Anti-VLA-4 antibody

Abstract

Providing an anti-VLA-4 antibody. [Solution] The present invention relates to alpha4-binding antibodies and fragments thereof. In one embodiment, a recombinant antibody molecule or alpha4-binding fragment thereof is provided, which comprises a variable light chain framework comprising a receptor sequence from IGKV4-1 and a variable heavy chain framework comprising a receptor sequence from IGHV1-f, and has heavy and light chain CDRs from murine antibody HP1 / 2 or murine antibody 21.6. In another embodiment, a recombinant antibody molecule or alpha4-binding fragment thereof is provided, which comprises a variable light chain framework comprising a receptor sequence from germline-engineered AAH70335.1 and a variable heavy chain framework comprising a receptor sequence from IGHV1-f, and is capable of binding to alpha4.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 61 / 324,944, filed Apr. 16, 2010, which is incorporated herein by reference in its entirety.

[0002] Technical Field The present invention relates to alpha4-binding antibodies and fragments thereof.

Background Art

[0003] Humanized antibodies can be used as therapeutic agents in place of mouse antibodies to avoid undesirable immune responses in humans, known as the HAMA (human anti-mouse antibody) response. Humanized antibodies are generally constructed by replacing the complementarity-determining regions (CDRs) of a human antibody with those of another species, typically a mouse antibody.

[0004] VLA-4 (also designated α4β1) is a member of the β1 integrin family of cell surface receptors. VLA-4 contains an α4 chain and a β1 chain and is involved in cell-cell interactions. Its expression is mainly restricted to lymphoid and myeloid cells. VLA-4 can bind to the endothelial cell ligand VCAM-1 (vascular cell adhesion molecule-1) and mediate the adhesion of T and B lymphocytes to the heparin II-binding fragment of human plasma fibronectin.

Summary of the Invention

Means for Solving the Problems

[0005] The inventors have discovered that germline variable region frameworks can be used to optimize CDR-grafted alpha4-binding antibodies such as anti-VLA-4 antibodies. Accordingly, the present invention features anti-VLA-4 variable heavy (VH) and variable light (VL) chains, and antibody molecules comprising such frameworks.

[0006] In one aspect, the present invention features a VH framework of an anti-α4 antibody having CDRs derived from a donor anti-α4 antibody (e.g., an anti-α4 antibody described herein), and having regions 1, 2, 3, and 4 derived from the sequence of the germline variable region sequence of the VH chain, or differing from it by 5, 10, or 15 or fewer. In one embodiment, variable framework region 4 (FR4) is a human consensus sequence. In one embodiment, the complete VH chain framework regions FR1, FR2, FR3, and FR4 are present. In another embodiment, the chain is an antigen-binding fragment of the VH region.

[0007] In one embodiment, the germline sequence is human IGHV1-f (SEQ ID NO: 2) as represented in FIG. 1. In certain embodiments, the VH framework sequence is at least 1, but 2, 3, 4, 5, 10, or 15 or fewer amino acid residues may differ from the germline sequence (e.g., SEQ ID NO: 2). In one embodiment, the VH framework further includes non-human residues other than the corresponding human residues. For example, the VH chain includes non-human residues at one or more of framework positions 24, 67, 76, 80, and 94 (Kabat numbering) of SEQ ID NO: 2.

[0008] In one embodiment, at least one or more of the complementarity determining regions (CDRs) of the variable domain are derived from a donor non-human α4-binding antibody. In one embodiment, the antigen-binding region of the CDR-grafted heavy chain variable domain includes CDRs corresponding to positions 26-34 (CDR1), positions 50-65 (CDR2), and positions 95-102 (CDR3) (Kabat numbering; Kabat et al., Sequences of Proteins of Immunological Interest ,5 th ed., vol. 4, 1991, U.S. Department of Health and Human Services, NIH, USA).

[0009] Accordingly, in one embodiment, the variable heavy chain (VH) framework has a receptor sequence derived from the human antibody germline sequence IGHV1-f.

[0010] In another embodiment, at least one amino acid in the FR1 region of VH, and two, three, four, five, or fewer than ten amino acid residues are other than the corresponding human germline residues. One or more of such residues can be identical to a non-human antibody framework region from which the CDR sequences are derived. In one embodiment, the amino acid residue at Kabat position 24 is mutated to be identical to a non-human antibody framework region.

