence identification number: 203 (NM_004001.3); the polynucleotide sequence of human FcγRIIIa is shown in sequence identification number: 204 (BC033678.1) or sequence identification number: 205 (NM_001127593.1); and the polynucleotide sequence of human FcγRIIIb is shown in sequence identification number: 206 (BC128562.1).

[0241] The amino acid sequences of human FcγRI are shown in sequence identification number 207 (NP_000557.1); the amino acid sequences of human FcγRIIa are shown in sequence identification number 208 (AAH20823.1), sequence identification number 209, sequence identification number 210 or sequence identification number 211; the amino acid sequences of human FcγRIIb are shown in sequence identification number 212 (AAI46679.1), sequence identification number 213 or sequence identification number 214; the amino acid sequences of human FcγRIIIa are shown in sequence identification number 215 (AAH33678.1), sequence identification number 216, sequence identification number 217 or sequence identification number 218; and the amino acid sequences of human FcγRIIIb are shown in sequence identification number 219 (AAI28563.1).

[0242] The amino acid sequences of rhesus monkey FcγRIIa are shown in sequence identification number 220 (FcγRIIa1), sequence identification number 221 (FcγRIIa2), or sequence identification number 222 (FcγRIIa3); the amino acid sequences of rhesus monkey FcγRIIb are shown in sequence identification number 223; and the amino acid sequences of rhesus monkey FcγRIIIa are shown in sequence identification number 224.

[0243] In one embodiment, compared to a corresponding reference FcγRIIb binding polypeptide, the invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb binding activity. In still other embodiments, the polypeptide of the invention comprises at least one amino acid modification at at least one position selected from the group consisting of: 231, 232, 233, 234, 235, 236, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334, and 396, according to EU designations. In some specific embodiments, the FcγRIIb has the sequence of rhesus monkey FcγRIIb (sequence identification number: 223). In some specific embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., sequence identification number: 212, 213, or 214).

[0244] In one embodiment, the invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb binding activity, comprising at least two amino acid modifications, including: (a) an amino acid modification at position 236; and (b) at least one amino acid modification at a position selected from the group consisting of: 231, 232, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334, and 396, according to EU designations. In some specific embodiments, FcγRIIb has the sequence of rhesus monkey FcγRIIb (Sequence Identification Number: 223). In some specific embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., sequence identification number: 212, 213 or 214).

[0245] In one embodiment, the invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb binding activity, which includes an amino acid modification at position 236, according to EU designation.

[0246] In one embodiment, the invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb binding activity, comprising at least two amino acid modifications, including: (a) an amino acid modification at position 236; and (b) at least one amino acid modification at a position selected from the group consisting of 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396, according to EU designations. In another embodiment, the variant Fc region comprises an amino acid modification at a position selected from the group consisting of 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396, according to EU designations. In yet another embodiment, the mutated Fc region includes an amino acid modification located at at least one position chosen from the group consisting of 268, 295, 326, and 330, according to EU numbers. In some specific embodiments, FcγRIIb has the sequence of rhesus monkey FcγRIIb (sequence identification number: 223). In some specific embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., sequence identification numbers: 212, 213, or 214).

[0247] In another embodiment, the invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb binding activity, comprising any of the following amino acid modifications (1)-(37): (1) positions 231, 236, 239, 268 and 330; (2) positions 231, 236, 239, 268, 295 and 330; (3) positions 231, 236, 268 and 330; (4) positions 231, 236, 268, 295 and 330; (5) positions 232, 236, 239, 268, 295 and 330; (6) positions 232, 236, 268, 295 and 330; (7) positions 232, 236 (8) Positions 235, 236, 268, 295, 326 and 330; (9) Positions 235, 236, 268, 295 and 330; (10) Positions 235, 236, 268 and 330; (11) Positions 235, 236, 268, 330 and 396; (12) Positions 235, 236, 268 and 396; (13) Positions 236, 239, 268, 295, 298 and 330; (14) Positions 236, 239, 268, 295, 326 and 330; (15) Positions 236, 239, 268, 295 and 330; (16) Positions 236, 239, 268 (17) Positions 236, 239, 268, 326 and 330; (18) Positions 236, 239, 268 and 330; (19) Positions 236, 239, 268, 330 and 396; (20) Positions 236, 239, 268 and 396; (21) Positions 236 and 268; (22) Positions 236, 268 and 295; (23) Positions 236, 268, 295, 298 and 330; (24) Positions 236, 268, 295, 326 and 330; (25) Positions 236, 268, 295, 326, 330 and 396; (26) Positions 236, 268, 295 (27) Positions 236, 268, 295, 330 and 396; (28) Positions 236, 268, 298 and 330; (29) Positions 236, 268, 298 and 396; (30) Positions 236, 268, 326 and 330; (31) Positions 236, 268, 326, 330 and 396; (32) Positions 236, 268 and 330; (33) Positions 236, 268, 330 and 396; (34) Positions 236, 268 and 396; (35) Positions 236 and 295; (36) Positions 236, 330 and 396; and (37) Positions 236 and 396, according to EU number. In some specific embodiments, FcγRIIb has the sequence of rhesus monkey FcγRIIb (sequence identification number: 223).In some specific embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., sequence identification number: 212, 213 or 214).

[0248] In another embodiment, the variant Fc region having enhanced FcγRIIb binding activity includes at least one amino acid selected from: (a) Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 231; (b) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 232; (c) Asp at position 233; (d) Trp, Tyr (e) Trp is at position 234; (f) Ala, Asp, Glu, His, Ile, Leu, Met, Asn, Gln, Ser, Thr, Val are at position 236; (g) Asp, Tyr are at position 237; (h) Glu, Ile, Met, Gln, Tyr are at position 238; (i) Ile, Leu, Asn, Pro, Val are at position 239; (j) Ile is at position 264; (k) Phe is at position 266; (l) Ala, His, Leu are at position 267; (m) Asp, Glu are at position 268; (n) Asp, Glu, Gly are at position 271; (o) Leu is at position 295 The group consisting of (p) Leu at position 298; (q) Glu, Phe, Ile, and Leu at position 325; (r) Thr at position 326; (s) Ile and Asn at position 327; (t) Thr at position 328; (u) Lys and Arg at position 330; (v) Glu at position 331; (w) Asp at position 332; (x) Asp, Ile, Met, Val, and Tyr at position 334; and (y) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 39, according to EU numbering. In some specific embodiments, FcγRIIb has the sequence of rhesus monkey FcγRIIb (sequence identification number: 223). In some specific embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., sequence identification number: 212, 213 or 214).

[0249] In yet another embodiment, the variant Fc region having enhanced FcγRIIb binding activity includes at least one amino acid modification (e.g., substitution) selected from the group consisting of (a) Gly, Thr at position 231; (b) Asp at position 232; (c) Trp at position 235; (d) Asn, Thr at position 236; (e) Val at position 239; (f) Asp, Glu at position 268; (g) Leu at position 295; (h) Leu at position 298; (i) Thr at position 326; (j) Lys, Arg at position 330; and (k) Lys, Met at position 396, according to EU numbering. In another embodiment, the variant Fc region with enhanced FcγRIIb binding activity includes amino acid modifications (e.g., substitutions) at Asn at position 236, Glu at position 268, Lys at position 330, and Met at position 396, according to EU designations. In yet another embodiment, the variant Fc region with enhanced FcγRIIb binding activity includes amino acid modifications (e.g., substitutions) at Asn at position 236, Asp at position 268, and Lys at position 330, according to EU designations. In yet another embodiment, the variant Fc region with enhanced FcγRIIb binding activity includes amino acid modifications (e.g., substitutions) at Asn at position 236, Asp at position 268, Leu at position 295, and Lys at position 330, according to EU designations. In another embodiment, the variant Fc region with enhanced FcγRIIb binding activity includes amino acid modifications (e.g., substitutions) at Thr at position 236, Asp at position 268, and Lys at position 330, according to EU designations. In yet another embodiment, the variant Fc region with enhanced FcγRIIb binding activity includes amino acid modifications (e.g., substitutions) at Asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, and Lys at position 330, according to EU designations. In yet another embodiment, the variant Fc region with enhanced FcγRIIb binding activity includes amino acid modifications (e.g., substitutions) at Trp at position 235, Asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, and Lys at position 330, according to EU designations.

[0250] In one embodiment, the invention provides a polypeptide comprising an Fc region having enhanced FcγRIIb binding activity, wherein an amino acid is modified at position 238, according to EU designation.

