Anti-VLA-4 antibody with reduced effector function

Abstract

This invention provides an anti-VLA-4 antibody and its binding fragment. [Solution] Disclosed are recombinant anti-alpha-4 antibodies having a specific sequence or alpha-4 binding fragments thereof. Further disclosure are polynucleotides encoding the antibody and its binding fragment, and methods for producing the antibody and its binding fragment. Further disclosure is a method for treating patients suffering from multiple sclerosis and / or epilepsy by administering the antibody and its binding fragment. Furthermore, a method for reducing the sensitivity of recombinant anti-alpha-4 antibodies or their binding fragments to scrambling is disclosed.

Description

[Technical Field]

[0001] This invention relates to an alpha-4 conjugated antibody and a fragment thereof.

[0002] Sequence List This application includes a sequence listing filed via EFS-Web, which is incorporated herein by reference in its entirety. The sequence listing was prepared on 12 April 2019, is named Anti_VLASequence PatentIN_ST25, and is 208 kilobytes in size. [Background technology]

[0003] Integrins are members of a large family of cell surface receptors that mediate interactions between cells and between cells and the cell matrix. Integrins exist as non-covalent αβ heterodimers, composed of various α and β chains, sharing high structural homology. Integrins mediate a wide variety of physiological processes and are associated with a wide range of pathological conditions. The α4 chain is primarily limited to leukocytes and can associate with two β chains, β1 and β7.

[0004] VLA-4 (also known as α4β1) and α4β7 play central roles in the pathophysiology of inflammatory diseases. VLA-4 is a member of the β1 integrin family of cell surface receptors. Containing α4 and β1 chains, VLA-4 is involved in intercellular interactions. Its expression is primarily limited to lymphoid cells, including T lymphocytes, and myeloid cells, including microglia and macrophages. VLA-4 binds to the endothelial cell ligand VCAM-1 (vascular cell adhesion molecule 1) and can mediate the adhesion of T and B lymphocytes to heparin II-binding fragments of human plasma fibronectin. VLA-4 regulates the transport of normal leukocytes (Lobb and Hemler, J. Clin. Invest. 94(5):1722-1728, 1994) and provides important costimulatory signals that aid in cell activation (Clark and Brugge, Science 268(5208):233-9, 1995). In inflammatory responses, VLA-4 has been recognized as an attractive therapeutic target because it modulates the migration of leukocytes to damaged tissue. In vivo studies using blocking monoclonal antibodies (Lobb and Hemler (above), Enders et al., Brain 121(Pt 7):1257-66, 1998, Ramos-Barbon et al., Am J Respir Crit Care Med. 163(1):101-8, 2001), inhibitory peptides (Molossi et al., J Clin Invest. 95(6):2601-10, 1995, Abraham, 1997, van der Laan et al., J Neurosci Res. 2002 Jan 15;67(2):191-9, 2002), and small molecule antagonists (Kudlacz et al., J. Pharmacol. Exp. Ther. 301(2):747-52, 2002) have demonstrated the important role of α4β1 integrin in leukocyte-mediated inflammation.

[0005] α4β1 mediates cell adhesion by binding to either VCAM-1 or fibronectin (e.g., fibronectin variants containing alternatively spliced ​​connecting segment 1 (CS1)) (Osborn et al., Cell 59(6):1203-11, 1989; Wayner et al., J. Cell Biol. 109(3):1321-30, 1989). Other potential ligands have also been identified (Bayless et al., J. Cell Sci. 111(Pt 9):1165-74, 1998), but the biological significance of these interactions is not very clear. The interaction between α4β1 and its ligands is of low affinity, and binding is thought to be regulated by polyvalent interactions. Although α4β1 expression is constitutive, its interactions with ligands include antigens, anti-T cell receptor mAbs, phorbol esters, and divalent cations Mn. 2+ This is greatly enhanced in the activated state, which can be induced by various stimuli, including specific β1-specific antibodies. These changes in affinity and / or avidity ultimately determine whether the interaction is productive and stabilizes the ligand / integrin complex (Humphries, Curr Opin Cell Biol. 8(5):632-40, 1996). α4β7 is expressed by a more limited group of leukocytes and is partially involved in lymphocyte homing to mucosal lymphoid tissue by binding to the mucosal addressin MadCAM, but also binds to VCAM-1 and fibronectin. Specific monoclonal antibodies (mAbs) against α4 can block the adhesion functions of both VLA4 and α4β7.

[0006] Using humanized antibodies as therapeutic agents instead of mouse antibodies can avoid an undesirable immune response in humans called the HAMA (human anti-mouse antibody) response. Humanized antibodies are generally constructed by substituting the complementarity-determining region (CDR) of a human antibody with the CDR of another species (typically a mouse antibody).

[0007] Antibodies possess the ability to bind to antigens via their variable region. Once an antibody binds to an antigen, the antigen becomes a target for destruction, which is often at least partially mediated by the antibody's constant region or Fc region. There are several effector functions or activities mediated by the antibody's Fc region. One effector function is the ability to bind to complement proteins, which can help lyse target antigens, such as intracellular pathogens, in a process called complement-dependent cell-mediated cytotoxicity (CDC). Another effector activity of the Fc region is binding to Fc receptors (e.g., FcγR) on the surface of immune cells, or so-called effector cells, that have the ability to trigger other immune actions. These immune actions (e.g., antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP)) function in the removal of pathogens / antigens, for example, by releasing immune activators and regulating antibody production, endocytosis, phagocytosis, and cell killing. In some clinical applications, these responses are crucial to the efficacy of the antibody, while in others, they can induce undesirable side effects. One example of an effector-mediated side effect is the release of inflammatory cytokines that trigger an acute fever response. Another example is the long-term absence of antigen-producing cells.

[0008] The effector function of antibodies can be avoided by using antibody fragments lacking the Fc region (e.g., Fab, F(ab')2, or single-stranded Fv(scFv)). However, these fragments have short half-lives due to rapid renal clearance. Fab and scFv fragments have only one antigen-binding site instead of two, which can impair the benefits derived from binding avidity and create manufacturing challenges. Alternative approaches aim to reduce the effector function of full-length antibodies while retaining other beneficial properties of the Fc region (e.g., long half-life and heterodimerization). One method for reducing effector function is to generate so-called aglycosylated antibodies by removing sugars bound to specific residues in the Fc region. Aglycosylated antibodies can be generated, for example, by deleting or altering sugar-bound residues, enzymatically removing sugars, producing antibodies in cells cultured in the presence of glycosylation inhibitors, or expressing antibodies in cells that cannot glycosylate proteins (e.g., bacterial host cells). Another method involves using the Fc region of IgG4 antibodies instead of IgG1 antibodies. It is well known that IgG4 antibodies exhibit lower levels of complement activation and antibody-dependent cell-mediated cytotoxicity compared to IgG1 antibodies.

[0009] Monoclonal antibodies against human α4 bind to epitopes A, B, and C, which have three functionally distinct tissue distributions (Pulido et al., J. Biol. Chem. 266:10241-10245, 1991; Schiffer et al., J. Biol. Chem. 270:14270-14273, 1995). Epitope B is subdivided into B1 and B2 epitopes, which are indistinguishable in terms of tissue distribution but are defined by the ability of mAbs to induce cell aggregation. Anti-α4 mAbs against epitopes A and B2 can induce isomorphic cell aggregation, but those against epitopes B1 and C cannot. [Overview of the project]

[0010] The present invention relates to an anti-VLA-4 antibody and its binding fragments comprising a germline variable region framework that can optimize CDR-grafted alpha-4 conjugated antibodies, such as anti-VLA-4 antibodies. Accordingly, the present invention comprises an anti-VLA-4 variable heavy chain (VH) and variable light chain (VL) and antibody molecule comprising such a framework. In some embodiments, VLA-4 is human VLA-4. In some embodiments, VLA-4 comprises an α4 chain (e.g., a human α4 chain).

[0011] The antibody may contain a modified Fc region that alters or reduces effector function and improves stability. Antibodies having a modified Fc region can improve the significant adverse effects observed with antibodies lacking oligosaccharides, particularly those affecting conformation and stability. Antibodies having a modified Fc region may also have altered or reduced effector function and improved stability. The present invention further relates to a method for producing the molecules described herein.

[0012] This antibody may improve upon the problems of "effector-less" antibodies in the prior art by providing an improved method for enhancing the stability of the Fc region. For example, the present invention provides a stability-engineered Fc polypeptide, such as a stabilized IgG antibody or other Fc-containing binding molecule, in which the Fc region of the polypeptide contains a stabilizing amino acid. In one embodiment, the present invention provides a method for introducing a mutation that enhances the stability of the Fc region at a specific amino acid residue position in the Fc region of a parent Fc polypeptide. In some embodiments, the stabilized Fc polypeptide has altered or reduced effector function (compared to a polypeptide that does not contain stabilizing amino acids) and exhibits enhanced stability compared to the parent Fc polypeptide. In some embodiments, the parent Fc polypeptide is human IgG1 (e.g., having the sequence of SEQ ID NO: 83). In some embodiments, the parent Fc polypeptide is human IgG4 (e.g., having the sequence of SEQ ID NO: 84).

[0013] Therefore, the antibodies disclosed herein provide several advantages including, but not limited to, the following. - Provision of a stabilized aglycosylated Fc polypeptide, such as a stabilized fusion protein or an aglycosylated IgG antibody, that includes a stabilized aglycosylated Fc region and is suitable as a therapeutic agent due to reduced effector function. - Provision of a stabilized Fc polypeptide that includes an Fc region derived from an IgG4 antibody and is suitable as a therapeutic agent due to reduced effector function, such as a stabilized glycosylated or aglycosylated fusion protein or an IgG4 antibody. - An efficient method for generating a stabilized Fc polypeptide with minimal polypeptide variation (e.g., by introducing changes into an unstabilized parent polypeptide or by expressing a nucleic acid molecule encoding the stabilized Fc polypeptide). - A method for enhancing the stability of an Fc polypeptide while avoiding an increase in immunogenicity and / or effector function. - A method for enhancing the scalability, manufacturability, and / or long-term stability of an Fc polypeptide, and - A method for treating a subject in need of treatment with a stabilized Fc polypeptide of the invention.

[0014] In one embodiment, the invention features an anti-α4 antibody VH chain that has a donor anti-α4 antibody, e.g., the CDRs of the anti-α4 antibodies described herein, and has regions 1, 2, 3, and 4 of the VH chain sequence or has a VH framework that has 5, 10, or fewer differences from the VH germline variable region sequence. In one embodiment, variable framework region 4 (FR4) is a human consensus sequence. In one embodiment, the complete VH chain has framework regions FR1, FR2, FR3, and FR4. In another embodiment, the chain is an antigen-binding fragment of the VH region.

[0015] In one embodiment, the germline sequence is human IGHV1-f (SEQ ID NO: 2) as shown in Figure 1. In certain embodiments, the VH framework sequence may differ from the germline sequence, e.g., SEQ ID NO: 2, by at least one to two, three, four, five, ten, or fifteen amino acid residues. In one embodiment, the VH framework further includes residues other than the corresponding human residues. For example, the VH chain includes one or more non-canonical residues at framework positions 24, 67, 76, 80, and 94 (Kabat numbering) of SEQ ID NO: 2.

[0016] In one embodiment, at least one of the complementarity-determining regions (CDRs) of the variable domain is derived from a donor non-human α4-binding antibody (i.e., a non-human antibody that specifically binds to α4). In one embodiment, the antigen-binding region of the CDR graft heavy chain variable domain includes CDRs corresponding to positions 26-34 (CDR1), 50-65 (CDR2), and 95-102 (CDR3) (Kabat numbering).

[0017] Therefore, in one embodiment, the variable heavy chain (VH) framework has an acceptor sequence derived from the human antibody germline sequence IGHV1-f.

[0018] In another embodiment, at least one amino acid and two, three, four, five, or fewer than ten amino acid residues in the FR1 region of VH are other than the corresponding human germline residues. One or more of these residues may be identical, for example, to the non-human antibody framework region from which the CDR sequence originates. In one embodiment, the amino acid residue at position 24 of Kabat is mutated to be identical to that of the non-human antibody framework region.

[0019] In another embodiment, at least one amino acid and two, three, four, five, or fewer than ten amino acid residues in the FR2 region of VH are other than the corresponding human germline residues. One or more of these residues may be identical, for example, to the non-human antibody framework region from which the CDR sequence originates.

[0020] In yet another embodiment, at least one amino acid and two, three, four, five, or fewer than ten amino acid residues in FR3 of the VH chain are other than the corresponding human germline residues. One or more of these residues may be identical, for example, to the non-human antibody framework region from which the CDR sequence originates. In one embodiment, the amino acid residue at Kabat position 94 is identical to the non-human antibody framework region. In yet another embodiment, the amino acid residues at Kabat positions 67, 76, 80, and 94 are identical to the non-human antibody framework region.

[0021] In certain embodiments, the VH chain of the antibody has the sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0022] In one embodiment, the present invention features a donor anti-VLA-4 antibody, for example, the CDR of the anti-VLA-4 antibody described herein, and an anti-VLA-4 VL chain having a VL framework having regions 1, 2, 3, and 4 of the VL chain sequence or 5, 10, or 15 or fewer differences (either per region or in total) from the germline variable region sequence of the VL chain. In one embodiment, variable framework region 4 (FR4) is the human consensus sequence. In one embodiment, framework regions FR1, FR2, FR3, and FR4 of the complete VL chain are present. In another embodiment, this chain is an antigen-binding fragment of the VL region.

[0023] In another embodiment, the germline sequence is IGKV4-1 (SEQ ID NO: 7) as shown in Figure 2. In yet another embodiment, the VL framework sequence may differ from the germline framework sequence, e.g., SEQ ID NO: 7, by at least one to two, three, four, five, ten, or fifteen amino acid residues. In yet another embodiment, the VL further includes non-human amino acid residues. For example, the VL chain further includes non-human residues at one or more of the framework positions 1, 73, and 87 (Kabat numbering) of SEQ ID NO: 7.

[0024] In one embodiment, this sequence is AAH7035.1 (SEQ ID NO: 12) or its germline-manipulated version (SEQ ID NO: 13) shown in Figure 2. In some embodiments, the VL framework sequence may differ from the germline-manipulated framework sequence, e.g., SEQ ID NO: 13, by at least one to five, ten, fifteen, twenty, or twenty-five amino acid residues. In one embodiment, the VL chain contains non-human residues. For example, the VL chain contains non-human residues at one or more of the framework positions 1 and 87 (Kabat numbering) of SEQ ID NO: 12. In another embodiment, the VL includes amino acid substitutions in the framework region to resemble a different human germline framework sequence, such as that of the germline sequence IGKV4-1. In certain embodiments, the VL framework sequence is modified to be identical to the IGKV4-1 germline sequence at positions 1-3, 5-23, 35-37, 39-42, 45-49, 57, 59-61, 63-64, 70-72, 74-84, 86-88, and 99-106 (Kabat numbering) of sequence number 12.

[0025] In one embodiment, at least one of the complementarity-determining regions (CDRs) of the variable domain is derived from a donor non-human α4-binding antibody. In another embodiment, the antigen-binding region of the CDR graft heavy chain variable domain includes CDRs corresponding to positions 24-31 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) (Kabat numbering). Thus, in one embodiment, the VL framework has an acceptor sequence constructed from the IGKV4-1 germline sequence, the antibody AAH70335.1, or the germline-manipulated antibody AAH70335.1.

[0026] In yet another embodiment, at least one amino acid and fewer than 2, 3, 4, 5, 10, or 15 residues in FR1 of the VL chain are non-human residues. One or more of these residues may be identical, for example, to the non-human antibody framework region from which the CDR sequence originates. In one embodiment, the amino acid residue at the N-terminal position of FR1 is mutated to be identical to that of the non-human antibody framework region.

[0027] In another embodiment, at least one amino acid and fewer than 2, 3, 4, 5, 10, or 15 residues in FR2 of the VL chain are non-human residues. One or more of these residues may be identical, for example, to the non-human antibody framework region from which the CDR sequence originates.

[0028] In yet another embodiment, at least one amino acid and fewer than 2, 3, 4, 5, 10, or 15 residues in FR3 of VL are non-human residues. One or more of these residues may be identical, for example, to the non-human antibody framework region from which the CDR sequence originates. In yet another embodiment, the amino acid residue at Kabat position 87 is mutated to be identical to the non-human antibody framework region. In yet another embodiment, the amino acid residues at Kabat positions 67 and 87 are mutated to be identical to the non-human antibody framework sequence. In yet another embodiment, the amino acid residues at Kabat positions 67, 73, and 87 of SEQ ID NO: 7 are mutated to be identical to the non-human antibody framework sequence.

[0029] In other embodiments, the VL chain of the antibody has the sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0030] In one embodiment, the VH chain of the antibody has the sequence of SEQ ID NO: 4, and the VL chain of the antibody has the sequence of SEQ ID NO: 11.

[0031] In one embodiment, the CDRs of the VH and VL acceptor framework sequences are selected to be similar to the CDR sequence of a non-human (e.g., mouse) antibody sequence, where the non-human antibody binds to integrin alpha 4 or a fragment thereof. In another embodiment, the CDR sequence is selected to be similar to the CDR sequence of a non-human antibody that binds to the B1 epitope of the VLA-4 α4 chain. In one embodiment, the CDR is selected to be similar to a mouse monoclonal antibody, e.g., HP1 / 2, HP2 / 1, HP2 / 4, L25, P4C2, or 21.6 (Pulido et al., J. Biol. Chem. 266:10241-10245, 1991, U.S. Patent No. 6,033,665). Modification may mean, for example, excision and insertion or alteration, e.g., by directional mutagenesis.

[0032] In another embodiment, the present invention relates to an antibody or an antigen-binding fragment thereof, - Anti-VLA-4 VL chains as described herein, for example, donor anti-VLA-4 antibodies, for example, CDRs of anti-VLA-4 antibodies as described herein, and anti-VLA-4 VL chains having LC framework regions 1, 2, and 3 of the VL chain sequence or having a VL framework with 5, 10, or 15 or fewer differences from the germline variable region sequence of the VL chain (in one embodiment, variable region 4 is the human consensus sequence), - Anti-VLA-4 VH chains as described herein, for example, donor anti-VLA-4 antibodies, for example, CDRs of anti-VLA-4 antibodies as described herein, and anti-VLA-4 VL chains having LC framework regions 1, 2, and 3 of the VL chain sequence or having a VL framework with 5, 10, or 15 or fewer differences from the germline variable region sequence of the VL chain (in one embodiment, variable region 4 is the human consensus sequence) and It features an antibody or its antigen-binding fragment, which includes [the specified substance].

[0033] In one embodiment, the antibody binds to one or both of α4β1 and α4β7.

[0034] In another embodiment, the VL chain or VH chain, or antibody, or fragment thereof, described herein is detectably labeled.

[0035] In another embodiment, the present invention relates to a recombinant anti-alpha-4 antibody or an alpha-4 binding fragment thereof, comprising: (a) a variable light chain containing the sequence of SEQ ID NO: 11; (b) a variable heavy chain containing the sequence of SEQ ID NO: 4; (c) a constant light chain of human kappa light chain (SEQ ID NO: 82); and (d) a constant heavy chain of human IgG1, wherein the constant region is S127C, K129R, G135E, G136S, Q203K, I207T, N21 according to the Kabat numbering scheme. The present invention provides a recombinant anti-alpha-4 antibody or an alpha-4 binding fragment thereof, comprising the constant heavy chain of human IgG1 containing at least one mutation selected from deletions of 1D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, or K478.

[0036] In some embodiments, the constant heavy chain of human IgG1 contains, consists of, or essentially consists of at least two mutations selected from deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, or K478 according to the Kabat numbering scheme.

[0037] In some embodiments, the constant heavy chain of human IgG1 contains, consists of, or essentially consists of at least three mutations selected from deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, or K478 according to the Kabat numbering scheme.

[0038] In some embodiments, the constant heavy chain of human IgG1 contains at least four, or at least five, or at least six mutations selected from deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, or K478 according to the Kabat numbering scheme. Or including, consisting of, or essentially consisting of, at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or at least 16, or at least 17, or at least 18, or at least 19, or at least 20, or at least 21, or at least 22, or at least 23, or at least 24.

[0039] In some embodiments, the steady-state heavy chain of human IgG1 includes, consists of, or is essentially composed of, mutations resulting from deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme.

[0040] In another embodiment, the present invention provides a recombinant anti-alpha-4 antibody or an alpha-4 binding fragment thereof, comprising: (a) a variable light chain comprising the sequence of SEQ ID NO: 11; (b) a variable heavy chain comprising the sequence of SEQ ID NO: 4; (c) a constant light chain of human kappa light chain (SEQ ID NO: 82); and (d) a constant heavy chain of human IgG4, wherein the constant region contains at least one mutation selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme.

[0041] In some embodiments, the constant heavy chain of human IgG4 contains, consists of, or is essentially composed of at least two mutations selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme.

[0042] In some embodiments, the constant heavy chain of human IgG4 contains, consists of, or essentially comprises at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme.

[0043] In some embodiments, the constant heavy chain of human IgG4 contains, consists of, or is essentially composed of one or more deletion mutations of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme.

[0044] It should be noted that the C'-terminal lysine residue (position 478 according to the Kabat numbering scheme) is typically deleted post-translation or through technical ingenuity. Therefore, in some embodiments, the heavy chain (or the Fc portion of its domain) of the antibodies described herein lacks C-terminal lysine. In other words, in some embodiments, the lysine at position 478 (Kabat numbering) is deleted in the heavy chain (or the Fc portion of its domain) of the antibodies described herein.

[0045] In some embodiments, the constant heavy chain of human IgG1 without mutations or substitutions has the sequence of SEQ ID NO: 83.

[0046] In some embodiments, the constant heavy chain of human IgG4 without mutations or substitutions has the sequence of SEQ ID NO: 84.

[0047] In yet another embodiment, the present invention features a vector comprising DNA encoding an antibody heavy chain or an α4-binding fragment thereof as described herein. In some embodiments, the DNA of the vector encodes a VH having the sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0048] In yet another embodiment, the present invention features a vector comprising DNA encoding an antibody light chain or an α4-binding fragment thereof as described herein. In some embodiments, the DNA of the vector encodes a VL chain having the sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0049] In yet another embodiment, the present invention features a vector comprising DNA encoding an antibody heavy chain or its α4-binding fragment as described herein and an antibody light chain or its α4-binding fragment as described herein.

[0050] In another embodiment, the present invention features host cells containing the vectors described herein, for example, those capable of expressing the heavy and / or light chain antibodies or antibody fragments described herein. In certain embodiments, the host cells are Chinese hamster ovary (CHO) cells.

[0051] In one embodiment, the present invention relates to a method for producing a recombinant anti-α4 antibody or its α4-binding fragment, comprising: preparing host cells, to which the host cells are transfected with (a) a DNA sequence encoding the antibody heavy chain or its α4-binding fragment as described herein, and (b) a DNA sequence encoding the antibody light chain or its α4-binding fragment; and culturing the transfected cells to produce a recombinant anti-α4 antibody molecule or its α4-binding fragment. The DNA encoding the antibody heavy chain and light chain may be present in the same vector or in different vectors.

[0052] In one embodiment, the present invention relates to a method for producing a recombinant anti-α4 antibody or its α4-binding fragment, comprising: preparing a host cell to which the host cell has been transfected with (a) a DNA sequence encoding the antibody heavy chain or its α4-binding fragment, for example, a DNA sequence having the sequence of SEQ ID NO: 4, and (b) a DNA sequence encoding the antibody light chain or its α4-binding fragment, for example, a DNA sequence having the sequence of SEQ ID NO: 11; and culturing the transfected cell line to produce a recombinant anti-α4 antibody molecule or its α4-binding fragment. The DNA encoding the antibody heavy chain and light chain may be present in the same vector or in different vectors.

[0053] In another embodiment, the present invention relates to a method for treating diseases or disorders mediated by α4 integrins, such as α4β1(VLA-4) or α4β7 integrins, characterized by administering an α4 antibody or antibody fragment described herein, or a pharmaceutical composition containing such antibody or fragment, to a subject requiring such treatment. Subjects include, for example, inflammatory disorders, immune disorders, or autoimmune disorders (e.g., inflammation of the central nervous system, e.g., multiple sclerosis, meningitis, neuromyelitis optica, neurosarcoidosis, CNS vasculitis, encephalitis, and transverse myelitis), tissue or organ graft rejection or graft-versus-host disease, acute CNS injury, e.g., stroke, traumatic brain injury (TBI), or spinal cord injury (SCI); chronic kidney disease; allergies, e.g., allergic asthma; type 1 diabetes mellitus; inflammatory bowel disorders, e.g., Crohn's disease, ulcerative colitis; myasthenia gravis; fibromuscular Pain; arthritis disorders, e.g., rheumatoid arthritis, psoriatic arthritis; inflammatory / immunocutaneous disorders, e.g., psoriasis, vitiligo, dermatitis, lichen planus; systemic lupus erythematosus; Sjögren's syndrome; hematological cancers, e.g., multiple myeloma, leukemia, lymphoma; solid cancers such as sarcomas or carcinomas, e.g., lung cancer, breast cancer, prostate cancer, brain cancer; and fibrous disorders, e.g., pulmonary fibrosis, myelofibrosis, cirrhosis, mesangial proliferative glomerulonephritis, crescentic glomerulonephritis, diabetic nephropathy, and interstitial renal fibrosis, which may be present or at risk of developing. In preferred embodiments, the present invention features methods for treating patients with multiple sclerosis or for alleviating symptoms in patients with multiple sclerosis, e.g., clinically isolated syndrome (CIS), relapsing-remitting multiple sclerosis, or active secondary progressive multiple sclerosis. In certain embodiments, multiple sclerosis is relapsing-type multiple sclerosis. In preferred embodiments, the present invention features a method for treating patients who have or are at risk of developing epilepsy, including drug-resistant epilepsy. In certain embodiments, the subject or patient is human.

[0054] In another embodiment, the present invention features a method of treating a patient by administering an α4-binding antibody or antibody fragment to the patient. In one embodiment, the patient has cancer, such as a solid tumor or a hematological malignancy. For example, the patient treated with the α4-binding antibody or antibody fragment may have acute myeloid leukemia (AML) or multiple myeloma (MM).

[0055] In another embodiment, the patient has an inflammatory disorder such as multiple sclerosis, asthma (e.g., moderate to severe asthma), rheumatoid arthritis, diabetes mellitus, or Crohn's disease. In yet another embodiment, the composition is administered as a regimen. In yet another embodiment, the method further includes selecting a patient suitable for treatment with the composition. A patient suitable for treatment is, for example, one who shows signs or symptoms of disease onset, such as signs or symptoms of MS. In a preferred embodiment, the patient has epilepsy.

[0056] In certain embodiments, the patient is administered a therapeutically effective dose of α4-conjugated antibody or antibody fragment. In certain embodiments, the patient is administered α4-conjugated antibody or antibody fragment in doses ranging from approximately 0.0003 mg / kg, 0.0004 mg / kg, 0.0005 mg / kg, 0.0006 mg / kg, 0.0007 mg / kg, 0.0008 mg / kg, 0.0009 mg / kg, 0.0010 mg / kg, 0.0015 mg / kg, or 0.0020 mg / kg to approximately 0.0025 mg / kg, 0.003 mg / kg, 0.005 mg / kg, 0.010 mg / kg, 0.0125 mg / kg, 0.025 mg / kg, 0 The antibody is administered in amounts ranging from 0.050 mg / kg, 0.0625 mg / kg, 0.080 mg / kg, 0.100 mg / kg, 0.200 mg / kg, 0.300 mg / kg, 0.3125 mg / kg, 0.40 mg / kg, 0.50 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg. In some embodiments, the patient is administered α4-conjugated antibody or antibody fragments in amounts ranging from about 0.025 mg / kg to about 10 mg / kg. In some embodiments, the patient is administered α4-conjugated antibody or antibody fragments in amounts ranging from about 0.0625 mg / kg to about 8.0 mg / kg. In some embodiments, the patient is administered an amount of α4-conjugated antibody or antibody fragment ranging from approximately 0.3 mg / kg to approximately 6.0 mg / kg. In some embodiments, the patient is administered an amount of α4-conjugated antibody or antibody fragment ranging from approximately 0.5 mg / kg to approximately 5.0 mg / kg.

[0057] In yet another embodiment, the method further includes administering a second therapeutic agent to the patient, such as a chemotherapeutic agent, a thrombolytic agent, a neuroprotective agent, an anti-inflammatory agent, a steroid, a cytokine, or a growth factor.

[0058] In one embodiment, the patient is administered a humanized anti-VLA-4 antibody or a fragment thereof as described herein, for example, HuHP1 / 2, H1L1, H1L2, or H1L3.

[0059] In one embodiment, the composition containing the α4-binding antibody is administered as a regimen, for example, at regular intervals. For example, the composition may be administered once a day, once a week, or once a month, once a week, twice a week, three times a week, four times a week, or more, or once every two weeks, once every three weeks, once every four weeks, or more.

[0060] In one embodiment, the dosage may be adjusted according to the patient's clearance rate after a previous administration of anti-α4 antibodies. For example, in one embodiment, the patient is not administered a second or subsequent dose until the level of anti-α4 antibodies in the patient's body falls below a predetermined level. In one embodiment, a sample of the patient (e.g., plasma, serum, blood, or urine) is assayed for the presence of anti-α4 antibodies, and if the level of anti-α4 antibodies exceeds a predetermined level, the patient is not administered a second or subsequent dose. If the level of anti-α4 antibodies in the patient's body is below a predetermined level, the patient is administered a second or subsequent dose.

[0061] In one embodiment, the composition is administered continuously, for example, over a period of more than 30 minutes to less than 1, 2, 4, or 12 hours. The composition comprising the antibody and the second agent may be administered by any suitable method, for example, subcutaneously, intramuscularly, or intravenously.

[0062] In some embodiments, the antibody and the second drug are administered in the same doses as those prescribed for monotherapy. In other embodiments, the antibody is administered in a dose less than or equal to the amount required for efficacy when administered alone. Similarly, the second drug may be administered in a dose less than or equal to the amount required for efficacy when administered alone.

[0063] Another feature of this disclosure is a method of evaluating a patient by determining whether the patient meets pre-selected criteria, and if the patient meets the pre-selected criteria, approving, providing, prescribing, or administering a VLA-4 conjugated antibody preparation described herein to the patient. In one embodiment, the pre-selected criterion is that the patient has not adequately responded to previous alternative therapeutic treatments or regimens, for example, for the treatment of MS. In another embodiment, the pre-selected criterion is the absence of any signs or symptoms of progressive multifocal leukoencephalopathy (PML), or the absence of any diagnosis of PML. In some cases, this selection is based on the absence of risk factors for PML, for example, that the subject does not test positive for JC virus DNA or JC virus antibody. In another embodiment, this criterion is incorporated herein by reference and is described in PCT / US07 / 75577 (published as WO2008 / 021954), which describes methods and systems for the distribution of drugs and delivery of drugs to patients.

[0064] In another embodiment, a method for distributing the composition described herein is provided. The composition comprises an alpha-4 conjugated antibody. The method comprises providing a recipient (e.g., an end-user, patient, physician, drug retailer or wholesaler, distributor, or hospital, nursing home clinic, or HMO dispensing department) with a package containing a unit dose of the drug sufficient to treat a patient for at least 6, 12, 24, 36, or 48 months. In another embodiment, the present invention features a method for evaluating the quality of a package or lot of a package of the composition described herein comprising an alpha-4 conjugated antibody (e.g., determining whether it is expired). The method comprises evaluating whether the package is expired. The expiration date is at least 6, 12, 24, 36, or 48 months from a pre-selected event such as manufacturing, assay, or packaging, for example, more than 24 or 36 months. In some embodiments, the determination or step is made as a result of analysis. For example, depending on whether the product has expired, appropriate analysis may determine whether the antibodies in the package are used or discarded, classified, selected, put on sale or held in reserve, shipped, moved to a new location, put into commercial transactions, sold or sold, withdrawn from commercial transactions, or delisted.

[0065] In another embodiment, the present invention features a package comprising at least two unit doses of an aqueous composition containing an α4-conjugated antibody. In one embodiment, all unit doses contain the same amount of antibody, while in other embodiments, there are two or more unit doses of different strengths, or two or more different formulations with different strengths or release characteristics, for example.

[0066] In another embodiment, the present invention includes a method for instructing a recipient on the administration of a formulation containing an α4-binding antibody. The method includes instructing the recipient (e.g., an end-user, patient, physician, drug retailer or wholesaler, distributor, or hospital, nursing home clinic, or HMO dispensing department) that the antibody should be administered to the patient according to the regimen described herein. The method may also include instructing the recipient that the antibody should be administered before its expiration date. The expiration date is at least 6, 12, 24, 36, or 48 months from a pre-selected event such as manufacturing, assay, or packaging, for example, more than 24 or 36 months. In one embodiment, the recipient also receives a supply of the antibody, for example, a supply of a unit dose of the antibody.

[0067] In one embodiment, the stabilized polypeptide comprises a chimeric Fc region, and the stabilized polypeptide comprises at least one constant domain derived from a human IgG4 antibody and at least one constant domain derived from a human IgG1 antibody.

[0068] In one embodiment, the Fc region is a glycosylated Fc region.

[0069] In one embodiment, the Fc region is an aglycosylated Fc region.

[0070] In one embodiment, the Fc region is an aglycosylated Fc region containing glutamine (Q) at positions 297 (EU numbering) and 314 (Kabat numbering).

[0071] In one embodiment, the chimeric Fc region includes the CH2 domain of an IgG4 isotype IgG antibody and the CH3 domain of an IgG1 isotype IgG antibody. In some embodiments, the chimeric Fc region, which includes the CH2 domain of an IgG4 isotype IgG antibody and the CH3 domain of an IgG1 isotype IgG antibody, further includes glutamine (Q) residues at positions 297 (EU numbering) and 314 (Kabat numbering) of the Fc region instead of naturally occurring arginine (N) residues.

[0072] In one embodiment, the chimeric Fc region comprises the hinge, CH1 domain, and CH2 domain of an IgG4 isotype IgG antibody, and the CH3 domain of an IgG1 isotype IgG antibody, which contains proline at amino acid positions 228 (EU numbering) and 241 (Kabat numbering). In some embodiments, the chimeric Fc region comprises the hinge, CH1 domain, and CH2 domain of an IgG4 isotype IgG antibody, and the CH3 domain of an IgG1 isotype IgG antibody, which contains proline at amino acid positions 228 (EU numbering) and 241 (Kabat numbering), and further comprises glutamine (Q) residues at positions 297 (EU numbering) and 314 (Kabat numbering) of the Fc region instead of naturally occurring arginine (N) residues.