[0011] In another embodiment, at least one amino acid in the FR2 region of VH, and two, three, four, five, or fewer than ten amino acid residues are other than the corresponding human germline residues. One or more of such residues can be identical to a non-human antibody framework region from which the CDR sequences are derived.

[0012] In yet another embodiment, at least one amino acid in the FR3 of the VH chain, and two, three, four, five, or fewer than ten amino acid residues are other than the corresponding human germline residues. One or more of such residues can be identical to a non-human antibody framework region from which the CDR sequences are derived. In one embodiment, the amino acid residue at Kabat position 94 is identical to a non-human antibody framework region. In yet another embodiment, the amino acid residues at Kabat positions 67, 76, 80, and 94 are identical to a non-human antibody framework region.

[0013] In one embodiment, the VH chain of the antibody has the sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0014] In one aspect, the present invention features a VL framework of an anti-VLA-4 VL chain having CDRs from a donor anti-VLA-4 antibody (e.g., the anti-VLA-4 antibodies described herein), and having regions 1, 2, 3, and 4 from the sequence of the germline variable region sequence of the VL chain, or differing from it by 5, 10, or 15 or less (per region or in total). In one embodiment, variable framework region 4 (FR4) is a human consensus sequence. In one embodiment, the complete VL chain framework regions FR1, FR2, FR3, and FR4 are present. In another embodiment, the chain is an antigen-binding fragment of the VL region.

[0015] In another embodiment, the germline sequence is IGKV4-1 (SEQ ID NO: 7), as represented in FIG. 2. In yet another embodiment, the VL framework sequence is at least 1, but can differ by 2, 3, 4, 5, 10, or 15 or less amino acid residues from the germline sequence (e.g., SEQ ID NO: 7). In another embodiment, the VL further contains something other than the corresponding human amino acid residues. For example, the VL chain further contains non-human residues at one or more of framework positions 1, 73, and 87 (Kabat numbering) of SEQ ID NO: 7.

[0016] In one embodiment, the sequence is AAH7035.1 (SEQ ID NO: 12) as represented in Table 2 or a germline engineered version thereof (SEQ ID NO: 13). In some embodiments, the VL framework sequence is at least 1, but can be 5, 10, 15, 20, or 25 or fewer amino acid residues different from the germline engineered framework sequence (e.g., SEQ ID NO: 13). In one embodiment, the VL chain contains non-human residues other than the corresponding human residues. For example, the VL chain contains non-human residues at one or more of framework positions 1 and 87 (Kabat numbering) of SEQ ID NO: 12. In another embodiment, the VL contains amino acid substitutions in the framework region such that it is similar to a human germline framework sequence different from the germline sequence such as IGKV4-1. In certain embodiments, the VL framework sequence is varied to be the same as 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, 99-106 (Kabat numbering) of SEQ ID NO: 12.

[0017] 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-grafted heavy chain variable domain contains the CDRs corresponding to positions 24-31 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) (Kabat numbering). Thus, in one embodiment, the VL framework has a receptor sequence constructed from the IGKV4-1 germline sequence, from antibody AAH70335.1, or from the germline engineered antibody AAH70335.1.

[0018] In yet another embodiment, at least one amino acid in FR1 of the VL chain, and two, three, four, five, ten, or fewer than fifteen residues are other than the corresponding human residues. One or more of such residues can be identical to a non-human antibody framework region from which the CDR sequences are derived. In one embodiment, the amino acid residue at the N-terminal position of FR1 is mutated to be identical to a non-human antibody framework region.

[0019] In another embodiment, at least one amino acid in FR2 of the VL chain, and two, three, four, five, ten, or fewer than fifteen residues are other than the corresponding human residues. One or more of such residues can be identical to a non-human antibody framework region from which the CDR sequences are derived.

[0020] In yet another embodiment, at least one amino acid in FR3 of the VL, and two, three, four, five, ten, or fewer than fifteen residues are other than the corresponding human residues. One or more of such residues can be identical to a non-human antibody framework region from which the CDR sequences are derived. In another embodiment, the amino acid residue at Kabat position 87 is mutated to be identical to a 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 a 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 a non-human antibody framework sequence.

[0021] 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.