[0251] In one embodiment, the invention provides a polypeptide comprising an Fc region having enhanced FcγRIIb binding activity, comprising at least one amino acid modification located at at least one position selected from the group consisting of 234, 238, 250, 264, 267, 307, and 330, according to EU designations. In still other embodiments, the polypeptide comprises at least one amino acid modification located at at least one position selected from the group consisting of 234, 250, 264, 267, 307, and 330, according to EU designations. In some specific embodiments, FcγRIIb has the sequence of rhesus monkey FcγRIIb (Sequence Identification Number: 223). In some specific embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., Sequence Identification Numbers: 212, 213, or 214).

[0252] In another embodiment, the invention provides a polypeptide comprising an Fc region having enhanced FcγRIIb binding activity, comprising any of the following amino acid modifications (1)-(9): (1) positions 234, 238, 250, 307 and 330; (2) positions 234, 238, 250, 264, 307 and 330; (3) positions 234, 238, 250, 264, 267, 307 and 330; 4) Positions 234, 238, 250, 267, 307, and 330; (5) Positions 238, 250, 264, 307, and 330; (6) Positions 238, 250, 264, 267, 307, and 330; (7) Positions 238, 250, 267, 307, and 330; (8) Positions 238, 250, and 307; and (9) Positions 238, 250, 307, and 330, according to EU numbers. In some specific embodiments, FcγRIIb has the sequence of rhesus monkey FcγRIIb (sequence identification number: 223). In some specific embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., sequence identification numbers: 212, 213, or 214).

[0253] In another embodiment, the variant Fc region having enhanced FcγRIIb binding activity includes at least one amino acid modification (e.g., substitution) selected from the group consisting of: (a) Tyr at position 234; (b) Asp at position 238; (c) Val at position 250; (d) Ile at position 264; (e) Ala at position 267; (f) Pro at position 307; and (g) Lys at position 330, according to EU designations. In another embodiment, the variant Fc region having enhanced FcγRIIb binding activity includes an amino acid modification (e.g., substitution) of Asp at position 238, according to EU designations. In yet another embodiment, the variant Fc region having enhanced FcγRIIb binding activity includes amino acid modifications (e.g., substitutions) of Asp at position 238, Val at position 250, and Pro at position 307, according to EU designations. In another embodiment, the variant Fc region with enhanced FcγRIIb binding activity includes amino acid modifications (e.g., substitutions) at Asp at position 238, Val at position 250, Pro at position 307, and Lys at position 330, according to EU designations. In yet another embodiment, the variant Fc region with enhanced FcγRIIb binding activity includes amino acid modifications (e.g., substitutions) at Asp at position 238, Val at position 250, Ala at position 264, Pro at position 307, and Lys at position 330, according to EU designations. In yet another embodiment, the variant Fc region with enhanced FcγRIIb binding activity includes amino acid modifications (e.g., substitutions) at Asp at position 238, Val at position 250, Ala at position 267, Pro at position 307, and Lys at position 330, according to EU designations. In yet another embodiment, the variant Fc region with enhanced FcγRIIb binding activity includes amino acid modifications (e.g., substitutions) at position 234 for Tyr, position 238 for Asp, position 250 for Val, position 307 for Pro, and position 330 for Lys, according to EU designations. In yet another embodiment, the variant Fc region with enhanced FcγRIIb binding activity includes amino acid modifications (e.g., substitutions) at position 234 for Tyr, position 238 for Asp, position 250 for Val, position 267 for Ala, position 307 for Pro, and position 330 for Lys, according to EU designations. In yet another embodiment, the variant Fc region having enhanced FcγRIIb binding activity includes amino acid modifications (e.g., substitutions) at Asp at position 238, Val at position 250, Ile at position 264, Ala at position 267, Pro at position 307, and Lys at position 330, according to EU designations.In another embodiment, the variant Fc region with enhanced FcγRIIb binding activity includes amino acid modifications (e.g., substitutions) at Tyr at position 234, Asp at position 238, Val at position 250, Ile at position 264, Pro at position 307, and Lys at position 330, according to EU numbering. In yet another embodiment, the variant Fc region with enhanced FcγRIIb binding activity includes amino acid modifications (e.g., substitutions) at Tyr at position 234, Asp at position 238, Val at position 250, Ile at position 264, Ala at position 267, Pro at position 307, and Lys at position 330, according to EU numbering. In some specific embodiments, FcγRIIb has the sequence of rhesus monkey FcγRIIb (sequence identification number: 223). In some specific embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., sequence identification number: 212, 213, or 214).

[0254] In another embodiment, the invention provides isolated polypeptides comprising a variant Fc region having an increased isoelectric point (pI). In some specific embodiments, the variant Fc region described herein comprises at least two amino acid modifications in the parental Fc region. In some specific embodiments, each amino acid modification increases the isoelectric point of the variant Fc region relative to the isoelectric point of the parental Fc region. These are based on the discovery that antibodies with increased pI due to modifications of at least two amino acid residues can enhance antigen elimination from plasma, for example, when the antibody is administered in vivo.

[0255] In this invention, pI can be theoretical or experimentally determined. The value of pI can be determined, for example, by isoelectric focusing, which is known to those skilled in the art. The theoretical pI value can be calculated, for example, using gene and amino acid sequence analysis software (Genetyx, etc.).

[0256] In one embodiment, the pI value may be increased compared to the original value, for example, by at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 or more, at least 0.6, 0.7, 0.8, 0.9 or more, at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 or more, or at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0 or more.

[0257] In some specific embodiments, the amino acid used to increase pI may be exposed on the surface of the variant Fc region. In this invention, an amino acid that can be exposed on the surface generally refers to an amino acid residue located on the surface of the polypeptide constituting the variant Fc region. An amino acid residue located on the surface of the polypeptide refers to an amino acid residue whose side chain can contact solvent molecules (which are generally and mostly water molecules). However, the side chain does not necessarily have to be in complete contact with the solvent molecules, and even when a portion of the side chain is in contact with the solvent molecules, the amino acid is defined as "an amino acid residue located on the surface". The amino acid residues located on the surface of the polypeptide also include amino acid residues that are close to the surface and thus can be contacted (even partially) with solvent molecules by another side chain, thus affecting the charge. Those skilled in the art can prepare homology models of the polypeptide, for example, using commercially available software. Alternatively, methods known to those skilled in the art, such as X-ray crystallography, may be used. Amino acid residues that can be exposed on a surface can be determined, for example, using the coordinates of a three-dimensional model, which utilizes computer programs such as InsightII (Accelrys). The location of exposed surfaces can be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. 55:379-400 (1971)); Connolly (J. Appl. Cryst. 16:548-558 (1983)). The location of exposed surfaces can also be determined using software suitable for protein models and three-dimensional structural information. Software suitable for such applications includes, for example, the SYBYL Biopolymer Module software (Tripos Associates). When the algorithm requires user input of size parameters, the "size" of the probe used for measurement can be set to a radius of approximately 1.4 Å or less. Furthermore, a method for determining the region of exposed surfaces using personal computer software has been described in Pacios (Comput. Chem. 18(4):377-386 (1994); J. Mol. Model. 1:46-53). (1995)). Based on the above information, suitable amino acid residues on the surface of the polypeptide constituting the variant Fc region can be selected.

[0258] In some specific embodiments, the polypeptide includes a variant Fc region and an antigen-binding domain. In still other embodiments, the antigen is a soluble antigen. In one embodiment, the antigen is present in the biological fluid of the object (e.g., plasma, tissue fluid, lymph, ascites, and pleural fluid). The antigen may also be a membrane antigen.

[0259] In some other embodiments, the antigen-binding activity of the antigen-binding domain varies depending on ion concentration conditions. In one embodiment, the ion concentration is not particularly limited and refers to either hydrogen ion concentration (pH) or metal ion concentration. Herein, metal ions refer to ions of Group I elements other than hydrogen, such as alkali metals and copper group elements, Group II elements such as alkaline earth metals and zinc group elements, Group III elements other than boron, Group IV elements other than carbon and silicon, Group VIII elements such as iron group and platinum group elements, elements belonging to subgroup A of groups V, VI, and VII, and metal elements such as antimony, bismuth, and polonium. In this invention, metal ions include, for example, calcium ions, as described in WO 2012 / 073992 and WO 2013 / 125667. In one embodiment, "ion concentration conditions" may refer to conditions that emphasize the different biological behaviors of the antigen-binding domain between low and high ion concentrations. Furthermore, "the antigen-binding activity of the antigen-binding domain changes according to ion concentration conditions" means that the antigen-binding activity of the antigen-binding domain changes between low and high ion concentrations (e.g., the antigen-binding domain is referred to herein as an "ion concentration-dependent antigen-binding domain"). The antigen-binding activity of the antigen-binding domain may be higher (stronger) or lower (weaker) under high ion concentration conditions compared to low ion concentration conditions. In one embodiment, an ion concentration-dependent antigen-binding domain (such as a pH-dependent antigen-binding domain or a calcium ion concentration-dependent antigen-binding domain) can be obtained by known methods, for example, as described in WO 2009 / 125825, WO 2012 / 073992, and WO 2013 / 046722.