[0073] In one embodiment, the IgG antibody is a human antibody.

[0074] In one embodiment, the aglycosylated Fc region includes a chimeric hinge domain.

[0075] In one embodiment, the chimeric hinge domain includes substitutions of proline residues at amino acid positions 228 (EU numbering) and 241 (Kabat numbering).

[0076] In one embodiment, the antibody comprises a VH chain having the sequence of SEQ ID NO: 4, a VL chain having the sequence of SEQ ID NO: 11, and a chimeric Fc region including the hinge, CH1 domain, and CH2 domain of an IgG4 isotype IgG antibody, and the CH3 domain of an IgG1 isotype IgG antibody, wherein the antibody includes substitutions to glutamine residues (Q) at positions 297 (EU numbering) and 314 (Kabat numbering) of the CH2 region, substitutions to proline residues (P) at amino acid positions 228 (EU numbering) and 241 (Kabat numbering) of the hinge region, and a deletion of lysine (K) at EU numbering position 447 (Kabat numbering position 478) of the CH3 region.

[0077] In one embodiment, the antibody comprises a heavy chain having the sequence of SEQ ID NO: 80 and a light chain having the sequence of SEQ ID NO: 81.

[0078] In one embodiment, the antibody comprises the heavy chain of SEQ ID NO: 86, the HP1 / 2 H1 heavy chain of IgG4 (wild type; glycosylated type).

[0079] In one embodiment, the antibody comprises the heavy chain of SEQ ID NO: 87, and the HP1 / 2 heavy chain of IgG4 S228P (EU numbering) (also known as IgG4P; glycosylated form).

[0080] In one embodiment, the antibody comprises the heavy chain of SEQ ID NO: 88, the HP1 / 2 H1 heavy chain of IgG4 S228P L235E (EU numbering) (also known as IgG4PE; glycosylated form).

[0081] In one embodiment, the antibody comprises the heavy chain of SEQ ID NO: 89 and the HP1 / 2 H1 heavy chain (aggregosyl) of IgG4 S228P T299A (EU numbering).

[0082] In one embodiment, the antibody comprises the heavy chain of SEQ ID NO: 90 and the HP1 / 2 H1 heavy chain (aggregosyl) of IgG1 N297Q (EU numbering).

[0083] In certain therapeutic antibodies, such as those possessing a constant IgG4 region, scrambling can result in a bispecific monoclonal antibody that is monovalent to a therapeutic antibody target (e.g., VLA4) and monovalent to another antigen. In one embodiment, the antibody can reduce PK / PD variability by eliminating scrambling or reducing the antibody's sensitivity to scrambling. In certain embodiments, the antibody can increase the potency of the bivalent monoclonal antibody and eliminate the bispecificity resulting from scrambling. In certain embodiments, the antibody can exhibit higher binding affinity and / or sustained higher receptor occupancy than its non-mutant counterpart.

[0084] Other features and advantages of the present invention will become apparent from the embodiments and claims for carrying out the invention described below.

[0085] This patent or application document includes at least one drawing drawn in color. Copies of this patent or patent application publication, including the color drawing(s), will be provided by the Japan Patent Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0086] [Figure 1] This shows three sequence variants of HP1 / 2 heavy chain (SEQ ID NO: 1) with the human heavy chain germline IGHV1-f (SEQ ID NO: 2). Lowercase letters above the sequence indicate insertions using the Kabat numbering scheme. [Figure 2] Four sequence variants of the HP1 / 2 light chain (SEQ ID NO: 6) are shown: one with the germline IGKV4-1 antibody sequence (SEQ ID NO: 7) (design L0-SEQ ID NO: 8, L1-SEQ ID NO: 9, and L2-SEQ ID NO: 10), and another with the human kappa germline manipulation AAH7033.1 antibody sequence (SEQ ID NO: 12) (design L3-SEQ ID NO: 11). Lowercase letters above the sequence indicate insertions according to the Kabat numbering scheme. [Figure 3] This is a graph showing the results of an ELISA assay. [Figure 4]This is a graph showing the results of an ELISA assay. [Figure 5] This is the amino acid sequence of IgG4 Fc (hinge + CH2 + CH3 domain) (SEQ ID NO: 14). The hinge region is shown in bold, and the CH3 domain is underlined. The "S" enclosed in a square is Ser228 (EU numbering, Ser241 in Kabat numbering). The "N" enclosed in a circle is Asn314 in Kabat numbering (Asn297 in EU numbering). [Figure 6] This graph shows flow cytometry data of HuHP1 / 2 binding to various tumor cell lines. "HP1 / 2" refers to humanized HP1 / 2. [Figure 7] A-C are a series of graphs showing the inhibition of AML cell line binding to fibronectin or VCAM1-Ig coated wells by HuHP1 / 2. A shows inhibition of HL60 and KG1 cell binding to FN-coated wells. B shows inhibition of KG1 cell binding to VCAM1-Ig-coated wells. C shows inhibition of HL60 cell binding to FN-coated and VCAM1-Ig-coated wells when incubated with 20 μg / mL of HuHP1 / 2 (black bar graph). Colorless bar graphs show cell adhesion rates in the presence of isotype controls. "HP1 / 2" refers to humanized HP1 / 2. [Figure 8] A-C constitute a series of graphs showing the inhibition of MM cell line binding to fibronectin or VCAM1-Ig coated wells by HuHP1 / 2. A shows inhibition of U266 and H929 cell binding to FN-coated wells. B shows inhibition of U266 and H929 cell binding to VCAM1-Ig-coated wells. C shows inhibition of U266 cell binding to FN-coated and VCAM1-Ig-coated wells when incubated with 20 μg / mL of HuHP1 / 2 (black bar graph). Colorless bar graphs show cell adhesion rates in the presence of isotype controls. "HP1 / 2" refers to humanized HP1 / 2. [Figure 9]A-C constitute a series of graphs showing the inhibition of CLL cell line binding to fibronectin or VCAM1-Ig coated wells by HuHP1 / 2. A shows inhibition of Mec1 and JM1 cell binding to FN-coated wells. B shows inhibition of Mec1 and JM1 cell binding to VCAM1-Ig-coated wells. C shows inhibition of Mec1 cell binding to FN-coated and VCAM1-Ig-coated wells when incubated with 20 μg / mL of HuHP1 / 2 (black bar graph). Colorless bar graphs show cell adhesion rates in the presence of isotype controls. "HP1 / 2" refers to humanized HP1 / 2. [Figure 10] This document shows the structure of a typical antigen-binding polypeptide (IgG antibody), as well as the functional characteristics of the antibody's antigen-binding and effector functions (e.g., Fc receptor (FcR) binding). It also demonstrates how the presence of sugar in the antibody's CH2 domain (glycosylation) alters the effector function (FcR binding) but does not affect antigen binding. [Figure 11A] The X-ray crystal structure of IgG1 (pdb code 1hzh) shows two interacting CH3 domains. IgG1 residues K409 / K440 (EU numbering / Kabat numbering) and D399 / D427 (EU numbering / Kabat numbering) are highlighted. [Figure 11B] Alignment of human IgG1 / kappa constant domain sequences using structure-based HMM (Wang et al, 2009, Proteins 76:99, 2009) is shown. The CL, CH1, and CH3 residue positions involved in interdomain interactions, as well as amino acid positions strongly covariant with amino acids in direct contact with carbohydrates, are highlighted in gray. Kabat and EU numbers are presented below the alignment. [Figure 11C] A ribbon diagram of the structure of the IgG1-CH2 domain is shown (Sondermann et al, 406(6793):267-73, 2000). The N-linked carbohydrate within the CH2 domain and the valine residue embedded in the characteristic 6-amino acid loop are shown. [Figure 11D]This shows the alignment of the natural IgG1-CH2 sequence with the fully modified IgG1-CH2 sequence. Modified residue positions are shown in black. The EU numbers for the modified positions are shown above the alignment. [Figure 12A] This shows the turbidity of an exemplary IgG Fc construct of the present invention after stirring. [Figure 12B] The monomer content (%) of the exemplary IgG Fc construct of the present invention after stirring is shown. [Figure 13] This shows the relative peak height over time of the IgG Fc construct while maintaining a low pH (pH 3.1). [Figure 14] The initial binding kinetics of exemplary IgG1 and IgG4 Fc constructs of the present invention to the Fcγ receptor, as measured by solution affinity surface plasmon resonance, are shown. [Figure 15] The titration curves used to calculate the IC50 of exemplary IgG1 and IgG4 Fc constructs of the present invention bound to CD64 (FcγRI) (Figure 6A) and CD16 (FcγRIIIa V158) (Figure 6B) are shown. [Figure 16] Figure 7A shows titration curves demonstrating reduced binding of IgG1 T318K (Kabat-numbered) (T299K, EU-numbered) to CD64 (FcγRI) compared to IgG1 T318A (Kabat-numbered) (T299A, EU-numbered) and IgG1 wild-type, as well as titration curves demonstrating reduced binding of other exemplary IgG4 Fc constructs incorporating the T318A (Kabat-numbered) (T299A, EU-numbered) mutation to CD64 (FcγRI) compared to IgG1 wild-type. [Figure 17] This shows the binding of an exemplary IgG4 Fc construct incorporating the T299K mutation to CD16 (FcγRIIIa V158) compared to other exemplary IgG4 Fc constructs incorporating the T299A mutation and to IgG1 wild type. [Figure 18] The titration curves used to evaluate the binding of exemplary IgG1 and IgG4 Fc constructs of the present invention to complement factor C1q are shown. [Figure 19A] The titration curves used to evaluate the binding of various Fc constructs to CD64 are shown. [Figure 19B] The titration curves used to evaluate the binding of various Fc constructs to CD16 are shown. [Figure 19C] This shows that the half-life of N314Q (Kabat numbered) (N297Q, EU numbered) IgG4-CH2 / IgG1-CH3 was the same as that of T318A (Kabat numbered) (T299A, EU numbered) antibody (slightly shorter than that of aglycosylated IgG1). [Figure 20A] The titration curves used to evaluate the binding of the T318X (Kabat numbered) (T299X, EU numbered) construct to CD64 are shown, and it is also shown that the positively charged side chains of T318R (Kabat numbered) (T299R, EU numbered) and T318KA (Kabat numbered) (T299K, EU numbered) result in a lower affinity for CD64. [Figure 20B] The titration curves used to evaluate the binding of CD64 to various alternative constructs are shown. [Figure 20C] The titration curve used to evaluate the binding of the construct to CD32aR is shown. [Figure 20D] The titration curve used to evaluate the binding of the construct to CD32aR is shown. [Figure 20E] This shows the binding of the structure to CD16. [Figure 20F] This shows the binding of the structure to CD16. [Figure 20G] The results of the C1q ELISA are shown. [Figure 20H] The results of the C1q ELISA are shown. [Figure 21] Panel A shows the titration curve used to evaluate the binding of the construct to CD64, and Panel B shows the titration curve used to evaluate the binding of the construct to CD16. [Figure 22A]This document contains the nucleotide and amino acid sequences of HP1 / 2 hG4P agly(N297Q, EU numbering)G1, kappa light chain (H1L3). The nucleotide sequence (DNA corresponding to SEQ ID NO: 91 is shown) and light chain sequence (SEQ ID NO: 92) are shown, with amino acids 1-233. Amino acids 1-19 (and coding DNA sequence), shown in bold italics, contain the synthetic LC signal peptide. The mature N-terminus begins with amino acid 20 (S). [Figure 22B] HP1 / 2 hG4P agly(N297Q, EU numbering)G1 contains the nucleotide and amino acid sequences of the heavy chain (H1L3). The nucleotide sequence of the heavy chain (SEQ ID NO: 94) (DNA is shown, corresponding to SEQ ID NO: 93) and amino acids 1-469 are shown. Amino acids 1-22 (DNA sequence is shown in italics) contain the recoded synthetic signal peptide. The mature N-terminus begins with amino acid 23 (E). [Figure 23A] HP1 / 2 hG4P agly(N297Q, EU numbering) G1 contains an amino acid sequence including the signal sequence (underlined) of the kappa light chain (H1L3). [Figure 23B] HP1 / 2 hG4P agly(N297Q, EU numbering)G1, containing the amino acid sequence of the kappa light chain (H1L3), has the mature light chain of SEQ ID NO: 81 and the mature heavy chain of SEQ ID NO: 80. [Figure 24] The UV spectra of proteins possessing the mature light chain of SEQ ID NO: 81 (the mature light chain of HP1 / 2 hG4P agly(N297Q, EU numbering)G1 H1L3 ND001) and the mature heavy chain of SEQ ID NO: 80 (the mature heavy chain of HP1 / 2 hG4P agly(N297Q, EU numbering)G1 H1L3 ND004 / ND006) are presented. [Figure 25]The SDS-PAGE results for proteins with the mature light chain of SEQ ID NO: 81 (HP1 / 2 hG4P agly(N297Q, EU numbering)G1 H1L3 ND001) and the mature heavy chain of SEQ ID NO: 80 (HP1 / 2 hG4P agly(N297Q, EU numbering)G1 H1L3 ND004 / ND006) are shown. Non-reducing conditions are shown in the two lanes on the left (columns 1 and 2), and reducing conditions are shown in the two lanes on the right (columns 3 and 4). Lane 1 is HP1 / 2 hG4P agly(N297Q, EU numbering)G1 H1L3 ND001) under non-reducing conditions, Lane 2 is the molecular weight marker under non-reducing conditions, Lane 3 is HP1 / 2 hG4P agly(N297Q, EU numbering)G1 H1L3 ND001) under reducing conditions, and Lane 4 is the molecular weight marker under reducing conditions. [Figure 26] The IEF results for proteins possessing the mature light chain of SEQ ID NO: 81 (the mature light chain of HP1 / 2 hG4P agly(N297Q, EU numbering)G1 H1L3 ND001) and the mature heavy chain of SEQ ID NO: 80 (the mature heavy chain of HP1 / 2 hG4P agly(N297Q, EU numbering)G1 H1L3 ND004 / ND006) are shown. [Figure 27] The results of analytical size exclusion chromatography of proteins possessing the mature light chain of SEQ ID NO: 81 (the mature light chain of HP1 / 2 hG4P agly(N297Q, EU numbering)G1 H1L3 ND001) and the mature heavy chain of SEQ ID NO: 80 (the mature heavy chain of HP1 / 2 hG4P agly(N297Q, EU numbering)G1 H1L3 ND004 / ND006) are shown. [Figure 28] The DSC results for proteins with the mature light chain of SEQ ID NO: 81 (mature light chain of HP1 / 2 hG4P agly(N297Q, EU numbering) G1 H1L3 ND001) and the mature heavy chain of SEQ ID NO: 80 (mature heavy chain of HP1 / 2 hG4P agly(N297Q) G1 H1L3 ND004 / ND006) are shown. The melting curve of the intact HP1 / 2 hG4P agly(N297Q, EU numbering) G1 H1L3 antibody is also shown. [Figure 29]The DSC results for proteins containing the mature light chain of SEQ ID NO: 81 (HP1 / 2 hG4P agly(N297Q, EU numbering)G1 H1L3 ND001) and the mature heavy chain of SEQ ID NO: 80 (HP1 / 2 hG4P agly(N297Q)G1 H1L3 ND004 / ND006) are shown. The melting curve of the Fc fragment is also shown. [Figure 30] The DSC results for proteins containing the mature light chain of SEQ ID NO: 81 (HP1 / 2 hG4P agly(N297Q, EU numbering)G1 H1L3 ND001) and the mature heavy chain of SEQ ID NO: 80 (HP1 / 2 hG4P agly(N297Q)G1 H1L3 ND004 / ND006) are shown. The melting curve for the Fab2 fragment is also shown. [Figure 31] The results of SDS-PAGE analysis of HP1 / 2 samples under non-reducing conditions are shown. Each sample was loaded at 6 and 3 μg / lane. Molecular weight markers are shown on the left side of the panel. SDS-PAGE was performed on Invitrogen 4-20% polyacrylamide gradient gels, and the gels were stained with Simply Blue dye (Invitrogen) and destained with distilled water. Lane 1 is HP1 / 2 hG4P agly (N297Q, EU numbering) G1 H1L3 at 6ug / lane, Lane 2 is HP1 / 2 hG4P agly (T299A) at 6ug / lane, Lane 3 is natalizumab G4P agly at 6ug / lane, Lane 4 is natalizumab G4P at 6ug / lane, Lane 5 is HP1 / 2 hG4P agly (N297Q, EU numbering) G1 H1L3 at 3ug / lane, Lane 6 is HP1 / 2 hG4P agly (T299A) at 3ug / lane, Lane 7 is natalizumab G4P agly at 3ug / lane, and Lane 8 is natalizumab G4P at 3ug / lane. [Figure 32A] The results of SEC analysis of HP1 / 2 samples are shown. The samples were passed through a Superdex Increase 5 / 150 GL column (GE 28-9909-45) at a flow rate of 0.2 ml / min in PBS (10 mM sodium phosphate, 0.15 M NaCl, pH 7.5). [Figure 32B] The results of SEC analysis for HP1 / 2 hG4P agly (N297Q, EU numbering) G1 H1L3, HP1 / 2 hG4P agly (T299A), natalizumab G4P agly, and natalizumab G4P are shown. Samples were flowed at a rate of 0.2 ml / min in PBS (10 mM sodium phosphate, 0.15 M NaCl, pH 7.5) on a Superdex 200 Increase 5 / 150 GL column (GE 28-9909-45). [Figure 33] A shows the DSC results for humanized HP1 / 2 H1 / L3 huIgG4P agly (N297Q, EU numbering) / IgG1 chimera (GP1 / 2 GrP agly / G1), and B shows the DSC results for humanized HP1 / 2 G1 / L3 huIgG4P agly (T299A, EU numbering) (HP1 / 2 hG4P). [Figure 34] This line graph shows the time course of pharmacokinetics in cynomolgus monkeys after a single IV bolus administration of 3 mg / kg HP1 / 2 H1 / L3 huIgG4P agly (N297Q, EU numbering) / IgG1 chimera (GP1 / 2 GrP agly / G1) (labeled HP12 in the figure; white circle), 30 mg / kg HP12 (black circle), and 3 mg / kg natalizumab (Tysabri, black triangle). [Figure 35] This line graph shows the time course of free receptors in cynomolgus monkeys after a single IV bolus administration of 3 mg / kg HP1 / 2 H1 / L3 huIgG4P agly (N297Q, EU numbering) / IgG1 chimera (GP1 / 2 GrP agly / G1) (labeled HP12 in the figure; white circle), 30 mg / kg HP12 (black circle), and 3 mg / kg natalizumab (Tysabri, black triangle). [Figure 36] This bar graph shows the expression levels of CD49 (alpha-4 integrin) in humans (Human 1 and Human 2) and cynomolgus monkeys (Cynomolgus monkey 1 and Cynomolgus monkey 2). [Figure 37] A to D are line graphs showing the time course of predicted receptor occupancy after a single IV dose of natalizumab (red line) and HP1 / 2 (black line) at the indicated dosages. [Figure 38] This graph shows the predicted receptor occupancy at Cmax after administration of specific doses of HP1 / 2 (labeled HP12 in the figure, white circle) and natalizumab (black triangle). [Modes for carrying out the invention]

[0087] Antibodies against VLA-4 have been demonstrated to be useful in the treatment of diseases. For example, the anti-VLA-4 antibody natalizumab (TYSABRI®) is used to treat relapsing-relapsing multiple sclerosis and Crohn's disease. However, in certain conditions, such as the treatment of acute conditions like spinal cord injury (SCI) or traumatic brain injury (TBI), or in treatments where the number of administrations is limited, such as cancer treatment, it may be advantageous to treat with an anti-VLA-4 antibody that has a different affinity than natalizumab, e.g., binds at a higher affinity and / or has a different pharmacological profile (e.g., has lower in vivo pharmacokinetic / pharmacodynamic variability). Such antibodies may also be useful in treating conditions such as multiple sclerosis, as this may reduce the frequency of required treatments or allow for more efficient administration by means other than infusion. Lower doses may also reduce the risk of adverse events such as PML. Therefore, the present invention provides an antibody having such desirable properties.

[0088] The specific antibodies of the present invention disclosed herein exhibited an unexpected characteristic: the newly designed humanized α4-binding antibody had a binding affinity to α4 that was 10 times higher than that of the anti-α4 antibody natalizumab.

[0089] This antibody also has a stabilized Fc region with reduced effector function, for example, by including one or more stabilizing amino acids in the Fc region of the Fc polypeptide. In some embodiments, the stabilizing amino acids stabilize the Fc region of the polypeptide without affecting the glycosylation and / or effector function of the polypeptide, and without significantly altering other desired functions of the polypeptide (e.g., antigen-binding affinity or half-life).

[0090] For the convenience of ensuring that this specification and the claims are clearly understood, the following definitions are provided below.

[0091] definition As used herein, the term “effector function” means the functional ability of an Fc region or portion thereof to bind to proteins and / or cells of the immune system and mediate various biological effects. Effector function may be antigen-dependent or antigen-independent. A decrease in effector function means that the antigen-binding activity of the variable region of the antibody (or fragment thereof) is maintained, but one or more effector functions are reduced. An increase or decrease in effector function, for example, Fc binding to an Fc receptor or complement protein, can be expressed in terms of a multiplier of change (e.g., a 1x change, a 2x change, etc.) and can be calculated, for example, based on the rate of change in binding activity determined using assays well known in the art. As used herein, the term “antigen-dependent effector function” means an effector function that is typically induced after an antibody has bound to a corresponding antigen. Typical antigen-dependent effector functions include the ability to bind to complement proteins (e.g., C1q). For example, when the Cl component of complement binds to an Fc region, the classical complement system is activated, which can lead to opsonization and lysis of cellular pathogens. This is a process called complement-dependent cell-mediated cytotoxicity (CDCC). Complement activation also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity.

[0092] Other antigen-dependent effector functions are mediated by antibodies binding to specific Fc receptors ("FcRs") on cells via their Fc region. Several Fc receptors exist that are specific to different classes of antibodies, including IgG (gamma receptor, i.e., IgγR), IgE (epsilon receptor, i.e., IgεR), IgA (alpha receptor, i.e., IgαR), and IgM (mu receptor, i.e., IgμR). When antibodies bind to Fc receptors on the cell surface, several important and diverse biological responses are induced, including endocytosis of immune complexes, phagocytosis and destruction of antibody-coated particles or microorganisms (also known as antibody-dependent phagocytosis, or ADCP), clearance of immune complexes, lysis of antibody-coated target cells by killer cells (also known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, regulation of immune system cell activation, and control of immunoglobulin placental cross-transit and production. The specific Fc receptor, the Fc gamma receptor (FcγR), plays a crucial role in suppressing or enhancing immune mobilization. FcγR is expressed in leukocytes and consists of three distinct classes: FcγRI, FcγRII, and FcγRIII (Gessner et al., Ann. Hematol., (1998), 76:231-48). Structurally, all FcγRs are members of the immunoglobulin superfamily, possessing an α-chain that binds to IgG, and their extracellular portion consists of two or three Ig-like domains. Human FcγRI (CD64) is expressed in human monocytes and exhibits high binding affinity (Ka=108~109M-1) to monomeric IgG1, IgG3, and IgG4. Human FcγRII(CD32) and FcγRIII(CD16) have low affinity (Ka<10⁷M⁻¹) for IgG1 and IgG3, respectively, and can only bind to complex or polymeric forms of these IgG isotypes. Furthermore, each class of FcγRII and FcγRIII includes both "A" and "B" types.FcγRIIa(CD32a) and FcγRIIIa(CD16a) bind to the surface of macrophages, NK cells, and some T cells via their transmembrane domains, while FcγRIIb(CD32b) and FcγRIIIb(CD16b) selectively bind to the cell surface of granulocytes (e.g., neutrophils) via phosphatidylinositol glycan (GPI) anchors. The human mouse homologs of FcγRI, FcγRII, and FcγRIII are FcγRIIa, FcγRIIb / 1, and FcγR1o, respectively.

[0093] As used herein, the term “antigen-independent effector function” means an effector function that can be induced by an antibody, regardless of whether the antibody is bound to the corresponding antigen. Typical antigen-independent effector functions include the cellular transport, circulating half-life, and clearance rate of immunoglobulins, as well as the enhancement of purification. The structurally unique Fc receptor, “neonatal Fc receptor” or “FcRn,” also known as the salvage receptor, plays a crucial role in regulating half-life and cellular transport. Other Fc receptors purified from microbial cells (e.g., Staphylococcus aureus protein A or G) can bind to the Fc region with high affinity and can be used to enhance the purification of Fc-containing polypeptides.

[0094] Unlike FcγR, which belongs to the immunoglobulin superfamily, human FcRn is structurally similar to major histocompatibility complex (MHC) class I polypeptides (Ghetie and Ward, Immunology Today, (1997), 18(12):592-8). FcRn is typically expressed as a heterodimer consisting of a transmembrane α chain or heavy chain complexed with a soluble β chain or light chain (β2 microglobulin). FcRn shares 22-29% sequence identity with class I MHC molecules and possesses a non-functional MHC peptide binding groove (Simister and Mostov, Nature, (1989), 337:184-7). Similar to MHC, the α chain of FcRn consists of three extracellular domains (α1, α2, α3), with a short cytoplasmic end that anchors the protein to the cell surface. The α1 and α2 domains interact with the FcR binding site within the antibody's Fc region (Raghavan et al, Immunity, (1994), 1:303-15). FcRn is expressed in the placental uterine region or yolk sac of mammals and is involved in the transfer of IgG from mother to fetus. FcRn is also expressed in the small intestine of neonatal rodents, where it is involved in the transfer of maternal IgG from ingested colostrum or milk through the brush border epithelium. FcRn is also expressed in numerous other tissues of many species, as well as in various endothelial cell lines. FcRn is also expressed in the vascular endothelium, muscular vascular structures, and hepatic sinusoids of adult humans. FcRn is thought to play an additional role in maintaining the circulating half-life or serum level of IgG by binding to IgG and recirculating it into the serum. The binding of FcRn to the IgG molecule is entirely pH-dependent, with optimal binding occurring at pH less than 7.0.

[0095] As used herein, the term “half-life” means the in vivo biological half-life of a particular conjugated polypeptide. Half-life can be expressed as the time required for half of the amount administered to a subject to be eliminated from the animal’s circulation and / or other tissues. When the clearance curve of a given conjugated polypeptide is constructed as a function of time, this curve is typically biphasic, consisting of a rapid α-phase and a longer β-phase. The α-phase typically represents the equilibrium between the intravascular and extravascular spaces of the administered Fc polypeptide and is partially determined by the size of the polypeptide. The β-phase typically represents the catabolism of the conjugated polypeptide in the intravascular space. Thus, in non-limiting embodiments, the term “half-life” as used herein means the half-life of the conjugated polypeptide in the β-phase. The typical β-phase half-life of a human antibody in humans is 21 days. As used herein, the term “polypeptide” means a polymer of two or more natural or non-natural amino acids. The term “Fc polypeptide” means a polypeptide containing an Fc region or a portion thereof (e.g., an Fc portion). In some embodiments, the Fc polypeptide is stabilized by the method of the present invention. In an optional embodiment, the Fc polypeptide further comprises binding sites operably linked or fused to the Fc region (or a portion thereof) of the Fc polypeptide.

[0096] As used herein, the term “protein” means a polypeptide (e.g., an Fc polypeptide) or a composition comprising two or more polypeptides. Therefore, a protein may be a monomer (e.g., a single Fc polypeptide) or a polymer. For example, in one embodiment, the protein of the present invention is a dimer. In one embodiment, the dimer of the present invention is a homodimer comprising two identical monomeric subunits or polypeptides (e.g., two identical Fc polypeptides). In another embodiment, the dimer of the present invention is a heterodimer comprising two non-identical monomeric subunits or polypeptides (e.g., two non-identical Fc polypeptides, or an Fc polypeptide and a second polypeptide other than an Fc polypeptide). The dimeric subunits may comprise one or more polypeptide chains, where at least one of the polypeptide chains is an Fc polypeptide. For example, in one embodiment, the dimer comprises at least two polypeptide chains (e.g., at least two Fc polypeptide chains). In one embodiment, the dimer comprises two polypeptide chains, where one or both of these chains are Fc polypeptide chains. In another embodiment, the dimer comprises three polypeptide chains, where one, two, or all of the polypeptide chains are Fc polypeptide chains. In yet another embodiment, the dimer comprises four polypeptide chains, where one, two, three, or all of the polypeptide chains are Fc polypeptide chains.

[0097] As used herein, the terms “conjugated,” “fused,” or “combined” are used synonymously. These terms mean the joining of two or more elements or components into one by any means, including chemical conjugation or recombinant means. Methods of chemical conjugation (e.g., using heterobifunctional crosslinking agents) are known in the art. As used herein, the terms “gene-fused” or “gene fusion” mean that two or more proteins, polypeptides, or fragments thereof collinearly covalently bonded or attached via their individual peptide backbones by gene expression of a single polynucleotide molecule encoding these proteins, polypeptides, or fragments. Such a gene fusion results in the expression of a single, continuous gene sequence. In some embodiments, the gene fusion is in-frame; that is, two or more ORFs fuse to form a continuous, longer ORF in a manner that maintains the correct reading frame of the original open reading frame (ORF). Thus, the resulting recombinant fusion protein is a single polypeptide containing two or more protein segments corresponding to polypeptides encoded by the original ORFs (these segments do not usually join in this manner naturally). Thus, even if the leading frame is continuous across the entire fused gene segment, the protein segments may be physically or spatially separated, for example, by an in-frame polypeptide linker.

[0098] As used herein, the term “Fc region” is defined as a portion of an immunoglobulin formed by two or more Fc parietes of an antibody heavy chain. In certain embodiments, the Fc region is a dimeric Fc region. “Dimerated Fc region” or “dcFc” means a dimer formed from Fc parietes of two distinct immunoglobulin heavy chains. A dimeric Fc region may be a homodimer consisting of two identical Fc parietes (e.g., Fc regions of naturally occurring immunoglobulins) or a heterodimer consisting of two non-identical Fc parietes. In other embodiments, the Fc region is a monomer or a “single-stranded” Fc region (i.e., an scFc region). A single-stranded Fc region consists of Fc parietes that are genetically related (i.e., encoded in a single continuous gene sequence) within a single polypeptide chain. An exemplary scFc region is disclosed in PCT application PCT / US2008 / 006260, filed on 14 May 2008 and incorporated herein by reference.

[0099] As used herein, the term “Fc moiety” refers to the portion of the immunoglobulin heavy chain that begins in the hinge region immediately upstream of the papain cleavage site (i.e., IgG residue 226, Kabat numbering / residue 216, EU numbering, with the first residue of the heavy chain constant region being 114) and ends at the C-terminus of the immunoglobulin heavy chain. Thus, the Fc moiety may be a complete Fc moiety or a portion thereof (e.g., a domain). A complete Fc moiety includes at least the hinge domain, the CH2 domain, and the CH3 domain (i.e., Kabat amino acids 226–477; EU amino acids 216–446). Additional lysine residues (K) may be present at the far C-terminus of the Fc moiety, but are often cleaved by the mature antibody. Unless otherwise noted, each amino acid position within the Fc region is numbered according to the EU numbering system recognized in the art. In certain embodiments, the Fc moiety includes at least one of the hinge domains (e.g., upper, middle, and / or lower hinge regions), CH2 domains, CH3 domains, or variants, parts, or fragments thereof. In some embodiments, the Fc moiety includes at least one CH2 domain or CH3 domain. In certain embodiments, the Fc moiety is a complete Fc moiety. In other embodiments, the Fc moiety includes one or more amino acid insertions, deletions, or substitutions compared to a naturally occurring Fc moiety. For example, at least one of the hinge domains, CH2 domains, or CH3 domains (or parts thereof) may be deleted. For example, the Fc portion may include, or consist of, (i) a hinge domain (or a part thereof) fused to a CH2 domain (or a part thereof), (ii) a hinge domain (or a part thereof) fused to a CH3 domain (or a part thereof), (iii) a CH2 domain (or a part thereof) fused to a CH3 domain (or a part thereof), (iv) a CH2 domain (or a part thereof), and (v) a CH3 domain or a part thereof.

[0100] Note that antibody amino acid residues have their own unique numbering scheme. See Kabat et al., Sequences of Proteins of Immunological Interest, 5. th The USD Department of Health and Human Services, NIH, USA, ed., vol. 4, 1991, presents a numbering scheme sometimes called the EU index numbering scheme, as described by Kabat et al. However, a new numbering system called the Kabat numbering scheme has since emerged.

[0101] In a non-restrictive example, the following table compares the EU numbering and Kabat numbering of the heavy chain CH1, CH2, and CH3 domains of human IgG1. The sequence of the constant region of human IgG1 is shown in Sequence ID No. 83. TIFF2026113464000002.tif180170TIFF2026113464000003.tif208170TIFF2026113464000004.tif172170

[0102] The amino acid residues in the table above are either naturally occurring or classical in human IgG1. Therefore, the classical residue at position 297 (EU numbering) (position 314 in Kabat numbering) is asparagine (N). When an amino acid residue changes from a classical residue, it is called a substitution or mutation.

[0103] Due to the similarity of human IgG molecules (e.g., IgG1, IgG2, IgG3, IgG4), the constant region of all IgG molecules begins with an alanine residue at EU position 118 (Kabat position 114) and ends with a lysine residue at the C-terminus at EU position 447 (Kabat position 478).

[0104] The table below shows the correlation between EU and Kabat numbering for human IgG1 hinges, starting from the glutamic acid residue at position 216 in EU numbering (position 226 in Kabat numbering). TIFF2026113464000005.tif53170

[0105] The table below shows the correlation between EU and Kabat numbering for human IgG4 hinges, starting from the glutamic acid residue at position 216 in EU numbering (position 226 in Kabat numbering). TIFF2026113464000006.tif38170

[0106] In some embodiments, if the substitutions referred to herein are present in either naturally occurring IgG1 or IgG4, the EU numbering or Kabat numbering indicating the substitution relates to the numbering of the naturally occurring parent molecule (or its domain). For example, if the serine at position 228 of the hinge of IgG4 is mutated to, for example, proline, this mutation is indicated by EU numbering S228P and Kabat numbering S241P.

[0107] The full-length sequence of the naturally occurring heavy chain of human IgG1 is presented here as Sequence ID No. 83, starting with the classic alanine residue at position 118 (EU numbering) [position 114 (Kabat numbering)].

[0108] The full-length sequence of the naturally occurring heavy chain of human IgG1 is presented here as Sequence ID No. 84, starting with the classic alanine residue at position 118 (EU numbering) [position 114 (Kabat numbering)].