[0022] In one embodiment, the CDRs of the VH and VL receptor framework sequences are selected to be similar to the CDR sequences of non-human (e.g., mouse) antibody sequences, and the non-human antibody binds to integrin alpha4 or a fragment thereof. In another embodiment, the sequence of the CDR is selected to be similar to the sequence of the CDR 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 can mean excision and insertion or change, for example, by site-directed mutagenesis.

[0023] In another aspect, the invention features an antibody or an antigen-binding fragment thereof, - an anti-VLA-4 VL chain as described herein (e.g., an anti-VLA-4 VL chain having CDRs derived from a donor anti-VLA-4 antibody (e.g., an anti-VLA-4 antibody as described herein)), and having light chain framework regions 1, 2, and 3 derived from the sequence of the germline variable region sequence of the VL chain, or differing from it by 5, 10, or 15 or less, and including a VL framework. In one embodiment, variable region 4 is a human consensus sequence, - an anti-VLA-4 VH chain as described herein (e.g., an anti-VLA-4 VL chain having CDRs derived from a donor anti-VLA-4 antibody (e.g., an anti-VLA-4 antibody as described herein)), and having light chain framework regions 1, 2, and 3 derived from the sequence of the germline variable region sequence of the VL chain, or differing from it by 5, 10, or 15 or less, and being a VL framework. In one embodiment, variable region 4 is a human consensus sequence.

[0024] In one embodiment, the antibody binds to one or both of α4β1 and α4β7.

[0025] In another aspect, the VL chain, VH chain, antibody, or fragment thereof described herein is labeled to a detectable extent.

[0026] In yet another aspect, the invention features a vector containing 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.

[0027] In yet another aspect, the invention features a vector containing 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.

[0028] In yet another aspect, the invention features a vector containing DNA encoding an antibody heavy chain or an α4-binding fragment thereof, as described herein, and DNA encoding an antibody light chain or an α4-binding fragment thereof, as described herein.

[0029] In another aspect, the invention features a host cell containing a vector described herein (e.g., one capable of expressing a heavy chain and / or light chain antibody, or antibody fragment, as described herein).

[0030] In one aspect, the invention provides a method for producing a recombinant anti-α4 antibody or an α4-binding fragment thereof, by (a) transfecting a host cell with a DNA sequence encoding an antibody heavy chain or an α4-binding fragment thereof, as described herein, and (b) a DNA sequence encoding an antibody light chain or an α4-binding fragment thereof, and culturing the transfected cells to produce a recombinant anti-α4 antibody molecule or an α4-binding fragment thereof. The DNA encoding the heavy and light chains of the antibody can be produced on the same vector or on different vectors.

[0031] In one aspect, the present invention provides a method for producing a recombinant anti-α4 antibody or an α4-binding fragment thereof, by (a) transfecting a host cell with a DNA sequence encoding an antibody heavy chain or an α4-binding fragment thereof, for example, having a DNA sequence of SEQ ID NO: 3, 4, or 5, and (b) a DNA sequence encoding an antibody light chain or an α4-binding fragment thereof, for example, having a DNA sequence of SEQ ID NO: 8, 9, 10, or 11, and culturing the transfected cell line to produce a recombinant anti-α4 antibody molecule or an α4-binding fragment thereof. The DNAs encoding the antibody heavy chain and light chain can be produced on the same vector or on different vectors.

[0032] In another aspect, the present invention features a method of treating a disease or disorder mediated by α4 integrin (e.g., α4β1 (VLA-4) or α4β7 integrin) by administering to a subject in need thereof an α4 antibody or antibody fragment described herein, or a pharmaceutical composition containing the antibody or fragment. The subject may have, or be at risk of developing, for example, inflammatory, immune, or autoimmune disorders (e.g., inflammation of the central nervous system such as multiple sclerosis, meningitis, neuromyelitis optica, neurosarcoidosis, CNS vasculitis, encephalitis, and transverse myelitis), tissue or organ transplant rejection or graft-versus-host disease, acute CNS injury (such as stroke, traumatic brain injury (TBI), or spinal cord injury (SCI)), chronic kidney disease, allergies (e.g., allergic asthma), type 1 diabetes mellitus, inflammatory bowel disease (such as Crohn's disease, ulcerative colitis, etc.), myasthenia gravis, fibromyalgia, arthritis disorders (such as rheumatoid arthritis, psoriatic arthritis, etc.), inflammatory / immune skin disorders (such as psoriasis, vitiligo, dermatitis, lichen planus, etc.), systemic lupus erythematosus, Sjogren's syndrome, blood cancers (such as multiple myeloma, leukemia, lymphoma, etc.), solid cancers of, for example, the lung, breast, prostate, brain (such as sarcoma or carcinoma, etc.), and fibrotic disorders (such as pulmonary fibrosis, myelofibrosis, cirrhosis, mesangial proliferative glomerulonephritis, crescentic glomerulonephritis, diabetic nephropathy, and interstitial renal fibrosis, etc.).