[0260] In this invention, the antigen-binding activity of the antigen-binding domain may be higher under high calcium ion concentration conditions than under low calcium ion concentration conditions. The high calcium ion concentration is not particularly limited, but may be selected from the range of 100 μM and 10 mM, 200 μM and 5 mM, 400 μM and 3 mM, 200 μM and 2 mM, 400 μM and 1 mM, or 500 μM and 2.5 mM, preferably close to the plasma (blood) concentration of calcium ions in vivo. Meanwhile, the low calcium ion concentration is not particularly limited, but may be selected from the range of 0.1 μM and 30 μM, 0.2 μM and 20 μM, 0.5 μM and 10 μM, 1 μM and 5 μM, or 2 μM and 4 μM, preferably close to the concentration of calcium ions in the early endosomes in vivo.

[0261] In one embodiment, the ratio of antibody binding activity under low calcium ion concentration conditions to that under high calcium ion concentration conditions is not particularly limited, but the ratio of the dissociation constant (KD) under low calcium ion concentration conditions to that under high calcium ion concentration conditions, i.e., KD(low calcium ion concentration conditions) / KD(high calcium ion concentration conditions), is 2 or greater, 10 or greater, or 40 or greater. The upper limit of the ratio can be 400, 1000, or 10000, provided that such antigen-binding domain can be manufactured using techniques known to those skilled in the art. Alternatively, for example, the dissociation rate constant (kd) can be used instead of KD. In this case, the ratio of kd under low calcium ion concentration conditions to that under high calcium ion concentration conditions, i.e., kd(low calcium ion concentration conditions) / kd(high calcium ion concentration conditions), is 2 or greater, 5 or greater, 10 or greater, or 30 or greater. The upper limit of the ratio can be 50, 100 or 200, as long as the antigen-binding domain can be manufactured based on the technical common sense of those with ordinary knowledge in the relevant field.

[0262] In this invention, the antigen-binding activity of the antigen-binding domain may be higher at low hydrogen ion concentrations (neutral pH) than at high hydrogen ion concentrations (acidic pH). The acidic pH may be, for example, selected from pH 4.0 to pH 6.5, pH 4.5 to pH 6.5, pH 5.0 to pH 6.5, or pH 5.5 to pH 6.5, preferably close to the in vivo pH of early endosomes. The acidic pH may also be, for example, pH 5.8 or pH 6.0. In some specific embodiments, the acidic pH is pH 5.8. Meanwhile, the neutral pH may be, for example, selected from pH 6.7 to pH 10.0, pH 6.7 to pH 9.5, pH 7.0 to pH 9.0, or pH 7.0 to pH 8.0, preferably close to the in vivo pH of plasma (blood). The neutral pH can also be, for example, pH 7.4 or pH 7.0. In some specific embodiments, the neutral pH is pH 7.4.

[0263] In one embodiment, the ratio of antibody binding activity under acidic pH conditions to that under neutral pH conditions is not limited, but the ratio of the dissociation constant (KD) under acidic pH conditions to the KD under neutral pH conditions, i.e., KD(acidic pH conditions) / KD(neutral pH conditions), is 2 or greater, 10 or greater, or 40 or greater. The upper limit of the ratio can be 400, 1000, or 10000, provided that such antigen-binding domain can be manufactured using techniques known to those skilled in the art. Alternatively, for example, the dissociation rate constant (kd) can be used instead of KD. In this case, the ratio of kd under acidic pH conditions to kd under neutral pH conditions, i.e., kd(acidic pH conditions) / kd(neutral pH conditions), is 2 or greater, 5 or greater, 10 or greater, or 30 or greater. The upper limit of the ratio can be 50, 100 or 200, as long as the antigen-binding domain can be manufactured based on the technical common sense of those with ordinary knowledge in the relevant field.

[0264] In one embodiment, for example, at least one amino acid residue is substituted with an amino acid residue having a side chain pKa of 4.0-8.0, and / or at least one amino acid having a side chain pKa of 4.0-8.0 is inserted into the antibody-binding domain, as described in WO 2009 / 125825. The amino acid can be substituted and / or inserted at any position, provided that the antigen-binding activity of the antigen-binding domain becomes weaker under acidic pH conditions than under neutral pH conditions compared to before substitution or insertion. When the antigen-binding domain has a variable region or CDR, the position can be within the variable region or CDR. The number of substituted or inserted amino acids can be suitably determined by known methods; and the number can be one or more. An amino acid having a side chain pKa of 4.0-8.0 can be used to modify the antigen-binding activity of the antigen-binding domain, depending on hydrogen ion concentration conditions. Such amino acids include, for example, neutral amino acids such as His(H) and Glu(E), and non-neutral amino acids such as histidine analogs (US2009 / 0035836), m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-I2-Tyr (pKa 7.38) (Heyl et al., Bioorg. Med. Chem. 11(17):3761-3768 (2003)). Amino acids having a side chain pKa of 6.0-7.0, including, for example, His(H), may also be used.

[0265] In another embodiment, for a variant Fc region with increased pI, a preferred antigen-binding domain has been described and can be obtained by the methods described in Japanese Patent Application Nos. JP2015-021371 and JP2015-185254.

[0266] In some specific embodiments, the variant Fc region with increased pI includes at least two amino acid modifications at positions selected from at least two groups consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422 and 431, according to EU numbers.

[0267] In some other embodiments, the variant Fc region with increased pI includes at least two amino acid modifications at positions selected from at least two groups consisting of: 311, 341, 343, 384, 399, 400, 401, 402 and 413, according to EU numbers.

[0268] In another embodiment, the invention provides a polypeptide comprising a variant Fc region having increased pI, comprising any of the following amino acid modifications (1)-(10): (1) positions 311 and 341; (2) positions 311 and 343; (3) positions 311, 343 and 413; (4) positions 311, 384 and 413; (5) positions 311 and 399; (6) positions 311 and 401; (7) positions 311 and 413; (8) positions 400 and 413; (9) positions 401 and 413; and (10) positions 402 and 413, according to EU designations.

[0269] Methods for increasing the pI of a protein include, for example, reducing the number of amino acids with negatively charged side chains (e.g., aspartic acid and glutamic acid) and / or increasing the number of amino acids with positively charged side chains (e.g., arginine, lysine, and histidine) under neutral pH conditions. Amino acids with negatively charged side chains have a negative charge, expressed as -1, at pH conditions sufficiently higher than their side chain pKa, a theory well known to those skilled in the art. For example, the theoretical pKa of the side chain of aspartic acid is 3.9, and the side chain has a negative charge, expressed as -1, under neutral pH conditions (e.g., in a solution at pH 7.0). Conversely, amino acids with positively charged side chains have a positive charge, expressed as +1, at pH conditions sufficiently lower than their side chain pKa. For example, the theoretical pKa of the side chain of arginine is 12.5, and the side chain has a positive charge represented as +1 under neutral pH conditions (e.g., in a solution at pH 7.0). Meanwhile, under neutral pH conditions (e.g., in a solution at pH 7.0), amino acids with uncharged side chains are known to include 15 neutral amino acids: alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, aspartic acid, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. It is understandable that amino acids that increase pI can be non-neutral amino acids.

[0270] As described above, increasing the pI of a protein under neutral pH conditions (e.g., in a solution at pH 7.0) can impart a +1 charge change to the protein of interest, for example, by replacing aspartic acid or glutamic acid (whose side chain has a negative charge of -1) in the protein's amino acid sequence with an amino acid whose side chain has no charge. Furthermore, a +1 charge change can be imparted to the protein, for example, by replacing an amino acid whose side chain has no charge with arginine or lysine (whose side chain has a positive charge of +1). Moreover, a +2 charge change can be simultaneously imparted to the protein by replacing aspartic acid or glutamic acid (whose side chain has a negative charge of -1) with arginine or lysine (whose side chain has a positive charge of +1). Alternatively, to increase the pI of a protein, amino acids with uncharged side chains and / or preferably positively charged side chains may be added to or inserted into the protein's amino acid column, or amino acids with uncharged side chains and / or preferably negatively charged side chains may be deleted from the protein's amino acid column. It is understood that, for example, in addition to their side-chain-derived charges, the N-terminal and C-terminal amino acid residues of a protein have main-chain-derived charges (NH3+ at the N-terminus and COO- at the C-terminus). Therefore, the pI of a protein can also be increased by performing additions, deletions, substitutions, or insertions into main-chain-derived functional groups.