[0109] As will be understood by those skilled in the art, the Fc moiety may be modified such that its amino acid sequence differs from that of the complete Fc moiety of a naturally occurring immunoglobulin molecule, while retaining at least one desirable function provided by the naturally occurring Fc moiety. For example, the Fc moiety may include, or consist of, at least a portion of an Fc moiety known in the art to be necessary for FcRn binding or a long half-life. In another embodiment, the Fc moiety includes at least a portion known in the art to be necessary for FcγR binding. In one embodiment, the Fc region of the present invention includes at least a portion known in the art to be necessary for protein A binding. In one embodiment, the Fc moiety of the present invention includes at least a portion of an Fc molecule known in the art to be necessary for protein G binding. In a particular embodiment, the Fc moieties of the Fc region are of the same isotype.

[0110] For example, the Fc portion may originate from an IgG1 or IgG4 isotype immunoglobulin (e.g., human immunoglobulin). However, the Fc region (or one or more Fc portions of an Fc region) may be chimeric. A chimeric Fc region may contain Fc portions derived from different immunoglobulin isotypes. In certain embodiments, at least two of the Fc portions of a dimeric or single-stranded Fc region may be from different immunoglobulin isotypes. In additional or alternative embodiments, a chimeric Fc region may contain one or more chimeric Fc portions. For example, a chimeric Fc region or chimeric Fc portion may contain one or more portions derived from an immunoglobulin of a first isotype (e.g., IgG1, IgG2, or IgG3 isotype), with the remainder of the Fc region or Fc portion being from different isotypes. For example, the Fc region or Fc portion of an Fc polypeptide may include a CH2 and / or CH3 domain derived from a first isotype (e.g., IgG1, IgG2, or IgG4 isotype) immunoglobulin and a hinge region derived from a second isotype (e.g., IgG3 isotype) immunoglobulin. In another embodiment, the Fc region or Fc portion includes a hinge and / or CH2 domain derived from a first isotype (e.g., IgG4 isotype) immunoglobulin and a CH3 domain derived from a second isotype (e.g., IgG1, IgG2, or IgG3 isotype) immunoglobulin. In another embodiment, the chimeric Fc region includes an Fc portion (e.g., a complete Fc portion) derived from a first isotype (e.g., IgG4 isotype) immunoglobulin and an Fc portion derived from a second isotype (e.g., IgG1, IgG2, or IgG3 isotype) immunoglobulin. In one exemplary embodiment, the Fc region or Fc portion includes a CH2 domain derived from IgG4 immunoglobulin and a CH3 domain derived from IgG1 immunoglobulin. In another embodiment, the Fc region or Fc portion includes CH1 and CH2 domains derived from an IgG4 molecule, as well as a CH3 domain derived from an IgG1 molecule.In another embodiment, the Fc region or Fc portion includes a portion of the CH2 domain of a particular isotype of antibody, for example, Kabat positions 309-360 and EU positions 292-340 of the CH2 domain. For example, in one embodiment, the Fc region or Fc portion includes amino acids 292-340 (EU numbering) and 309-360 (Kabat numbering) of CH2 derived from the IgG4 portion, with the remainder of CH2 derived from the IgG1 portion (alternatively, positions 292-340 (EU numbering) and 309-360 (Kabat numbering) of CH2 may be derived from the IgG1 portion, and the remainder of CH2 may be derived from the IgG4 portion).

[0111] In other embodiments, the Fc region or Fc portion may include a chimeric hinge region. The chimeric hinge may be partly derived from an IgG1, IgG2, or IgG4 molecule (e.g., upper and lower intermediate hinge sequences) and partly derived from an IgG3 molecule (e.g., intermediate hinge sequence). In another example, the Fc region or Fc portion may include a chimeric hinge that is partly derived from an IgG1 molecule and partly from an IgG4 molecule. In certain embodiments, the chimeric hinge may include upper and lower hinge domains derived from an IgG4 molecule, as well as an intermediate hinge domain derived from an IgG1 molecule. Such a chimeric hinge may be created by introducing proline substitutions (Ser228Pro (EU numbering); Ser241Pro (Kabat numbering)) at the EU228 and Kabat241 positions of the intermediate hinge domain of the IgG4 hinge region. In another embodiment, the chimeric hinge may include the amino acids at positions 246-249 of the Kabat IgG2 antibody and / or the Ser228Pro(EU numbering)Ser241Pro(Kabat numbering) mutation, where the remaining amino acids of the hinge are derived from the IgG4 antibody (e.g., the chimeric hinge of sequence ESKYGPPCPPCPAPPVAGP). Further chimeric hinges are described in U.S. Patent Application No. 10 / 880,320, which is incorporated herein by reference in whole.

[0112] The definition of "Fc region" explicitly includes "aglycosylated Fc region." As used herein, "aglycosylated Fc region" refers to an Fc region in which, for example, one or more of its Fc portions lack a covalently bonded oligosaccharide or glycan at the N-glycosylation site at EU297 and Kabat314. In certain embodiments, the aglycosylated Fc region is fully aglycosylated; that is, the entire Fc portion lacks a carbohydrate. In other embodiments, the aglycosylation is partial aglycosylation (i.e., semi-glycosylation). The aglycosylated Fc region may also be a deglycosylated Fc region, which is an Fc region in which the Fc carbohydrate has been removed, for example, chemically or enzymatically. Alternatively, the aglycosylated Fc region may be non-glycosylated or unglycosylated. In other words, an antibody expressed without the Fc carbohydrate, for example, by expression in organisms that do not naturally bind carbohydrates to proteins (e.g., bacteria), or by expression in host cells or organisms in which the glycosylation mechanism is lost by genetic engineering or the addition of glycosylation inhibitors (e.g., glycosyltransferase inhibitors), for example, by mutations in one or more residues encoding the glycosylation pattern at the N-glycosylation site at EU position 297 or 299, Kabat position 314 or 318 (e.g., Asn-X-Ser or Asn-X-Thr). In alternative embodiments, the Fc region is a "glycosylated Fc region," i.e., fully glycosylated at all available glycosylation sites.

[0113] The term “parent Fc polypeptide” includes polypeptides (e.g., IgG antibodies) that contain an Fc region for which stabilization is desired. In some embodiments, the parent Fc polypeptide is an effector-less Fc polypeptide. Stabilized Fc polypeptides with reduced effector function have been reported (see, for example, PCT Publication WO2010 / 085682, which is incorporated herein by reference in whole). Thus, the parent Fc polypeptide represents the original Fc polypeptide on which the method of the present invention is performed or which can be used as a reference point for stability comparison. The parent polypeptide may include a native (i.e., naturally occurring) Fc region or Fc moiety (e.g., the Fc region or Fc moiety of human IgG4), or an Fc region having an existing amino acid sequence modification (e.g., insertions, deletions, and / or other changes) of a naturally occurring sequence but lacking one or more stabilizing amino acids.

[0114] The terms “mutation” or “mutate” are understood to include physically creating a mutation in the parent Fc polypeptide (for example, by altering a codon of a nucleic acid molecule encoding one amino acid, for example, by site-directed mutagenesis, to result in a codon encoding a different amino acid), or synthesizing a variant Fc region having an amino acid not found in the parent Fc region (for example, by knowing the nucleotide sequence of the nucleic acid molecule encoding the parent Fc region and designing the synthesis of a nucleic acid molecule containing a nucleotide sequence encoding a variant of the parent Fc region without the need to mutate one or more nucleotides of the nucleic acid molecule encoding the stabilized polypeptide of the present invention).

[0115] In one exemplary embodiment, the parent Fc polypeptide comprises the Fc region of the effectorless Fc polypeptide. As used herein, the term “effectorless Fc polypeptide” means an Fc polypeptide having altered or reduced effector function compared to a wild-type, aglycosylated antibody of the IgG1 isotype. In some embodiments, the reduced or altered effector function is antibody-dependent effector function, e.g., ADCC and / or ADCP. In one embodiment, the effectorless Fc polypeptide has reduced effector function as a result of modification or reduction of glycosylation in the Fc region of the Fc polypeptide, e.g., the aglycosylated Fc region. In another embodiment, the effectorless Fc polypeptide has reduced effector function due to the incorporation of the IgG4 Fc region or a portion thereof (e.g., the CH2 and / or CH3 domains of the IgG4 antibody).

[0116] The terms “variant Fc polypeptide” or “Fc variant” include Fc polypeptides derived from a parent Fc polypeptide. An Fc variant differs from a parent Fc polypeptide in that it includes stabilization of one or more stabilizing amino acid residues, for example, due to the introduction of at least one Fc stabilizing mutation. In certain embodiments, the Fc variant of the present invention includes an Fc region (or Fc moiety) that is sequence-identical to the parent polypeptide except for the presence of one or more stabilizing Fc amino acids. In some embodiments, the Fc variant has enhanced stability compared to the parent Fc polypeptide and, in some cases, has equivalent or reduced effector function compared to the parent Fc polypeptide.

[0117] When a polypeptide or amino acid sequence is said to "derived from" a given polypeptide or protein, it means the origin of the polypeptide. In some embodiments, a polypeptide or amino acid sequence derived from a particular sequence has an amino acid sequence that is essentially identical to that sequence or a portion thereof (this portion consisting of at least 10-20 amino acids, or at least 20-30 amino acids, or at least 30-50 amino acids, or another number of amino acids that a person skilled in the art could identify as originating from that sequence). In relation to polypeptides, "linear sequence" or "sequence" means the order of amino acids in the polypeptide in the direction from the amino terminus to the carboxyl terminus, where adjacent residues in the sequence are consecutive in the primary structure of the polypeptide. A polypeptide derived from another polypeptide (e.g., a parent Fc polypeptide) (e.g., a variant Fc polypeptide) may have one or more amino acid residues that have one or more mutations compared to the starting polypeptide or parent polypeptide, e.g., substitution with another amino acid residue, or insertion or deletion of one or more amino acid residues. In some embodiments, the polypeptide includes an amino acid sequence that does not occur naturally. Such variants necessarily have less than 100% sequence identity or similarity to the starting polypeptide. In a non-limiting embodiment, the variant has an amino acid sequence such that, for example, over the entire length or a portion of the variant molecule (e.g., the Fc region or Fc portion), the amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide is about 75% to less than 100%, or about 80% to less than 100%, or about 85% to less than 100%, or about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), or about 95% to less than 100%. In one embodiment, one amino acid differs between the sequence of the starting polypeptide (e.g., the Fc region of the parent Fc polypeptide) and the sequence derived therefrom (e.g., the Fc region of the variant Fc polypeptide).In other embodiments, the starting polypeptide sequence and the variant polypeptide sequence differ by 2 to 10 amino acids (e.g., about 2 to 20, 2 to 15, 2 to 10, 5 to 20, 5 to 15, and 5 to 10 amino acids). For example, there may be fewer than 10 amino acids that differ (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids). This sequence identity or similarity is defined herein as the percentage of amino acid residues that are identical (i.e., the same residues) to the starting amino acid residues in the candidate sequence after aligning these sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage.

[0118] The Fc polypeptide of the present invention may contain an amino acid sequence derived from a human immunoglobulin sequence (e.g., at least one Fc region or Fc moiety) (e.g., an Fc region or Fc moiety of a human IgG molecule). However, the polypeptide may also contain one or more amino acids derived from another mammalian species. For example, a primate Fc moiety or primate binding site may be included in the target polypeptide. Alternatively, one or more mouse amino acids may be present in the Fc polypeptide. In some embodiments, the Fc polypeptide of the present invention is non-immunogenic.

[0119] Furthermore, it will be understood by those skilled in the art that the Fc polypeptide of the present invention may be modified so that its amino acid sequence differs from that of the parent polypeptide from which it originates, while retaining one or more desirable activities of the parent polypeptide (e.g., reduced effector function). In certain embodiments, nucleotide or amino acid substitutions are made to stabilize the Fc polypeptide. In one embodiment, an isolated nucleic acid molecule encoding an Fc variant can be produced by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence of the parent Fc polypeptide so that one or more amino acid substitutions, additions, or deletions are introduced into the encoding protein. Mutations (e.g., stabilizing mutations) can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0120] As used herein, the term “protein stability” means a recognized measure in the art relating to the maintenance of one or more physical properties of a protein in response to environmental conditions (e.g., rising or falling temperature). In one embodiment, this physical property is the maintenance of the protein’s covalent structure (e.g., the absence of proteolytic cleavage, unwanted oxidation, or deamidation). In another embodiment, this physical property is the presence of the protein in a properly folded state (e.g., the absence of soluble or insoluble aggregates or precipitates). In one embodiment, protein stability is measured by assaying the protein’s biophysical properties, such as thermal stability, pH unfolding profile, stable removal of glycosylation, solubility, biochemical function (e.g., the ability to bind to proteins (e.g., ligands, receptors, antigens, etc.) or chemical moieties), and / or combinations thereof. In another embodiment, biochemical function is indicated by the binding affinity of interactions. In one embodiment, the measure of protein stability is thermal stability, i.e., resistance to thermal load. Stability can be measured using methods known in the art and / or specified herein. For example, the "transition temperature," also known as "Tm," may be measured. Tm is the temperature at which 50% of a polymer, such as a binding molecule, denatures, and is considered a standard parameter representing the thermal stability of a protein.

[0121] The term "amino acid" includes alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (VaI or V). Non-classical amino acids are also within the scope of the present invention, and these include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs such as those described in Ellman et al. Meth. Enzym. 202:301-336 (1991). To generate such non-spontaneous amino acid residues, the procedures of Noren et al. Science 244:182 (1989) and Ellman et al. (above) can be used. Briefly, these procedures involve chemically activating a suppressor tRNA with a non-spontaneous amino acid residue, then transcribing it in vitro and translating the RNA. The introduction of non-classical amino acids can also be achieved using peptide chemistry known in the art. As used herein, the term “polar amino acid” includes amino acids (e.g., M, F, W, S, Y, N, Q, C) that have a net charge of zero but have partial charges at various parts of their side chains that are not zero. These amino acids may be involved in hydrophobic and electrostatic interactions. As used herein, the term “charged amino acid” includes amino acids that may have a non-zero net charge in their side chains (e.g., R, K, H, E, D). These amino acids may be involved in hydrophobic and electrostatic interactions. As used herein, the term “amino acid with sufficient stereovolume” includes amino acids that have side chains occupying a relatively large three-dimensional space.Examples of amino acids having a side-chain chemical structure with sufficient stereovolume include tyrosine, tryptophan, arginine, lysine, histidine, glutamic acid, glutamine, and methionine, or their analogues or mimics.

[0122] "Amino acid substitution" means that at least one amino acid residue present in the default amino acid sequence (the amino acid sequence of the starting polypeptide) is replaced by a second, different "substituting" amino acid residue. "Amino acid insertion" means that at least one additional amino acid is incorporated into the default amino acid sequence. Insertions typically consist of the insertion of one or two amino acid residues, but in the present invention, larger "peptide insertions," such as the insertion of about 3 to about 5, or even up to about 10, 15, or 20 amino acid residues, may be made. The inserted residue(s) may be naturally occurring or non-naturally occurring, as disclosed above. "Amino acid deletion" means that at least one amino acid residue is removed from the default amino acid sequence. As shown above, these terms include actual changes that occur to an existing physical nucleic acid molecule, or modifications to an existing nucleic acid sequence that are made during the design process (e.g., on paper or on a computer). In certain embodiments, the polypeptide of the present invention is a conjugated polypeptide. As used herein, the term “binding polypeptide” (e.g., binding antibody) means a polypeptide (e.g., Fc polypeptide) comprising at least one target binding site or binding domain that specifically binds to a particular target molecule (e.g., an antigen or binding partner). For example, in one embodiment, the binding polypeptide of the present invention comprises an immunoglobulin antigen binding site or a portion of a receptor molecule involved in ligand binding, or a portion of a ligand molecule involved in receptor binding. The binding polypeptide of the present invention comprises at least one binding site. In one embodiment, the binding polypeptide of the present invention comprises at least two binding sites. In one embodiment, the binding polypeptide comprises two binding sites. In another embodiment, the binding polypeptide comprises three binding sites. In another embodiment, the binding polypeptide comprises four binding sites. In one embodiment, the binding sites are linked in series with each other. In other embodiments, the binding sites are located at different positions on the binding polypeptide, for example, at one or more N-terminuses or C-terminuses of the Fc region of an Fc polypeptide.For example, if the Fc region is an scFc region, the binding site may be attached to the N-terminus, C-terminus, or both ends of the scFc region. If the Fc region is a dimeric Fc region, the binding site may be attached to one or both N-terminuses and / or one or both C-terminuses.

[0123] As used herein, the terms “binding domain,” “binding site,” or “binding portion” mean a portion, region, or site of a binding polypeptide that has biological activity (excluding biological activity by Fc) that mediates specific binding to a target molecule (e.g., an antigen, ligand, receptor, substrate, or inhibitor). Exemplary binding domains include biologically active proteins or portions, antigen-binding sites, receptor-binding domains of ligands, ligand-binding domains of receptors, or enzymatic domains. In another example, the term “binding portion” means a biologically active molecule or portion that binds to a component of a biological system (e.g., a protein in serum, or on a cell surface, or in the cell matrix) and this binding results in a biological effect (e.g., measured by changes in the active portion and / or the binding component (e.g., cleavage of the active portion and / or the binding component, signal transduction, or enhancement or inhibition of a biological response in a cell or object)).

[0124] As used herein, the term “ligand-binding domain” means a region or derivative thereof that retains at least a portion (or most) of the qualitative ligand-binding ability and / or biological activity of a native receptor (e.g., a cell surface receptor) or a corresponding native receptor. As used herein, the term “receptor-binding domain” means a region or derivative thereof that retains at least a portion of the qualitative receptor-binding ability and / or biological activity of a native ligand or a corresponding native ligand. In one embodiment, the binding polypeptide of the present invention has at least one binding domain that is specific to a target molecule to be reduced or eliminated, e.g., a cell surface antigen or a soluble antigen. In some embodiments, the binding domain includes or comprises an antigen-binding site (e.g., a variable heavy chain sequence and a variable light chain sequence or six CDRs derived from an antibody, located in an alternative framework region (e.g., a human framework region optionally containing one or more amino acid substitutions)). As used herein, the term “binding affinity” includes the strength of the binding interaction and therefore includes both actual binding affinity and apparent binding affinity. Actual binding affinity is the ratio of the association rate to the dissociation rate. Therefore, conferring or optimizing binding affinity involves varying one or both of these components to achieve a desired level of binding affinity. Apparent affinity may include, for example, interaction avidity.

[0125] As used herein, the term “binding free energy” (or “free energy of binding”) includes the meanings recognized in the art, particularly those applied to binding-site-ligand interactions or Fc-FcR interactions in solvents. A decrease in binding free energy enhances affinity, while an increase in binding free energy reduces affinity.

[0126] The term "specificity" includes the number of potential binding sites that specifically bind to a given target (e.g., to trigger an immune response). A binding polypeptide may be monospecific, containing one or more binding sites that specifically bind to the same target (e.g., the same epitope), or it may be polyspecific, containing two or more binding sites that specifically bind to different regions of the same target (e.g., different epitopes) or different targets. In one embodiment, a polyspecific binding polypeptide (e.g., a bispecific polypeptide) can be constructed that has binding specificity to two or more target molecules (e.g., two or more antigens or two or more epitopes on the same antigen). In another embodiment, a polyspecific binding polypeptide has at least one binding domain specific to the target molecule to be reduced or eliminated, and at least one binding domain specific to the target molecule on the cell. In yet another embodiment, a polyspecific binding polypeptide has at least one binding domain specific to the target molecule to be reduced or eliminated, and at least one binding domain specific to the drug. In yet another embodiment, the polyspecific conjugating polypeptide has at least one binding domain specific to a target molecule to be reduced or eliminated, and at least one binding domain specific to a prodrug. In yet another embodiment, the polyspecific conjugating polypeptide is a tetravalent antibody having two binding domains specific to one target molecule and two binding sites specific to a second target molecule.

[0127] As used herein, the term “binding value” means the number of potential binding domains in a binding polypeptide or binding protein. Each binding domain specifically binds to one target molecule. If a binding polypeptide contains two or more binding domains, each binding domain may specifically bind to the same molecule or different molecules (e.g., different ligands or different antigens, or different epitopes on the same antigen). In one embodiment, the binding polypeptide of the present invention is monovalent. In another embodiment, the binding polypeptide of the present invention is polyvalent. In another embodiment, the binding polypeptide of the present invention is divalent. In yet another embodiment, the binding polypeptide of the present invention is trivalent. In a further embodiment, the binding polypeptide of the present invention is tetravalent. In certain embodiments, the binding polypeptide of the present invention utilizes a polypeptide linker. As used herein, the term “polypeptide linker” means a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) that connects two domains of a polypeptide chain with a linear amino acid sequence. For example, a polypeptide linker may be used to connect a binding site to the Fc region (or Fc portion) of the Fc polypeptide of the present invention. In some embodiments, such a polypeptide linker provides flexibility to the polypeptide molecule. For example, in one embodiment, a VH domain or VL domain is fused to or linked to a polypeptide linker, the N-terminus or C-terminus of the polypeptide linker is bound to the C-terminus or N-terminus of the Fc region (or Fc moiety), and the N-terminus of the polypeptide linker is bound to the N-terminus or C-terminus of the VH or VL domain. In certain embodiments, the polypeptide linker is used to link (e.g., gene fusion) two Fc moieties or domains of an scFc polypeptide. Such polypeptide linkers are also referred to herein as Fc-linked polypeptides. As used herein, the term "Fc-linked polypeptide" specifically means a linking polypeptide that links (e.g., gene fusion) two Fc moieties or domains. The linking molecule of the present invention may comprise two or more peptide linkers.

[0128] As used herein, the term “properly folded polypeptide” includes polypeptides in which all functional domains constituting the polypeptide are clearly active (e.g., the conjugated polypeptide of the present invention). As used herein, the term “improperly folded polypeptide” includes polypeptides in which at least one of the functional domains of the polypeptide is inactive. As used herein, “properly folded Fc polypeptide” or “properly folded Fc region” includes an Fc region (e.g., an scFc region) in which at least two component Fc portions are properly folded such that the resulting Fc region contains at least one effector function.

[0129] As used herein, the term “immunoglobulin” includes polypeptides having a combination of two heavy chains and two light chains, regardless of whether or not they possess the specific immunoreactivity associated with them.

[0130] As used herein, the term “antibody” means an aggregate (e.g., an intact antibody molecule, an antibody fragment, or a variant thereof) that has significant known specific immune response activity against an antigen of interest (e.g., a tumor-associated antigen). Antibodies and immunoglobulins include light and heavy chains, which may or may not be linked by interchain covalent bonds. The basic structures of immunoglobulins in vertebrate systems are relatively well understood.

[0131] As will be described in more detail below, the general term "antibody" includes five different classes of antibodies that can be biochemically distinguished. The Fc portion of each class of antibody is clearly within the scope of this invention, and the following description generally relates to the IgG class of immunoglobulin molecules. With respect to IgG, immunoglobulins consist of two identical light-chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chains with molecular weights of 53,000 to 70,000. These four chains are linked by disulfide bonds to form a "Y" structure, with the light chains flanking the heavy chains, starting from the branching point of the "Y" and extending throughout the variable domain.

[0132] The light chains of immunoglobulins are classified as either kappa or lambda (K, λ). Each heavy chain class can have either a kappa or lambda light chain attached to it. Generally, the light and heavy chains are covalently bonded to each other, and the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds, depending on whether the immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell. In the heavy chain, the amino acid sequence extends from the N-terminus at both ends of the Y-constituent branching to the C-terminus at the end of each chain. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses within these (e.g., γ1-γ4). It is the properties of this chain that determine the "class" of an antibody as IgG, IgM, IgA IgG, or IgE, respectively. Subclasses (isotypes) of immunoglobulins, such as IgG1, IgG2, IgG3, IgG4, and IgA1, have been thoroughly studied and are known to lead to functional specialization. Variations of these classes and isotypes are readily recognizable to those skilled in the art in light of the present disclosure and therefore fall within the scope of the present invention.

[0133] Both the light and heavy chains are divided into structurally and functionally homologous regions. The term “region” refers to a portion or sub-portion of a single immunoglobulin (as in the term “Fc region”) or a single antibody chain, including constant or variable regions, as well as more individual portions or sub-portions of the domain. For example, the light chain variable domain includes “complementarity-determining regions” (CDRs) scattered within the “framework region” (FRs), as defined herein. A particular region of an immunoglobulin may be defined as a “constant” (C) region or a “variable” (V) region based on whether there is relatively little sequence diversity within the region of various class members in the case of a “constant region,” or significant diversity within the region of various class members in the case of a “variable region.” The terms “constant region” and “variable region” may also be used functionally. In this regard, it will be understood that the variable region of an immunoglobulin or antibody determines antigen recognition and specificity. Conversely, the constant region of an immunoglobulin or antibody gives rise to important effector functions such as secretion, transplacental mobility, Fc receptor binding, and complement binding. The subunit structures and three-dimensional configurations of the constant region of various immunoglobulin classes are well known.

[0134] The constant and variable regions of immunoglobulin heavy and light chains fold to form domains. The term "domain" refers to an independently folding spherical region of a heavy or light chain polypeptide, including, for example, a β-pleated sheet and / or peptide loops stabilized by intrachain disulfide bonds (e.g., containing 3-4 peptide loops). The constant region domain of an immunoglobulin light chain is synonymous with "light chain constant region domain," "CL region," or "CL domain." The constant domain of a heavy chain (e.g., hinge, CH1, CH2, or CH3 domain) is synonymous with "heavy chain constant region domain," "CH region domain," or "CH domain." The variable domain of a light chain is synonymous with "light chain variable region domain," "VL region domain," or "VL domain." The variable domain of a heavy chain is synonymous with "heavy chain variable region domain," "VH region domain," or "VH domain."

[0135] By convention, the numbering of variable and constant domains increases distal to the antigen-binding site or amino terminus of the immunoglobulin or antibody. The N-terminus of each of the heavy and light chains of an immunoglobulin is a variable region, and the C-terminus is a constant region. The CH3 and CL domains actually contain the carboxyl terms of the heavy and light chains, respectively. Thus, the domains of the light chain immunoglobulin are oriented in the VL-CL direction, and the domains of the heavy chain are oriented in the VH-CH1-hinge-CH2-CH3 direction. The amino acid positions within the heavy chain constant region, including the amino acid positions within the CH1, hinge, CH2, and CH3 domains, are numbered herein according to the EU indexing system unless otherwise specified. In contrast, the amino acid positions within the light chain constant region (e.g., the CL domain) are numbered herein according to the Kabat indexing system (Kabat et al., ibid.).

[0136] As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "VL domain" includes the amino-terminal variable domain of an immunoglobulin light chain as assigned by the Kabat indexing system.

[0137] As used herein, the term “CH1 domain” includes, for example, the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain extending around EU118–215. The CH1 domain is adjacent to the VH domain, located on the amino-terminal side of the hinge region of the immunoglobulin heavy chain molecule, and does not form part of the Fc region of the immunoglobulin heavy chain. In one embodiment, the conjugated polypeptide of the present invention includes a CH1 domain derived from an immunoglobulin heavy chain molecule (e.g., a human IgG1 or IgG4 molecule).

[0138] As used herein, the term “hinge region” refers to the portion of a heavy chain molecule that binds the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, allowing the two antigen-binding regions at the N-terminus to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al. J.Immunol. 1998, 161:4083).

[0139] As used herein, the term “CH2 domain” includes, for example, a portion of a heavy-chain immunoglobulin molecule extending around EU231–340. The CH2 domain is unique in that it does not form a close pair with another domain. Rather, two N-linked branched carbohydrate chains interpose between two CH2 domains in an intact, natural IgG molecule. In one embodiment, the conjugated polypeptide of the present invention includes a CH2 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule). In another embodiment, the conjugated polypeptide of the present invention includes a CH2 domain derived from an IgG4 molecule (e.g., a human IgG4 molecule). In one exemplary embodiment, the polypeptide of the present invention includes a CH2 domain (EU231–340) or a portion thereof.

[0140] As used herein, the term “CH3 domain” refers to the portion of a heavy-chain immunoglobulin molecule that extends approximately 110 residues from the N-terminus of the CH2 domain, for example, around positions 341–446b (EU numbering system). Additional lysine residues (K) may be present at the far C-terminus of the CH3 domain, but are often cleaved by the mature antibody. The CH3 domain typically forms the C-terminal portion of the antibody. However, in some immunoglobulins, further domains may extend from the CH3 domain to form the C-terminal portion of the molecule (e.g., the CH4 domain in the μ-chain of IgM and the ε-chain of IgE). In one embodiment, the conjugated polypeptide of the present invention comprises a CH3 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule). In another embodiment, the conjugated polypeptide of the present invention comprises a CH3 domain derived from an IgG4 molecule (e.g., a human IgG4 molecule).

[0141] As used herein, the term "CL domain" includes, for example, the first (most amino-terminal) constant region domain of an immunoglobulin light chain extending from around Kabat107A to 216. The CL domain is adjacent to the VL domain. In one embodiment, the conjugated polypeptide of the present invention includes a CL domain derived from a kappa light chain (e.g., a human kappa light chain).

[0142] As described above, the variable region of an antibody allows the antibody to selectively recognize and specifically bind to the epitope of an antigen. Antibodies that specifically bind to a target are sometimes called target-binding antibodies (for example, an α4-binding antibody is an antibody that specifically binds to α4). "Specifically binding" means that the antibody (or its variable region) binds to its target (for example, the epitope of α4 integrin) by approximately 5 × 10⁻¹⁴ units. -5 (i.e., less than 50 μM), or about 1 × 10⁻⁶ -5 (i.e., less than 10 μM), or about 5 × 10⁻¹⁰ -6 (i.e., less than 5 μM), or about 1 × 10⁻¹⁶ -6 (i.e., less than 1 μM), or about 5 × 10⁻¹⁶ -7Less than (i.e., 500 nM), or less than about 1×10-7 (i.e., 100 nM), or about 5×10 -8 Less than (i.e., 50 nM), or less than about 1×10-8 (i.e., 10 nM), or less than about 5×10-9 (i.e., 5 nM), or about 1×10 -9 Less than (i.e., 1 nM), or about 5×10 -10 Less than (i.e., 500 pM), or about 1×10 -10 Less than (i.e., 100 pM), or about 5×10 -11 Less than (50 pM), or about 1×10 -11 Less than (10 pM), or about 5×10 -12 Less than (5 pM), or about 1×10 -12 Less than (1 pM), or about 5×10 -13 Less than (500 fM), or about 1×10 -13 Less than (100 fM), or about 5×10 -14 Less than (50 fM), or about 1×10 -14 Less than (10 fM), or about 5×10 -15 Less than (5 fM), or less than about 1×10-15 (1 fM), of the equilibrium dissociation constant (K D ) means specifically binding. That is, a subset of the VL domain and VH domain, or complementarity determining regions (CDRs) of the antibody combine to form a variable region that determines the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding sites present at the ends of each arm of the Y. More specifically, the antigen-binding site is determined by three CDRs in each of the VH and VL chains.

[0143] As used herein, the term “antigen-binding site” includes a site that specifically binds to (immunely reacts to) an antigen, such as a cell surface or a soluble antigen. In one embodiment, the binding site includes immunoglobulin heavy and light chain variable regions, and the binding site formed by these variable regions determines the specificity of the antibody. The antigen-binding site is formed by different variable regions depending on the polypeptide. In one embodiment, the binding polypeptide of the present invention includes an antigen-binding site comprising at least one heavy or light chain CDR of an antibody molecule (e.g., a sequence known in the art or described herein). In another embodiment, the binding polypeptide of the present invention includes an antigen-binding site comprising at least two CDRs of one or more antibody molecules. In another embodiment, the binding polypeptide of the present invention includes an antigen-binding site comprising at least three CDRs of one or more antibody molecules. In another embodiment, the binding polypeptide of the present invention includes an antigen-binding site comprising at least four CDRs of one or more antibody molecules. In another embodiment, the binding polypeptide of the present invention includes an antigen-binding site comprising at least five CDRs of one or more antibody molecules. In another embodiment, the binding polypeptide of the present invention includes an antigen-binding site comprising six CDRs of an antibody molecule. Exemplary antibody molecules containing at least one CDR that may be included in the conjugated polypeptide of the present invention are known in the art, and such exemplary molecules are described herein.

[0144] As used herein, the term “CDR” or “complementarity-determining region” refers to non-contiguous antigen-binding sites found within the variable regions of both heavy-chain and light-chain polypeptides. These specific regions are described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), as well as Chothia et al., J. MoI. Biol. 196:901-917 (1987) and MacCallum et al., J. MoI. Biol. 262:732-745 (1996), and their definitions include overlaps or subsets of amino acid residues when compared to one another. For comparison, the amino acid residues included in a CDR as defined by each of the above references are shown. In some embodiments, the term “CDR” refers to a CDR as defined by Kabat based on sequence comparison. TIFF2026113464000007.tif57170

[0145] As used herein, the terms “framework region” or “FR region” include amino acid residues that are part of the variable region but not part of the CDR (for example, when using Kabat’s definition of CDR). Therefore, the variable region framework is approximately 100–120 amino acids long but contains only amino acids outside the CDR. Specific examples of heavy chain variable regions and CDRs as defined by Kabat et al. include: framework region 1 corresponds to the variable region domain containing amino acids 1–30; framework region 2 corresponds to the variable region domain containing amino acids 36–49; framework region 3 corresponds to the variable region domain containing amino acids 66–94; and framework region 4 corresponds to the variable region domain from amino acid 103 to the end of the variable region. The light chain framework regions are similarly separated by each of the light chain variable region CDRs. Similarly, using the definition of CDRs by Chothia et al. or McCallum et al., the boundaries of the framework regions are separated by each of the aforementioned CDR ends. In some embodiments, the CDRs are defined by Kabat.

[0146] In naturally occurring antibodies, the six CDRs present in each monomeric antibody are short, discontinuous sequences of amino acids specifically positioned to form antigen-binding sites when the antibody takes on a three-dimensional configuration in an aqueous environment. The remainder of the variable domains of the heavy and light chains exhibit little intermolecular variation in amino acid sequence and are called framework regions. The framework regions mostly take on a β-sheet three-dimensional structure, and the CDRs form loops, which connect the β-sheet structure and, in some cases, form part of it. Thus, these framework regions function to form a scaffold for positioning the six CDRs correctly through non-covalent interactions between the chains. The antigen-binding sites formed by the positioned CDRs determine a surface complementary to the epitope of the immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to the immunoreactive antigen epitope. The positions of the CDRs can be readily identified by those skilled in the art.