[0033] In another aspect, 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, a patient being treated with an α4-binding antibody or antibody fragment may have acute myeloid leukemia (AML) or multiple myeloma (MM).

[0034] In another embodiment, the patient has an inflammatory disorder, such as multiple sclerosis, asthma (e.g., moderate to severe asthma), rheumatoid arthritis, diabetes, or Crohn's disease. In another embodiment, the composition is administered as part of a dosing schedule. In yet another embodiment, the method further comprises selecting a patient suitable for treatment with the composition. A patient suitable for treatment exhibits signs or symptoms indicative of the disease occurrence, such as signs or symptoms indicative of MS.

[0035] In yet another embodiment, the method further comprises administering to the patient a second therapeutic agent, such as a chemotherapeutic agent, a thrombolytic agent, a neuroprotective agent, an anti-inflammatory agent, a steroid, a cytokine, or a growth factor.

[0036] In one embodiment, the patient is administered a humanized anti-VLA-4 antibody or fragment thereof as described herein, such as HuHP1 / 2, H1L1, H1L2, or H1L3.

[0037] In one embodiment, a composition containing an α4-binding antibody is administered as part of a dosing schedule at regular intervals, such as once a day, once a week, or once a month, once a week, twice a week, three times a week, four or more times a week, or once every two weeks, once every three weeks, once every four or more weeks.

[0038] In one embodiment, dosing can be adjusted according to the clearance rate of the pre-administered anti-α4 antibody in the patient. For example, in one embodiment, the second or subsequent doses are not administered until the level of the α4 antibody in the patient's body falls below a predetermined level. In one embodiment, a sample from the patient (e.g., a plasma, serum, blood, or urine sample) is assayed for the presence of the anti-α4 antibody, and if the level of the anti-α4 antibody exceeds a predetermined level, the second or subsequent doses are not administered to the patient. When the level of the anti-α4 antibody in the patient's body falls below a predetermined level, the second or subsequent doses are administered to the patient.

[0039] In one embodiment, the composition is administered continuously over a period of, for example, greater than 30 minutes but less than 1, 2, 4, or 12 hours. The composition containing the antibody and the second agent can be administered by any suitable method (e.g., subcutaneously, intramuscularly, or intravenously).

[0040] In some embodiments, each of the antibody and the second agent is administered at the same dose as the dose prescribed for each for monotherapy. In other embodiments, the antibody is administered at a dose that is less than the amount necessary for efficacy when administered alone. Similarly, the second agent can be administered at a dose that is less than the amount necessary for efficacy when administered alone.

[0041] Another aspect of the present disclosure features a method of evaluating a patient by determining whether the patient meets a preselected criterion and, if the patient meets the preselected criterion, permitting, providing, prescribing, or administering to the patient a VLA-4 binding antibody formulation as described herein. In one embodiment, the preselected criterion is, for example, an inadequate response of the patient to prior alternative therapies or dosing regimens for the treatment of MS. In another embodiment, the preselected criterion is the absence of any signs or symptoms of progressive multifocal leukoencephalopathy (PML) or the absence of a diagnosis of PML. In some cases, the selection is based on the absence of risk factors for PML, for example, the patient does not test positive for JC virus DNA or does not test positive for JC virus antibodies. In another embodiment, the criterion is as described in International Application No. PCT / US07 / 75577 (published as International Publication No. WO2008 / 021954), which is incorporated herein by reference and which describes methods and systems for drug distribution and for providing drugs to patients.