[0271] The amino acid substitutions used to increase pI include, for example, replacing an amino acid with a negatively charged side chain with an amino acid with an uncharged side chain, replacing an amino acid with an uncharged side chain with an amino acid with a positively charged side chain, and replacing an amino acid with a negatively charged side chain with an amino acid with a positively charged side chain in the amino acid sequence of the parent Fc region, which may be implemented alone or in a suitable combination.

[0272] The amino acid insertion or addition used to increase pI includes, for example, the insertion or addition of an amino acid with an uncharged side chain and / or the insertion or addition of an amino acid with a positively charged side chain in the amino acid sequence of the parent Fc region, which may be carried out alone or in a suitable combination.

[0273] The amino acid deletion used to increase pI includes, for example, deletion of amino acids with uncharged side chains and / or deletion of amino acids with negatively charged side chains in the amino acid sequence of the parent Fc region, which may be carried out alone or in a suitable combination.

[0274] In one embodiment, the natural amino acids used to increase pI can be classified as follows: (a) amino acids with negatively charged side chains can be Glu (E) or Asp (D); (b) amino acids with uncharged side chains can be Ala (A), Asn (N), Cys (C), Gln (Q), Gly (G), His (H), Ile (I), Leu (L), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), or Val (V); and (c) amino acids with positively charged side chains can be His (H), Lys (K), or Arg (R). In one embodiment, the modified amino acid is inserted or substituted with Lys (K) or Arg (R).

[0275] In another embodiment, the invention provides isolated polypeptides comprising a variant Fc region having enhanced FcγRIIb binding activity and increased pI. In some specific embodiments, the variant Fc region described herein comprises at least two amino acid modifications in the parental Fc region.

[0276] In one embodiment, the invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb binding activity and increased pI, comprising at least three amino acid modifications, including: (a) at least one amino acid modification located at a position selected from the group consisting of: 231, 232, 233, 234, 235, 236, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334 and 396, according to EU designations; and (b) at At least two amino acid modifications, which are located in at least two groups selected from the group consisting of 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422 and 431, according to EU designations.

[0277] In one embodiment, the invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb binding activity and increased pI, and comprising at least three amino acid modifications, including: (a) at least one amino acid modification at a position in at least one group consisting of: 231, 232, 235, 236, 239, 268, 295, 298, 326, 330 and 396, according to EU numbers; and (b) at least two amino acid modifications at positions in at least two groups consisting of: 311, 341, 343, 384, 399, 400, 401, 402 and 413, according to EU numbers.

[0278] In another embodiment, the invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb binding activity and increased pI, comprising any of the following amino acid modifications (1)-(9): (1) positions 235, 236, 268, 295, 311, 326, 330 and 343; (2) positions 236, 268, 295, 311, 326, 330 and 343; (3) positions 236, 268, 295, 311, 330 and 413; 4) Positions 236, 268, 311, 330, 396, and 399; (5) Positions 236, 268, 311, 330, and 343; (6) Positions 236, 268, 311, 330, 343, and 413; (7) Positions 236, 268, 311, 330, 384, and 413; (8) Positions 236, 268, 311, 330, and 413; and (9) Positions 236, 268, 330, 396, 400, and 413, according to EU numbers. In some specific embodiments, FcγRIIb has a sequence of rhesus monkey FcγRIIb (sequence identification number: 223). In some specific embodiments, FcγRIIb has a sequence of human FcγRIIb (e.g., sequence identification numbers: 212, 213, or 214).

[0279] In one embodiment, the invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb binding activity and increased pI, comprising at least three amino acid modifications, including: (a) at least one amino acid modification at a position selected from the group consisting of 234, 238, 250, 264, 267, 307 and 330, according to EU numbers; and (b) at least two amino acid modifications at positions selected from the group consisting of 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422 and 431, according to EU numbers. In some other embodiments, the polypeptide includes at least two amino acid modifications located in at least two positions chosen from the group consisting of 311, 341, 343, 384, 399, 400, 401, 402, and 413, according to EU designations. In some specific embodiments, FcγRIIb has the sequence of rhesus monkey FcγRIIb (sequence identification number: 223). In some specific embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., sequence identification numbers: 212, 213, or 214).

[0280] In another embodiment, the invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb binding activity and increased pI, comprising any of the following amino acid modifications (1)-(16): (1) positions 234, 238, 250, 264, 307, 311, 330 and 343; (2) positions 234, 238, 250, 264, 307, 311, 330 and 413; (3) positions 234, 238, 250, 264, 267, 307, 311, 330 and 343; (4) Positions 234, 238, 250, 264, 267, 307, 311, 330 and 413; (5) Positions 234, 238, 250, 267, 307, 311, 330 and 343; (6) Positions 234, 238, 250, 267, 307, 311, 330 and 413; (7) Positions 234, 238, 250, 307 (8) Positions 234, 238, 250, 307, 311, 330 and 413; (9) Positions 238, 250, 264, 267, 307, 311, 330 and 343; (10) Positions 238, 250, 264, 267, 307, 311, 330 and 413; (11) Positions 238, 250, 264, 307, 311, 330 and 343; (12) Position 23 8, 250, 264, 307, 311, 330 and 413; (13) positions 238, 250, 267, 307, 311, 330 and 343; (14) positions 238, 250, 267, 307, 311, 330 and 413; (15) positions 238, 250, 307, 311, 330 and 343; and (16) positions 238, 250, 307, 311, 330 and 413, according to EU numbers.

[0281] In yet another embodiment, the variant Fc region includes an amino acid modification selected from a single modification, a combination of single modifications, or a combination of modifications as described in Tables 14-30.

[0282] In some embodiments, the polypeptide includes the variant Fc region of the present invention. In another embodiment, the polypeptide is a constant region of an antibody heavy chain. In yet another embodiment, the polypeptide is an antibody heavy chain. In yet another embodiment, the polypeptide is an antibody. In yet another embodiment, the polypeptide is an Fc fusion protein.

[0283] In yet another embodiment, the invention provides a polypeptide comprising an amino acid sequence of any one of sequence identification numbers 229-381.

[0284] As used herein, “parental Fc region” refers to the Fc region prior to the introduction of the amino acid modification described herein. Preferred examples of parental Fc regions include Fc regions derived from natural antibodies. Antibodies include, for example, IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM. Antibodies may be derived from humans or monkeys (e.g., rhesus monkeys, macaques, marmosets, chimpanzees, or baboons). Natural antibodies may also contain naturally occurring mutations. Many heteromorphic sequences of IgGs arising from genetic homogeneity have been described in “Sequences of Proteins of Immunological Interest,” NIH Publication No. 91-3242, and any of them may be used in this invention. In particular, for human IgG1, the amino acid sequence at positions 356 to 358 (EU number) may be DEL or EEM. Preferred examples of parental Fc regions include Fc regions derived from the heavy chain constant regions of human IgG1 (Sequence Identification Number: 195), human IgG2 (Sequence Identification Number: 196), human IgG3 (Sequence Identification Number: 197), and human IgG4 (Sequence Identification Number: 198). Another preferred example of a parental Fc region is an Fc region derived from the heavy chain constant region SG1 (Sequence Identification Number: 9). Furthermore, the parental Fc region can be an Fc region created by adding amino acid modifications other than those described herein to an Fc region derived from a natural antibody.