[0147] In certain embodiments, the binding polypeptide of the present invention comprises at least two antigen-binding domains that result in the association of the binding polypeptide with a selected antigen (for example, within the same binding polypeptide (e.g., both the N-terminus and C-terminus of a single polypeptide), or bound to each binding polypeptide that is a component of the multimer-binding protein of the present invention). The antigen-binding domains do not need to originate from the same immunoglobulin molecule. In this regard, the variable region may or may not originate from any type of animal that can be induced to initiate a humoral response to a desired antigen and produce immunoglobulins. Thus, the variable region may be mammalian in origin, for example, from humans, mice, non-human primates (e.g., cynomolgus macaques, macaques, etc.), wolves, camelids (e.g., camels, llamas, and related species).

[0148] The terms “antibody variant” or “modified antibody” include antibodies that do not occur naturally and whose amino acid sequence or amino acid side-chain chemical structure differs from that of naturally occurring antibodies by at least one amino acid or an amino acid modification as described in this invention. As used herein, the term “antibody variant” includes synthetic antibodies that have been altered to not occur naturally, for example, antibodies that contain at least two heavy chain portions but do not contain two complete heavy chains (e.g., domain deletion antibodies or minibodies), polyspecific antibodies that have been altered to bind to two or more different antigens or different epitopes on a single antigen (e.g., bispecific, trispecific, etc.), heavy chain molecules bound to scFv molecules, single-chain antibodies, diabodies, triabodies, and antibodies having altered effector function.

[0149] As used herein, the term “scFv molecule” includes a binding molecule comprising one light chain variable domain (VL) or a portion thereof and one heavy chain variable domain (VH) or a portion thereof, wherein each variable domain (or portion thereof) is derived from the same or different antibodies. The scFv molecule may include an scFv linker interposed between the VH domain and the VL domain. The scFv molecule is known in the art and is described, for example, in U.S. Patent No. 5,892,019, Ho et al. 1989. Gene 77:51, Bird et al. 1988. Science 242:423, Pantoliano et al. 1991. Biochemistry 30:10117, Milenic et al. 1991. Cancer Research 51:6363, and Takkinen et al. 1991. Protein Engineering 4:837.

[0150] As used herein, "scFv linker" refers to the portion interposed between the VL and VH domains of scFv. In some embodiments, the scFv linker can maintain the scFv molecule in an antigen-binding conformation. In one embodiment, the scFv linker comprises or consists of an scFv linker peptide. In certain embodiments, the scFv linker peptide comprises or consists of a gly-ser polypeptide linker. In other embodiments, the scFv linker comprises a disulfide bond.

[0151] As used herein, the term “gly-ser polypeptide linker” means a peptide consisting of a glycine residue and a serine residue. An exemplary gly / ser polypeptide linker comprises the amino acid sequence (Gly4Ser)n. In one embodiment, n=1. In another embodiment, n=2. In a different embodiment, n=3, i.e., (Gly4Ser)3. In another embodiment, n=4, i.e., (Gly4Ser)4. In another embodiment, n=5. In yet another embodiment, n=6. In another embodiment, n=7. In yet another embodiment, n=8. In another embodiment, n=9. In yet another embodiment, n=10. Another exemplary gly / ser polypeptide linker comprises the amino acid sequence Ser(Gly4Ser)n. In one embodiment, n=1. In one embodiment, n=2. In a certain embodiment, n=3. In another embodiment, n=4. In another embodiment, n=5. In yet another embodiment, n=6.

[0152] As used herein, the term “natural cysteine” means a cysteine ​​amino acid that occurs naturally at a particular amino acid position in a polypeptide and is not artificially modified, introduced, or altered. The term “engineered cysteine ​​residue or analogue” means a non-natural cysteine ​​residue or cysteine ​​analogue (e.g., thiol-containing analogues such as thiazoline-4-carboxylic acid and thiazolidinedione-4-carboxylic acid (thioproline, Th)) that is introduced at an amino acid position in a polypeptide that does not naturally contain a cysteine ​​residue or analogue at that amino acid position by synthetic means (e.g., by recombinant technology, in vitropeptide synthesis, enzymatic or chemical coupling of peptides, or any combination thereof).

[0153] As used herein, the term “disulfide bond” includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a natural disulfide bond, and the two heavy chains are linked by two natural disulfide bonds at positions 239 and 242 in the Kabat numbering system (positions 226 or 229 in the EU numbering system).

[0154] As used herein, the term “conjugated cysteine” means a native or engineered cysteine ​​residue within a polypeptide that forms a disulfide bond or other covalent bond with a second native or engineered cysteine ​​or other residue present in the same or a different polypeptide. “Intrachain conjugated cysteine” means a conjugated cysteine ​​that is covalently bonded (i.e., intrachain disulfide bonded) with a second cysteine ​​present in the same polypeptide. “Interchain conjugated cysteine” means a conjugated cysteine ​​that is covalently bonded (i.e., interchain disulfide bonded) with a second cysteine ​​present in a different polypeptide.

[0155] As used herein, the term “free cysteine” means a naturally occurring or engineered cysteine ​​amino acid residue (and its analogues or mimics, e.g., thiazoline-4-carboxylic acid and thiazolidined-4-carboxylic acid (thioproline, Th)) in a polypeptide sequence that exists in a substantially reduced form. In some embodiments, free cysteine ​​can be modified by the effector portion of the present invention.

[0156] The term "thiol-modifying reagent" refers to a chemical factor that can selectively react with the thiol group of an manipulated cysteine ​​residue or its analogue within a bound polypeptide (e.g., within the polypeptide linker of the bound polypeptide), thereby providing a means for site-specific chemical addition or crosslinking of the effector moiety to the bound polypeptide, and thus forming a modified bound polypeptide. In some embodiments, the thiol-modifying reagent utilizes the thiol or sulfhydryl functional group present on a free cysteine ​​residue. Exemplary thiol-modifying reagents include maleimides, alkyl and aryl halides, α-haloacyls, and pyridyl disulfides.

[0157] The term “functional moiety” includes, in some embodiments, a moiety that adds a desired function to the binding polypeptide. In some embodiments, this function is added without significantly altering the polypeptide’s inherent desired activity, such as the molecule’s antigen-binding activity. The binding polypeptide of the present invention may include one or more functional moieties, which may be the same or different. Examples of useful functional moieties include, but are not limited to, effector moieties, affinity moieties, and blocking moieties. Exemplary blocking moieties include moieties with sufficient stereovolume and / or charge to reduce the glycosylation that occurs, for example, by blocking the ability of glycosidases to glycosylate the polypeptide. Blocking moieties may further, or alternatively, reduce effector function, for example, by inhibiting the ability of an Fc region to bind to a receptor or complement protein. Non-limiting blocking moieties include cysteine ​​adducts, cysteine, mixed disulfide adducts, and PEG moieties. Exemplary detectable moieties include fluorescent moieties, radioisotope moieties, radiopaque moieties, and the like. With respect to the conjugation of chemical moieties, the term “conjugation moiety” includes a portion that can link a functional moiety to the remainder of the conjugation polypeptide. Conjugation moieties may be selected to be cleavable or incleavable. Incleavable conjugation moieties generally have high in vivo stability, but may also have undesirable pharmacokinetic properties.

[0158] The term "spacer portion" refers to a non-protein portion designed to introduce space within a molecule. In one embodiment, the spacer portion may be a optionally substituted chain of 0 to 100 atoms selected from carbon, oxygen, nitrogen, sulfur, etc. In one embodiment, the spacer portion is selected to be water-soluble. In another embodiment, the spacer portion is a polyalkylene glycol, such as polyethylene glycol or polypropylene glycol.

[0159] The terms “PEGylated moiety” or “PEG moiety” include polyalkylene glycol compounds or derivatives thereof, with or without coupling agents or derivatization by coupling or activating moieties (e.g., by thiols, triflates, torecylates, aziridines, oxiranes, or maleimide moieties, e.g., PEG-maleimide). Other suitable polyalkylene glycol compounds include not only maleimide monomethoxyPEG and activated PEG polypropylene glycol, but also charged or neutral polymers of the type of dextran, colomic acid, or other carbohydrate polymers, amino acid polymers, and biotin derivatives. As used herein, the term “effector moiety” (E) may include diagnostic and therapeutic agents having biological or other functional activity (e.g., proteins, nucleic acids, lipids, drug moieties, and fragments thereof). For example, a conjugated polypeptide containing an effector moiety conjugated to it has at least one additional function or property compared to an unconjugated polypeptide. For example, conjugating a cytotoxic drug moiety (e.g., an effector moiety) to a binding polypeptide (e.g., via its polypeptide linker) results in the formation of a modified polypeptide having a second function (i.e., cytotoxicity of the drug in addition to antigen binding). In another example, conjugation of a second binding polypeptide to a first binding polypeptide may result in additional binding properties. In one embodiment, if the effector moiety is a gene-encoded therapeutic or diagnostic protein or nucleic acid, the effector moiety can be synthesized or expressed by either peptide synthesis or recombinant DNA methods, which are well known in the art. In another embodiment, if the effector is a peptide or drug moiety not encoded by a gene, the effector moiety can be artificially synthesized or purified from a natural source.

[0160] As used herein, the term “drug portion” includes anti-inflammatory agents, anticancer agents, anti-infective agents (e.g., antifungal agents, antibacterial agents, antiparasitic agents, antiviral agents, etc.), and anesthetic therapeutic agents. In further embodiments, the drug portion is an anticancer agent or a cytotoxic agent. A suitable drug portion may also include a prodrug.

[0161] As used herein, the term “prodrug” means a precursor or derivative form of a pharmaceutically active substance that, compared to the parent drug, is less active, reactive, or prone to side effects, and can be enzymatically activated or converted into a more active form in vivo. Examples of prodrugs suitable for the present invention include, but are not limited to, phosphate-containing prodrugs, amino acid-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine, and other 5-fluorouridine prodrugs, which can be converted into more active cytotoxic free drugs. Those skilled in the art may chemically modify the desired drug moiety or its prodrug to facilitate the reaction of the relevant compound for the purpose of preparing the modified binding proteins of the present invention. The drug moiety also includes derivatives of the drug moieties described herein, pharmaceutically acceptable salts, esters, amides, and ethers.

[0162] An "affinity resin" is a chemical surface that binds with high affinity to affinity domains, thereby facilitating the separation of proteins bound to affinity domains from other components of the reaction mixture. Affinity resins can be coated onto the surface of a solid support or a portion thereof. Alternatively, the affinity resin may constitute the solid support. Such solid supports may include suitably modified chromatography columns, microtiter plates, beads, or biochips (e.g., glass wafers). Exemplary affinity resins consist of nickel, chitin, amylase, and the like. The terms "vector" or "expression vector" are used herein to mean a vector used in accordance with the invention as a vehicle for introducing and expressing a desired polynucleotide in cells. As is known to those skilled in the art, such vectors can be readily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, vectors compatible with the invention include a selection marker, appropriate restriction sites to facilitate the cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0163] Those skilled in the art will understand that when a polynucleotide (e.g., a DNA sequence) encoding an antibody (or fragment or chain thereof) described herein is included in an expression vector that also includes regulatory elements such as a promoter, enhancer, and / or poly-A tail, this polynucleotide will be expressed as a protein in the host cell into which the vector has been introduced. Therefore, such a polynucleotide inserted into an expression vector is sometimes said to be "positioned for expression" in the vector and, consequently, in the host cell into which the vector has been introduced. However, a polynucleotide (e.g., a DNA sequence) does not need to be inserted into an expression vector to be positioned for expression in a host cell. Well-known methods are available for positioning polynucleotides for expression in cells. For example, a DNA sequence can be inserted into the genome of a host cell, for example, using homologous recombination, so that the expression of the inserted DNA sequence is regulated by the host cell's endogenous promoter and other regulatory elements. Such homologous recombination techniques are well known.

[0164] In the present invention, a number of expression vector systems may be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Other vectors involve the use of polycistronic systems having an internal ribosome binding site. Exemplary vectors include those described in U.S. Patent Nos. 6,159,730 and 6,413,777, and U.S. Patent Application No. 20030157641A1. Furthermore, cells in which the DNA has been incorporated into the chromosome may be selected by introducing one or more markers that enable selection of transfected host cells. These markers may confer prototrophicity to a trophic-prone host, or confer resistance to biocides (e.g., antibiotics) or heavy metals such as copper. The genes of the selectable markers may be directly ligated to the DNA sequence to be expressed, or they may be introduced into the same cells by co-transformation. In one embodiment, an inducible expression system may be used. For optimal mRNA synthesis, additional elements may be required. These elements may include signal sequences, splice signals, and transcription promoters, enhancers, and termination signals. In one embodiment, optimal secretion of the polypeptide can be achieved by in-frame fusion of a secretion signal, such as one of several well-analyzed bacterial leader peptides (e.g., pelB, phoA, or ompA), to the N-terminus of the polypeptide of the present invention (Lei et al. (1988), Nature, 331:543; Better et al. (1988), Science, 240:1041; Mullinax et al, (1990), PNAS, 87:8095).

[0165] The term "host cell" refers to a cell constructed using recombinant DNA technology and transformed with a vector encoding at least one heterologous gene. In descriptions of the process of isolating proteins from recombinant hosts, the terms "cell" and "cell culture" are used synonymously for the purpose of indicating the origin of the protein unless otherwise specified. In other words, recovery of a protein from "cells" may mean recovery from either whole cells spun down or from a cell culture containing both culture medium and suspended cells. The host cell lines used for protein expression are, for example, derived from mammals. Those skilled in the art are expected to have the ability to preferentially determine the particular host cell line best suited for expressing the desired gene product. Exemplary host cell lines include, but are not limited to, DG44 and DUXBII (Chinese hamster ovary cell line, DHFR-negative), HELA (human cervical cancer), CVI (monkey kidney cell line), COS (a derivative of CVI with SV40 T antigen), R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney cell line), SP2 / O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-IcIBPT (bovine endothelial cell), RAJI (human lymphocyte), and 293 (human kidney). CHO cells (and their variants, including DHFR-deficient CHO-K1 cells) are useful host cells. Host cell lines are typically available from commercial services, the American Tissue Culture Collection, or public literature. The polypeptides of the present invention can also be expressed in bacterial cells, or non-mammalian cells such as yeast cells, or plant cells. In this regard, it will be understood that various non-mammalian single-celled microorganisms, such as bacteria, that can grow under culture or fermentation, can also be transformed. Easily transformable bacteria include members of the Enterobacteriaceae family, such as Escherichia coli or Salmonella strains, and members of the Bacillaceae family, such as Bacillus subtilis, Pneumococcus, Streptococcus, and Haemophilus influenzae.Furthermore, it will be understood that when polypeptides are expressed in bacteria, they typically become part of inclusion bodies. These polypeptides need to be isolated, purified, and then assembled into functional molecules.

[0166] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism, but several other strains, including Pichia pastoris, are also commonly available. For expression in Saccharomyces, for example, plasmid YRp7 (Stinchcomb et al, (1979), Nature, 282:39; Kingsman et al, (1979), Gene, 7:141; Tschemper et al, (1980), Gene, 10:157) is commonly used. This plasmid already contains the TRP1 gene, which serves as a selection marker for yeast mutant strains lacking the ability to grow in tryptophan, such as ATCC number 44076 or PEP4-1 (Jones, (1977), Genetics, 85:12). The presence of trp1 dysfunction, a characteristic of the yeast host cell genome, provides an effective environment for detecting transformation through proliferation in the absence of tryptophan.

[0167] In vitro production allows for scale-up to obtain large quantities of the desired modified conjugated polypeptides of the present invention. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include, for example, homogeneous suspension culture in an airlift reactor or a continuous stirring reactor, or culture of immobilized or captured cells in, for example, hollow fibers, microcapsules, agarose microbeads, or ceramic cartridges. If necessary and / or desired, the polypeptide solution may be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, hydrophobic interaction chromatography (HIC), chromatography in DEAE-cellulose, or affinity chromatography.

[0168] As used herein, the expression “subjects to which administration of the conjugated polypeptide is effective” includes, for example, mammalian subjects to which administration of the conjugated polypeptide is effective for the detection of an antigen recognized and specifically bound to the conjugated polypeptide of the present invention (e.g., in a diagnostic technique), and / or treatment with the conjugated polypeptide is effective for reducing or eliminating a target recognized and specifically bound to the conjugated polypeptide. For example, in one embodiment, the subject may benefit from reduction or elimination of soluble or particulate molecules (e.g., toxins or pathogens) in circulation or serum, or from reduction or elimination of a population of cells expressing the target (e.g., tumor cells). As described above, the conjugated polypeptide may be used in an unconjugated form, or it may be conjugated to, for example, a drug, prodrug, or isotope to form a modified conjugated polypeptide for administration to the subject.

[0169] The terms “PEGylation,” “polyethylene glycol,” or “PEG” include polyalkylene glycol compounds or derivatives thereof, with or without coupling agents or derivatization by coupling or activating moieties (e.g., by thiols, triflates, torecylates, aziridines, oxiranes, or maleimide moieties, e.g., PEG-maleimide). Other suitable polyalkylene glycol compounds include, but are not limited to, maleimide monomethoxyPEG and activated PEG polypropylene glycol, as well as charged or neutral polymers of the type of dextran, colomic acid, or other carbohydrate polymers, amino acid polymers, and biotin derivatives.

[0170] I. Variable Region The present invention provides alpha-4 conjugated antibodies and fragments thereof, wherein the variable light chain (VL) and variable heavy chain (VH) frameworks have acceptor sequences constructed from the sequences of germline or germline-manipulated antibodies such as IGKV4-1 antibody, geAAH70335.1 antibody, or IGHV1-f antibody. The CDR sequences are derived from non-human anti-α4 conjugated antibodies such as anti-VLA-4 antibody HP1 / 2. See PCT Publication WO2011 / 130603, which is incorporated herein by reference in its entirety. The antibodies described herein may have affinity increased by at least 1.5, 2.0, 2.5, or 3.0 times compared to their mouse parent. In one embodiment, the affinity increase is at least 1.5, 2.0, 2.5, or 3.0 times, but less than 25, 20, or 15 times, respectively.

[0171] The mouse monoclonal antibody (mAb) HP1 / 2 is a member of the B1 subgroup of α4 mAbs and inhibits in vitro cell adhesion to both VCAM1 and fibronectin with a potency of approximately 0.1 nM. The cryo-electron microscopy (cryo-EM) structure of the HP1 / 2 Fab was analyzed as a complex with the α4β1 external domain. HP1 / 2 binds to the α4β-propeller domain outside the ligand-binding groove, non-competitively weakening ligand binding. The epitope and conformation of the α4 integrin recognized and specifically bound by HP1 / 2 are similar to those observed in the natalizumab α4 integrin crystal structure.

[0172] mAbs targeting α4 integrin effectively block the migration of lymphocytes, eosinophils, and monocytes into tissues in vivo, exhibiting disease-modifying effects. In particular, mAb HP1 / 2 has been shown to exacerbate allergic reactions in sheep (Lobb et al., Ann. NYAcad. Sci 796:113-123, 1996), bronchial airway hyperresponsiveness in guinea pigs (Pretolani et al., J. Ex. Med. 180(3):795-805, 1994, Kraneveld et al., J. Allergy Clin. Immunol. 100(2):242-50, 1997), ulcerative colitis in primates (Podolsky et al., J. Clin Invest. 92(1):372-380, 1993), and to perform endarterectomy for carotid intatimal hyperplasia in NHP (Lumsden et al. HP1 / 2 has been shown to be effective in various animal models, including those described in al., J. Vasc. Surg. 26(1):87-93, 1997. It reduces neoembryoform formation after balloon injury and subsequent luminal stenosis (Labinaz et al., Can J. Cardiol. 16(2):187-96, 2000), reduces the inflammatory response to stent placement in rabbits (Ma et al., 2004), and prevents transplant heart rejection in rabbits (Sadahiro et al., Am. J. Pathol. 142(3):675-683, 1993). Furthermore, HP1 / 2 stimulates the recruitment of hematopoietic progenitor cells from the bone marrow stroma to the peripheral blood in baboons (Papayannopoulou and Nakamoto, Proc. Natl. Acad. Sci 90(20):9374-9378, 1993).

[0173] HP1 / 2 antibodies were produced in Balb / c mice injected with JM (immature T-cell leukemia) cell lines (Sanchez-Madrid et al., Eur.J.Immunol.16:1343-1349, 1986). Splenocytes from two mice were fused with mouse myeloma cells of SP2 or P3-X63Ag8.653. This antibody was humanized in 1993, but re-humanized in 2010 to take advantage of advances in humanization design.

[0174] The alpha-4 conjugated antibody or its fragment may contain a variable light chain having the sequence of SEQ ID NOs: 8, 9, 10, or 11. The alpha-4 conjugated antibody or its fragment may contain a variable heavy chain having the sequence of SEQ ID NOs: 3, 4, or 5.

[0175] In a particular embodiment, the alpha-4 conjugated antibody comprises a variable light chain having the sequence of SEQ ID NO: 11 and a variable heavy chain having the sequence of SEQ ID NO: 4.

[0176] The alpha-4 conjugated antibody may further include a variant Fc region, which is described in more detail herein.

[0177] II. Parent Fc polypeptide A variant Fc polypeptide may be derived from a parent or starting Fc polypeptide known in the art. In one embodiment, the parent Fc polypeptide is an antibody, such as IgG immunoglobulin, including all subtypes of IgG and combinations thereof. In some embodiments, the parent Fc polypeptide of an IgG antibody is of one IgG subtype or a combination of different portions of the Fc region of two or more IgG subtypes. In humans, IgG subtypes include IgG1, IgG2, IgG3, and IgG4. The parent Fc polypeptide contains an Fc region derived from immunoglobulin, but may optionally further contain binding sites operably linked or fused to the Fc region. In some embodiments, the aforementioned polypeptide binds to an antigen, such as a ligand, cytokine, receptor, cell surface antigen, or cancer cell antigen. Although the examples herein use an IgG antibody, it is understood that the method may be equally applicable to the Fc region of any Fc polypeptide. If the Fc polypeptide is an antibody, this antibody may be synthetic, naturally occurring (e.g., serum-derived), produced by a cell line (e.g., a hybridoma), or produced in a transgenic organism.

[0178] In certain embodiments, the Fc polypeptide of the present invention comprises one Fc moiety of an Fc region. In other embodiments, the Fc polypeptide is a dcFc polypeptide. A dcFc polypeptide means a polypeptide comprising a dimeric Fc (or dcFc) region. In other embodiments, the Fc polypeptide of the present invention is an scFc polypeptide. As used herein, the term scFc polypeptide means a polypeptide comprising a single-stranded Fc(scFc) region, such as an scFc polypeptide comprising at least two Fc moieties fused via a mobile polypeptide linker interposed between at least two Fc moieties. An exemplary scFc region is disclosed in PCT application PCT / US2008 / 006260, filed on 14 May 2008 and incorporated herein by reference.

[0179] In certain embodiments, the polypeptide of the present invention may include an Fc region (hereinafter referred to as the “homomer Fc region”) containing an Fc portion having the same or substantially the same sequence composition. In other embodiments, the polypeptide of the present invention may include an Fc region (i.e., a “heteromeric Fc region”) containing at least two Fc portions with different sequence compositions. In certain embodiments, the conjugated polypeptide of the present invention includes an Fc region containing at least one insertion or amino acid substitution. In one exemplary embodiment, the heteromeric Fc region contains an amino acid substitution in the first Fc portion, but not in the second Fc portion.

[0180] In one embodiment, the conjugated polypeptide of the present invention may include two or more Fc regions that are independently selected from the Fc regions described herein. In one embodiment, the Fc regions are the same. In another embodiment, at least two of the Fc regions are different. For example, the Fc regions of the Fc polypeptide of the present invention may contain the same number of amino acid residues or may differ in length by one or more amino acid residues (e.g., about five amino acid residues (e.g., amino acid residues 1, 2, 3, 4, or 5), about ten, about fifteen, about 20, about 30, about 40, or about 50). In yet another embodiment, the Fc regions may differ in sequence at one or more amino acid positions. For example, at least two of the Fc regions may differ at about five amino acid positions (e.g., amino acid positions 1, 2, 3, 4, or 5), about ten, about fifteen, about 20, about 30, about 40, or about 50).

[0181] Parental Fc polypeptides may combine or aggregate with other polypeptides to form a multimeric Fc polypeptide or protein (also referred to herein as a “multimer”). The multimeric Fc polypeptide or protein of the present invention comprises at least one parental Fc polypeptide of the present invention. Therefore, the parental polypeptide includes, but is not limited to, monomers as well as multimeric (e.g., dimers, trimers, tetramers, and hexamers) Fc polypeptides or proteins. In certain embodiments, the Fc polypeptides that make up the multimer are the same (i.e., homomeric multimers, e.g., homodimers, homotrimers, homotetramers). In other embodiments, at least two of the Fc polypeptides that make up the multimeric protein of the present invention are different (i.e., heteromeric multimers, e.g., heterodimers, heterotrimers, heterotetramers). In certain embodiments, at least two of the Fc polypeptides can form a dimer.

[0182] In another embodiment, the Fc polypeptide of the present invention comprises a dimeric Fc region (either a single-stranded polypeptide that forms a dimer or a double-stranded polypeptide that forms a dimer) and is monomeric with respect to the biologically active moiety present in the molecule. For example, such an Fc construct may contain only one biologically active moiety. Stabilized single-stranded or double-stranded Fc monomer constructs are desirable, for example, when crosslinking of the target molecule is not desired (e.g., in the case of certain antibodies, e.g., anti-CD40 antibodies). In another embodiment, such an Fc construct may contain two different biologically active moieties. In yet another embodiment, such an Fc construct may contain two identical biologically active moieties. In yet another embodiment, such an Fc construct may contain three or more identical biologically active moieties.

[0183] A.Fc part The Fc moiety useful for generating the parent Fc polypeptide of the present invention can be obtained from several different origins. In some embodiments, the Fc moiety of the conjugated polypeptide is derived from human immunoglobulin. However, it is understood that the Fc moiety may be derived from immunoglobulin of another mammalian species, including, for example, various rodents (e.g., mice, rats, rabbits, guinea pigs) or non-human primates (e.g., chimpanzees, macaques). Furthermore, Fc may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4. For example, Fc may be derived from IgG1 but may have a specific mutation in its sequence. In some embodiments, human isotype IgG1 or IgG4 is used. In some embodiments, Fc may be a chimera comprising a moiety derived from one type of Ig immunoglobulin (e.g., the CH3 domain of IgG1) and another moiety derived from another type of Ig immunoglobulin (e.g., the CH1 domain of IgG4).

[0184] Genetic sequences of various Fc regions (e.g., human constant region sequences) are available in the form of publicly accessible deposits. Constant region domains containing Fc sequences that have (or lack) specific effector functions, or specific modifications that reduce immunogenicity, may be selected. Many sequences of antibodies and antibody-coding genes are publicly available, and suitable Fc sequences (e.g., hinge, CH2, and / or CH3 sequences, or parts thereof) can be obtained from these sequences using techniques recognized in the art. Genetic material obtained using any of the aforementioned methods can then be modified or synthesized to obtain the Fc polypeptide of the present invention. Furthermore, it will be understood that the scope of the present invention encompasses alleles, variants, and mutations of constant region DNA sequences.

[0185] The Fc region sequence can be cloned, for example, using polymerase chain reaction and primers selected to amplify the domain of interest. To clone the Fc region sequence from an antibody, mRNA can be isolated from hybridomas, spleens, or lymphocytes, reverse transcribed into DNA, and the antibody gene can be amplified by PCR. PCR amplification methods are described in detail in U.S. Patents 4,683,195, 4,683,202, 4,800,159, 4,965,188, and, for example, “PCR Protocols: A Guide to Methods and Applications” Innis et al. eds., Academic Press, San Diego, CA (1990), Ho et al. 1989. Gene 77:51, Horton et al. 1993. Methods Enzymol. 217:270). PCR can be initiated with consensus constant-region primers or with more specific primers based on publicly available heavy and light chain DNA and amino acid sequences. As described above, PCR can also be used to isolate DNA clones encoding the light and heavy chains of the antibody. In this case, the library may be screened with consensus primers or larger homologous probes such as mouse constant-region probes. Numerous primer sets suitable for amplifying antibody genes are known in the art (e.g., 5' primers based on the N-terminal sequence of purified antibodies (Benhar and Pastan. 1994. Protein Engineering 7:1509), rapid amplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods 173:33), antibody leader sequences (Larrick et al. 1989 Biochem. Biophys. Res. Commun. 160:1250). For antibody sequence cloning, see U.S. Patent No. 5,658,570 by Newman et al., filed on January 25, 1995, which is incorporated herein by reference).

[0186] The parent Fc polypeptide of the present invention may comprise a single Fc moiety or multiple Fc moieties. If two or more Fc moieties are present (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fc moieties), at least two of the Fc moieties may associate to form a properly folded Fc region (e.g., a dimeric Fc region or a single-stranded Fc region (scFc)). In one embodiment, the Fc moieties may be of different types. In one embodiment, at least one Fc moiety present in the parent Fc polypeptide comprises a hinge domain or a portion thereof. In another embodiment, the parent Fc polypeptide comprises at least one Fc moiety comprising at least one CH2 domain or a portion thereof. In another embodiment, the parent Fc polypeptide comprises at least one Fc moiety comprising at least one CH3 domain or a portion thereof. In another embodiment, the parent Fc polypeptide comprises at least one Fc moiety comprising at least one CH4 domain or a portion thereof. In another embodiment, the parent Fc polypeptide comprises at least one Fc moiety containing at least one hinge domain or portion thereof and at least one CH2 domain or portion thereof (for example, in the hinge-CH2 direction). In another embodiment, the parent Fc polypeptide comprises at least one Fc moiety containing at least one CH2 domain or portion thereof and at least one CH3 domain or portion thereof (for example, in the CH2-CH3 direction). In another embodiment, the parent Fc polypeptide comprises at least one Fc moiety containing at least one hinge domain or portion thereof, at least one CH2 domain or portion thereof and at least one CH3 domain or portion thereof, for example, in the hinge-CH2-CH3, hinge-CH3-CH2, or CH2-CH3-hinge direction.

[0187] In certain embodiments, the parent Fc polypeptide comprises at least one complete Fc region derived from one or more immunoglobulin heavy chains (e.g., an Fc moiety containing hinge, CH2, and CH3 domains, but these do not necessarily have to be derived from the same antibody). In other embodiments, the parent Fc polypeptide comprises at least two complete Fc regions derived from one or more immunoglobulin heavy chains. In some embodiments, the complete Fc moieties are derived from human IgG immunoglobulin heavy chains (e.g., human IgG1 or human IgG4).

[0188] In another embodiment, the parent Fc polypeptide comprises at least one Fc moiety containing a complete CH3 domain (around amino acids 341-438 of the antibody Fc region according to EU numbering). In another embodiment, the parent Fc polypeptide comprises at least one Fc moiety containing a complete CH2 domain (around amino acids 231-340 of the antibody Fc region according to EU numbering). In another embodiment, the parent Fc polypeptide comprises at least one Fc moiety containing at least a CH3 domain, as well as at least one of a hinge region (around amino acids 216-230 of the antibody Fc region according to EU numbering) and a CH2 domain. In one embodiment, the parent Fc polypeptide comprises at least one Fc moiety containing a hinge and a CH3 domain. In another embodiment, the parent Fc polypeptide comprises at least one Fc moiety containing a hinge, CH2, and CH3 domains. In some embodiments, the Fc moiety is derived from a human IgG immunoglobulin heavy chain.

[0189] The constant region domain or a portion thereof constituting the Fc portion may be derived from different immunoglobulin molecules. For example, the parent Fc polypeptide may include a hinge and / or CH2 domain or a portion thereof derived from the IgG4 molecule, and a CH3 region or a portion thereof derived from the IgG1 molecule. In another embodiment, the parent Fc polypeptide may include a chimeric hinge domain. For example, the chimeric hinge may include a hinge domain that is partly derived from the IgG1 molecule and partly derived from the IgG3 molecule. In another embodiment, the chimeric hinge includes an intermediate hinge domain derived from the IgG1 molecule, and upper and lower hinge domains derived from the IgG4 molecule.

[0190] As will be understood by those skilled in the art, the parent Fc portion may be identical to the corresponding Fc portion of a naturally occurring immunoglobulin, or it may be modified to have a different amino acid sequence. In certain embodiments, the parent Fc polypeptide is modified, for example, by amino acid mutations (e.g., additions, deletions, or substitutions). For example, the parent Fc polypeptide may be an Fc portion having at least one amino acid substitution compared to the wild-type Fc from which the Fc portion originates. For example, if the Fc portion is derived from a human IgG1 antibody, the variant includes at least one amino acid mutation (e.g., substitution) at the corresponding position of the human IgG1 Fc region compared to the wild-type amino acid.

[0191] Amino acid substitutions may be located within the Fc region of an antibody at positions that "correspond" to positional numbers assigned to the corresponding residues (defined using the convention of EU numbering). Those skilled in the art can easily generate alignments to determine what EU numbers "correspond" to the positions within the Fc region.

[0192] In one embodiment, the substitution is located at an amino acid position within the hinge domain or a portion thereof. In another embodiment, the substitution is located at an amino acid position within the CH2 domain or a portion thereof. In yet another embodiment, the substitution is located at an amino acid position within the CH3 domain or a portion thereof. In yet another embodiment, the substitution is located at an amino acid position within the CH4 domain or a portion thereof.

[0193] In certain embodiments, the parent Fc polypeptide contains two or more amino acid substitutions. The parent Fc polypeptide may contain, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions compared to the wild-type Fc region. In some embodiments, the amino acid substitutions are spatially arranged with at least one amino acid position between them, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid positions apart. In some embodiments, the manipulated amino acids are spatially arranged with at least 5, 10, 15, 20, or 25 or more amino acid positions apart.

[0194] In certain embodiments, the substitution results in a change in at least one effector function provided by the Fc region containing the wild-type Fc moiety (e.g., a reduction in the Fc region's ability to bind to an Fc receptor (e.g., FcγRI, FcγRII, or FcγRIII) or a complement protein (e.g., C1q), or its ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or complement-dependent cell-mediated cytotoxicity (CDC)).