[0042] In another aspect, a method of dispensing the compositions described herein is provided. The compositions contain an alpha4-binding antibody. The method includes providing a package containing a unit dosage of the drug sufficient to treat a patient over at least 6, 12, 24, 36, or 48 months to a recipient (e.g., an end user, patient, physician, retail or wholesale pharmacy, distributor, hospital pharmacy, clinic in a care facility, or HMO). In another aspect, the invention includes assessing the quality of a single or multiple packages of the compositions described herein that contain an alpha4-binding antibody (e.g., to determine whether its expiration date has passed). The method includes assessing whether the expiration date of the package has passed. The expiration date is at least 6, 12, 24, 36, or 48 months from a preselected event such as manufacture, assay, or packaging (e.g., greater than 24 or 36 months). In some embodiments, the determination or step is made as a result of an analysis. For example, depending on the accurate analysis, the antibody in the package is used or discarded, classified, selected, published or withheld, shipped, moved to a new location, marketed, sold, offered for sale, recalled from the market, or the market entry is aborted, depending on whether the expiration date of the product has passed.

[0043] In another aspect, the invention features a package containing at least two unit dosages of an aqueous composition containing an alpha4-binding antibody. In one embodiment, all unit dosages contain the same amount of antibody, and in other embodiments, there are unit dosages of two or more strengths, or two or more different formulations having, for example, different strengths or release properties.

[0044] In another aspect, the invention includes a method of instructing a recipient regarding the administration of a formulation containing an α4-binding antibody. The method includes instructing the recipient (e.g., an end user, patient, physician, retail or wholesale pharmacy, distributor, hospital pharmacy, clinic in a care facility, or HMO) that the antibody should be administered to a patient according to a dosing schedule described herein. The method can 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 preselected event such as manufacture, assay, or packaging (e.g., greater than 24 or 36 months). In one embodiment, the recipient also receives a supply of the antibody, e.g., a unit dose of the antibody.

[0045] In another aspect, the invention features a method of making an antibody that includes CDRs from a donor antibody such as a non-human (e.g., a mouse antibody) and one or both of a heavy and a light chain variable region from one or more human germline variable region frameworks. The method includes one or both of 1 and 2, where 1 and 2 are as follows. 1. Identify or select a stable human receptor heavy chain variable framework having the same residues as the non-human donor heavy chain in one or more of the residues in 1.a), 1.b), and 1.c). a) VH Kabat positions 2, 4, 24, 26, 27, 29, 36, 38, 46, 47, 48, 49, 66, 67, 69, 71, 78, 93, and 94 (which are thought to be important for maintaining CDR conformation, although not bound by theory). b) VH Kabat positions 1, 2, 27, 28, 30, 43, 66, 68, 70, 72, 73, 74, and 75 (which are thought to be able to interact with an antigen, although not bound by theory). c) VH Kabat positions 37, 39, 44, 45, 47, 91, 93, and 103 (which are thought to be important for the integrity of the VH / VL interface, although not bound by theory). Identify or select a stable receptor light chain variable framework having the same residues as the donor light chain in one or more of the residues in 2.a), b), and c). a) VL Kabat positions 2, 4, 38, 43, 44, 48, 58, 64, 71, and 73 (although not bound by theory, considered important for maintaining the CDR conformation). b) VL Kabat positions 1, 2, 49, 57, 60, 63, 65, 66, 67, 68, 69, and 70 (although not bound by theory, potentially considered capable of interacting with the antigen). c) VL Kabat positions 36, 38, 43, 44, 46, 49, 87, and 98 (although not bound by theory, considered important for the integrity of the VH / VL interface). Select the germline sequence to further maximize the matches at the residues identified in 3.1 and 2, and provide a variable region having a donor CDR and a selected germline framework having matching residues identified in 1 or 2, for example, by backmutating additional residues identified in 1 or 2 of the germline to the mouse sequence. Evaluate each matching position by 3D structure analysis or modeling, etc., and reintroduce an equivalent mouse residue or a common human antibody residue that is compatible with the antibody structure if a given position meets the criteria for interfering with, for example, the conformation of the CDR, the interaction with the antigen, or the integrity of the VH / VL interface. In one embodiment, at least 3, 4, or 5 of the residues identified in (1.a) match. For example, in one embodiment, residues 24, 29, or 94 match.

[0046] In one embodiment, at least 3, 4, or 5 of the residues identified in (1.b) match. For example, in one embodiment, residues 1, 73, or 75 match.

[0047] In one embodiment, at least three, four, or five of the residues identified in (1.c) match. For example, in one embodiment, residues 37, 93, or 103 match.

[0048] In one embodiment, at least three, four, or five of the residues identified in (2.a) match. For example, in one embodiment, residues 2, 71, and 73 match.