[0285] In addition, amino acid modifications performed for other purposes may be incorporated into the variant Fc region described herein. For example, amino acid substitutions that improve FcRn binding activity can be added (Hinton et al., J. Immunol. 176(1):346-356 (2006); Dall'Acqua et al., J. Biol. Chem. 281(33):23514-23524 (2006); Petkova et al., Intl. Immunol. 18(12):1759-1769 (2006); Zalevsky et al., Nat. Biotechnol. 28(2):157-159 (2010); WO 2006 / 019447; WO 2006 / 053301; and WO 2009 / 086320), as well as amino acid substitutions to improve antibody heterogeneity or stability (WO 2009 / 041613). Alternatively, peptides with enhanced antigen clearance properties described in WO 2011 / 122011, WO 2012 / 132067, WO 2013 / 046704, or WO 2013 / 180201; peptides with specific binding properties to target tissues described in WO 2013 / 180200; and peptides with the ability to repeatedly bind to multiple antigen molecules described in WO 2009 / 125825, WO 2012 / 073992, or WO 2013 / 047752 may be bound to the variant Fc region described herein. Alternatively, to impart other antigen-binding capabilities, amino acids disclosed in EP1752471 and EP1772465 may be modified and bound to the CH3 region of the variant Fc region described herein. Alternatively, for the purpose of increasing plasma retention, an amino acid modification that reduces the pI of the constant region (WO 2012 / 016227) can be incorporated into the variant Fc region described herein. Alternatively, for the purpose of increasing cellular uptake, an amino acid modification that increases the pI of the constant region (WO 2014 / 145159) can be incorporated into the variant Fc region described herein. Alternatively, for the purpose of increasing the elimination of target molecules from plasma, an amino acid modification that increases the pI of the constant region (Japanese Patent Applications JP2015-021371 and JP2015-185254) can be incorporated into the variant Fc region described herein. In one embodiment, such modification may include, for example, substitution at at least one position selected from the group consisting of 311, 343, 384, 399, 400, and 413, according to EU numbers. In yet another embodiment, such substitution may be an amino acid substituted with Lys or Arg at each position.

[0286] Amino acid modifications that enhance human FcRn binding activity at acidic pH can also bind to the variant Fc regions described herein. Specifically, such modifications may include, for example, replacing Met at position 428 with Leu and Asn at position 434 with Ser, according to EU numbers (Zalevsky et al., Nat. Biotechnol. 28:157-159 (2010)); replacing Asn at position 434 with Ala (Deng et al., Metab. Dispos. 38(4):600-605 (2010)); replacing Met at position 252 with Tyr, replacing Ser at position 254 with Thr and replacing Thr at position 256 with Glu (Dall'Acqua et al., J. Biol. Chem. 281:23514-23524 (2006)); replacing Thr at position 250 with Gln and replacing Met at position 428 with Leu (Hinton et al., J. Immunol. 176(1):346-356). (2006)); replace As in position 434 with His (Zheng et al., Clin. Pharmacol. Ther. 89(2):283-290 (2011), and WO 2010 / 106180, WO 2010 / 045193, WO 2009 / 058492, WO 2008 / 022152, WO 2006 / 050166, WO 2006 / 053301, WO 2006 / 031370, WO 2005 / 123780, WO 2005 / 047327, WO 2005 / 037867, WO 2004 / 035752 or WO The modifications described in 2002 / 060919. Such modifications may include, for example, at least one of the following: replacing Met at position 428 with Leu, replacing Asn at position 434 with Ala, and replacing Tyr at position 436 with Thr. Those modifications may further include replacing Gln at position 438 with Arg and / or replacing Ser at position 440 with Glu (Japanese Patent Application Nos. JP2015-021371 and JP2015-185254).

[0287] Two or more polypeptides including the variant Fc region described herein may be contained in a molecule, wherein the two polypeptides of the variant Fc region are associated, much like antibodies. The type of antibody is not limited and may include IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM, etc.

[0288] Two binding peptides including a variant Fc region may be peptides including a variant Fc region with the same amino acid modification introduced (hereinafter referred to as homologous variant Fc regions), peptides including variant Fc regions with different amino acid modifications introduced, or peptides including a variant Fc region with only one amino acid modification introduced (hereinafter referred to as heterologous peptides including variant Fc regions). One preferred amino acid modification is a modification in the cyclic structure from positions 233 to 239 (EU number) in the CH2 domain of the Fc region, which relates to binding to FcγRIIb and FcγRIIa. Preferably, the modification is introduced in the cyclic structure of the CH2 domain of one of the Fc regions to enhance the binding activity and / or selectivity of FcγRIIb, while the other modification is introduced in the cyclic structure of the CH2 domain of the other Fc region to destabilize it. Examples of amino acid modifications that can destabilize the cyclic structure of the CH2 domain include replacing at least one amino acid selected from positions 235, 236, 237, 238, and 239 with other amino acids. Specifically, destabilization can be achieved, for example, by modifying the amino acid at position 235 to Asp, Gln, Glu, or Thr; by modifying the amino acid at position 236 to Asn; by modifying the amino acid at position 237 to Phe or Trp; by modifying the amino acid at position 238 to Glu, Gly, or Asn; and by modifying the amino acid at position 239 to Asp or Glu, according to EU designations.

[0289] Regarding the binding of heteropeptides including the variant Fc region, techniques for inhibiting unplanned binding of homopeptides including the variant Fc region can be employed by introducing an interface with electrostatic repulsion at the CH2 or CH3 domain of the Fc region, as described in WO 2006 / 106905.

[0290] For example, the amino acid residues in contact with the interface of the CH2 or CH3 domain of the Fc region include residues at position 356 (EU number), residues at position 439 (EU number), residues at position 357 (EU number), residues at position 370 (EU number), residues at position 399 (EU number), and residues at position 409 (EU number) in the CH3 domain.

[0291] More specifically, for example, an Fc region may be made in which one to three pairs of amino acid residues having the same charge are selected from (1) to (3) as shown below: (1) amino acid residues at positions 356 and 439 in the CH3 domain (EU number); (2) amino acid residues at positions 357 and 370 in the CH3 domain (EU number); and (3) amino acid residues at positions 399 and 409 in the CH3 domain (EU number).

[0292] In addition, a heterologous polypeptide comprising a variant Fc region can be manufactured, wherein one to three pairs of amino acid residues selected from (1) to (3) above have the same charge in the CH3 domain of the first Fc region, and the amino acid residue pairs selected from the aforementioned first Fc region also have the same charge in the CH3 domain of the second Fc region, but the charges in the first and second Fc regions are opposite.

[0293] In the Fc region described above, for example, the negatively charged amino acid residues are preferably selected from glutamic acid (E) and aspartic acid (D), while the positively charged amino acid residues are preferably selected from lysine (K), arginine (R) and histidine (H).

[0294] Additional known techniques may be used for the binding of heterologous polypeptides including variant Fc regions. Specifically, this technique involves replacing the amino acid side chains present in one of the Fc regions with larger side chains (knobs; which stand for "bumps") and replacing the amino acid side chains present in the Fc regions with smaller side chains (holes; which stand for "cavities") to place the knobs within the holes. This can improve the efficient binding between Fc region-containing polypeptides that have different amino acid sequences from each other (WO 1996 / 027011; Ridgway et al., Prot. Eng. 9:617-621 (1996); Merchant et al., Nat. Biotech. 16, 677-681 (1998)).

[0295] In addition, other known techniques can be used for heterologous binding of peptides including variant Fc regions. The binding of peptides including Fc regions can be effectively induced using the strand-exchange engineered domain CH3 heterodimer (Davis et al., Prot. Eng. Des. & Sel., 23:195-202 (2010)). This technique can also be used to effectively induce binding between peptides containing variant Fc regions with different amino acid sequences.

[0296] Alternatively, heterodimeric antibody manufacturing technology utilizing the binding of antibodies CH1 and CL and the binding of VH and VL, as described in WO 2011 / 028952, may also be used.

[0297] It is also possible to use the methods described in WO 2008 / 119353 and WO 2011 / 131746 to produce heterodimeric antibodies by pre-producing two homodimeric antibodies, culturing the antibodies under reducing conditions to separate them, and then recombinizing them.

[0298] It is also possible to use the method described in Strop (J. Mol. Biol. 420:204-219 (2012)) to produce heterodimeric antibodies by introducing charged residues such as Lys, Arg, Glu and Asp so that electrostatic repulsion is introduced into the CH3 domain.

[0299] In addition, it is also possible to use the method described in WO 2012 / 058768 to manufacture heterodimeric antibodies by adding modifications to the CH2 and CH3 domains.

[0300] When two polypeptides containing variant Fc regions with different amino acid sequences are expressed simultaneously, polypeptides containing homologous variant Fc regions are often generated as impurities in order to produce a polypeptide containing a heterologous variant Fc region. In this case, polypeptides containing heterologous variant Fc regions can be efficiently obtained by separating and purifying them from the polypeptide containing the homologous variant Fc region using conventional techniques. A method for efficiently separating and purifying heterodimeric antibodies from homodimeric antibodies using ion exchange chromatography has been reported, which involves introducing amino acid modifications into the variable regions of the heavy chains of the two antibodies to create a difference in isoelectric point between the homodimeric and heterodimeric antibodies (WO 2007 / 114325). Another method for purifying heterodimeric antibodies using Protein A chromatography has been reported, which involves constructing heterodimeric antibodies comprising two heavy chains derived from mouse IgG2a, which binds to Protein A, and rat IgG2b, which does not bind to Protein A (WO 1998 / 050431 and WO 1995 / 033844).