[0195] The parent Fc polypeptide may use substitutions recognized in the art that are known to result in alterations of effector function. In particular, the parent Fc polypeptide of the present invention may be, for example, PCT International Publications WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 / 58572A1, WO00 / 09560A2, WO00 / 32767A1, WO00 / 42072A2, WO02 / 44215A2, WO02 / 060919A2, WO03 / 074569A2, WO04 / 016 750 A2, WO04 / 029207 A2, WO04 / 035752 A2, WO04 / 063351 A2, WO04 / 074455 A2, WO04 / 099249 A2, WO05 / 040217 A2, WO04 / 044859, WO05 / 070963 A1, WO05 / 077981 A2, WO05 / 092925 A2, WO05 / 123780 A2, WO06 / 019447 A1, WO06 / 047350 A2, and WO0 6 / 085967A2, U.S. Patent Publications US2007 / 0231329, US2007 / 0231329, US2007 / 0237765, US2007 / 0237766, US2007 / 0237767, US2007 / 0243188, US20070248603, US20070286859, US20080057056, or U.S. Patent Nos. 5,648,260, 5,739,277, 5,834,250, and 5,869,046 This may include changes (e.g., substitutions) at one or more amino acid positions disclosed in Patent Nos. 6,096,871, 6,121,022, 6,194,551, 6,242,195, 6,277,375, 6,528,624, 6,538,124, 6,737,056, 6,821,505, 6,998,253, 7,083,784, and 7,317,091 (the portions of each document relating to Fc mutations are incorporated herein by reference).In one embodiment, a specific change (e.g., a specific substitution of one or more amino acids disclosed in the Art) may be made at one or more of the disclosed amino acid positions. In another embodiment, a different change (e.g., a different substitution of one or more amino acid positions disclosed in the Art) may be made at one or more of the disclosed amino acid positions.

[0196] In some embodiments, the parent Fc polypeptide may include an Fc moiety containing amino acid substitutions at amino acid positions corresponding to EU amino acid positions within the "15-angstrom contact zone" of the Fc moiety. The 15-angstrom zone includes residues located at EU positions 243–261, 275–280, 282–293, 302–319, 336–348, 367, 369, 372–389, 391, 393, 408, and 424–440 of the full-length wild-type Fc moiety.

[0197] In another embodiment, the parent Fc polypeptide includes an Fc region containing one or more Fc moieties that, despite being truncated, are sufficient to impart one or more functions to the Fc region. For example, the portion of the Fc moiety that binds to FcRn (i.e., the FcRn binding portion) contains amino acids approximately 282-438 (EU numbering). Thus, the Fc moiety of the parent Fc polypeptide may contain or consist of an FcRn binding portion. The FcRn binding portion may originate from the heavy chain of any isotype, including IgG1, IgG2, IgG3, and IgG4. In one embodiment, the FcRn binding portion of an antibody of human isotype IgG1 is used. In another embodiment, the FcRn binding portion of an antibody of human isotype IgG4 is used. In certain embodiments, the FcRn binding portion is aglycosylated. In other embodiments, the FcRn binding portion is glycosylated.

[0198] In certain embodiments, the parental Fc polypeptide contains amino acid substitutions of the Fc moiety that alter the antigen-independent effector function of the antibody, particularly the circulating half-life of the antibody. Such polypeptides exhibit increased or decreased binding to FcRn compared to polypeptides lacking these substitutions, and therefore have increased or decreased half-lives in serum, respectively. Parental Fc polypeptides with improved affinity for FcRn are expected to have a longer serum half-life, and such molecules are useful in methods of treating mammals where a longer half-life of the administered polypeptide is desired, for example, to treat a chronic disease or disorder. In contrast, parental Fc polypeptides with decreased FcRn binding affinity are expected to have a shorter half-life, and such molecules are also useful, for example, for administration to mammals where a shortened circulating time may be advantageous, such as for in vivo diagnostic imaging, or in situations where prolonged presence of the starting polypeptide in circulation would cause toxic side effects. Parental Fc polypeptides with reduced FcRn binding affinity are also less likely to cross the placenta and are therefore useful in the treatment of diseases or disorders in pregnant women. Furthermore, other applications where reduced FcRn binding affinity may be desired include applications where localization to the brain, kidneys, and / or liver is desired. In one exemplary embodiment, the parental Fc polypeptide exhibits reduced transport from vascular structures through the epithelium of the renal glomeruli. In another embodiment, the binding polypeptide of the present invention exhibits reduced transport from the brain to the intervascular space across the blood-brain barrier (BBB). In one embodiment, the parental Fc polypeptide with altered FcRn binding comprises at least one Fc moiety (e.g., one or two Fc moieties) having one or more amino acid substitutions within the “FcRn binding loop” of the Fc moiety. The FcRn binding loop consists of amino acid residues 280-299 (according to EU numbering) of the full-length wild-type Fc moiety. In other embodiments, the parental Fc polypeptide having altered FcRn binding affinity comprises at least one Fc moiety (e.g., one or two Fc moieties) having one or more amino acid substitutions within the 15A FcRn "contact zone".

[0199] As used herein, the term 15A FcRn “contact zone” includes the residues at positions 243–261, 275–280, 282–293, 302–319, 336–348, 367, 369, 372–389, 391, 393, 408, 424, and 425–440 (EU numbering) of the full-length Fc moiety of the wild type. In some embodiments, the parental Fc polypeptide having altered FcRn binding affinity comprises at least one Fc moiety (e.g., one or two Fc moieties) having one or more amino acid substitutions at any one of the amino acid positions corresponding to EU256, 277-281, 283-288, 303-309, 313, 338, 342, 376, 381, 384, 385, 387, 434 (e.g., N434A or N434K), and 438. Exemplary amino acid substitutions that alter FcRn binding activity are disclosed in PCT International Publication WO05 / 047327, incorporated herein by reference. In other embodiments, the parental Fc polypeptide comprises at least one Fc moiety having an engineered cysteine ​​residue or an analogue located on the solvent-exposed surface. In some embodiments, the manipulated cysteine ​​residue or its analogues does not interfere with the effector function provided by the Fc region. In some embodiments, the Fc polypeptide includes an Fc moiety comprising at least one manipulated free cysteine ​​residue or its analogue that substantially does not contain a disulfide bond with a second cysteine ​​residue. In some embodiments, the Fc polypeptide may include an Fc moiety having one or more manipulated cysteine ​​residues or their analogues at positions 349-371, 390, 392, 394-423, 441-446, and 446b (EU numbering) within the CH3 domain. In some further embodiments, the Fc polypeptide includes an Fc variant having a cysteine ​​residue or an analogue manipulated to one of the following positions: 350, 355, 359, 360, 361, 389, 413, 415, 418, 422, 441, 443, and EU446b (EU numbering).Any of the above-described manipulated cysteine ​​residues or their analogues may subsequently be conjugated to a functional portion using techniques recognized in the art (e.g., conjugated with a thiol-reactive heterobifunctional linker).

[0200] B. Effectorless Fc polypeptide In certain embodiments, the parent Fc polypeptide is an "effectorless" Fc polypeptide having altered or reduced effector function. In some embodiments, the reduced or altered effector function is antigen-dependent effector function. For example, the parent Fc polypeptide may contain sequence diversity (e.g., amino acid substitutions) that reduces the polypeptide's antigen-dependent effector function, particularly ADCC or complement activation, compared to, for example, a wild-type Fc polypeptide. Unfortunately, such parent Fc polypeptides often have reduced stability and are ideal candidates for stabilization by the methods of the present invention.

[0201] Fc polypeptides with reduced FcγR binding affinity are expected to reduce effector function, and such molecules are also useful in treating conditions where, for example, destruction of target cells is undesirable, e.g., normal cells may express the target molecule, or chronic administration of polypeptides may result in immune system activation that is not required. In one embodiment, Fc polypeptides exhibit reduced antigen-dependent effector function, selected from the group consisting of opsonization, phagocytosis, complement-dependent cytotoxicity, antibody-dependent cytotoxicity (ADCC), or modulation of effector cells, compared to Fc polypeptides containing a wild-type Fc region. In one embodiment, Fc polypeptides exhibit altered binding to activated FcγR (e.g., FcγRI, FcγRIIa, or FcγRIIIa). In another embodiment, Fc polypeptides exhibit altered binding affinity to inhibitory FcγR (e.g., FcγRIIb). In other embodiments, an Fc polypeptide having reduced FcγR binding affinity (e.g., reduced FcγRI, FcγRII, or FcγRIIIa binding affinity) includes at least one Fc moiety (e.g., one or two Fc moieties) having amino acid substitutions at amino acid positions corresponding to one or more of the following positions: 234, 236, 239, 241, 251, 252, 261, 265, 268, 293, 294, 296, 298, 299, 301, 326, 328, 332, 334, 338, 376, 378, and 435 (EU numbering). In other embodiments, an Fc polypeptide having reduced complement binding affinity (e.g., reduced C1q binding affinity) includes an Fc moiety (e.g., one or two Fc moieties) having amino acid substitutions at one or more amino acid positions corresponding to one or more of the following positions: 239, 294, 296, 301, 328, 333, and 376 (EU numbering).

[0202] Exemplary amino acid substitutions that alter FcγR or complement-binding activity are disclosed in PCT International Publication WO05 / 063815, incorporated herein by reference. In certain embodiments, the binding polypeptide of the present invention may comprise one or more of the specific substitutions of S239D, S239E, M252T, H268D, H268E, I332D, I332E, N434A, and N434K (i.e., one or more of these substitutions at amino acid positions corresponding to one or more of these EU-numbered positions within the antibody Fc region).

[0203] In certain exemplary embodiments, the effector function of a parent “effectorless” polypeptide may be altered or reduced due to an aglycosylated Fc region within the parent Fc polypeptide. In certain embodiments, the aglycosylated Fc region is generated by an amino acid substitution that alters the glycosylation of the Fc region. For example, asparagine at EU297 in the Fc region may be altered (e.g., by substitution, insertion, deletion, or chemical modification) to inhibit its glycosylation. In another exemplary embodiment, the amino acid residue at EU299 (e.g., threonine (T)) is substituted (e.g., with alanine (A)) to reduce the glycosylation of the adjacent residue 297. Exemplary amino acid substitutions that reduce or alter glycosylation are disclosed in PCT International Publication WO05 / 018572 and U.S. Patent Publication 2007 / 0111281, which are incorporated herein by reference. In other embodiments, the aglycosylated Fc region is generated by enzymatic or chemical removal of oligosaccharides, or by expression of Fc polypeptides in host cells that cannot glycosylate the Fc region (e.g., bacterial or mammalian host cells with impaired glycosylation mechanisms).

[0204] In certain embodiments, the aglycosylated Fc region is partially aglycosylated or semi-glycosylated. For example, the Fc region may include a first glycosylated Fc portion (e.g., a glycosylated CH2 region) and a second aglycosylated Fc portion (e.g., an aglycosylated CH2 region). In other embodiments, the Fc region may be completely aglycosylated; that is, none of its Fc portions are glycosylated.

[0205] The aglycosylated Fc region of the “effectorless” polypeptide may be that of any IgG isotype (e.g., IgG1, IgG2, IgG3, or IgG4). In one exemplary embodiment, the parent Fc polypeptide may comprise the aglycosylated Fc region of an IgG4 antibody, such as “agly IgG4.P”. agly IgG4.P is an engineered form of IgG4 antibody containing a proline substitution (Ser228Pro) in the hinge region and a Thr299Ala mutation in the CH2 domain, so that an aglycosylated Fc region (EU numbering) is generated. agly IgG4.P has been shown to have no immunoeffector function measurable in vitro. In another exemplary embodiment, the parent Fc polypeptide comprises the aglycosylated Fc region of an IgG1 antibody, such as “agly IgG1”. agly IgG1 is an aglycosylated form of IgG immunoglobulin IgG1 with a Thr299Ala mutation (EU numbering) resulting in a low effector function profile. Both agly IgG4.P antibody and agly IgG1 antibody represent an important class of therapeutic reagents for cases where immunoeffector function is not desired.

[0206] In certain exemplary embodiments, the “effectorless” parental Fc polypeptide includes an Fc region derived from an IgG4 antibody. The IgG4 Fc region may be identical to the wild-type Fc region or may have one or more modifications to the wild-type IgG4 sequence. Such an IgG4-like Fc polypeptide has reduced effector function as a result of the inherently reduced ability of the IgG4 antibody to bind to complement and / or Fc receptors. The parental Fc polypeptide of an IgG4 isotype may be glycosylated or aglycosylated. Furthermore, the Fc region of an IgG4-like Fc polypeptide may include the complete Fc portion of an IgG4 antibody, or it may include a chimeric Fc portion in which a portion of the Fc portion is derived from an IgG4 antibody and the remainder from an antibody of another isotype. In one exemplary embodiment, the chimeric Fc portion includes a CH3 domain derived from an IgG1 antibody and a CH2 domain derived from an IgG4 antibody. In another embodiment, the IgG4 antibody contains a chimeric hinge in which the upper and lower hinge domains are derived from the IgG4 antibody, but the intermediate hinge domain is derived from the IgG1 antibody as a result of a proline substitution (Ser228Pro) within the hinge region. In yet another embodiment, the parental chimeric IgG4 antibody contains a chimeric hinge in which the upper and lower hinge domains are derived from the IgG4 antibody, but the intermediate hinge domain is derived from the IgG1 antibody as a result of a proline substitution (Ser228Pro) within the hinge region, a CH1 domain derived from the IgG1 or IgG4 antibody, a CH2 domain (or EU numbering positions 292-340) derived from the IgG4 antibody, and a CH1 domain and / or CH3 domain derived from the IgG1 antibody.

[0207] In certain embodiments, the reduction in effector function of an “effectorless” Fc polypeptide is a reduction in binding to Fc receptors (FcRs) such as FcγRI, FcγRII, FcγRIII, and / or FcγRIIIb receptors, or to complement proteins, such as complement protein C1q. This change in binding may be about 1-fold or greater, for example, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 50, or 100-fold or greater, or any interval or range thereof. Such reductions in effector function, such as the reduction in Fc binding to Fc receptors or complement proteins, can be readily calculated based on the rate of reduction in binding activity determined, for example, using the assay described herein or an assay known in the art.

[0208] In one embodiment of the present invention, the stabilized Fc polypeptide comprises a single-stranded Fc region. Such single-stranded Fc regions are known in the art (see, for example, WO200801243, WO2008131242, and WO2008153954) and can be prepared using known methods. The stabilizing amino acids taught herein can be incorporated into one or more Fc moieties of such constructs using methods known to those skilled in the art. Such a single-stranded Fc region or a gene-fused Fc region is a synthetic Fc region composed of Fc domains (or Fc moieties) that are genetically related (i.e., encoded in a single continuous gene sequence) within a single polypeptide chain. Thus, a gene-fused Fc region (i.e., an scFc region) is monomeric in that it contains a single polypeptide chain, but the appropriate portion of the molecule dimerizes to form an Fc region. It will be understood that the teachings herein regarding Fc moieties are applicable to both double-stranded Fc dimers and single-stranded Fc dimers. For example, either type of Fc region construct may be derived from, for example, an IgG1 antibody or an IgG4 antibody, or it may be a chimeric construct (e.g., containing a chimeric hinge and / or containing a CH2 domain derived from an IgG4 antibody and a CH3 domain derived from an IgG1 antibody).

[0209] III. Variant Fc polypeptide having a stabilized Fc region In certain embodiments, the present invention provides a variant Fc polypeptide comprising an amino acid sequence that is a variant of any one of the parent Fc polypeptides described above. In particular, the variant Fc polypeptide of the present invention comprises an Fc region (or Fc moiety) having an amino acid sequence derived from the Fc region (or Fc moiety) of the parent Fc polypeptide. In some embodiments, the variant Fc polypeptide differs from the parent Fc polypeptide by the presence of at least one of the stabilizing Fc mutations described herein. In certain embodiments, the Fc variant may include further amino acid sequence changes. In some embodiments, the Fc variant has enhanced stability compared to the parent Fc polypeptide and, optionally, altered effector function compared to the parent Fc polypeptide. For example, the variant Fc polypeptide may have antigen-dependent effector function equivalent to or lower than that of the parent Fc polypeptide (e.g., ADCC and / or CDC). Furthermore, or alternatively, the variant Fc polypeptide may have antigen-independent effector function (e.g., long half-life) compared to the parent Fc polypeptide.

[0210] In certain embodiments, the variant Fc polypeptide includes an Fc region (or Fc moiety) that is essentially identical to the Fc region (Fc moiety) of the parent Fc polypeptide, except for one or more amino acid residues that are substituted for another amino acid residue or have one or more amino acid residues inserted or deleted. In certain embodiments, the variant Fc polypeptide has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations compared to the starting polypeptide or parent polypeptide. In some embodiments, the variant polypeptide contains an amino acid sequence that does not occur naturally.

[0211] Such variants necessarily have less than 100% sequence identity or similarity with the starting polypeptide. In some embodiments, the variant has an amino acid sequence that, for example, over the entire length or a portion of the variant molecule (e.g., the Fc region or Fc portion), has about 75% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide, for example, about 80% to less than 100%, or about 85% to less than 100%, or about 90% to less than 100% (e.g., 91-99%, 92-99%, 93-99%, 94-99%, 95-99%, 96-99%, 97-99%, 98-99%, or 99%), or about 95% to less than 100%. In one embodiment, one amino acid differs between the sequence of the starting polypeptide (e.g., the Fc region of the parent Fc polypeptide) and the sequence derived therefrom (e.g., the Fc region of the variant Fc polypeptide).

[0212] In certain embodiments, the variant Fc polypeptide of the present invention is a stabilized Fc polypeptide. That is, a stabilized polypeptide contains at least one sequence variation or stabilizing Fc mutation. As used herein, the term “stabilizing Fc mutation” includes mutations within the Fc region of a variant Fc polypeptide that result in enhanced protein stability (e.g., thermal stability) in the variant Fc polypeptide compared to its parental Fc polypeptide of origin. In some embodiments, the stabilizing mutation includes the substitution of a destabilizing amino acid within the Fc region with a substitute amino acid (hereinafter referred to as a “stabilizing amino acid”) that gives the Fc region enhanced protein stability. In one embodiment, the stabilized Fc polypeptide of the present invention contains one or more amino acid stabilizing Fc mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 stabilizing mutations). The stabilizing Fc mutation may be introduced, for example, into the CH2 domain, the CH3 domain, or both the CH2 and CH3 domains of the Fc region.

[0213] In certain exemplary embodiments, the variant Fc polypeptide of the present invention is a stabilized variant of the “effectorless” parent Fc polypeptide described above. That is, the stabilized variant has enhanced stability compared to the “effectorless parent Fc polypeptide.” In one exemplary embodiment, the variant Fc polypeptide is a stabilized variant of the parent Fc polypeptide that includes an aglycosylated Fc region of an IgG1 antibody, for example, an aglycosylated IgG1 Fc region containing the T299A mutation (EU numbering). In another exemplary embodiment, the variant Fc polypeptide is a stabilized variant of the parent Fc polypeptide that includes an Fc region of a glycosylated or aglycosylated IgG4 antibody. For example, the variant Fc polypeptide may include a stabilizing mutation in the Fc region derived from an “agly IgG4.P” antibody.

[0214] In some embodiments, the stabilized Fc polypeptide of the present invention exhibits enhanced stability under the same measurement conditions compared to the variant Fc polypeptide. However, it will be recognized that the degree to which the stability of the Fc variant polypeptide is enhanced compared to its parent Fc polypeptide may differ under selected measurement conditions. For example, the enhancement of stability may be observed at specific pH values, e.g., acidic, neutral, or basic pH values. In one embodiment, the enhanced stability is observed at an acidic pH of less than about 6.0 (e.g., about 6.0, about 5.5, about 5.0, about 4.5, or about 4.0). In another embodiment, the enhanced stability is observed at a neutral pH of about 6.0 to about 8.0 (e.g., about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0). In yet another embodiment, the enhanced stability is observed at a basic pH of about 8.0 to about 10.0 (e.g., about 8.0, about 8.5, about 9.0, about 9.5, or about 10.0).

[0215] The enhancement of the thermal stability of the variant Fc polypeptide can be evaluated, for example, using one of the methods described later. In certain embodiments, the stabilized Fc polypeptide has an Fc region (or Fc moiety) that has about 0.1, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 40, or about 50°C higher thermal stability (e.g., melting temperature, i.e., Tm) than the parent polypeptide from which it originates. In certain embodiments, the stabilized Fc polypeptide variant of the present invention is expressed as a monomeric soluble protein, of which 25% or less (e.g., about 25%, about 20%, about 15%, about 10%, or less than about 5%) are in dimer, tetramer, or otherwise aggregated form.

[0216] In another embodiment, the stabilized Fc polypeptide has a T50 above 40°C (e.g., 40, 41, 42, 43, 44, 45, 46, 47, 48, 49°C or higher) in a thermal load assay (see U.S. Patent Application No. 11 / 725,970, incorporated herein by reference, and Example 4 below). In some embodiments, the stabilized Fc molecule of the present invention has a T50 above 50°C (e.g., 50, 51, 52, 53, 54, 55, 56, 57, 58°C or higher). In some embodiments, the stabilized Fc molecule of the present invention has a T50 above 60°C (e.g., 60, 61, 62, 63, 64, 65°C or higher). In yet another embodiment, the stabilized Fc molecule of the present invention has a T50 above 65°C (e.g., 65, 66, 67, 68, 69, 70°C or higher). In a further embodiment, the stabilized Fc molecule of the present invention has a T50 above 70°C (e.g., 70, 71, 73, 74, 75°C or higher).

[0217] In certain embodiments, the stabilized Fc molecule of the present invention has a CH2 domain with a Tm value of about 60°C (e.g., about 61, 62, 63, 64, 65°C or higher), about 65°C (e.g., 65, 66, 67, 68, 69°C or higher), or about 70°C (e.g., 71, 72, 73, 74, 75°C or higher). In other embodiments, the stabilized Fc molecule of the present invention has a CH3 domain with a Tm value of about 70°C (e.g., 71, 72, 73, 74, 75°C or higher), about 75°C (e.g., 76, 77, 78, 79, 80°C or higher), or about 80°C (e.g., 81, 82, 83, 84, 85°C or higher). In certain embodiments, the stabilized Fc polypeptide is a variant of a parent Fc polypeptide containing an aglycosylated or glycosylated Fc region of an IgG4 antibody (e.g., aglyIgG4.P). In other embodiments, the stabilized Fc polypeptide is a variant of a parent Fc polypeptide containing an aglycosylated Fc region of an IgG1 antibody (e.g., aglyIgG1). In yet another embodiment, the stabilized Fc molecule of the present invention has an Fc region or Fc moiety (e.g., CH2 and / or CH3 domains) that has substantially the same or higher thermal stability as the glycosylated IgG1 antibody.

[0218] In certain embodiments, the variant Fc polypeptide of the present invention results in reduced aggregation compared to the parent Fc polypeptide from which it originates. In one embodiment, the stabilized Fc molecule produced by the method of the present invention has at least 1% reduced aggregation compared to the parent Fc molecule. In other embodiments, the stabilized Fc polypeptide has at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 100% reduced aggregation compared to the parent molecule.

[0219] In other embodiments, the stabilized Fc polypeptide of the present invention exhibits increased long-term stability or shelf life compared to its parental Fc polypeptide from which it originates. In one embodiment, the stabilized Fc molecule produced by the method of the present invention has a shelf life increased by at least one day compared to the unstabilized bound molecule. This means that the preparation of the stabilized Fc polypeptide contains substantially the same amount of biologically active variant Fc polypeptide as that present the previous day, and there is no recognizable aggregation or degradation of the variant polypeptide in the preparation. In other embodiments, the stabilized Fc molecule has a shelf life increased by at least two days, at least five days, at least one week, at least two weeks, at least one month, at least two months, at least six months, or at least one year compared to the unstabilized Fc molecule.

[0220] In certain embodiments, the stabilized Fc polypeptide of the present invention is expressed in an increased yield compared to its parent Fc polypeptide. In one embodiment, the stabilized Fc polypeptide of the present invention has a yield increased by at least 1% compared to the parent Fc molecule. In other embodiments, the stabilized Fc polypeptide has a yield increased by at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% compared to the parent Fc molecule.

[0221] In exemplary embodiments, the stabilized Fc polypeptide of the present invention is expressed in host cells, e.g., bacterial or eukaryotic (e.g., yeast or mammalian) host cells, in increased yield (compared to its parental Fc polypeptide). Exemplary mammalian host cells that may be used to express nucleic acid molecules encoding the stabilized Fc polypeptide of the present invention include Chinese hamster ovary (CHO) cells, HELA (human cervical cancer) cells, CVI (monkey kidney) cells, COS (a derivative of CVI having the SV40 T antigen) cells, R1610 (Chinese hamster fibroblast) cells, BALBC / 3T3 (mouse fibroblast) cells, HAK (hamster kidney) cells, SP2 / O (mouse myeloma) cells, BFA-1c1BPT cells (bovine endothelial cells), RAJI (human lymphocyte) cells, PER.C6® (human retina-derived cell line, Crucell, The Netherlands), and 293 cells (human kidney).

[0222] In other embodiments, the stabilized Fc polypeptide of the present invention is expressed in host cells in increased yield (compared to its parent Fc polypeptide) under large-scale (e.g., commercial-scale) conditions. In an exemplary embodiment, the stabilized Fc molecule has an increased yield when expressed in at least 10 liters of culture medium. In other embodiments, the stabilized Fc-binding molecule has an increased yield when expressed from host cells in at least 20 liters, at least 50 liters, at least 75 liters, at least 100 liters, at least 200 liters, at least 500 liters, at least 1000 liters, at least 2000 liters, at least 5,000 liters, or at least 10,000 liters of culture medium. In one exemplary embodiment, at least 10 mg (e.g., 10 mg, 20 mg, 50 mg, or 100 mg) of stabilized Fc molecule is produced per liter of culture medium.

[0223] (i) Stabilized Fc amino acids In certain embodiments, the stabilized Fc polypeptide of the present invention comprises the CH2 domain of an IgG4 molecule (or its amino acids 292-340) and the CH3 domain of an IgG1 molecule, having a Gln(Q) residue at position 297. In other embodiments, the stabilized Fc polypeptide of the present invention comprises the CH2 and CH3 domains of an IgG1 molecule, as well as a Lys(K) residue at position 299, either alone or in combination with an Asp(D) residue at position 297.

[0224] (ii) Example of the stabilized Fc portion Examples of the stabilized Fc portion of the present invention can be found throughout this specification, in the examples, and in the sequence listing.

[0225] In certain exemplary embodiments, the stabilized Fc polypeptide of the present invention comprises a stabilized IgG4 Fc region containing one, two, or more of the Fc amino acid sequences shown in Table 1.1 below. Stabilized Fc mutations are shown in bold and underlined.

[0226] TIFF2026113464000008.tif167170TIFF2026113464000009.tif208170TIFF2026113464 000010.tif200170TIFF2026113464000011.tif202170TIFF2026113464000012.tif86170

[0227] In certain exemplary embodiments, the stabilized Fc polypeptide of the present invention comprises a stabilized chimeric Fc region having one, two, or more of the chimeric Fc partial amino acid sequences shown in Table 1.2 below.

[0228] TIFF2026113464000013.tif83170TIFF2026113464000014.tif112170

[0229] In other exemplary embodiments, the stabilized Fc polypeptide of the present invention comprises a stabilized aglycosylated IgG1 Fc region having one, two, or more of the IgG1 Fc partial amino acid sequences shown in Table 1.3 below.

[0230] TIFF2026113464000015.tif56170TIFF2026113464000016.tif206170TIFF2026113464000017.tif172170

[0231] IV. Methods for stabilizing variant Fc polypeptides In a particular embodiment, the present invention relates to a method for stabilizing a polypeptide comprising an Fc region (e.g., an aglycosylated Fc region), comprising (a) selecting one or more amino acid positions for mutation within at least one Fc portion of a starting Fc region, and (b) stabilizing the polypeptide by mutating one or more of the selected amino acid positions.

[0232] In one embodiment, the starting Fc region is an IgG1 Fc region. In another embodiment, the starting Fc region is an IgG4 Fc region. In yet another embodiment, the starting Fc region is a chimeric Fc region. In one embodiment, the starting Fc region is an aglycosylated IgG1 Fc region. In yet another embodiment, the starting Fc region is an aglycosylated IgG4 Fc region.

[0233] In some embodiments, the Fc region of the antibody (or fragment thereof) described herein has the sequence shown in SEQ ID NO: 85.

[0234] In one embodiment, the amino acid location selected for mutation is within the elongation loop in the Fc region of the starting IgG molecule (e.g., IgG4 molecule). In another embodiment, the amino acid location selected for mutation is located at the interface between CH3 domains. In yet another embodiment, the amino acid location selected for mutation is near the carbohydrate contact site in the 1Hz crystal structure (e.g., V264, R292, or V303). In yet another embodiment, the amino acid location may be near the CH3 / CH2 interface, or near the CH3 / CH2 interface (e.g., H310). In yet another embodiment, one or more mutations that change the overall surface charge of the Fc region may occur, for example, at one or more of the surface-exposed glutamine residues (Q268, Q274, or Q355). In yet another embodiment, the amino acid location is a valine residue found in the CH2 and CH3 "valine core". The "valine core" of CH2 consists of five valine residues (V240, V255, V263, V302, and V323), all positioned toward the same proximal inner core of the CH2 domain. A similar "valine core" is observed in CH3 (V348, V369, V379, V397, V412, and V427). In another embodiment, the amino acid position chosen for mutation is one that is predicted to interact with or contact an N-linked carbohydrate at amino acid 297. Such amino acid positions can be identified by examining the crystal structure of the Fc region bound to a congeneral Fc receptor (e.g., FcγRIIIa). Exemplary amino acids that interact with N297 include a loop formed by residues 262–270.

[0235] Exemplary amino acid positions include positions 240, 255, 262–266, 267–271, 292–299, 302–309, 379, 397–399, 409, 412, and 427 according to EU numbering convention. In a particular embodiment, one or more amino acid positions selected for mutation are one or more amino acid positions selected from the group consisting of positions 240, 255, 262, 263, 264, 266, 268, 274, 292, 299, 302, 303, 307, 309, 323, 348, 355, 369, 379, 397, 399, 409, 412, and 427. In a particular embodiment, one or more amino acid positions selected for mutation are selected from the group consisting of positions 240, 262, 264, 266, 297, 299, 307, 309, 399, 409, and 427. In another embodiment, one or more amino acid positions are selected from the group consisting of positions 297, 299, 307, 309, 409, and 427. In another embodiment, one or more amino acid positions are selected from amino acid residues 240, 262, 264, and 266. In another embodiment, at least one of the amino acid positions is at EU297. In another embodiment, at least one of the amino acid positions is at EU299. In another embodiment, at least one of the amino acid positions is at EU307. In another embodiment, at least one of the amino acid positions is at EU309. In another embodiment, at least one of the amino acid positions is at EU399. In another embodiment, at least one of the amino acid positions is at EU409. In yet another embodiment, at least one of the amino acid positions is at EU427.

[0236] In certain embodiments, the Fc region is an IgG1 Fc region. In certain embodiments where the Fc region is an IgG1 Fc region, one or more amino acid positions are selected from amino acid residues 240, 262, 264, 299, 297, and 266 according to EU numbering. In other embodiments where the Fc region is an IgG4 Fc region, one or more amino acid positions are selected from amino acid residues 297, 299, 307, 309, 399, 409, and 427 according to EU numbering.

[0237] In one embodiment, the mutation reduces the size of the amino acid side chain at that amino acid position (e.g., substitution with alanine (A), serine (S), or threonine (T)). In another embodiment, the mutation is a substitution with an amino acid having a nonpolar side chain (e.g., substitution with glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), and tryptophan (W)). In yet another embodiment, the mutation adds hydrophobicity to the CH3 interface, for example, by increasing the association between two interacting domains (e.g., Y349F, T350V, and T394V) or by increasing the volume of the side chain at the interface (e.g., F405Y). In yet another embodiment, one or more amino acids in the "valine core" are substituted with isoleucine or phenylalanine to increase their stability. In another embodiment, the amino acids (e.g., L351 and / or L368) are mutated into higher-order branched hydrophobic side chains.

[0238] In one embodiment, the mutation is a substitution by alanine (A). In another embodiment, the mutation is a substitution by phenylalanine (F). In yet another embodiment, the mutation is a substitution by leucine (L). In one embodiment, the mutation is a substitution by threonine (T). In yet another embodiment, the mutation is a substitution by lysine (K). In one embodiment, the mutation is a substitution by proline (P). In yet another embodiment, the mutation is a substitution by phenylalanine (F).

[0239] In one embodiment, the mutation comprises one or more of the mutations or substitutions shown in Table 5.1, Table 5.2, Table 5.3, and / or Table 5.4 below.

[0240] In certain embodiments, the mutation comprises one or more substitutions selected from the group consisting of 240F, 262L, 264T, 266F, 297Q, 297S, 297D, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, 409M, and 427F (in accordance with the EU numbering convention). In another embodiment, the mutation comprises one or more substitutions selected from the group consisting of 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399E, 399S, 409K, 409M, and 427F. In another embodiment, one or more amino acid positions are selected from amino acid residues 240F, 262L, 264T, and 266F. In another embodiment, at least one of the substitutions is 299A. In another embodiment, at least one of the substitutions is 299K. In another embodiment, at least one of the substitutions is 307P. In another embodiment, at least one of the substitutions is 309K. In another embodiment, at least one of the substitutions is 309M. In another embodiment, at least one of the substitutions is 309P. In another embodiment, at least one of the substitutions is 323F. In another embodiment, at least one of the substitutions is 399S. In another embodiment, at least one of the substitutions is 399E. In another embodiment, at least one of the substitutions is 409K. In another embodiment, at least one of the substitutions is 409M. In another embodiment, at least one of the substitutions is 427F.

[0241] In another embodiment, the mutation comprises two or more substitutions (e.g., 2, 3, 4, or 5 substitutions). In another embodiment, the mutation comprises three or more substitutions (e.g., 3, 4, 5, or 6 substitutions). In yet another embodiment, the stabilized Fc region comprises four or more substitutions (e.g., 4, 5, 6, or 7 substitutions).

[0242] In another embodiment, the present invention relates to a method for producing a stabilized binding molecule containing a stabilized Fc region, comprising fusing a polypeptide containing the stabilized Fc region of the present invention to the amino-terminus or carboxy-terminus of the binding site with a gene. In a particular embodiment, the stabilized Fc region is stabilized by the method of the present invention.