[0049] In one embodiment, at least three, four, or five of the residues identified in (2.b) match. For example, in one embodiment, residues 1, 68, or 70 match.

[0050] In one embodiment, at least three, four, or five of the residues identified in (2.c) match. For example, in one embodiment, residues 46, 87, or 98 match.

[0051] In one embodiment, residue 6 in (1.a), residue 2 in (1.b), and residue 4 in (1.c) match.

[0052] In another embodiment, residue 4 in (2.a), residue 2 in (2.b), and residue 4 in (2.c) match.

[0053] In one embodiment, the heavy chain germline sequences are VH3, VH1, and VH5 germline classes. In another embodiment, the light chain germline sequences are Vkappa or Vlambda sequences.

[0054] The term "treating" refers to treating in an amount, manner, and / or mode effective to improve a condition, symptom, or parameter associated with a disorder, or to prevent progression of the disorder, to a statistically significant degree or to a degree detectable by one of ordinary skill in the art. The effective amount, manner, or mode can be varied depending on the subject and can be tailored to the subject.

[0055] "α4-binding antibody" refers to an antibody that binds to the α4 subunit of VLA-4 (α4β1) integrin and at least partially inhibits the activity of VLA-4, particularly the binding activity or signal transduction activity of VLA-4 integrin (e.g., the ability to transduce VLA-4-mediated signals). For example, a VLA-4-binding antibody can inhibit the binding of VLA-4 to its cognate ligand (e.g., a cell surface protein such as VCAM-1 (vascular cell adhesion molecule-1)) or to an extracellular matrix component (fibronectin or osteopontin). An alpha4-binding antibody can bind to both α4β1 and α4β7. Generally, the antibody binds to the B1 epitope of α4. An α4-binding antibody can bind to VLA-4 with a K of less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, or less than about 10 -11 M of K d and can bind to VLA-4.

[0056] As used herein, the term "antibody" refers to a protein that includes at least one immunoglobulin variable region (e.g., an immunoglobulin variable domain or an amino acid sequence that provides an immunoglobulin variable domain sequence). For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another embodiment, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The light chain of the immunoglobulin can be of the kappa or lambda type. In one embodiment, the antibody is glycosylated. The antibody can be functional with respect to antibody-dependent cytotoxicity and / or complement-mediated cytotoxicity, or can be non-functional with respect to one or both of these activities.

[0057] The VH and VL regions can be further subdivided into regions of hypervariability called "complementary determining regions" ("CDRs"), which are interspersed with more conserved regions called "framework regions" (FRs). The extent of FRs and CDRs is precisely defined (e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition , U.S. Department of Health and Human Services, NIH Publication No. 91 - 3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196 :901 - 917. See also). The Kabat definition is used herein. Each VH and VL generally consists of three CDRs and four FRs, and is arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus.

[0058] An "immunoglobulin domain" refers to a domain derived from a variable or constant domain of an immunoglobulin molecule. An immunoglobulin domain generally contains two β - sheets formed by about seven β - strands and conserved disulfide bonds (e.g., A.F. Williams and A.N. Barclay (1988) Ann. Rev. Immunol. 6 :381 - 405. See also).

[0059] As used herein, an "immunoglobulin variable domain sequence" refers to an amino acid sequence that can form the structure of an immunoglobulin variable domain. For example, this sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, this sequence may omit one or two or more N - terminal or C - terminal amino acids, internal amino acids may include one or more insertions or additional terminal amino acids, or may include other changes. In one embodiment, a polypeptide containing an immunoglobulin variable domain sequence can be associated with another immunoglobulin variable domain sequence to form a target - binding structure (or "antigen - binding site") (e.g., a structure that interacts with VLA - 4).

[0060] The VH or VL chain of an antibody can further include all or part of the heavy or light chain constant region, respectively, so as to form the heavy or light immunoglobulin chain thereby. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains. The heavy and light immunoglobulin chains can be connected by disulfide bonds. The heavy chain constant region generally includes three constant domains, namely CH1, CH2, and CH3. The light chain constant region generally includes the CL domain. The heavy and light chain variable regions contain a binding region that interacts with the antigen. Certain regions of the antibody generally mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0061] The term "immunoglobulin" includes a variety of broad classes of polypeptides that can be biochemically discriminated. Those skilled in the art will understand that heavy chains are classified into gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) in some of their subclasses (e.g., γ1-γ4). This property of the chain determines the "class" of the antibody, as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, etc. are well-characterized and are known to confer functional differentiation. Each modified version of these classes and isotypes can be readily identified by those skilled in the art in view of the present disclosure and is thus within the scope of the present invention. All immunoglobulin classes are clearly within the scope of the present invention. The light chain is classified into either kappa or lambda (κ, λ). Each heavy chain class may bind to a kappa or lambda light chain.