[0301] In addition, Protein A chromatography can be used to replace amino acid residues (EU number) at positions 435 and 436 with amino acids such as Tyr or His, which are located at the Protein A binding position of the antibody heavy chain, to generate different Protein A binding affinities, thereby effectively purifying heterodimeric antibodies.

[0302] In this invention, amino acid modification refers to any one or a combination of substitution, deletion, addition, insertion, and modification. In this invention, amino acid modification can be rewritten as amino acid mutation.

[0303] When substituted amino acid residues, different amino acid residues may be substituted to target the following aspects as described in (a)-(c): (a) the polypeptide backbone structure in the sheet-like or helical region; (b) the charge or hydrophobicity at the target site; or (c) the size of the side chain.

[0304] Based on their general side chain properties, amino acid residues are classified into the following groups: (a) hydrophobic: leucine, Met, Ala, Val, Leu and Ile; (b) neutral hydrophilic: Cys, Ser, Thr, Asn and Gln; (c) acidic: Asp and Glu; (d) basic: His, Lys and Arg; (e) residues that affect chain orientation: Gly and Pro; and (f) aromatic: Trp, Tyr and Phe.

[0305] Amino acid modification using various methods known to those skilled in the art. Such methods include, but are not limited to, localized mutagenesis (Hashimoto-Gotoh et al., Gene 152:271-275 (1995); Zoller, Meth. Enzymol. 100:468-500 (1983); Kramer et al., Nucleic Acids Res. 12: 9441-9456 (1984)); Kramer and Fritz, Methods Enzymol. 154: 350-367 (1987); and Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985)), PCR mutagenesis, and cartridge mutagenesis.

[0306] The number of amino acid modifications introduced into the Fc region is not limited. In some specific embodiments, it may be 1, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 8 or less, 10 or less, 12 or less, 14 or less, 16 or less, 18 or less, or 20 or less.

[0307] Amino acid modifications include post-translational modifications. Specific post-translational modifications can be the addition or deletion of glycans. For example, an amino acid residue at position 297 (EU number) in the constant region of IgG1 can be modified with a glycan. The modified glycan structure is not limited. For example, sialic acid can be added to a glycan in the Fc region (MAbs 2010 Sep-Oct, 2(5): 519-527). Generally, antibodies expressed in eukaryotic cells involve glycosylation in the constant region. For example, certain types of glycans are known to be commonly added to antibodies expressed in cells, such as naturally occurring antibody-producing cells of mammals or eukaryotic cells transformed with an expression vector containing antibody-encoding DNA.

[0308] The eukaryotic cells described herein include yeast and animal cells. For example, CHO cells and HEK293 cells are representative animal cells used for transformation with expression vectors containing DNA encoding antibodies. On the other hand, unglycosylated constant regions are also included in this invention. Antibodies with unglycosylated constant regions can be obtained by expressing a gene encoding the antibody in prokaryotic cells such as Escherichia coli.

[0309] Furthermore, the polypeptide including the variant Fc region of the present invention can be chemically modified with various molecules, such as polyethylene glycol (PEG) and cytotoxic substances. Methods for such chemical modification of polypeptides have been established in the relevant art.

[0310] In one embodiment, the invention provides an isolated polypeptide comprising a variant Fc region having enhanced FcγRIIb binding activity. In some embodiments, the polypeptide is an antibody. In some embodiments, the polypeptide is an Fc fusion protein. In one embodiment, the invention provides an isolated polypeptide comprising a variant Fc region having enhanced FcγRIIb binding activity. Furthermore, in some embodiments, the polypeptide is an antibody. In some specific embodiments, the antibody is a chimeric antibody or a humanized antibody. The source of the antibody is not particularly limited, but includes, for example, human antibodies, mouse antibodies, rat antibodies, and rabbit antibodies. In some embodiments, the polypeptide is an Fc fusion protein.

[0311] The variable region of an antibody, including the variant Fc region described herein and the protein-binding motif of an Fc fusion protein including the variant Fc region, can recognize any antigen. Examples of antigens that can be bound by such antibodies and fusion proteins include, but are not limited to, ligands (cytokines, chemokines, etc.), receptors, cancer antigens, MHC antigens, differentiation antigens, immunoglobulins, and some immune complexes containing immunoglobulins.

[0312] Examples of cytokines that may bind to antibodies or fusion proteins that may be recombinantly fused with the variant Fc region of the invention and / or with a polypeptide that includes the disclosed variant Fc region include, but are not limited to, interleukins 1 to 18, community-stimulating factors (G-CSF, M-CSF, GM-CSF, etc.), interferons (IFN-α, IFN-β, IFN-γ, etc.), growth factors (EGF, FGF, IGF, NGF, PDGF, TGF, HGF, etc.), tumor necrosis factors (TNF-α and TNF-β), lymphotoxin, erythropoietin, receptor agonists, SCF, TPO, MCAF, and BMP.

[0313] Examples of chemokines that may bind to antibodies or fusion proteins that include the variant Fc region of the invention and / or a polypeptide recombinantly fused with the disclosed variant Fc region include, but are not limited to, CC chemokines such as CCL1 to CCL28, CXC chemokines such as CXCL1 to CXCL17, C chemokines such as XCL1 to XCL2, and CX3C chemokines such as CX3CL1.

[0314] Examples of receptors that may bind to antibodies or fusion proteins that are recombinantly fused with the variant Fc region of the invention and / or with a polypeptide that includes the disclosed variant Fc region include, but are not limited to, receptors belonging to receptor families, such as the hematopoietic growth factor receptor family, cytokine receptor family, tyrosine kinase receptor family, serine / threonine kinase receptor family, TNF receptor family, G protein-coupled receptor family, GPI-anchored receptor family, tyrosine phosphatase receptor family, adhesion factor family, and hormone receptor family. Receptors belonging to these receptor families and their characteristics have been described in numerous publications, for example, Cooke (ed.), New Comprehesive Biochemistry Vol. 18B "Hormones and their Actions Part II" pp. 1-46 (1988) Elsevier Science Publishers BV; Patthy (Cell 61(1): 13-14 (1990)); Ullrich (Cell 61(2): 203-212 (1990)); Massagué (Cell 69(6): 1067-1070 (1992)); Miyajima et al. (Annu. Rev. Immunol. 10: 295-331 (1992)); Taga et al. (FASEB J. 6: 3387-3396 (1992)); Fantl et al. (Annu. Rev. Biochem. 62: 453-481). (1993)); Smith et al. (Cell 76(6):959-962 (1994)); and Flower (Biochim. Biophys. Acta 1422(3): 207-234 (1999)).

[0315] Examples of specific receptors belonging to the above receptor family include human or mouse erythropoietin (EPO) receptors (Jones et al., Blood 76(1):31-35 (1990); D'Andrea et al., Cell 57(2):277-285 (1989)), human or mouse granulocyte colony-stimulating factor (G-CSF) receptors (Fukunaga et al., Proc. Natl. Acad. Sci. USA 87(22):8702-8706 (1990), mG-CSFR; Fukunaga et al., Cell 61(2): 341-350 (1990)), human or mouse thrombopoietin (TPO) receptors (Vigon et al., Proc. Natl. Acad. Sci. USA. 89(12):5640-5644 (1992); Skoda et al., EMBO J.12(7):2645-2653 (1993)), human or mouse insulin receptor (Ullrich et al., Nature 313(6005):756-761 (1985)), human or mouse Flt-3 ligand receptor (Small et al., Proc. Natl. Acad. Sci. USA. 91(2):459-463 (1994)), human or mouse platelet-derived growth factor (PDGF) receptor (Gronwald et al., Proc. Natl. Acad. Sci. USA. 85(10):3435-3439 (1988)), human or mouse interferon (IFN)-α and β receptors (Uze et al., Cell 60(2): 225-234 (1990); Novick et al., Cell 77(3):391-400). (1994)), human or mouse leptin receptor, human or mouse growth hormone (GH) receptor, human or mouse interleukin (IL)-10 receptor, human or mouse insulin-like growth factor (IGF)-I receptor, human or mouse leukemia inhibitory factor (LIF) receptor and human or mouse ciliary neurotrophic factor (CNTF) receptor.