[0243] In another embodiment, the therapeutic antibody can eliminate scrambling or reduce the antibody's sensitivity to scrambling. Certain antibodies, for example, those containing the wild-type IgG4 constant region (e.g., including the hinge region), are sensitive to scrambling with endogenous IgG4 antibodies and may produce two copies of a functionally monovalent product (see, e.g., Aalberse and Schuurman, Immunology 105:9-19, 2002). The scrambling rate may depend on the endogenous level of IgG4, which is variable. As a result, therapeutic antibodies containing the wild-type human IgG4 constant region have a variable pharmacokinetic / pharmacodynamic (PK / PD) profile. Scrambling of antibodies containing the wild-type IgG4 constant region may result in a bispecific antibody that is monovalent to VLA4 and monovalent to another antigen. Therefore, in some embodiments, the therapeutic antibodies described herein have a more consistent PK profile than antibodies containing the wild-type IgG4 constant region, even though they have the same specificity (e.g., specific binding to α4). In some embodiments, the consistent PK profile of the therapeutic antibodies described herein may increase the safety, purity, and / or potency of the antibodies described herein compared to antibodies having the same specificity (e.g., specific binding to α4) but containing a wild-type IgG4 constant region.

[0244] In some embodiments, therapeutic antibodies comprising a heavy chain hinge and Fc region that are chimeric or hybrid of human IgG1 antibody and human IgG4 antibody can reduce or eliminate scrambling that occurs with antibodies comprising a hinge and Fc region derived from a human IgG4 molecule. In one embodiment, the antibody described herein having a chimeric or hybrid heavy chain constant region can reduce PK / PD variability by eliminating scrambling or reducing the antibody's sensitivity to scrambling. In some embodiments, the therapeutic antibody described herein has lower PK / PD variability than an antibody having the same specificity (e.g., specific binding to α4) but containing a wild-type IgG4 constant region. In certain embodiments, the antibody described herein can increase the potency of a bivalent monoclonal antibody and / or eliminate the bispecificity caused by scrambling. In certain embodiments, the antibody described herein can exhibit higher binding affinity and / or sustained high receptor occupancy than its non-mutant counterpart or than an antibody having the same specificity (e.g., specific binding to α4) but containing a wild-type IgG4 constant region. In some embodiments, therapeutic antibodies described herein that exhibit low PK / PK variability and / or high binding affinity and / or reduced or absent scrambling may increase the safety, purity, and / or potency of the antibodies described herein compared to antibodies having the same specificity (e.g., specific binding to α4) but containing a wild-type IgG4 constant region. In preferred embodiments, an antibody that abolishes scrambling or reduces the sensitivity of the antibody to scrambling is a recombinant anti-alpha 4 antibody comprising (a) a heavy chain containing, essentially derived from, or consisting of the sequence of SEQ ID NO: 80, and (b) a light chain containing, essentially derived from, or consisting of the sequence of SEQ ID NO: 81.

[0245] V. Methods for evaluating protein stability The stability properties of the compositions of the present invention can be analyzed using methods known in the art. Stability parameters acceptable to those skilled in the art may be used. Exemplary parameters are described in more detail below. In exemplary embodiments, thermal stability is evaluated. In some embodiments, the expression level of the compositions of the present invention (e.g., measured by yield %) is evaluated. In some embodiments, the aggregation level of the compositions of the present invention is evaluated.

[0246] In certain embodiments, the stability properties of the Fc polypeptide are compared to those of a suitable control. Exemplary controls include parental Fc polypeptides, such as wild-type Fc polypeptides and wild-type (glycosylated) IgG1 or IgG4 antibodies. Another exemplary control is an aglycosylated Fc polypeptide and an aglycosylated IgG1 or IgG4 antibody. In one embodiment, one or more parameters, as described below, are measured.

[0247] In one embodiment, one or more of these parameters are measured after expression in mammalian cells. In one embodiment, one or more of the parameters described later are measured under large-scale manufacturing conditions (e.g., expression of Fc polypeptide or a molecule containing Fc polypeptide in a bioreactor).

[0248] A. Thermal stability The thermal stability of the compositions of the present invention can be analyzed using several non-limiting biophysical or biochemical techniques known in the art. In certain embodiments, thermal stability is evaluated by analytical spectroscopy. An exemplary analytical spectroscopy is differential scanning calorimetry (DSC). DSC uses a calorimeter that is sensitive to the thermal absorption associated with the unfolding of most proteins or protein domains (see, for example, Sanchez-Ruiz, et al., Biochemistry, 27:1648-52, 1988). To determine the thermal stability of a protein, a sample of the protein is inserted into a calorimeter and the temperature is increased until the Fc polypeptide (or its CH2 or CH3 domain) unfolds. The temperature at which the protein unfolds indicates the overall protein stability.

[0249] Another exemplary analytical spectroscopy is circular dichroism (CD) spectroscopy. CD spectroscopy measures the optical activity of a composition as a function of increasing temperature. Circular dichroism (CD) spectroscopy measures the difference in absorbance between left-handed and right-handed polarized light resulting from structural asymmetry. Disordered or unfolded structures yield CD spectra that are quite different from those of ordered or folded structures. CD spectra reflect the sensitivity of proteins to the denaturing effect of increasing temperature and therefore indicate the thermal stability of proteins (see van Mierlo and Steemsma, J. Biotechnol, 79(3):281-98, 2000).

[0250] Another exemplary analytical spectroscopic method for measuring thermal stability is fluorescence emission spectroscopy (see van Mierlo and Steemsma above). Yet another exemplary analytical spectroscopic method for measuring thermal stability is nuclear magnetic resonance (NMR) spectroscopy (see, for example, van Mierlo and Steemsma).

[0251] In other embodiments, the thermal stability of the compositions of the present invention is measured biochemically. An exemplary biochemical method for evaluating thermal stability is a thermal load assay. In a "thermal load assay," the compositions of the present invention are subjected to a series of high temperatures over a period of time. For example, in one embodiment, a test Fc polypeptide containing an Fc region is subjected to a series of gradually increasing temperatures for, for example, 1 to 1.5 hours. The ability of the Fc region to bind to an Fc receptor (e.g., FcγR, protein A, or protein G) is then assayed by the relevant biochemical assay (e.g., ELISA or DELFIA). An exemplary thermal load assay is described in Embodiment 4 below.

[0252] In one embodiment, such assays may be performed in a high-throughput format. In another embodiment, a library of Fc variants may be prepared using methods known in the art. Fc expression may be induced, and Fc may be subjected to thermal loading. The loaded test samples may be assayed for binding, and a stable Fc polypeptide may be scaled up and further analyzed.

[0253] In certain embodiments, thermal stability is evaluated by measuring the melting temperature (Tm) of the composition of the present invention using one of the techniques described above (e.g., analytical spectroscopy). The melting temperature is the temperature at the midpoint of the thermal transition curve, where 50% of the molecules of the composition are folded.

[0254] In other embodiments, thermal stability is assessed by measuring the specific heat or heat capacity (Cp) of the composition of the present invention using analytical spectroscopy techniques (e.g., DSC). The specific heat of a composition is the energy (e.g., in kcal / mol units) required to raise the temperature of 1 mole of water by 1°C. A large Cp is characteristic of a denatured or inert protein composition. In certain embodiments, the change in the heat capacity of the composition (ΔCp) is measured by determining the specific heat of the composition before and after the thermal transition. In other embodiments, thermal stability may be assessed by measuring or determining other parameters of thermodynamic stability, such as the Gibbs free energy of unfolding (ΔG), the enthalpy of unfolding (ΔH), or the entropy of unfolding (ΔS).

[0255] In other embodiments, one or more of the biochemical assays described above (e.g., a thermal load assay) is used to determine the temperature (i.e., Tc value) at which 50% of the composition retains its activity (e.g., binding activity).

[0256] B. Agglomeration % In certain embodiments, the stability of the compositions of the present invention is determined by measuring their tendency to aggregate. Aggregation can be measured by several non-limiting biochemical or biophysical techniques. For example, the aggregation of the compositions of the present invention can be evaluated using chromatography, for example, size exclusion chromatography (SEC). SEC separates molecules based on size. The column is packed with semi-solid beads of a polymer gel that accepts ions and small molecules internally but not larger ones. When the protein composition is added to the top of the column, small folded proteins (i.e., non-aggregating proteins) are distributed to a larger volume than larger protein aggregates available in the solvent. As a result, larger aggregates move faster through the column, thus allowing the mixture to be separated or fractionated into its components. Each fraction can be quantified separately (e.g., by light scattering) as it elutes from the gel. Thus, the aggregation % of the compositions of the present invention can be determined by comparing the concentration of the fraction with the total concentration of the protein added to the gel. A stable composition elutes from the column essentially as a single fraction and appears essentially as a single peak in the elution profile or chromatogram.

[0257] In some embodiments, SEC is used in conjunction with inline light scattering (e.g., classical or dynamic light scattering) to determine the aggregation percentage of the composition. In certain embodiments, static light scattering is used to measure the mass of each fraction or peak, independently of molecular shape or elution position. In some embodiments, dynamic light scattering is used to measure the hydrodynamic size of the composition. Other exemplary methods for evaluating protein stability include fast SEC (see, e.g., Corbet et al., Biochemistry. 23(8):1888-94, 1984).

[0258] In non-limiting embodiments, the aggregation percentage is determined by measuring the proportion of protein aggregates in a protein sample. In some embodiments, the aggregation percentage of a composition is measured by determining the proportion of folded proteins in a protein sample.

[0259] C. Yield % In other embodiments, the stability of the compositions of the present invention is evaluated by measuring the amount of protein recovered (herein “% yield”) after protein expression (e.g., recombinant expression). For example, % yield can be measured by determining the number of milligrams of protein recovered per milliliter of host culture medium (i.e., mg / ml of protein). In some embodiments, % yield is evaluated after expression in mammalian host cells (e.g., CHO cells).

[0260] D. Loss % In still other embodiments, the stability of the compositions of the present invention is evaluated by monitoring protein loss at a series of temperatures (e.g., -80 to 25° C.) after storage over a predetermined period of time. The amount or concentration of protein recovered is determined using any protein quantification method known in the art and can be compared to the initial concentration of the protein. Exemplary protein quantification methods include SDS-PAGE analysis or Bradford assay (Bradford, et al., Anal. Biochem. 72, 248, (1976)). Non-limiting methods for assessing % loss utilize any of the analytical SEC methods described above. The % loss measurement can be determined under any desired storage conditions or storage formulations, e.g., in a lyophilized protein preparation.

[0261] E. Proteolysis % In further embodiments, the stability of the compositions of the present invention is assessed by determining the amount of protein that has been proteolytically degraded after storage under standard conditions. In one exemplary embodiment, proteolysis is determined by SDS-PAGE of a protein sample, where the amount of intact protein is compared to the amount of low molecular weight fragments that appear on the SDS-PAGE gel. In another exemplary embodiment, proteolysis is determined by mass spectrometry (MS), where the amount of protein of expected molecular weight is compared to the amount of low molecular weight protein fragments in the sample.

[0262] F. Binding affinity In further embodiments, the stability of the compositions of the present invention can be evaluated by determining their target binding affinity. A wide variety of methods for determining binding affinity are known in the art. An exemplary method for determining binding affinity is to use surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that enables real-time analysis of biomolecular-specific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further explanation, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26, Jonsson, U., et al. (1991) Biotechniques 11:620-627, Johnsson, B., et al. (1995) / . MoI. Recognit. 8:125-131, and Johnsson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0263] G. Other binding tests In yet another embodiment, the compositional stability of the present invention can be assessed by quantifying the binding of the labeled compound to denatured or unfolded portions of the binding molecule. Such molecules may be hydrophobic, because they are likely to bind to or interact with large hydrophobic patches of amino acids that are normally embedded within the native protein but are exposed in denatured or unfolded binding molecules. An exemplary labeled compound is the hydrophobic fluorescent dye 1-anilino-8-naphthalenesulfonic acid (ANS).

[0264] VI. Stabilized binding polypeptides containing a stabilized Fc region In one embodiment, the polypeptide of the present invention comprises a CH1 domain derived from an IgG4 antibody, a CH2 domain derived from an IgG4 antibody, and a CH3 domain derived from an IgG1 antibody. In one embodiment, the polypeptide further comprises a Ser228Pro substitution. The polypeptide may further comprise a mutation at amino acids 297 and / or 299, for example, 297Q and / or 299K, or 297S and / or 299K. The polypeptide may also comprise a CH1 domain derived from an IgG1 or IgG4 antibody, a CH2 domain derived from an IgG4 antibody, and a CH3 domain derived from an IgG1 antibody, and this polypeptide may comprise one or more of the Ser228Pro, 297Q, or 299K substitutions. The amino acid sequence of the Fc region consisting of a CH1 domain derived from an IgG4 molecule (including the Ser228Pro substitution), a CH2 domain derived from an IgG4 antibody, and a CH3 domain derived from an IgG1 antibody is presented in SEQ ID NO: 43. In one embodiment, the stabilized Fc polypeptide of the present invention comprises the amino acid sequence shown in SEQ ID NO: 40. In one embodiment, the stabilized Fc polypeptide of the present invention comprises the amino acid sequence shown in SEQ ID NO: 74. In one embodiment, the stabilized Fc polypeptide of the present invention comprises the amino acid sequence shown in SEQ ID NO: 75. In one embodiment, the stabilized Fc polypeptide of the present invention comprises the amino acid sequence shown in SEQ ID NO: 76. In one embodiment, the stabilized Fc polypeptide of the present invention comprises the amino acid sequence shown in SEQ ID NO: 77.

[0265] In one embodiment, the Fc region of the polypeptide of the present invention is single-stranded (scFc). In one embodiment, the molecule containing the Fc region described in this paragraph is monovalent. In one embodiment, the molecule containing the Fc region described in this paragraph is monovalent, and the Fc region is scFc. The molecule containing the Fc region described herein may also contain scFv.

[0266] VII. Stabilized Fc-containing polypeptides containing functional components The variant Fc-containing polypeptide of the present invention may be further modified to produce a desired effect. For example, the Fc region of the variant Fc polypeptide may be covalently bonded to additional parts, i.e., functional parts such as blocking parts, detectable parts, diagnostic parts, and / or therapeutic parts. Exemplary functional parts are first described below, followed by chemistry useful for bonding such functional parts to different amino acid side-chain chemical structures.

[0267] Examples of useful functional parts include, but are not limited to, blocking parts, detectable parts, diagnostic parts, and therapeutic parts. Exemplary blocking parts include parts with sufficient stereovolume and / or charge to reduce effector function, for example, by inhibiting the ability of an Fc region to bind to a receptor or complement protein. Non-limiting blocking parts include polyalkylene glycol parts, such as PEG parts or PEG-maleimide parts. Non-limiting PEGylated moieties (or associated polymers) may include, for example, polyethylene glycol ("PEG"), polypropylene glycol ("PPG"), polyoxyethylated glycerol ("POG") and other polyoxyethylated polyols, polyvinyl alcohol ("PVA") and other polyalkylene oxides, polyoxyethylated sorbitol, or polyoxyethylated glucose. The polymer may be a homopolymer, random copolymer, or block copolymer or terpolymer based on the above monomers, as long as it has at least one active sulfone moiety, and may be linear or branched, substituted or unsubstituted. The polymer moieties may have any length or molecular weight, but these characteristics may affect the biological properties. The average molecular weight of the polymer is important for clearance in pharmaceutical applications. It is particularly useful for reducing the rate, and is in the range of 2,000 to 35,000 Daltons. Furthermore, when two groups are bonded one to each end of the polymer, the length of the polymer can affect the effective distance between the two groups and other spatial relationships. Thus, those skilled in the art can modify the length of the polymer to optimize or confer desired biological activity. PEG is useful in biological applications for several reasons. PEG is typically clear, colorless, odorless, water-soluble, thermally stable, inert to many chemical factors, does not hydrolyze, and is non-toxic. PEGylation can improve the pharmacokinetic performance of a molecule by increasing its apparent molecular weight. The increase in apparent molecular weight reduces the rate of clearance from the body after subcutaneous or systemic administration. Often, PEGylation can reduce antigenicity and immunogenicity.Furthermore, PEGylation can increase the solubility of biologically active molecules.

[0268] PEGylated antibodies and antibody fragments can generally be used to treat conditions that can be alleviated or modified by administration of the antibodies and antibody fragments described herein. Generally, PEGylated aglycosylated antibodies and antibody fragments have an increased half-life compared to unPEGylated aglycosylated antibodies and antibody fragments. PEGylated aglycosylated antibodies and antibody fragments can be used alone, together, or in combination with other pharmaceutical compositions. Examples of detectable moieties useful in the methods and polypeptides of the present invention include fluorescent moieties, radioisotope moieties, radiopaque moieties, and detectable labels such as biotin, fluorophores, chromophores, spin resonance probes, or radiolabels. Exemplary fluorophores include fluorescent dyes (e.g., fluorescein, rhodamine, etc.) and other luminescent molecules (e.g., luminol). Fluorophores may be environmentally sensitive such that their fluorescence changes when located near one or more residues in a modified protein that undergo a structural change upon binding to a substrate (e.g., a dansyl probe). Examples of radiolabeled molecules include small molecules containing atoms with one or more low-sensitivity nuclei (such as 13C, 15N, 2H, 125I, 123I, 99Tc, 43K, 52Fe, 67Ga, 68Ga, 111In, etc.). Other useful parts are known in the art.

[0269] Examples of useful diagnostic portions in the methods and polypeptides of the present invention include detectable portions suitable for revealing the presence of a disease or disorder. Typically, the diagnostic portion allows for the determination of the presence, absence, or level of molecules associated with the disease or disorder, such as a target peptide, one protein, or multiple proteins. Such diagnostic agents are also suitable for prognosis and / or diagnosis of a disease or disorder and its progression.

[0270] Examples of therapeutic portions useful in the methods and polypeptides of the present invention include, for example, anti-inflammatory agents, anticancer agents, antineurodegenerative agents, and anti-infective agents. Functional portions may also have one or more of the above-mentioned functions.

[0271] Exemplary therapeutic agents include radionuclides with high-energy ionizing radiation that can induce multiple strand breaks in nuclear DNA and are therefore suitable for inducing cell death (e.g., cancer cell death). Exemplary high-energy radionuclides include 90Y, 125I, 1311, 123I, 111In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re, and 188Re. These isotopes typically produce high-energy OC or β particles with short pathway lengths. Such radionuclides kill adjacent cells, such as newly formed cells to which a conjugate has attached or invaded. Radionuclides have little to no effect on cells to which they are not localized and are essentially non-immunogenic.

[0272] Examples of therapeutic agents include cytotoxic agents, such as cell division inhibitors (e.g., alkylating agents, DNA synthesis inhibitors, DNA intercalators or crosslinkers, or DNA-RNA transcription regulators), enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin binders, hormones and hormone antagonists, and anti-angiogenic agents.

[0273] Exemplary therapeutic agents also include alkylating agents such as drugs of the anthracycline family (e.g., adriamycin, carminomycin, cyclosporine A, chloroquine, metopterin, mithramycin, porphyromycin, streptonigrin, porphyromycin, anthracendione, and aziridine). In another embodiment, the chemotherapeutic agent portion is a cell division inhibitor, such as a DNA synthesis inhibitor. Examples of DNA synthesis inhibitors include, but are not limited to, methotrexate and dichloromethotrexate, 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosine β-D-arabinofuranoside, 5-fluoro-5'-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, actinomycin-D, and mitomycin C. Exemplary DNA intercalators or crosslinking agents include, but are not limited to, bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cis-diammineplatin(II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin. Exemplary therapeutic agents also include transcription factors such as actinomycin D, daunorubicin, doxorubicin, homohalintonin, and idarubicin. Other exemplary cell division inhibitors compatible with the present invention include ansamycin benzoquinone, quinonoid derivatives (e.g., quinolones, genistein, bactacyclines), busulfan, ifosfamide, mechloretamine, triadicone, diadicone, carbazylquinone, indoloquinone EO9, diaziridinyl-benzoquinone methyl DZQ, triethylene phosphoramide, and nitrosourea compounds (e.g., carmustine, lomustine, semstine).

[0274] Examples of therapeutic agents include cytotoxic nucleosides such as adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, futraful, and 6-mercaptopurine; taxoids (e.g., paclitaxel, docetaxel, taxane); nocodazole; rhizoxin; dorastatin (e.g., dorastatin-10, -11, or -15); colchicine; and corticinoids (e.g., ZD6126). This also includes tubulin binders such as combretastatin (e.g., combretastatin A-4, AVE-6032) and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine (navelbine)); and anti-angiogenic compounds such as angiostatin K1-3, DL-α-difluoromethyl-ornithine, endostatin, fumagiline, genistein, minocycline, staurosporine, and (±)-thalidomide. Exemplary therapeutic agents include hormones and hormone antagonists, such as corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone or medroprogesterone), estrogens (e.g., diethylstilbestrol), antiestrogenic agents (e.g., tamoxifen), androgens (e.g., testosterone), aromatase inhibitors (e.g., aminoglutethimide), 17-(allylamino)-17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide, apigenin, brefelzin A, cimetidine, dichloromethylenediphosphonic acid, leuprolide (leuprorelin), luteinizing hormone-releasing hormone, pifislin-α, rapamycin, sex hormone-binding globulin, and thapsigardin.

[0275] Exemplary therapeutic agents also include enzyme inhibitors, such as S(+)-camptothecin, curcumin, (-)-deguerin, 5,6-dichlorobenzaimidazole 1-β-D-ribofuranoside, etoposide, formestan, fostoriesin, hispidin, 2-imino-1-imidazolidinedioacetic acid (cyclocreatine), mebinoline, trichostatin A, thyrofostine AG34, and thyrofostine AG879. Examples of therapeutic agents include gene regulators, such as 5-aza-2'-deoxycytidine, 5-azacitidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifen, transretinal (vitamin A aldehyde), retinoic acid, vitamin A acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone.

[0276] Exemplary therapeutic agents include, for example, pteridine family drugs, diynenes, and cytotoxic agents such as podophyllotoxins. Particularly useful members of these classes include, for example, metopterin, podophyllotoxin, or podophyllotoxin derivatives, such as etoposide or etoposide phosphate, leulosidine, vindesine, and leulosine.

[0277] Other cytotoxics compatible with the teachings contained herein include auristatins (e.g., auristatin E and monomethyl auristan E), calicheamicin, gramicidin D, mytansinoids (e.g., mytansin), neocarlutinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, tenoposide, colchicine, dihydroxyanthracine dione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and their analogs or homologs. Other types of functional components are known in the art and can be readily used in the methods and compositions of the present invention based on the teachings contained herein.

[0278] Regardless of whether the functional portion is a small molecule, nucleic acid, polymer, peptide, protein, chemotherapeutic agent, or other type of molecule, the chemistry for attaching the aforementioned functional portion to a specific amino acid side chain is well known in the art (for a detailed review of specific linkers, see, for example, Hermanson, GT, Bioconjugate Techniques, Academic Press (1996)).

[0279] VIII. Pharmaceutical Compositions Alpha-4 conjugates, such as VLA-4-binding antibodies having a stabilized Fc region, can be formulated as pharmaceutical compositions. Typically, the pharmaceutical composition includes a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any physiologically compatible solvent, dispersion medium, coating, antimicrobial and antifungal agents, isotonic agents and absorption retarders, and the like.

[0280] A "pharmaceutically acceptable salt" means a salt that retains the desired biological activity of the parent compound without producing any undesirable toxic effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, and hydroiodic acid, as well as those derived from non-toxic organic acids such as aliphatic monocarboxylic acids and dicarboxylic acids, phenyl-substituted alkanos, hydroxyalkanoics, aromatic acids, and aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, as well as those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0281] The antibody compositions described herein may be formulated according to methods known in the art. The formulation of pharmaceuticals is a well-established art and is further described in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472), Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727), and Kibbe (ed.), Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, 3rd ed. (2000) (ISBN: 091733096X).

[0282] In one embodiment, the α4 antibody may be formulated with excipient materials such as sodium chloride, dibasic sodium phosphate heptahydrate, monobasic sodium phosphate, and polysorbate 80. In another embodiment, the α4 antibody may be formulated in citrate buffer at, for example, pH 5, 5.5, 6, 6.5, 7, or 7.5. In yet another embodiment, the α4 antibody may be formulated in a solution containing 2, 4, 5, 6, 8, 10, 12, 14, or 15% sucrose. The α4 antibody may be supplied in buffer at, for example, a concentration of about 20 mg / ml and stored at 2-8°C.

[0283] Pharmaceutical compositions may also be in various other forms. These include liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injections and infusions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The form may depend on the intended mode of administration and therapeutic use. Typically, the drug compositions described herein are in the form of injections or infusions.

[0284] Such compositions may be administered parenterally (e.g., by intravenous, subcutaneous, intraperitoneal, or intramuscular injection). As used herein, the terms “parenteral administration” and “administered parenterally” mean, but are not limited to, injections, other than enteral and topical administration, and include intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0285] Pharmaceutical compositions typically must be sterile and stable under manufacturing and storage conditions. They may also be tested to ensure compliance with dosage regulations and industry standards.

[0286] The composition may be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of the drug described herein, along with one or a combination of the components listed above as needed, into a suitable solvent, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the drug described herein into a sterile vehicle containing a basic dispersion medium and other necessary components listed above. For sterile powders for the preparation of sterile injectable solutions, typical preparation methods are vacuum drying and freeze-drying, from which a powder of the drug described herein and any additional desired components is obtained from a pre-sterilized filtered solution. Appropriate fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of a surfactant. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents, such as monostearate and gelatin, in the composition.

[0287] IX. Administration Alpha-4 conjugated antibodies containing a stabilized Fc region can be administered to subjects, such as human subjects, by various methods. In many applications, the route of administration is one of intravenous injection or infusion, subcutaneous injection, or intramuscular injection. Alpha-4 conjugated antibodies may be administered as a fixed dose or in doses of mg / kg. Antibodies may be administered intravenously (IV) or subcutaneously (SC). For example, antibodies may be administered in fixed unit doses of approximately 50-600 mg IV every four weeks, or approximately 50-100 mg SC (e.g., 75 mg) at least once a week (e.g., twice a week). In one embodiment, antibodies are administered IV in fixed unit doses of 50 mg, 60 mg, 80 mg, 100 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 180 mg, 200 mg, 300 mg, 400 mg, 500 mg, or 600 mg or more. The IV dose may be administered once, twice, or three or more times per week, or once every two, three, four, or five weeks, or less frequently.

[0288] In one embodiment, the antibody is administered via SC in fixed unit doses of 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 100 mg, or 120 mg or more. The SC dose may be administered once, twice, or three or more times per week, or once every two, three, four, or five weeks, or at a less frequent frequency.

[0289] Anti-α4 antibodies with a stabilized Fc region can also be administered as bolus doses of approximately 1–10 mg / kg, for example, approximately 6.0 mg / kg, 4.0 mg / kg, 3.0 mg / kg, 2.0 mg / kg, or 1.0 mg / kg. Modified dose ranges typically include doses lower than approximately 600 mg / subject, 400 mg / subject, 300 mg / subject, 250 mg / subject, 200 mg / subject, or 150 mg / subject, administered every four weeks or once a month. α4-binding antibodies may be administered, for example, every three–five weeks, every four weeks, or monthly.

[0290] Medication may be adjusted according to the patient's clearance rate after previous administration of anti-α4 antibodies. For example, a patient may not receive a second or subsequent dose until the level of anti-α4 antibodies in their body has decreased to below a predetermined level. In one embodiment, a patient sample (e.g., plasma, serum, blood, urine, or cerebrospinal fluid (CSF)) is assayed for the presence of anti-α4 antibodies, and if the level of anti-α4 antibodies exceeds a predetermined level, the patient does not receive a second or subsequent dose. If the level of anti-α4 antibodies in the patient's body is below the predetermined level, the patient receives a second or subsequent dose. Patients determined to have excessively high anti-α4 levels (above the predetermined level) may be retested after one, two, three, or one week, and if the level of anti-α4 antibodies in the patient sample has decreased to below the predetermined level, the patient may receive a second or subsequent dose of the antibody.

[0291] This dose may also be selected to reduce or avoid antibody production against α4-binding antibodies to achieve α4 subunit saturation greater than 40, 50, 70, 75, or 80%, or to achieve α4 subunit saturation less than 80, 70, 60, 50, or 40%, or to prevent an increase in circulating leukocyte levels.

[0292] In certain embodiments, the activator may be prepared with a carrier that prevents the rapid release of the compound, such as in a sustained-release formulation including an implant and a microcapsule-encapsulated delivery system. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Many methods for preparing such formulations are patented or generally known. See, for example, Controlled Drug Delivery (Drugs and the Pharmaceutical Sciences), Second Edition, J. Robinson and VHLLee, eds., Marcel Dekker, Inc., New York, 1987.

[0293] The pharmaceutical composition may be administered by a medical device. For example, the pharmaceutical composition may be administered by a needle-free subcutaneous injection device such as the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Well-known examples of implants and modules are described, for example, in U.S. Patent No. 4,487,603 disclosing an implantable microinfusion pump for dispensing pharmaceuticals at a controlled rate; U.S. Patent No. 4,486,194 disclosing a therapeutic device for administering pharmaceuticals through the skin; U.S. Patent No. 4,447,233 disclosing a pharmaceutical infusion pump for delivering pharmaceuticals at a precise infusion rate; U.S. Patent No. 4,447,224 disclosing a variable flow rate implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196 disclosing an osmotic drug delivery system having a multi-chamber compartment; and U.S. Patent No. 4,475,196 disclosing an osmotic drug delivery system. Naturally, many other such implants, delivery systems, and modules are known.

[0294] This disclosure also features a device for administering the first and second drugs. This device may include, for example, one or more compartments for storing pharmaceutical preparations and may be configured to deliver unit doses of the first and second drugs. The first and second drugs may be stored in the same or separate compartments. For example, the device may allow the drugs to be combined before administration. It is also possible to administer the first and second drugs using different devices.

[0295] The administration regimen is adjusted to produce a desired response, such as a therapeutic response or a combined therapeutic effect. Generally, any combination doses of the VLA-4 conjugate and the second drug (either separately or in combination) can be used to deliver both drugs to the subject in bioavailable amounts.

[0296] As used herein, unit dosage form or “fixed dose” means a physically separate unit suitable as a dose unit for treating a subject, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier and, if applicable, to other agents.

[0297] A pharmaceutical composition may contain the agents described herein in a “therapeutic dose.” Such a dose may be determined based on the combined effect of the first and second agents administered. The therapeutic dose of an agent may also vary depending on factors such as the individual’s medical condition, age, sex, and weight, as well as the compound’s ability to induce a desired response in the individual, such as improvement in at least one disorder parameter, e.g., multiple sclerosis parameter, or improvement in at least one symptom of the disorder, e.g., multiple sclerosis symptoms such as muscular atrophy, ataxia, and tremor. The therapeutic dose is also such that the therapeutically beneficial effect outweighs any toxic or adverse effect of any of the compositions.

[0298] X. Devices and kits Preparations containing the antibodies described herein may be administered by a medical device. This device may be designed with features such as portability, room temperature storage, and ease of use so that it can be used in emergencies, for example, by untrained subjects or on-site emergency responders, and can be moved to medical facilities and other medical devices. The device may include, for example, one or more compartments for storing pharmaceutical preparations containing α4-binding antibodies, and may be configured to deliver one or more unit doses of the drug.

[0299] For example, the pharmaceutical composition may be administered by a transcutaneous delivery device such as a syringe including a subcutaneous syringe or a multi-chamber syringe. Other suitable delivery devices include stents, catheters, microneedles, and implantable sustained-release devices. The composition may also be administered intravenously using a standard IV device, for example, an IV tube with or without an in-line filter. In certain embodiments, this device is a syringe used for SC administration or IM administration.

[0300] The pharmaceutical composition may be administered by a medical device. For example, the pharmaceutical composition may be administered by a needle-free subcutaneous injection device such as the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of well-known implants and modules are described, for example, in U.S. Patent No. 4,487,603 disclosing an implantable microinfusion pump for dispensing pharmaceuticals at a controlled rate; U.S. Patent No. 4,486,194 disclosing a therapeutic device for administering pharmaceuticals through the skin; U.S. Patent No. 4,447,233 disclosing a pharmaceutical infusion pump for delivering pharmaceuticals at a precise infusion rate; U.S. Patent No. 4,447,224 disclosing a variable flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196 disclosing an osmotic drug delivery system having a multi-chamber compartment; and U.S. Patent No. 4,475,196 disclosing an osmotic drug delivery system. Therapeutic compositions may be in the form of biodegradable or non-biodegradable sustained-release formulations for subcutaneous or intramuscular administration. Methods for such compositions are known in the art. Continuous delivery can also be achieved using implantable or external pumps. Administration may be intermittent, for example, by once-daily injection, or continuously in low doses, such as with a sustained-release formulation. The delivery device may be modified to be optimal for the administration of the α4-binding antibody. For example, the syringe may be silicone-treated to an extent that is optimal for antibody storage and delivery. Of course, many other such implants, delivery systems, and modules are known.

[0301] The disclosure also features a device for administering a first and a second drug (e.g., an antibody and a second drug). This device may include, for example, one or more compartments for storing pharmaceutical preparations and may be configured to deliver unit doses of the first and second drugs. The first and second drugs may be stored in the same or separate compartments. In one embodiment, the device combines the drugs before administration. In some embodiments, the first and second drugs are administered by different devices.

[0302] The α4-binding antibody may be provided in a kit. In one embodiment, the kit includes (a) a container containing a composition comprising a high concentration of VLA-4-binding antibody, optionally (b) a container containing a composition comprising a second drug, and optionally (c) informational material. The informational material may be explanatory material, instructional material, marketing material, or other material relating to the use of the drug for the method and / or therapeutic utility described herein. In one embodiment, the kit also includes a second drug. For example, the kit includes a first container containing a composition comprising an α4-binding antibody, and a second container containing a second drug.

[0303] The format of the kit's informational materials is not limited. In one embodiment, the informational materials may include information on antibody production, concentration, expiration date, batch, or production site information. In one embodiment, the informational materials relate to a method of treating subjects with acute injuries such as spinal cord injury or traumatic brain injury, or inflammatory diseases (e.g., MS), or subjects at risk of experiencing episodes related to inflammatory diseases, by administering α4-conjugated antibodies, for example, in a preferred dose, dosage form, or mode of administration (e.g., the dose, dosage form, or mode of administration described herein). The information may be provided in various formats, including printed text, computer-readable materials, video recordings, or audio recordings, or information providing links or addresses to substantial materials.

[0304] In addition to the drug, the kit composition may include other components such as solvents or buffers, stabilizers, or preservatives. The drug may be supplied in any form, e.g., liquid, dry, or lyophilized, and may be substantially pure and / or sterile. If the drug is supplied as a liquid solution, this solution is typically an aqueous solution. If the drug is supplied in dry form, reconstitution is generally by adding a suitable solvent. A solvent, e.g., sterile water or a buffer, may optionally be provided in the kit.