[0316] Cancer antigens are antigens that express cells becoming malignant, and they are also called tumor-specific antigens. When cells become cancerous, the abnormal glycans that appear on the cell surface or protein molecules are also cancer antigens, and they are also called glycan cancer antigens. Examples of cancer antigens that can be bound by antibodies or fusion proteins containing the variant Fc region of the invention include, but are not limited to, GPC3, which is a receptor belonging to the aforementioned GPI-anchored receptor family and is also expressed in many cancers, including liver cancer (Midorikawa et al., Int. J. Cancer 103(4):455-465 (2003)), and EpCAM, which is expressed in many cancers, including lung cancer (Linnenbach et al., Proc. Natl. Acad. Sci. USA 86(1):27-31 (1989)), CA19-9, CA15-3, and sialic acid SSEA-1 (SLX).

[0317] MHC antigens are broadly classified into MHC type I antigens and MHC type II antigens. MHC type I antigens include HLA-A, -B, -C, -E, -F, -G and -H, while MHC type II antigens include HLA-DR, -DQ and -DP.

[0318] Examples of differentiation antigens that may bind to antibodies or fusion proteins that are recombinantly fused with the variant Fc region of the invention and / or peptides including the disclosed variant Fc region include, but are not limited to, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15s, CD16, CD18, CD19, CD20, CD21, CD23, CD25, CD28, CD29, CD30, CD32, CD33, CD34, CD35, C D38, CD40, CD41a, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, C D54, CD55, CD56, CD57, CD58, CD61, CD62E, CD62L, CD62P, CD64, CD69, CD71, CD73, CD95, CD102, CD106, CD122, CD126 and CDw130.

[0319] Immunoglobulins include IgA, IgM, IgD, IgG, and IgE. Immunocomplexes contain at least one of the components of an immunoglobulin.

[0320] Other examples of antigens that may bind to antibodies or fusion proteins that are recombinantly fused with the variant Fc region of the invention and / or with a polypeptide including the disclosed variant Fc region include, but are not limited to, 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxy-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, and activin RIB. ALK-4, activator RIIA, activator RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, α-1-antitrypsin, α-V / β-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, AR T, artemin, anti-Id, ASPARTIC, atrial natriuretic peptide, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4, BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, β-NGF, BOK, bombesin, bone-derived nutritional factor, BPDE, BPDE-DNA, BTC, complement factor 3(C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, autolysin A, autolysin B, autolysin C / DPPI, autolysin D, autolysin E, autolysin H, autolysin L, autolysin O, autolysin S, autolysin V, autolysin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CC L26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR11, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, C D4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, complement regulator (decay accelerator), des(1-3)-IGF-I (Brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, hepatocyte glycosides (ephrin) B2 / EphB4, EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, Fibroblast Activating Protein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrinogen, FL, FLIP, Flt-3, Flt-4, Follicle Stimulin, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF8 (myosin), GDF-9, GDF-15 (MIC-1), GDNF, GFAP, GFRa-1, GFR-α1, GFR-α2, GFR-α3, GITR, glucagon, Glut4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone-releasing hormone, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB-encapsulated glycoprotein, HCMVgH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV), gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL- IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, Interferon (IFN)-α, IFN-β, IFN-γ, Inhibin, iNOS, Insulin A chain, Insulin B chain, Insulin-like growth factor 1, Integrin α2, Integrin α3, Integrin α4, Integrin α4 / β1, Integrin α4 / β7, Integrin α5 (α V), Integrin α5 / β1, Integrin α5 / β3, Integrin α6, Integrin β1, Integrin β2, Interferon γ, IP-10, I-TAC, JE, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, Kallikrein L3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), Basement Mucin 5, LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1bp1, LBP, LDGF, LECT2, lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin β receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-α, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MM P-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Mullerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, N-calcinin, NCA 90, NCAM, Enkephalin, Neurotrophic Factor-3,-4 or-6, Neuroturin, Nerve Growth Factor (NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroxine, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PD K-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, pro-diastolic hormone, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, diastolic hormone A chain, diastolic hormone B chain, renin, respiratory syncytial virus (RSV) F, RSV, Fgp, Ret, Rheumatoid Factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, Serine, Serum Albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72(Tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptors (e.g., T-cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β Pan-specific, TGF-βRI (ALK-5), TGF-βRII, TGF-βRIIb, TGF-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1) Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60), TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb) TNFC, p33), TNFSF4 (OX4 ligand, gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand, CD70), TNFSF8 (CD30 ligand, CD153), TNFSF9 (4-1BB ligand, CD137 ligand), TP-1, t-PA, Tpo, TRIL, TRIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expression Lewis-Y related carbohydrates, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, virus antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand disease (von Willebrand disease)Willebrand) factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9 A. WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR 1. DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, Oxidized LDL, PCSK9, Prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, Tau, VAP1, High Molecular Weight Kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9 EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, Factor B, Factor D, Factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, Factor V, Factor Va, Factor VII, Factor VIIa, Factor VIII, Factor VIIIa, Factor IX, Factor IXa, factor Factors X, Xa, XI, XIa, XII, XIIa, XIII, XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, Syndecan-1, Syndecan-2, Syndecan-3, Syndecan-4, LPA and S1P; and receptors for hormones and growth factors.

[0321] As discussed in the text, modifications to one or more amino acid residues in the amino acid sequence constituting the variable region are permissible, provided that their antigen-binding activity is maintained. There are no particular limitations on the location or number of amino acids modified when modifying the amino acid sequence in the variable region. For example, amino acids appearing in the CDR and / or FR may be appropriately modified. When the modified amino acid is in the variable region, binding activity is preferably maintained and is not particularly limited; for example, the binding activity may be 50% or greater, 80% or greater, or 100% or greater compared to before the modification. Furthermore, binding activity may be increased by amino acid modification. For example, the binding activity may be 2, 5, or 10 times higher than before the modification. Modifications to the amino acid sequence may be at least any of the following: amino acid residue substitution, addition, deletion, and modification.

[0322] For example, modifying the N-terminus of the variable region by pyroglutamylation to pyroglutamic acid is a modification well known to those skilled in the art. Therefore, when the N-terminus of the heavy chain is glutamylamine, the antibody described herein may include a variable region in which glutamylamine is modified to pyroglutamic acid.

[0323] The antibody variable regions described herein may have any sequence and may be antibody variable regions of any origin, such as mouse antibodies, rat antibodies, rabbit antibodies, goat antibodies, camel antibodies, humanized antibodies derived from these non-human antibodies through humanization, and human antibodies. Furthermore, these antibodies may have various amino acid substitutions introduced into their variable regions to improve their antigen binding, pharmacokinetics, stability, and immunogenicity. Due to their pH dependence in antigen binding, the variable regions may be able to repeatedly bind to antigens (WO 2009 / 125825).

[0324] The κ and λ chains are present in the constant region of the antibody light chain, and either one is acceptable. In addition, they may have some amino acid modifications, such as substitution, deletion, addition, and / or insertion.

[0325] Furthermore, the polypeptides comprising the mutated Fc region described herein can be linked with other proteins, such as bioactive peptides, to form Fc fusion proteins. Such fusion proteins can be polymers of at least two polypeptides comprising the mutated Fc region. Examples of other proteins include, but are not limited to, receptors, adhesion molecules, ligands, and enzymes.

[0326] Examples of Fc fusion proteins include proteins that fuse the Fc region to a receptor that binds to the target molecule, including TNFR-Fc fusion protein, IL1R-Fc fusion protein, VEGFR-Fc fusion protein and CTLA4-Fc fusion protein (Economides et al., Nat. Med. 9(1):47-52 (2003); Dumont et al., BioDrugs. 20(3):151-60 (2006)). In addition, the fused protein can be other molecules with target binding activity, such as scFvs (WO 2005 / 037989), single-domain antibodies (WO 2004 / 058821; WO 2003 / 002609), antibody-like molecules (Davinder, Curr. Op. Biotech. 17:653-658 (2006); Current Opinion in Biotechnology 18:1-10 (2007); Nygren et al., Curr. Op. Struct. Biol. 7:463-469 (1997); and Hoss. Protein Science 15:14-27 (2006)), such as DARPins (WO 2002 / 020565), affinity molecules (WO 1995 / 001937), Avimer (WO 2004 / 044011; WO 2005 / 040229) and Adnectin (WO 2002 / 032925). Furthermore, antibodies and Fc fusion proteins can be multispecific and can bind to various types of target molecules or antigenic determinants. B. Recombinant methods and compositions

[0327] Antibodies can be generated using recombinant methods and compositions, for example, as described in US 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-myosin antibody as described herein is provided. In another embodiment, an isolated nucleic acid encoding a polypeptide comprising a variant Fc region or a parental Fc region as described herein is provided. Such nucleic acids may encode an amino acid sequence comprising VL and / or an amino acid sequence comprising VH of the antibody (e.g., the light and / or heavy chains of the antibody). In yet another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In yet another embodiment, a host cell comprising such nucleic acids is provided. In one embodiment, the host cell comprises (e.g., having been transformed to have): (1) a vector comprising nucleic acids encoding an amino acid sequence comprising VL of the antibody and an amino acid sequence comprising VH of the antibody, or (2) a first vector comprising nucleic acids encoding an amino acid sequence comprising VL of the antibody, and a second vector comprising nucleic acids encoding an amino acid sequence comprising VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as Chinese hamster ovary (CHO) cells or lymphocytes (e.g., Y0, NSO, and Sp20 cells). In one embodiment, a method for preparing an anti-myosin antibody is provided, wherein the method includes culturing a host cell comprising a nucleic acid encoding an antibody, as provided above, under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium). In another embodiment, a method for preparing a polypeptide comprising a variant Fc region or a parental Fc region is provided, wherein the method includes culturing a host cell comprising a nucleic acid encoding a polypeptide, such as the antibody, Fc region, or variant Fc region provided above, under conditions suitable for polypeptide expression, and optionally recovering the polypeptide from the host cell (or host cell culture medium).

[0328] For the recombinant production of antimyosin antibodies, for example, nucleic acids encoding the antibodies as described above are isolated and inserted into one or more vectors for further selection and / or expression in host cells. For the recombinant production of the Fc region, nucleic acids encoding the Fc region are isolated and inserted into one or more vectors for further selection and / or expression in host cells. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibodies).

[0329] Suitable host cells for the selection, colonization, or expression of the vector encoding the antibody include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and peptides in bacteria, see, for example, US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (edited by BKC Lo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in *E. coli*). After expression, the antibody can be separated from the soluble fraction of bacterial cell paste and can be further purified.

[0330] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable hosts for the colonization or expression of antibody-encoding vectors. These include fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0331] Host cells suitable for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Several baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for the transfection of fall armyworm (Spodoptera frugiperda) cells.

[0332] Plant cell cultures can also be used as hosts. Please refer, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (description of PLATNIBODIES™ technology for generating antibodies in genetically modified plants).

[0333] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension can be used. Other examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 cell line (COS-7); the human embryonic kidney cell line (293 or, for example, 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse support cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumors (MMT 060562); for example, Mather et al., Annals NY Acad. Sci. 383:44-68. TRI cells, as described in (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA77:4216 (1980)); and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For reviews of specific mammalian host cell lines suitable for antibody production, please refer to, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKC Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0334] Antibodies with pH-dependent properties can be obtained, for example, by screening and / or mutation methods as described in WO 2009 / 125825. Screening methods may include any procedure for identifying antibodies with pH-dependent binding properties within a population of antibodies specific to a particular antigen. In some specific embodiments, screening methods may include measuring one or more binding parameters (e.g., KD or kd) of a single antibody in an original population of antibodies at acidic and neutral pH conditions. Measurement of antibody binding parameters may utilize, for example, surface plasma resonance or any other analytical method that allows for quantitative or qualitative assessment of the binding properties of an antibody to a particular antigen. In some specific embodiments, screening methods may include identifying antibodies that bind to an antigen at an acidic / neutral KD ratio of 2 or greater. In still other embodiments, screening methods may include identifying antibodies that bind to an antigen at a pH 5.8 / pH 7.4 KD ratio of 2 or greater. Alternatively, screening methods may include identifying antibodies that bind to an antigen at an acidic / neutral KD ratio of 2 or greater. In some other embodiments, the screening method may include identifying antibodies that bind to the antigen at a ratio of 2 or greater for pH 5.8 / pH 7.4 kd.

[0335] In another embodiment, the mutation method may include introducing a deletion, substitution, or addition of an amino acid into the heavy chain and / or light chain of the antibody to enhance the pH-dependent binding of the antibody to the antigen. In some specific embodiments, mutations may be performed in one or more variable domains of the antibody, for example, in one or more HVRs (e.g., CDRs). For example, the mutation may include replacing an amino acid in one or more HVRs (e.g., CDRs) of the antibody with another amino acid. In some specific embodiments, the mutation may include replacing one or more amino acids in at least one HVR (e.g., CDR) of the antibody with histidine. In some specific embodiments, “enhanced pH-dependent binding” means that the mutated form of the antibody exhibits a larger acid / neutral KD ratio or a larger acid / neutral KD ratio compared to the original “parent” form of the antibody before mutation (i.e., the lower pH-dependent form). In some specific embodiments, the mutated form of the antibody has an acid / neutral KD ratio of 2 or greater. In some specific embodiments, the mutant form of the antibody has a pH 5.8 / pH 7.4 kD ratio of 2 or greater. Alternatively, the mutant form of the antibody has an acidic / neutral kD ratio of 2 or greater. In still other embodiments, the mutant form of the antibody has a pH 5.8 / pH 7.4 kD ratio of 2 or greater.

[0336] Polyclonal antibodies are preferably produced by repeated subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant in animals. It is useful to use bifunctional or derivatized reagents (e.g., maleimidobenzoyl sulfosuccinimide ester (conjugated via cysteine ​​residues), N-hydroxysuccinimide (conjugated via lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N═C═NR, where R and R1 are different alkyl groups) to conjugate the relevant antigen to a protein that is immunizing against the species to be immunized (e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitors).

[0337] Animals (typically non-human mammals) are immunized against an antigen, immunogenic conjugate, or derivative by combining, for example, 100 µg or 5 µg of protein or conjugate (for rabbits and mice, respectively) with three times the volume of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, animals are boosted by subcutaneous injection at multiple sites using peptides or conjugates from 1 / 5 to 1 / 10 of the original amount of Freund's complete adjuvant. Animals are bled between 7 and 14 days post-boost injection to determine serum antibody titration concentrations. Booster immunizations are continued until titer plateaus are reached. Preferably, animals are boosted with conjugates of the same antigen, but conjugated to different proteins and / or using different cross-linking agents. Conjugates can also be generated in recombinant cell cultures as protein fusions. Aggregating agents such as alum are also suitable for enhancing immune responses.

[0338] Monoclonal antibodies can be obtained from substantially homogeneous antibody populations, meaning that the individual antibodies in the population are identical, except for small amounts of naturally occurring mutations and / or post-translational modifications (e.g., isomerization, acetylation). Therefore, the modifier “monoclonal” indicates the characteristic of the antibody, which is not a mixture of dispersed antibodies.

[0339] For example, monoclonal antibodies can be formed using the fusion tumor method first described by Kohler et al., Nature 256(5517):495-497 (1975). In the fusion tumor method, mice or other suitable host animals, such as hamsters, are immunized as described herein to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunization protein. Alternatively, lymphocytes can be immunized in vitro.

[0340] Immunoassay reagents typically contain proteins containing antigens or fusion variants thereof. Generally, peripheral blood lymphocytes (PBLs) are used if human-derived cells are required, or spleen cells or lymph node cells are used if non-human mammalian-derived cells are required. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent, such as polyethylene glycol, to form fusion tumor cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0341] Immortalized cell lines are typically transformed mammalian cells, particularly rodent, bovine, and human myeloma cells. Rat or mouse myeloma cell lines are commonly used. The resulting fusion tumor cells are seeded and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused, parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the fusion tumor culture medium will typically contain hypoxanthine, aminopterin, and thymine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0342] Preferred immortalized myeloma cells are those that can be efficiently fused, support the stable and high production of antibodies by selected antibody-producing cells, and are sensitive to culture media (such as HAT medium). Among these, preferred are mouse myeloma lines, such as those obtained from MOPC-21 and MPC-11 mouse tumors, available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 cells (and their derivatives, such as X-63-Ag8-653) available from the American Type Culture Collection, Manassas, Virginia USA. Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor et al., J Immunol. 133(6):3001-3005 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications; Marcel Dekker, Inc., New York, pp. 51-63 (1987)).

[0343] Determine whether monoclonal antibodies against the anti-antigen are produced in the culture medium for the growth of fusion tumor cells. Preferably, the binding specificity of the monoclonal antibodies produced by the fus...

Claims

1. Use of a polypeptide for manufacturing an anti-myosin antibody, wherein the polypeptide comprises an amino acid region at positions 81-100 of the myosinogen peptide corresponding to sequence identification number: 78, the antibody binding to an antigenic determinant of a fragment consisting of amino acids at positions 81-100 of the myosinogen peptide (sequence identification number: 78).

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