[0305] A kit may include one or more containers for one or more compositions containing a drug. In some embodiments, the kit includes separate containers, dividers, or compartments for the composition and informational material. For example, the composition may be contained in a bottle, vial, or syringe, and the informational material may be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial, or syringe with the informational material attached in the form of a label. In some embodiments, the kit includes a plurality of individual containers (e.g., packs), each containing one or more unit dosage forms (e.g., dosage forms described herein) of the drug. The containers may contain combined unit doses, for example, units containing both an α4-binding antibody and a second drug, for example, in a desired ratio. For example, the kit may include a plurality of syringes, ampoules, foil packets, blister packs, or medical devices, each containing, for example, a single combined unit dose. The kit container may be airtight, waterproof (e.g., resistant to changes in moisture or evaporation), and / or light-blocking.

[0306] The kit may optionally include a device suitable for administering the composition, such as a syringe or other suitable delivery device. This device may be supplied pre-loaded with one or both of the drugs, or it may be empty but suitable for loading.

[0307] XI. Oncology The α4-binding antibodies and methods described herein may be used to treat cancers, including solid tumors and hematological malignancies. Exemplary solid tumors include sarcomas and carcinomas of the lung, breast, pancreas, colon, prostate, bladder, and brain. Hematological malignancies include cancers such as multiple myeloma, leukemia, and lymphoma.

[0308] A method is provided for treating patients with hematological malignancies with a composition comprising an α4-binding antibody, such as the anti-VLA-4 antibody described herein. Hematological malignancies are cancers of the body's hematopoietic and immune systems. This type of cancer affects the blood, bone marrow, and / or lymph nodes. Hematological malignancies include leukemias such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia, acute erythroleukemia, and hairy cell leukemia (HCL), lymphomas such as Hodgkin's disease and non-Hodgkin's lymphoma, as well as multiple myeloma, Waldenström macroglobulinemia, myelodysplastic syndromes (MDS) (which can lead to AML), myeloproliferative disorders such as polycythemia vera (PV, PCV, or primary polycythemia vera (PRV)), essential thrombocytosis (ET), myelofibrosis, heavy chain disease, and amyloidosis resulting from light chain disease.

[0309] Patients with hematological malignancies can be identified by analyzing blood cell counts and blood smears, for example, using a light microscope useful for identifying malignant cells. Malignant cells can also be identified using biopsies of bone marrow, and lymph node biopsies may be useful for identifying lymphadenopathy.

[0310] Alpha-4 binding antibodies (e.g., humanized anti-VLA-4 antibodies such as HuHP1 / 2, H1L0, H1L1, H1L2, or H1L3) are useful in treating leukemias such as AML. Leukemia is a cancer that originates in the bone marrow, and the malignant cells are white blood cells. AML (also known as acute myeloid leukemia, acute myeloblastic leukemia, acute granulocytic leukemia, and acute nonlymphoblastic leukemia) is a malignant tumor that arises from either granulocytes or monocytes. AML is characterized by the uncontrolled and excessive proliferation and accumulation of cells called leukemic blasts, which cannot function as normal blood cells, as well as the blockage of the production of normal bone marrow cells, resulting in a deficiency of red blood cells (anemia), platelets (thrombocytopenia), and normal white blood cells (especially neutrophils, i.e., neutropenia) in the blood.

[0311] All subtypes of AML are suitable for treatment with VLA-4 conjugated antibodies. AML subtypes are classified based on the developmental stage at which myeloblasts have reached at the time of diagnosis. This classification and subset allows physicians to determine what treatment will best work for that cell type and how quickly the disease may develop. These subsets are M0, myeloblastic by special analysis; M1, myeloblastic, unmatured; M2, myeloblastic, matured; M3, promyelocytic; M4, myelomonocytic; M5, monocytic; M6, erythroleukemia; and M7, megakaryocytic. VLA-4 antibodies may be administered in conjunction with secondary agents that are particularly suitable for the subtype of AML. For example, acute promyelocytic leukemia (APL) and acute monocytic leukemia are subtypes of AML that require different treatment than other subtypes of AML. A second agent for the treatment of APL may include an antimetabolite such as total trans retinoic acid (ATRA) or cytarabine. A second agent for the treatment of acute monocytic leukemia may include a deoxyadenosine analog such as 2-chloro-2'-deoxyadenosine (2-CDA).

[0312] Risk factors for AML include the presence of certain genetic disorders, such as Down syndrome, Fanconi anemia, Schwakman-Diamond syndrome, and others. Patients with AML and a genetic disorder may be administered a VLA-4 conjugate antibody and a second drug to treat the symptoms of the genetic disorder. For example, patients with AML and Fanconi anemia may be administered a VLA-4 conjugate antibody and an antibiotic.

[0313] Other risk factors for AML include chemotherapy or radiation therapy for different cancer treatments, tobacco smoke, and exposure to large amounts of benzene.

[0314] Other cancers suitable for treatment with α4-binding antibodies include solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, ovarian cancer, squamous cell carcinoma, basal Examples include cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic lung carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminomas, embryonic carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, Schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0315] XII. Other Disorders The formulations and methods described herein are for the treatment of other inflammatory disorders, immune disorders, or autoimmune disorders, e.g., inflammation of the central nervous system (e.g., meningitis, neuromyelitis optica, neurosarcoidosis, CNS vasculitis, encephalitis, and transverse myelitis, in addition to multiple sclerosis); tissue or organ graft rejection or graft-versus-host disease; acute CNS injury, e.g., stroke or spinal cord injury (SCI); chronic kidney disease; allergies, e.g., allergic asthma, moderate to severe allergic rhinitis, ocular allergies; type 1 diabetes mellitus; inflammatory bowel disorders, e.g., Crohn's disease, ulcerative colitis (e.g., for treatment or maintenance of remission); epilepsy; eosinophilic gastroenteritis; myasthenia gravis; fibromyalgia; rheumatology / immunology-related disorders, e.g., arthritis disorders, e.g. It can also be used to treat rheumatoid arthritis, psoriatic arthritis; dermatological disorders, e.g., inflammatory / immunodermatological disorders, e.g., psoriasis, vitiligo, dermatitis (e.g., atopic dermatitis), lichen planus, moderate to severe chronic urticaria; systemic lupus erythematosus (SLE; e.g., lupus nephritis); scleroderma (e.g., progressive systemic sclerosis (PSS), e.g., pulmonary PSS); acute or chronic primary eosinophilic pneumonia; Sjögren's syndrome; acute coronary syndrome (ACS); acute myocardial infarction; atherosclerosis; and fibrous disorders, e.g., pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), pulmonary fibrosis (e.g., XRT-induced), myelofibrosis, cirrhosis, mesangial proliferative glomerulonephritis, crescentic glomerulonephritis, diabetic nephropathy, and interstitial renal fibrosis. In preferred embodiments, the formulation may be used to treat epilepsy.

[0316] The formulations and methods described herein may also be used to treat neurological disorders such as cerebral ischemia, including the prevention of transient ischemic attacks and / or arterial stenosis in patients. Other exemplary neurological disorders include chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barré syndrome (GBS), macular degeneration (e.g., exudative macular degeneration), ocular diseases such as anterior ischemic optic neuropathy, neuropathic pain (e.g., symptomatic neuropathic pain), Alzheimer's disease, amyotrophic lateral sclerosis (ALS) (e.g., disease-modifying ALS), and Parkinson's disease.

[0317] The formulations and methods described herein may also be used to treat patients who have undergone transplants, such as kidney, heart, or bone marrow transplants.

[0318] XIII. Multiple sclerosis The formulations containing the alpha-4 conjugate antibody described herein are useful for the treatment of inflammatory diseases such as multiple sclerosis (MS). Multiple sclerosis is a central nervous system disorder characterized by inflammation and loss of myelin sheath.

[0319] MS patients can be identified by criteria that establish a clinically clear diagnosis of MS, as defined by the Research Meeting on the Diagnosis of MS (Poser et al., Ann. Neurol. 13:227, 1983). For example, an individual with clinically clear MS has had two seizures, as well as either clinical evidence of two lesions, or clinical evidence of one lesion and evidence of another distinct lesion in a clinically relevant area. Clear MS can also be diagnosed by evidence of two seizures and oligoclonal bands of IgG in the cerebrospinal fluid, or a combination of seizures, clinical evidence of two lesions, and oligoclonal bands of IgG in the cerebrospinal fluid. MS can also be diagnosed using the McDonald criteria (McDonald et al., 2001, “Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the Diagnosis of Multiple Sclerosis,” Ann. Neurol. 50:121-127). The McDonald criteria include the use of MRI evidence of CNS dysfunction over a period of time, which is used to diagnose MS in the absence of multiple clinical seizures. The effectiveness of treatment for multiple sclerosis can be evaluated in several different ways. The following parameters may be used to measure the effectiveness of treatment. Two exemplary criteria include the EDSS (Global Disability Scale) and the appearance of exacerbations on MRI (Magnetic Resonance Imaging). The EDSS is a method for grading clinical impairment due to MS (Kurtzke, Neurology 33:1444, 1983). Eight functional systems are evaluated for the type and severity of neurological impairment. Briefly, before treatment, the patient is evaluated for impairments in the pyramidal system, cerebellar system, brainstem system, sensory system, intestinal and bladder system, visual system, cerebral system, and other functional systems. Follow-up is performed at predetermined intervals. The scale ranges from 0 (normal) to 10 (death due to MS). A reduction of one entire step indicates effective treatment (Kurtzke, Ann. Neurol. 36:573-79, 1994). Patients may be diagnosed using other criteria used by those skilled in the art.

[0320] An exacerbation is defined as the appearance of new symptoms attributable to MS, accompanied by reasonable new neurological abnormalities (IFNB MS Study Group, see above). Furthermore, an exacerbation must last for at least 24 hours and be preceded by at least 30 days of stagnation or improvement. Briefly, the patient undergoes a standard neurological examination by a clinician. Exacerbations are classified as mild, moderate, or severe according to changes in the Neurological Rating Scale (NRS) (Sipe et al., Neurology 34:1368, 1984). The annual exacerbation rate and exacerbation-free patient rate are determined.

[0321] A therapy may be considered effective if there is a statistically significant difference between the treatment group and the placebo group in any of these measures regarding the rate or proportion of patients with progression-free or relapse-free status. Furthermore, the time to the first exacerbation, as well as the duration and severity of the exacerbation, may also be measured. A measure of the therapy's effectiveness in this regard is a statistically significant difference in the time to or duration and severity of the first exacerbation between the treatment group and the control group. A progression-free or relapse-free period exceeding one year, 18 months, or 20 months is particularly noteworthy. Effectiveness may also be assessed using any method known in the art for evaluating symptoms of MS, including improved mobility, for example, using a timed walk test used alone or in combination with other criteria.

[0322] The efficacy of the first agent, and optionally the second agent, may also be evaluated based on one or more of the following criteria: the frequency of MBP-responsive T cell occurrence determined by limiting dilution, the proliferation response of MBP-responsive T cell lines and clones, and the cytokine profiles of T cell lines and clones to MBP established from patients. Efficacy is indicated by a reduction in the frequency of reactive cells, a reduction in thymidine incorporation by the modified peptide compared to the native type, and a reduction in TNF and IFN-α.

[0323] Clinical measures include recurrence rates at 1- and 2-year intervals, and changes in EDSS, including the time to progression of 1.0 unit from baseline to a 6-month-lasting EDSS. In the Kaplan-Meier curve, a delay in the sustained progression of disability indicates efficacy. Other criteria include changes in area and volume on T2 images on MRI, and the number and volume of lesions as determined by gadolinium-enhanced images.

[0324] Using MRI, active lesions can be measured using gadolinium-DTPA enhanced imaging (McDonald et al. Ann. Neurol. 36:14, 1994), or the location and extent of lesions can be measured using T2 weighting techniques. Briefly, a baseline MRI is obtained. The same imaging plane and patient position are used in each subsequent trial. Positioning and imaging sequences may be selected to maximize lesion detection and facilitate lesion tracking. The same positioning and imaging sequences may be used in subsequent trials. The presence, location, and extent of MS lesions may be determined by the radiologist. Lesion area may be estimated per slice and summed up to obtain the total lesion area. Three analyses may be performed: evidence of new lesions, incidence of active lesions, and rate of change in lesion area (Paty et al., Neurology 43:665, 1993). Therapeutic improvement may be established by statistically significant improvement compared to the individual patient's baseline or in comparison to the treatment group versus the placebo group.

[0325] Illustrative symptoms associated with multiple sclerosis that can be treated by the methods described herein include optic neuritis, diplopia, nystagmus, ataxia, internuclear ophthalmoplegia, photopsia due to motion and sound, concentric pupillary disorder, paresis, parenoplegia, parparaplegia, parhemiplegia, quadriplegia, paraplegia, hemiplegia, quadriplegia, spasticity, dysarthria, muscular atrophy, spasms, muscle cramps, hypotonia, clonus, myoclonus, myokymia, restless legs syndrome, foot drop, reflex dysfunction, paresthesia, anesthesia, Symptoms include neuralgia, neuropathic pain and neurogenic pain, L'Hermitte's sign, proprioceptive dysfunction, trigeminal neuralgia, ataxia, intention tremor, dysmetria, vestibular ataxia, dizziness, speech ataxia, dystonia, antagonistic repetition failure, frequent urination, bladder spasm, flaccid bladder, detrusor-sphincter ataxia, erectile dysfunction, anorgasmia, frigidity, constipation, defecation urgency, fecal incontinence, depression, cognitive impairment, dementia, mood swings, emotional instability, euphoria, bipolar disorder, anxiety, aphasia, speech disorders, fatigue, Uthoff's symptoms, gastroesophageal reflux, and sleep disorders.

[0326] Each case of MS exhibits one of several presentation patterns and subsequent courses. Most commonly, MS first presents as a series of attacks, followed by miraculous periods of complete or partial remission as symptoms subside, but eventually relapses after a period of stability. This is called relapsing-remitting (RR) MS. Clinically isolated syndrome (CIS) is another type of relapsing MS. CIS refers to an initial episode of neurological symptoms lasting at least 24 hours and caused by inflammation or demyelination (loss of myelin, the membrane covering nerve cells) in the central nervous system (CNS). Primary progressive (PP) MS is characterized by gradual clinical decline without clear remission, although there may be temporary periods of stability or slight relief of symptoms. Active primary progressive MS occurs when a patient experiences occasional relapses or when there is evidence of new lesions on MRI. "Inactive" or "progressive" means there is evidence of worsening symptoms over time, regardless of relapses or whether new lesions are shown on MRI. Secondary progressive (SP) MS begins with a relapsing-remitting course, followed by a primary progressive course. Active secondary progressive MS is considered a type of relapsing MS. Rarely, patients may follow a progressive-relapsing (PR) course, in which the disease progresses with intermittent acute attacks. PP, SP, and PR are sometimes collectively referred to as chronic progressive MS.

[0327] Therefore, relapsing-type multiple sclerosis (or simply relapsing multiple sclerosis) includes, but is not limited to, clinically isolated syndrome (CIS), relapsing-remitting multiple sclerosis (RRMS), and active secondary progressive multiple sclerosis (active SPMS).

[0328] A small number of patients experience malignant MS, defined as a rapid and severe debilitation that leads to significant disability or even death shortly after the onset of the disease. This debilitation may be suppressed or slowed by the administration of the therapeutic agents described herein.

[0329] The administration of anti-α4 antibodies featured herein may be effective in alleviating one or more symptoms of MS, such as one or more of the symptoms described above. For example, the administration of anti-α4 antibodies described herein can be used to treat primary progressive or secondary progressive multiple sclerosis (PPMS or SPMS, respectively), and treatment with anti-α4 antibodies may be effective in preventing relapses.

[0330] In addition to, or prior to, human trials, the efficacy of using the two drugs may be evaluated using animal models. Exemplary animal models of multiple sclerosis are experimental autoimmune encephalitis (EAE) mouse models, e.g., those described by Tuohy et al. (J. Immunol. (1988) 141:1126-1130), Sobel et al. (J. Immunol. (1984) 132:2393-2401), and Traugott (Cell Immunol. (1989) 119:114-129). Mice may be administered the first and second drugs described herein before induction of EAE. The mice are then evaluated against characteristic criteria to determine the efficacy of using the two drugs in the model.

[0331] XIV. Epilepsy The formulations containing the alpha-4 conjugate antibody described herein are useful for the treatment of epilepsy.

[0332] Epileptic seizures are paroxysmal episodes resulting from abnormal neuronal discharges that can be detected by electroencephalography or clinical means, with specific areas of the brain influencing their clinical manifestation. Epileptic seizures fundamentally arise from an imbalance in the excitability of neurons, which may be related to the neuronal membrane or processes of excitation and inhibition. See also Holmes et al., Epilepsia 45(12):1568-1579, 2004. Conceptually, epilepsy is defined as a brain disorder characterized by a persistent predisposition to epileptic seizures, as well as the neurobiological, cognitive, psychological, and social consequences of this condition (Fisher RS, Curr Opin Neurol. 28(2):130-5, 2015). Recent studies on the prevalence and incidence of epilepsy have reported a lifetime prevalence of 7.6 per 1,000 people and an annual cumulative incidence of 67.77 per 100,000 people (Fiest et al., Neurology 88(3):296-303, 2017).

[0333] Prior to this invention, the common treatment for epilepsy patients was antiepileptic drugs (AEDs), and it is estimated that approximately 70% of patients achieved good seizure control with AED therapy. Patients who fail two (or more) valid trials of an AED that has demonstrated tolerance and is appropriately selected and used are defined as having drug-resistant epilepsy (see the errata for Kwan et al., Epilepsia. 51(6):1069-77, 2010 and Epilepsia. 51(9):1922, 2010). For these patients, other therapies are considered, including epilepsy surgery, neurostimulatory devices, special diets, behavioral therapy, and other experimental treatments. AEDs typically function by modulating voltage-gated or ligand-opening ion channels, or by effects on inhibitory or excitatory neurotransmitter systems. Currently, more than 20 types of AEDs are commercially available. Despite the release of many newer AEDs in recent years, the effectiveness of new and older drugs in managing epilepsy is generally equal, and it remains rare for patients with a history of drug-resistant epilepsy to be freed from seizures by new therapies.

[0334] The need to develop therapies that improve seizure control or eliminate seizures in drug-resistant epilepsy remains high and unmet. Targeting the potential roles of the immune system and inflammation in the pathogenesis of epilepsy represents an area that has not been adequately investigated in AED development and clinical trials. Evidence from both experimental models of epilepsy and human patients with epilepsy suggests a role of inflammation. The formulations disclosed herein can be used to treat epilepsy. For example, a formulation comprising a recombinant anti-alpha-4 antibody or its alpha-4 binding fragment, comprising (a) a heavy chain containing the sequence of SEQ ID NO: 80 and (b) a light chain containing the sequence of SEQ ID NO: 81, can be administered to patients with epilepsy. It is hypothesized that blocking α4 integrin reduces leukocyte-vascular interaction and stabilizes the integrity of the blood-brain barrier. Furthermore, it is hypothesized that seizure-induced, epilepsy-reducing leukocyte-vascular interaction reduces the frequency and severity of seizures.

[0335] XV. Antibody generation Recombinant antibodies that bind to alpha-4 can be generated in vivo or in vitro by methods such as phage display. These methods can be used to supply anti-alpha-4 CDRs used in the CDR graft antibodies described herein. Furthermore, such CDRs can be selected in relation to the germline frameworks disclosed herein by using methods such as phage display, for example, by using a library whose framework is a germline framework.

[0336] EP 239 400 (Winter et al.) describes the modification of antibodies by substituting the complementarity-determining region (CDR) of one species with that of another species (within a given variable region). Because CDR-substituted antibodies contain significantly fewer non-human components compared to true chimeric antibodies, they may be less likely to induce an immune response in humans (Riechmann et al., 1988, Nature 332, 323-327; Verhoeyen et al., 1988, Science 239, 1534-1536). Typically, recombinant nucleic acid technology is used to substitute the CDR of a mouse antibody with the corresponding region of a human antibody to obtain a sequence encoding the desired substituted antibody. A human constant region gene segment of the desired isotype (usually CH for gamma I, CL for kappa) may be added, and the heavy and light chain genes can be co-expressed in mammalian cells to produce a soluble antibody. Large non-immunophage display libraries can also be used to isolate high-affinity antibodies that could be developed as human therapeutics using standard phage techniques (see, for example, Hoogenboom et al. (1998) Immunotechnology 4:1-20, and Hoogenboom et al. (2000) Immunol Today 2:371-8, US2003-0232333).

[0337] The anti-α4 antibodies or antibody fragments described herein can recognize and specifically bind to epitopes of α4 subunits involved in binding to congeneral ligands, such as VCAM-1 or fibronectin. In some embodiments, the anti-α4 antibody or antibody fragment recognizes and specifically binds to the B epitope of α4 integrin. The binding of the antibodies described herein can inhibit the binding of α4 integrin to one or more congeneral ligands (e.g., VCAM-1 and fibronectin).

[0338] In some embodiments, the antibodies featured herein may interact with cells, such as the VLA-4 of lymphocytes, but without causing cell aggregation.

[0339] An exemplary α4-binding antibody has one or more CDRs, for example, all three heavy chain (HC) CDRs and / or all three light chain (LC) CDRs of a particular antibody disclosed herein, or CDRs that are at least 80, 85, 90, 92, 94, 95, 96, 97, 98, 99% identical to such antibody. In one embodiment, the H1 and H2 hypervariable loops have the same classical structure as those of the antibody described herein. In one embodiment, the L1 and L2 hypervariable loops have the same classical structure as those of the antibody described herein.

[0340] In one embodiment, the amino acid sequence of the HC and / or LC variable domain sequence is at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to the amino acid sequence of the HC and / or LC variable domain of the antibody described herein. The amino acid sequence of the HC and / or LC variable domain sequence may differ from the corresponding sequence of the antibody described herein by at least one amino acid, and by 10, 8, 6, 5, 4, 3, or 2 or fewer amino acids. For example, the difference may be mainly or entirely within the framework region.

[0341] The amino acid sequences of the HC and LC variable domain sequences may be encoded by nucleic acid sequences that hybridize to the nucleic acid sequences described herein under high stringency conditions, or by sequences that encode the variable domains or amino acid sequences described herein. In one embodiment, the amino acid sequences of one or more framework regions (e.g., FR1, FR2, FR3, and / or FR4) of the HC and / or LC variable domains are at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to the corresponding framework regions of the HC and LC variable domains of the antibody described herein. In one embodiment, one or more heavy or light chain framework regions (e.g., HCFR1, FR2, and FR3) are at least 70, 80, 85, 90, 95, 96, 97, 98, or 100% identical to the sequences of the corresponding framework regions of a human germline antibody.

[0342] The calculation of “homology” or “sequence identity” (these terms are used synonymously herein) between two sequences is performed as follows: The sequences are aligned for the purpose of best comparison (for example, gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for best alignment, and non-homologous sequences may be ignored for comparison purposes). The best alignment is determined as the best score obtained using the GAP program of the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Then, the amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then these molecules are identical at that position (as used herein, amino acid or nucleic acid “identity” is equal to amino acid or nucleic acid “homology”). The percentage of identity between the two sequences is a function of the number of identical positions common to the sequences.

[0343] As used herein, the term “hybridize under high stringency conditions” refers to the conditions for hybridization and washing. Guidelines for carrying out the hybridization reaction can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1–6.3.6, which are incorporated herein by reference. This reference describes both aqueous and non-aqueous methods, either of which may be used. High stringency hybridization conditions include hybridization in 6×SSC at approximately 45°C followed by one or more washes in 0.2×SSC and 0.1% SDS at 65°C, or substantially similar conditions.

[0344] XVI. Antibody Production Antibodies can be produced in prokaryotic and eukaryotic cells. In one embodiment, the antibody (e.g., scFv) is expressed in yeast cells such as Pichia (e.g., Powers et al. (2001) J. Immunol. Methods 251:123-35), Hanseula, or Saccharomyces.

[0345] In one embodiment, antibodies, particularly full-length antibodies, such as IgG, are produced in mammalian cells. Exemplary mammalian host cells for recombinant expression include Chinese hamster ovary (CHO cells) (including, for example, dhfr-CHO cells described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR-selectable marker, such as those described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621), lymphocyte cell lines, such as NS0 myeloma cells and SP2 cells, COS cells, K562, and cells of transgenic animals, such as transgenic mammals. For example, the cells are mammary epithelial cells.

[0346] In addition to the nucleic acid sequence encoding the immunoglobulin domain, recombinant expression vectors can carry additional nucleic acid sequences, such as sequences that regulate vector replication in host cells (e.g., origin of replication) and selectable marker genes. Selectable marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Patents 4,399,216, 4,634,665, and 5,179,017). Exemplary selectable marker genes include the dihydrofolate reductase (DHFR) gene (for methotrexate selection / amplification in dhfr-host cells) and the neo gene (for G418 selection).

[0347] In an exemplary recombinant expression system for an antibody (e.g., a full-length antibody or its antigen-binding portion), a recombinant expression vector encoding both the antibody heavy chain and antibody light chain is introduced into dhfr-CHO cells by calcium phosphate transfection. In the recombinant expression vector, the genes for the antibody heavy and light chains are operably linked to enhancer / promoter regulatory elements (e.g., CMV enhancer / AdMLP promoter regulatory elements or SV40 enhancer / AdMLP promoter regulatory elements, derived from SV40, CMV, adenovirus, etc.) to promote high levels of gene transcription. The recombinant expression vector also carries the DHFR gene, enabling selection of vector-transfected CHO cells using methotrexate selection / amplification. Culturing the selected transformant host cells enables expression of the antibody heavy and light chains, and intact antibodies are recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect host cells, select transformants, culture host cells, and recover antibodies from the culture medium. For example, some antibodies can be isolated by affinity chromatography using protein A or protein G. For instance, purified α4-binding antibodies can be concentrated to approximately 100 mg / mL to approximately 200 mg / mL using protein concentration techniques known in the art.

[0348] Antibodies may also include modifications, such as altering Fc function to reduce or eliminate interactions with, for example, the Fc receptor or C1q, or both. For example, the constant region of human IgG4 may have a Ser-to-Pro mutation at residue 228, which fixes the hinge region. The amino acid sequence of IgG4 Fc (hinge + CH2 + CH3 domain) is shown in Figure 5.

[0349] In another example, the human IgG1 constant region may be mutated in one or more residues, for example, one or more of residues 234 and 237, as numbered in U.S. Patent No. 5,648,260. Other exemplary modifications include those described in U.S. Patent No. 5,648,260.

[0350] For some antibodies containing an Fc domain, the antibody production system may be designed to synthesize antibodies in which the Fc region is glycosylated. In another example, the Fc domain of the IgG molecule is glycosylated at asparagine 297 within the CH2 domain (see Figure 5). This asparagine is a modification site with a branched oligosaccharide. This glycosylation is involved in effector functions mediated by the Fcγ receptor and complement C1q (Burton and Woof (1992) Adv. Immunol. 51:1-84, Jefferis et al. (1998) Immunol. Rev. 163:59-76). The Fc domain can be produced in mammalian expression systems that appropriately glycosylate the residue corresponding to asparagine 297. The Fc domain may also include other eukaryotic post-translational modifications.

[0351] Other suitable Fc domain modifications include those described in WO2004 / 029207. For example, the Fc domain may be XMAB®Fc(Xencor,Monrovia,CA). The Fc domain or fragment may have substitutions in the Fcγ receptor (FcγR) binding region, as in the domains and fragments described in WO05 / 063815. In some embodiments, the Fc domain or fragment has substitutions in the neonatal Fc receptor (FcRn) binding region, as in the domains and fragments described in WO05047327. In other embodiments, the Fc domain is single-stranded, a fragment thereof, or a modified form thereof, for example, as described in WO2008143954. Other suitable Fc modifications are known and described in the art.

[0352] Antibodies may be produced by transgenic animals. For example, U.S. Patent No. 5,849,992 describes a method for expressing antibodies in the mammary glands of a transgenic mammal. A transgene is constructed comprising a milk-specific promoter, the antibody of interest, e.g., the antibody described herein, and a nucleic acid sequence encoding a signal sequence for secretion. The milk produced by such a transgenic female mammal contains the secreted antibody of interest, e.g., the antibody described herein. The antibody may be purified from the milk or used directly in some applications.

[0353] The antibody may be modified, for example, by a moiety that improves its stabilization and / or retention in circulation, for example, in blood, serum, lymph, bronchoalveolar lavage fluid, or other tissues, by, for example, at least 1.5, 2, 5, 10, or 50 times.

[0354] For example, the VLA-4 conjugated antibody may associate with a polymer, such as a substantially non-antigenic polymer, such as a polyalkylene oxide or polyethylene oxide. Suitable polymers vary substantially by weight. Polymers having a number-average molecular weight in the range of about 200 to about 35,000 daltons (or about 1,000 to about 15,000, or 2,000 to about 12,500) can be used.

[0355] For example, the VLA-4 conjugated antibody may be conjugated to a water-soluble polymer, such as a hydrophilic polyvinyl polymer, such as polyvinyl alcohol or polyvinylpyrrolidone. A non-limiting list of such polymers includes polyalkylene oxide homopolymers, such as polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylene-conjugated polyols, their copolymers and their block copolymers, provided that the water solubility of the block copolymer is maintained. Further useful polymers include polyoxyalkylenes, such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (pluronic); polymethacrylic acid; carbomers; lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextran sulfate, dextran, dextrin, glycogen, or homopolysaccharides and heteropolysaccharides such as acid mucopolysaccharides, such as the polysaccharide subunits of hyaluronic acid. Examples include branched or unbranched polysaccharides containing the sugar monomers D-mannose, D-galactose and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid (e.g., polymannuronic acid or alginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminic acid; polymers of sugar alcohols such as polysorbitol and polymannitol; and heparin or heparon.

[0356] XVII. Exemplary Second Drug In some cases, the formulations described herein, for example, formulations containing an alpha-4 conjugated antibody, are administered in combination with a second agent or a formulation containing a second agent.

[0357] In one embodiment, the α4-conjugated antibody and the second drug are prepared as a combination, and this combination is administered to the subject. Furthermore, for example, one dose of the α4-conjugated antibody preparation and then one dose of the preparation containing the second drug can be administered separately at least 24 hours before or after the administration of the combination. In another embodiment, the antibody and the second drug are prepared as separate preparations, and the administration step includes administering the antibody and the second drug in succession. The succession administration may be performed on the same day (for example, within one hour of each other, or at least 3, 6, or 12 hours apart) or on different days.

[0358] Generally, the antibody and the second drug are administered in multiple doses spaced apart by time. Generally, the antibody and the second drug are administered according to a regimen. One or both regimens may have a regular periodicity. The antibody regimen may have a different periodicity from the second drug regimen; for example, one may be administered more frequently than the other. In one embodiment, one of the antibody and the second drug is administered once a week, and the other once a month. In another embodiment, one of the antibody and the second drug is administered consecutively, for example, over a period ranging from more than 30 minutes to less than 1, 2, 4, or 12 hours, and the other is administered as a bolus. The antibody and the second drug may be administered by any suitable method, for example, subcutaneously, intramuscularly, or intravenously.

[0359] In some embodiments, the antibody and the second drug are administered in the same doses as those prescribed for monotherapy. In other embodiments, the antibody is administered in a dose less than or equal to the amount required for efficacy when administered alone. Similarly, the second drug may be administered in a dose less than or equal to the amount required for efficacy when administered alone.

[0360] Non-limiting examples of second drugs for treating multiple sclerosis in combination with α4-binding antibodies include the following: Interferons, e.g., human interferon beta-1a (e.g., AVONEX® or REBIF®) and interferon beta-1b (BETASERON®; human interferon beta substituted at position 17; Berlex / Chiron), • Glaciramer acetate (also known as copolymer 1, Cop-1; COPAXONE®; Teva Pharmaceutical Industries, Inc.) • RITUXAN® (rituximab), or another anti-CD20 antibody containing an antibody that binds to an epitope that competes with or overlaps with rituximab. • Mitoxantrone (NOVANTRONE®, Lederle), • Myelin regeneration agents such as opicinumab, • Chemotherapy drugs, such as clabribine (LEUSTATIN®), azathioprine (IMURAN®), cyclophosphamide (CYTOXAN®), cyclosporine-A, methotrexate, 4-aminopyridine, and tizanidine. • Corticosteroids, for example, methylprednisolone (MEDRONE®, Pfizer), prednisone, • Immunoglobulins, for example, RITUXAN® (rituximab); CTLA4Ig; alemtuzumab (MABCAMPATH®) or daclizumab (an antibody that binds to CD25), • Statins, and TNF antagonist.

[0361] Glutiramer acetate is a protein formed from random chains of the amino acids glutamic acid, lysine, alanine, and tyrosine (hence the name GLATiramer). Glutiramer acetate can be synthesized in solution using N-carboxyamino acid anhydrides from these amino acids in a ratio of approximately 5 parts alanine, 3 parts lysine, 1.5 parts glutamic acid, and 1 part tyrosine.

[0362] Further second agents include antibodies or antagonists of other human cytokines or growth factors, such as TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-15, IL-16, IL-18, EMAP-11, GM-CSF, FGF, and PDGF. Even more exemplary second agents include antibodies against cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, and CD90, or their ligands. For example, daclizumab is an anti-CD25 antibody that can improve multiple sclerosis.

[0363] Further exemplary antibodies include antibodies that contribute to the activity of the agents described herein, such as antibodies that recruit interferon receptors, for example, interferon-beta receptors. Typically, in embodiments in which the second agent comprises an antibody, it specifically binds to target proteins other than VLA-4 or α4 integrin, or to epitopes on VLA-4 other than those recognized and specifically bound by at least the first agent.

[0364] Further additional exemplary second agents include FK506, rapamycin, mycophenolate mofetil, leflunomide, nonsteroidal anti-inflammatory drugs (NSAIDs), such as phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, complement inhibitors, adrenergic agonists, agents that interfere with signaling by inflammatory cytokines as described herein, IL-1β-converting enzyme inhibitors (e.g., Vx740), anti-P7, PSGL, TACE inhibitors, T cell signaling inhibitors, such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurine, angiotensin-converting enzyme inhibitors, soluble cytokine receptors and their derivatives as described herein, and anti-inflammatory cytokines (e.g., IL-4, IL-10, IL-13, and TGF).

[0365] In some embodiments, a second agent may be used to treat one or more symptoms or side effects of MS. Such agents include, for example, amantadine, baclofen, papaverine, meclizine, hydroxyzine, sulfamethoxazole, ciprofloxacin, doxart, pemoline, dantrolene, desmopressin, dexamethasone, tolterodine, phenytoin, oxybutynin, bisacodyl, venlafaxine, amitriptyline, methenamine, clonazepam, isoniazid, vardenafil, nitrofurantoin, cytoplasmic viscous Many of the second type of drug, which are small molecules, have molecular weights ranging from 150 to 5000 daltons.

[0366] Examples of TNF antagonists include chimeric antibodies, humanized antibodies, human antibodies, or in vitro-produced antibodies (or their antigen-binding fragments) against TNF (e.g., human TNFα), e.g., D2E7 (human TNFα antibody, U.S. Patent No. 6,258,562; BASF), CDP-571 / CDP-870 / BAY-10-3356 (humanized anti-TNFα antibody; Celltech / Pharmacia), cA2 (chimeric anti-TNFα antibody; REMICADE®, Centocor); anti-TNF antibody fragments (e.g., CPD870); TNF receptors, e.g., soluble fragments of p55 or p75 human TNF receptors, or their derivatives, e.g., 75kD TNFR-IgG (75kD TNF receptor-IgG fusion protein, ENBREL®; Immunex; see, e.g., Arthritis & Rheumatism (1994) Vol. 37, S295, J. Invest. Med. (1996) Vol. 44, 235A), p55kdTNFR-IgG (55kD TNF receptor-IgG fusion protein (LENERCEPT®)); enzyme antagonists, e.g., TNFα-converting enzyme (TACE) inhibitors (e.g., alpha-sulfonylhydroxamic acid derivatives, WO01 / 55112, and N-hydroxyformamide TACE inhibitors GW3333, -005, or -022); and TNF-bp / s-TNFR (soluble TNF-binding protein; see, e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S284, Amer. J. Physiol. - Heart and See Circulatory Physiology (1995) Vol.268, pp.37-42.

[0367] In addition to the second drug, other drugs may also be delivered to the target. However, in some embodiments, no proteins or biologics other than the α4-conjugated antibody and the second drug are administered to the target as part of the pharmaceutical composition. The α4-conjugated antibody and the second drug may be the only drugs delivered by injection. In embodiments where the second drug is a recombinant protein, the α4-conjugated antibody and the second drug may be the only recombinant drugs administered to the target, or the only recombinant drugs that modulate at least the immune or inflammatory response. In yet another embodiment, the α4-conjugated antibody alone is the only recombinant drug, or the only biologic drug administered to the target.

[0368] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which the present invention pertains. Methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present invention, but preferred methods and materials are described below. All published documents, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification shall prevail, including definitions. Furthermore, the materials, methods, and examples are illustrative and not intended to be limiting. [Examples]

[0369] Unless otherwise specified, in these embodiments, all amino acid numbering uses EU numbering.

[0370] Example 1. The variant anti-VLA-4 antibody is potent compared to the humanized HP1 / 2 antibody. Anti-VLA-4 antibodies were constructed using the germline framework IGKV4-1 (or designs L1 and L2) or germline manipulation AAH7033.1 (design L3) for the VL chain, and the germline framework IGHV1-f for the VH chain. These antibodies had fewer reverse mutations than the humanized HP1 / 2 antibodies described in U.S. Patent No. 6,602,503.

[0371] Variations of heavy chains Three variations of the heavy chain sequence are shown in Figure 1 as Design H0, Design H1, and Design H2. In each design, the mouse HP1 / 2 CDR is grafted onto the IGHV1-f framework. Design H0 does not contain reverse mutations in the framework region, while designs H1 and H2 have varying degrees of reverse mutations in the framework region sequence to optimize the affinity of the humanized antibody.

[0372] Variations of light chains The sequences of four light chain variations are shown in Figure 2 as designs L0, L1, L2, and L3 (also referred to as L0, L1, L2, and L3). In each design, the mouse HP1 / 2 CDR is grafted onto the germline framework. The IGKV4-1 germline framework was used for designs L0, L1, and L2, and the AAH70335 germline manipulation framework was used for design L3. Design L0 does not contain reverse mutations in the framework region, while designs L1, L2, and L3 have varying degrees of reverse mutations in the framework region to optimize the affinity of the humanized antibody.

[0373] The results of the competitive ELISA assay are shown in Table 2 and Figure 3. In this experiment, α4β1 was pre-incubated with the test mAb, and then mouse HP1 / 2 was used as the competitive reagent. The results of this experiment showed that antibodies with light chain L2 or L3 were potenter than the antibodies described in U.S. Patent No. 6,602,503. The results are shown in Table 2 and Figure 3 below. In this assay, the antibody heavy chain (H1) has the "designed H1" sequence shown in Figure 1, while L1 refers to the designed L1 in Figure 2.

[0374] TIFF2026113464000018.tif39170

[0375] In Table 2, the chimeric mAb is a chimeric HP1 / 2 antibody in which the mouse variable heavy and light chains are genetically fused to the human IgG1 constant region. This antibody is essentially identical in binding affinity to the original mouse HP1 / 2 antibody (Sanchez-Madrid et al., Eur.J.Immunol.16:1343-1349, 1996). The experimental results demonstrate that the affinity of a monoclonal antibody can be improved compared to its mouse parent sequence by humanizing the germline manipulation acceptor framework.

[0376] Another competitive assay compares the binding affinity of the novel antibody to that of the humanized 21.6 anti-α4 antibody (TYSABRI® (natalizumab)) described in US5,840,299. This experiment assayed the binding of a mixture of mouse HP1 / 2 and the test mAb to α4β1. The results of this experiment are shown in Figure 4 and Table 3 below, demonstrating that the newly designed antibody is approximately 10 times potent than natalizumab.

[0377] TIFF2026113464000019.tif40170

[0378] Example 2. Humanized HP1 / 2 (HuHP1 / 2) binds to VLA-4 in tumor cell lines. The binding of the anti-VLA-4 antibody HuHP1 / 2 to various cell lines was tested by flow cytometry. Binding was tested on the CLL (chronic lymphocytic leukemia) cell lines Mec1 and JM1, the MM (multiple myeloma) cell lines U266 and H929, and the AML (acute myeloid leukemia) cell lines HL60 and KG1. HuHP1 / 2 bound to all tested tumor cell lines (Figure 6). The EC50 values ​​for antibody binding to each of the different cell lines were calculated using flow cytometry data. This information is shown in Table 4 below.

[0379] HuHP1 / 2 was also found to block the adhesion of AML cell lines to fibronectin (FN) and VCAM1-Ig fusion proteins. To test whether the antibody could block adhesion, AML cell lines HL60 or KG1 were adhered to FN coat wells (Figure 7A) or VCAM1-Ig coat wells (Figure 7B) in the presence of gradually increasing concentrations of HP1 / 2 or isotype control antibodies. HuHP1 / 2 blocked the adhesion of both cell types to the FN and VCAM1-Ig coat wells. Maximum inhibition of HL60 cell binding to both ligands was achieved with 20 ug / ml of HuHP1 / 2 (Figure 7C).

[0380] HuHP1 / 2 was also found to block the adhesion of MM cell lines to FN and VCAM1-Ig fusion proteins. MM cell lines U266 and H929 were adhered to FN coat wells (Figure 8A) or VCAM1-Ig coat wells (Figure 8B) in the presence of gradually increasing concentrations of HP1 / 2 or isotype control antibodies. HuHP1 / 2 blocked the adhesion of both types of cell lines to the FN and VCAM1-Ig coat wells. Maximum inhibition of U266 cell binding to both ligands was achieved with 20 μg / mL of HuHP1 / 2 (Figure 8C).

[0381] HuHP1 / 2 was also found to block the adhesion of CLL cell lines to FN and VCAM1-Ig fu...

Claims

1. Recombinant anti-alpha-4 antibody or its alpha-4 binding fragment, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A steady-state heavy chain of human IgG1, wherein the steady-state region contains at least one mutation selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme, and The recombinant anti-alpha-4 antibody or its alpha-4 binding fragment, comprising the above.

2. The recombinant anti-alpha-4 antibody or its alpha-4 binding fragment according to claim 1, wherein the constant heavy chain of human IgG1 contains at least two mutations selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme.

3. The constant heavy chain of the human IgG1 has at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme. Recombinant anti-alpha-4 antibody or its alpha-4 binding fragment according to claim 1, comprising 1, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or at least 16, or at least 17, or at least 18, or at least 19, or at least 20, or at least 21, or at least 22, or at least 23, or at least 24, or at least 25 fragments.

4. The recombinant anti-alpha-4 antibody or its alpha-4 binding fragment according to claim 1, wherein the constant heavy chain of human IgG1 contains a mutation selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme.

5. Recombinant anti-alpha-4 antibody or its alpha-4 binding fragment, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A chimeric Fc region comprising the hinge, CH1 domain, and CH2 domain of an IgG4 isotype IgG antibody, and the CH3 domain of an IgG1 isotype IgG antibody, wherein the chimeric Fc region contains at least one mutation selected from the group consisting of a substitution of glutamine (Q) at EU-numbered position 297 (Kabat-numbered position 314) of the CH2 region, a substitution of proline (P) at EU-numbered amino acid position 228 (Kabat-numbered position 241) of the hinge region, and a deletion of lysine (K) at EU-numbered position 447 (Kabat-numbered position 478) of the CH3 region. The recombinant anti-alpha-4 antibody or its alpha-4 binding fragment, comprising the above.

6. A recombinant anti-alpha-4 antibody or alpha-4 binding fragment according to claim 5, having increased thermal stability compared to an antibody comprising: (a) a variable light chain comprising the sequence of SEQ ID NO: 11; (b) a variable heavy chain comprising the sequence of SEQ ID NO: 4; (c) a constant light chain of human kappa light chain (SEQ ID NO: 82); and (d) an Fc region comprising the hinge, CH1 domain, CH2 domain, and CH3 domain of the IgG4 isotype IgG antibody, and comprising or consisting of a substitution to a non-asparagine residue at EU-numbered position 297 (Kabat-numbered position 314).

7. Recombinant anti-alpha-4 antibody or its alpha-4 binding fragment, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A steady-state heavy chain of human IgG4, wherein the steady-state region contains at least one mutation selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme, and The recombinant anti-alpha-4 antibody or its alpha-4 binding fragment, comprising the above.

8. The recombinant anti-alpha-4 antibody or alpha-4 binding fragment according to claim 7, wherein the constant heavy chain of human IgG4 contains at least two mutations selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme.

9. The recombinant anti-alpha-4 antibody or alpha-4 binding fragment according to claim 7, wherein the constant heavy chain of human IgG4 contains at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme.

10. The recombinant anti-alpha-4 antibody or alpha-4 binding fragment according to claim 7, wherein the constant heavy chain of human IgG4 includes deletion mutations of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme.

11. Recombinant anti-alpha-4 antibody or its alpha-4 binding fragment, (a) A heavy chain containing the sequence of sequence number 80, (b) Light chain containing the sequence of Sequence ID No. 81 and The recombinant anti-alpha-4 antibody or its alpha-4 binding fragment, comprising the above.

12. A polynucleotide that codes for a protein, The aforementioned protein, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A steady-state heavy chain of human IgG1, wherein the steady-state region contains at least one mutation selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme, and The polynucleotide comprising the above.

13. A polynucleotide that codes for a protein, The aforementioned protein, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A steady-state heavy chain of human IgG1, wherein the steady-state region contains at least two, at least three, at least four, or at least five mutations selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme. The steady-state heavy chain of human IgG1 comprises at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or at least 16, or at least 17, or at least 18, or at least 19, or at least 20, or at least 21, or at least 22, or at least 23, or at least 24, or at least 25 of the same human IgG1. The polynucleotide comprising the above.

14. A polynucleotide that codes for a protein, The aforementioned protein, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A chimeric Fc region comprising the hinge, CH1 domain, and CH2 domain of an IgG antibody of the IgG4 isotype, and the CH3 domain of an IgG antibody of the IgG1 isotype, wherein the chimeric Fc region includes a substitution of glutamine (Q) at EU-numbered position 297 (Kabat-numbered position 314) of the CH2 region, a substitution of proline (P) at EU-numbered amino acid position 228 (Kabat-numbered position 241) of the hinge region, and a deletion of lysine (K) at EU-numbered position 447 (Kabat-numbered position 478) of the CH3 region. The polynucleotide comprising the above.

15. A polynucleotide that codes for a protein, The aforementioned protein, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A steady-state heavy chain of human IgG4, wherein the steady-state region contains at least one mutation selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme, and The polynucleotide comprising the above.

16. A polynucleotide that codes for a protein, The aforementioned protein, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A steady-state heavy chain of human IgG4, wherein the steady-state region contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme, and The polynucleotide comprising the above.

17. A polynucleotide that codes for a protein, The aforementioned protein, (a) A heavy chain containing the sequence of sequence number 80, (b) Light chain containing the sequence of Sequence ID No. 81 and The polynucleotide comprising the above.

18. A method for alleviating the symptoms of a patient suffering from multiple sclerosis, wherein the method is The procedure involves administering a therapeutically effective dose of recombinant anti-alpha-4 antibody to the patient. The recombinant anti-alpha-4 antibody, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A steady-state heavy chain of human IgG1, wherein the steady-state region contains at least one mutation selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme, comprising: The aforementioned method.

19. A method for alleviating the symptoms of a patient suffering from multiple sclerosis, wherein the method is The procedure involves administering a therapeutically effective dose of recombinant anti-alpha-4 antibody to the patient. The recombinant anti-alpha-4 antibody, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A steady-state heavy chain of human IgG1, wherein the steady-state region contains at least two, at least three, at least four, or at least five mutations selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme. or comprising the steady-state heavy chain of human IgG1, including at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or at least 16, or at least 17, or at least 18, or at least 19, or at least 20, or at least 21, or at least 22, or at least 23, or at least 24, or at least 25 The aforementioned method.

20. A method for alleviating the symptoms of a patient suffering from multiple sclerosis, wherein the method is The procedure involves administering a therapeutically effective dose of recombinant anti-alpha-4 antibody to the patient. The recombinant anti-alpha-4 antibody, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A chimeric Fc region comprising the hinge, CH1 domain, and CH2 domain of an IgG4 isotype IgG antibody, and the CH3 domain of an IgG1 isotype IgG antibody, wherein the chimeric Fc region includes a substitution of glutamine (Q) at EU-numbered position 297 (Kabat-numbered position 314) of the CH2 region, a substitution of proline (P) at EU-numbered amino acid position 228 (Kabat-numbered position 241) of the hinge region, and a deletion of lysine (K) at EU-numbered position 447 (Kabat-numbered position 478) of the CH3 region, The aforementioned method.

21. A method for alleviating the symptoms of a patient suffering from multiple sclerosis, wherein the method is The procedure involves administering a therapeutically effective dose of recombinant anti-alpha-4 antibody to the patient. The recombinant anti-alpha-4 antibody, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A steady-state heavy chain of human IgG4, wherein the steady-state region contains at least one mutation selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme, comprising the steady-state heavy chain of human IgG4, The aforementioned method.

22. A method for alleviating the symptoms of a patient suffering from multiple sclerosis, wherein the method is The procedure involves administering a therapeutically effective dose of recombinant anti-alpha-4 antibody to the patient. The recombinant anti-alpha-4 antibody, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A steady-state heavy chain of human IgG4, wherein the steady-state region contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme, comprising: The aforementioned method.

23. A method for alleviating the symptoms of a patient suffering from multiple sclerosis, wherein the method is The procedure involves administering a therapeutically effective dose of recombinant anti-alpha-4 antibody to the patient. The recombinant anti-alpha-4 antibody, (a) A heavy chain containing, essentially having, or consisting of the sequence of sequence number 80, (b) A light chain comprising, essentially having, or consisting of, the sequence of sequence number 81 The aforementioned method.

24. The method according to claim 18, 19, 20, 21, 22, or 23, wherein the multiple sclerosis is relapsing-type multiple sclerosis.

25. The method according to claim 24, wherein the relapsing-type multiple sclerosis is clinically isolated syndrome (CIS), relapsing-remitting multiple sclerosis, or active secondary progressive multiple sclerosis.

26. The method according to claim 18, 19, 20, 21, 22, or 23, wherein the multiple sclerosis is primary progressive multiple sclerosis or progressive relapsing multiple sclerosis.

27. The method according to claim 18, 19, 20, 21, 22, or 23, wherein the multiple sclerosis is active multiple sclerosis, including active relapsing multiple sclerosis.

28. The method according to claim 18, 19, 20, 21, 22, or 23, wherein the patient is a human being.

29. It is a host cell, (a) A polynucleotide encoding an antibody heavy chain, wherein the antibody heavy chain comprises a variable heavy chain containing the sequence of SEQ ID NO: 4 and a constant heavy chain of human IgG1, and the constant region contains at least one mutation selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme, (b) A polynucleotide encoding an antibody light chain, wherein the antibody light chain includes a variable light chain containing the sequence of SEQ ID NO: 11 and a constant light chain of human kappa light chain (SEQ ID NO: 82), and the polynucleotide is transfected with this polynucleotide. The aforementioned host cells.

30. It is a host cell, (a) A polynucleotide encoding an antibody heavy chain, wherein the antibody heavy chain comprises a variable heavy chain containing the sequence of Sequence ID No. 4 and a constant heavy chain of human IgG1, and the constant region contains at least two, or fewer, mutations selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme. Each of the polynucleotides comprises three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten, or at least eleven, or at least twelve, or at least thirteen, or at least fourteen, or at least fifteen, or at least sixteen, or at least seventeen, or at least eighteen, or at least nineteen, or at least twenty, or at least twenty-one, or at least twenty-two, or at least twenty-three, or at least twenty-five, (b) A polynucleotide encoding an antibody light chain, wherein the antibody light chain includes a variable light chain containing the sequence of SEQ ID NO: 11 and a constant light chain of human kappa light chain (SEQ ID NO: 82), and the polynucleotide is transfected with this polynucleotide. The aforementioned host cells.

31. It is a host cell, (a) A polynucleotide encoding an antibody heavy chain, wherein the antibody heavy chain comprises a variable heavy chain containing the sequence of SEQ ID NO: 4, and a chimeric Fc region containing the hinge, CH1 domain, and CH2 domain of an IgG4 isotype IgG antibody, and the CH3 domain of an IgG1 isotype IgG antibody, wherein the chimeric Fc region includes a substitution of glutamine (Q) at EU-numbered position 297 (Kabat-numbered position 314) of the CH2 region, a substitution of proline (P) at EU-numbered amino acid position 228 (Kabat-numbered position 241) of the hinge region, and a deletion of lysine (K) at EU-numbered position 447 (Kabat-numbered position 478) of the CH3 region, (b) A polynucleotide encoding an antibody light chain, wherein the antibody light chain includes a variable light chain containing the sequence of SEQ ID NO: 11 and a constant light chain of human kappa light chain (SEQ ID NO: 82), and the polynucleotide is transfected with this polynucleotide. The aforementioned host cells.

32. It is a host cell, (a) A polynucleotide encoding an antibody heavy chain, wherein the antibody heavy chain comprises a variable heavy chain containing the sequence of SEQ ID NO: 4 and a constant heavy chain of human IgG4, and the constant region contains at least one mutation selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme, (b) A polynucleotide encoding an antibody light chain, wherein the antibody light chain includes a variable light chain containing the sequence of SEQ ID NO: 11 and a constant light chain of human kappa light chain (SEQ ID NO: 82), and the polynucleotide is transfected with this polynucleotide. The aforementioned host cells.

33. It is a host cell, (a) A polynucleotide encoding an antibody heavy chain, wherein the antibody heavy chain comprises a variable heavy chain containing the sequence of Sequence ID No. 4 and a constant heavy chain of human IgG4, and the constant region contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme, (b) A polynucleotide encoding an antibody light chain, wherein the antibody light chain includes a variable light chain containing the sequence of SEQ ID NO: 11 and a constant light chain of human kappa light chain (SEQ ID NO: 82), and the polynucleotide is transfected with this polynucleotide. The aforementioned host cells.

34. It is a host cell, (a) A polynucleotide encoding an antibody heavy chain containing the sequence of Sequence ID No. 80, (b) A polynucleotide that has been transfected with a light chain encoding the sequence of sequence number 81, The aforementioned host cells.

35. The host cell according to claim 29, 30, 31, 32, 33, or 34, wherein the host cell is a Chinese hamster ovary cell.

36. A method for producing a recombinant anti-α4 antibody or its α4-binding fragment by preparing host cells, In the aforementioned host cells, (a) A polynucleotide encoding an antibody heavy chain, arranged for expression in the cells, wherein the antibody heavy chain comprises a variable heavy chain containing the sequence of SEQ ID NO: 4 and a constant heavy chain of human IgG1, and the constant region contains at least one mutation selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme, (b) A polynucleotide encoding an antibody light chain, arranged for expression in the cell, wherein the antibody light chain is transfected with the polynucleotide, the polynucleotide comprising a variable light chain containing the sequence of SEQ ID NO: 11 and a constant light chain of human kappa light chain (SEQ ID NO: 82). The aforementioned method.

37. A method for producing a recombinant anti-α4 antibody or its α4-binding fragment by preparing host cells, In the aforementioned host cells, (a) A polynucleotide encoding an antibody heavy chain, arranged for expression in the cells, wherein the antibody heavy chain comprises a variable heavy chain containing the sequence of SEQ ID NO: 4 and a constant heavy chain of human IgG1, wherein the constant region contains as few mutations as possible selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme. The polynucleotide also includes 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or at least 16, or at least 17, or at least 18, or at least 19, or at least 20, or at least 21, or at least 22, or at least 23, or at least 24, or at least 25, (b) A polynucleotide encoding an antibody light chain, arranged for expression in the cell, wherein the antibody light chain is transfected with the polynucleotide, the polynucleotide comprising a variable light chain containing the sequence of SEQ ID NO: 11 and a constant light chain of human kappa light chain (SEQ ID NO: 82), The aforementioned method.

38. A method for producing a recombinant anti-α4 antibody or its α4-binding fragment by preparing host cells, In the aforementioned host cells, (a) A polynucleotide encoding an antibody heavy chain, arranged for expression in the cell, wherein the antibody heavy chain comprises a variable heavy chain containing the sequence of SEQ ID NO: 4, and a chimeric Fc region containing the hinge, CH1 domain, and CH2 domain of an IgG4 isotype IgG antibody, and the CH3 domain of an IgG1 isotype IgG antibody, wherein the chimeric Fc region includes a substitution of glutamine (Q) at EU-numbered position 297 (Kabat-numbered position 314) of the CH2 region, a substitution of proline (P) at EU-numbered amino acid position 228 (Kabat-numbered position 241) of the hinge region, and a deletion of lysine (K) at EU-numbered position 447 (Kabat-numbered position 478) of the CH3 region, (b) A polynucleotide encoding an antibody light chain, arranged for expression in the cell, wherein the antibody light chain is transfected with the polynucleotide, the polynucleotide comprising a variable light chain containing the sequence of SEQ ID NO: 11 and a constant light chain of human kappa light chain (SEQ ID NO: 82). The aforementioned method.

39. A method for producing a recombinant anti-α4 antibody or its α4-binding fragment by preparing host cells, In the aforementioned host cells, (a) A polynucleotide encoding an antibody light chain, arranged for expression in the cell, wherein the antibody light chain includes a variable light chain containing the sequence of SEQ ID NO: 11 and a constant light chain of human kappa light chain (SEQ ID NO: 82), (b) A polynucleotide encoding an antibody heavy chain, arranged for expression in the cells, wherein the antibody heavy chain comprises a variable heavy chain containing the sequence of SEQ ID NO: 4 and a constant heavy chain of human IgG4, and the constant region contains at least one mutation selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme, and is transfected with the polynucleotide. The aforementioned method.

40. A method for producing a recombinant anti-α4 antibody or its α4-binding fragment by preparing host cells, In the aforementioned host cells, (a) A polynucleotide encoding an antibody light chain, arranged for expression in the cell, wherein the antibody light chain includes a variable light chain containing the sequence of SEQ ID NO: 11 and a constant light chain of human kappa light chain (SEQ ID NO: 82), (b) A polynucleotide encoding an antibody heavy chain, arranged for expression in the cells, wherein the antibody heavy chain comprises a variable heavy chain containing the sequence of Sequence ID No. 4 and a constant heavy chain of human IgG4, and the constant region contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme, and is transfected with the polynucleotide. The aforementioned method.

41. A method for producing a recombinant anti-α4 antibody or its α4-binding fragment, This involves preparing host cells. In the aforementioned host cells, (a) A polynucleotide encoding an antibody heavy chain containing the sequence of Sequence ID No. 80, arranged for expression in the cell, (b) A polynucleotide encoding a light chain containing the sequence of Sequence ID No. 81, which is transfected for expression in the cell, The above preparations, and The method for generating the recombinant anti-α4 antibody molecule or its α4-binding fragment by culturing the transfected host cells.

42. The method according to claim 36, 37, 38, 39, 40, or 41, wherein the host cells are Chinese hamster ovary cells.

43. Recombinant anti-alpha-4 antibody or its alpha-4 binding fragment, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A steady-state heavy chain of human IgG1, wherein the steady-state region contains at least one mutation selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme, and Includes, (e) Compared to antibodies or binding fragments containing the wild-type IgG4 constant region, the sensitivity to scrambling is reduced. The recombinant anti-alpha-4 antibody or its alpha-4 binding fragment.

44. The recombinant anti-alpha-4 antibody or its alpha-4 binding fragment according to claim 43, wherein the constant heavy chain of human IgG1 contains at least two mutations selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme.

45. The constant heavy chain of the human IgG1 has at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme. Recombinant anti-alpha-4 antibody or its alpha-4 binding fragment according to claim 43, comprising 1, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or at least 16, or at least 17, or at least 18, or at least 19, or at least 20, or at least 21, or at least 22, or at least 23, or at least 24, or at least 25 fragments.

46. The recombinant anti-alpha-4 antibody or its alpha-4 binding fragment according to claim 43, wherein the constant heavy chain of human IgG1 contains a mutation selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme.

47. Recombinant anti-alpha-4 antibody or its alpha-4 binding fragment, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A chimeric Fc region comprising the hinge, CH1 domain, and CH2 domain of an IgG4 isotype IgG antibody, and the CH3 domain of an IgG1 isotype IgG antibody, wherein the chimeric Fc region contains at least one mutation selected from the group consisting of a substitution of glutamine (Q) at EU-numbered position 297 (Kabat-numbered position 314) of the CH2 region, a substitution of proline (P) at EU-numbered amino acid position 228 (Kabat-numbered position 241) of the hinge region, and a deletion of lysine (K) at EU-numbered position 447 (Kabat-numbered position 478) of the CH3 region. Includes, (e) Compared to antibodies or binding fragments containing the wild-type IgG4 constant region, the sensitivity to scrambling is reduced. The recombinant anti-alpha-4 antibody or its alpha-4 binding fragment.

48. The recombinant anti-alpha-4 antibody or alpha-4 binding fragment according to claim 47, having increased thermal stability compared to an antibody comprising: (a) a variable light chain comprising the sequence of SEQ ID NO: 11; (b) a variable heavy chain comprising the sequence of SEQ ID NO: 4; (c) a constant light chain of human kappa light chain (SEQ ID NO: 82); and (d) an Fc region comprising the hinge, CH1 domain, CH2 domain, and CH3 domain of the IgG4 isotype IgG antibody, and comprising or consisting of a substitution to a non-asparagine residue at EU-numbered position 297 (Kabat-numbered position 314).

49. Recombinant anti-alpha-4 antibody or its alpha-4 binding fragment, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A steady-state heavy chain of human IgG4, wherein the steady-state region contains at least one mutation selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme, and Includes, (e) Compared to antibodies or binding fragments containing the wild-type IgG4 constant region, the sensitivity to scrambling is reduced. The recombinant anti-alpha-4 antibody or its alpha-4 binding fragment.

50. The recombinant anti-alpha-4 antibody or alpha-4 binding fragment according to claim 49, wherein the constant heavy chain of human IgG4 contains at least two mutations selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme.

51. The recombinant anti-alpha-4 antibody or alpha-4 binding fragment according to claim 49, wherein the constant heavy chain of human IgG4 contains at least three, at least four, at least five, at least six, at least 49, at least eight, or at least nine mutations selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme.

52. The recombinant anti-alpha-4 antibody or alpha-4 binding fragment according to claim 49, wherein the constant heavy chain of human IgG4 includes deletion mutations of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme.

53. Recombinant anti-alpha-4 antibody or its alpha-4 binding fragment, (a) A heavy chain containing the sequence of sequence number 80, (b) Light chain containing the sequence of Sequence ID No. 81 and Includes, (c) Compared to antibodies or binding fragments containing the wild-type IgG4 constant region, the sensitivity to scrambling is reduced. The recombinant anti-alpha-4 antibody or its alpha-4 binding fragment.

54. A method for reducing the sensitivity of a recombinant anti-alpha-4 antibody or its alpha-4 binding fragment to scrambling compared to an antibody or binding fragment containing a wild-type IgG4 constant region, wherein the method is This involves preparing host cells. In the aforementioned host cells, (a) A polynucleotide encoding an antibody light chain, arranged for expression in the cell, wherein the antibody light chain includes a variable light chain containing the sequence of SEQ ID NO: 11 and a constant light chain of human kappa light chain (SEQ ID NO: 82), (b) A polynucleotide encoding an antibody heavy chain, arranged for expression in the cells, wherein the antibody heavy chain is transfected with the polynucleotide, the polynucleotide comprising a variable heavy chain containing the sequence of SEQ ID NO: 4 and a constant heavy chain of human IgG4. The above preparations, and To introduce at least one mutation selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme into the constant heavy chain of human IgG4. The method, including the method described above.

55. A method for reducing the sensitivity of a recombinant anti-alpha-4 antibody or its alpha-4 binding fragment to scrambling compared to an antibody or binding fragment containing a wild-type IgG4 constant region, wherein the method is This involves preparing host cells. In the aforementioned host cells, (a) A polynucleotide encoding an antibody light chain, arranged for expression in the cell, wherein the antibody light chain includes a variable light chain containing the sequence of SEQ ID NO: 11 and a constant light chain of human kappa light chain (SEQ ID NO: 82), (b) A polynucleotide encoding an antibody heavy chain, arranged for expression in the cells, wherein the antibody heavy chain is transfected with the polynucleotide, the polynucleotide comprising a variable heavy chain containing the sequence of SEQ ID NO: 4 and a constant heavy chain of human IgG4. The above preparations, and To introduce at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme into the constant heavy chain of the human IgG4. The method, including the method described above.

56. A method for producing a recombinant anti-alpha-4 antibody or alpha-4 binding fragment that has reduced sensitivity to scrambling compared to an antibody or binding fragment containing the wild-type IgG4 constant region, wherein the method is This involves preparing host cells. In the aforementioned host cells, (a) A polynucleotide encoding an antibody heavy chain containing the sequence of Sequence ID No. 80, arranged for expression in the cell, (b) A polynucleotide encoding a light chain containing the sequence of Sequence ID No. 81, which is transfected for expression in the cell, The above preparations, and By culturing the transfected host cells, the recombinant anti-α4 antibody molecule or its α4-binding fragment is generated. The method, including the method described above.

57. The method according to claim 53, 54, 55, or 56, wherein the host cells are Chinese hamster ovary cells.

58. A method for treating a patient suffering from epilepsy, wherein the method is The procedure involves administering a therapeutically effective dose of recombinant anti-alpha-4 antibody to the patient. The recombinant anti-alpha-4 antibody, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A steady-state heavy chain of human IgG1, wherein the steady-state region contains at least one mutation selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme, comprising: The aforementioned method.

59. A method for treating a patient suffering from epilepsy, wherein the method is The procedure involves administering a therapeutically effective dose of recombinant anti-alpha-4 antibody to the patient. The recombinant anti-alpha-4 antibody, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A steady-state heavy chain of human IgG1, wherein the steady-state region contains at least two, at least three, at least four, or at least five mutations selected from the group consisting of deletions of S127C, K129R, G135E, G136S, Q203K, I207T, N211D, K222R, P227S, S232Y, C233G, D234, K235, T236, H237P, T238P, L247F, H281Q, K287Q, Y313F, N314Q, A346G, A349S, P350S, and K478 according to the Kabat numbering scheme. or comprising the steady-state heavy chain of human IgG1, including at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or at least 16, or at least 17, or at least 18, or at least 19, or at least 20, or at least 21, or at least 22, or at least 23, or at least 24, or at least 25 The aforementioned method.

60. A method for treating a patient suffering from epilepsy, wherein the method is The procedure involves administering a therapeutically effective dose of recombinant anti-alpha-4 antibody to the patient. The recombinant anti-alpha-4 antibody, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A chimeric Fc region comprising the hinge, CH1 domain, and CH2 domain of an IgG4 isotype IgG antibody, and the CH3 domain of an IgG1 isotype IgG antibody, wherein the chimeric Fc region includes a substitution of glutamine (Q) at EU-numbered position 297 (Kabat-numbered position 314) of the CH2 region, a substitution of proline (P) at EU-numbered amino acid position 228 (Kabat-numbered position 241) of the hinge region, and a deletion of lysine (K) at EU-numbered position 447 (Kabat-numbered position 478) of the CH3 region, The aforementioned method.

61. A method for treating a patient suffering from epilepsy, wherein the method is The procedure involves administering a therapeutically effective dose of recombinant anti-alpha-4 antibody to the patient. The recombinant anti-alpha-4 antibody, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A steady-state heavy chain of human IgG4, wherein the steady-state region contains at least one mutation selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme, comprising the steady-state heavy chain of human IgG4, The aforementioned method.

62. A method for treating a patient suffering from epilepsy, wherein the method is The procedure involves administering a therapeutically effective dose of recombinant anti-alpha-4 antibody to the patient. The recombinant anti-alpha-4 antibody, (a) A variable light chain containing the sequence of sequence number 11, (b) A variable heavy chain containing the sequence of sequence number 4, (c) The steady-state light chain of the human kappa light chain (SEQ ID NO: 82), (d) A steady-state heavy chain of human IgG4, wherein the steady-state region contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of deletions of S241P, N314Q, Q376R, E377D, M381L, R440K, E450Q, L476P, and K478 according to the Kabat numbering scheme, comprising: The aforementioned method.

63. A method for treating a patient suffering from epilepsy, wherein the method is The procedure involves administering a therapeutically effective dose of recombinant anti-alpha-4 antibody to the patient. The recombinant anti-alpha-4 antibody, (a) A heavy chain containing, essentially having, or consisting of the sequence of sequence number 80, (b) A light chain comprising, essentially having, or consisting of, the sequence of sequence number 81 The aforementioned method.

64. The method according to claim 58, 59, 60, 61, 62, or 63, wherein the epilepsy is drug-resistant epilepsy.

65. The method according to claim 58, 59, 60, 61, 62, or 63, wherein the patient is a human.

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