[0062] The term "antigen-binding fragment" of a full-length antibody refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to a target of interest (e.g., VLA-4). Examples of binding fragments included within the scope of "antigen-binding fragments" of full-length antibodies include (i) Fab fragments, i.e., monovalent fragments consisting of the VL, VH, CL, and CH1 domains, (ii) F(ab')2 fragments, i.e., divalent fragments containing two Fab fragments linked by disulfide bridges in the hinge region, (iii) Fd fragments consisting of the VH and CH1 domains, (iv) Fv fragments consisting of the VL and VH domains of one arm of the antibody, (v) dAb fragments consisting of the VH domain (Ward et al., (1989) Nature 341:544-546), and (vi) isolated complementarity-determining regions (CDRs) that retain function. Further, two of the domains of Fv, VL, and VH are encoded by genes, but they can be joined by a synthetic linker that enables them to be made as a single protein chain that pairs the VL region and VH region to form a monovalent molecule known as a single-chain Fv (scFv) using recombinant methods. See, e.g., Bird et al. (1988) Science 242:423-426, and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

[0063] In some embodiments, the foregoing antibodies are pegylated.

[0064] In some embodiments, the foregoing antibodies or fragments thereof are multispecific. In further embodiments, the foregoing antibodies or fragments thereof are monovalent or bispecific.

[0065] Details of one or more embodiments of the invention are set forth in the following accompanying drawings and description. Other features, objects, and advantages of the invention will be apparent from the following description, drawings, and claims. Accordingly, the invention provides the following items: (Item 1) A recombinant antibody molecule or its α4-binding fragment, comprising a variable light chain framework containing a receptor sequence derived from IGKV4-1 and a variable heavy chain framework containing a receptor sequence derived from IGHV1-f, and having heavy and light chain CDRs derived from mouse antibody HP1 / 2 or mouse antibody 21.6. (Item 2) A recombinant antibody molecule or its α4-binding fragment capable of binding to α4, comprising a variable light chain framework containing a receptor sequence (SEQ ID NO: 15) derived from AAH70335.1 with manipulated germline and a variable heavy chain framework containing a receptor sequence derived from IGHV1-f. (Item 3) The recombinant antibody molecule or its α4-binding fragment according to Item 1 or 2, further comprising CDRs derived from mouse antibody HP1 / 2. (Item 4) The recombinant antibody molecule or its α4-binding fragment according to Item 1 or 2, further comprising CDRs derived from mouse antibody 21.6. (Item 5) The recombinant antibody molecule or its α4-binding fragment according to Item 1 or 2, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. (Item 6) The recombinant antibody molecule or its α4-binding fragment according to Item 1, wherein the light chain variable region comprises the sequence of SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. (Item 7) The recombinant antibody molecule or its α4-binding fragment according to Item 2, wherein the light chain variable region comprises the sequence of SEQ ID NO: 11. (Item 8) The antibody according to Item 1 or 2, which binds to VLA-4. (Item 9) A vector comprising DNA encoding an antibody heavy chain or its α4-binding fragment, comprising the sequence of SEQ ID NO: 3, 4, or 5. (Item 10) A vector comprising DNA encoding an antibody light chain or its α4-binding fragment, comprising SEQ ID NO: 8, 9, 10, or 11. (Item 11) A method for producing a recombinant anti-α4 antibody or its α4-binding fragment, comprising: (a)(i) A DNA sequence comprising the sequence of SEQ ID NO: 3, 4, or 5, which encodes an antibody heavy chain or its α4-binding fragment, and (ii) a DNA sequence comprising the sequence of SEQ ID NO: 8, 9, 10, or 11, which encodes an antibody light chain or its α4-binding fragment, to provide a host cell; (b) Culturing the above cells to produce a recombinant anti-α4 antibody molecule or its α4-binding fragment; A method comprising. (Item 12) A method for treating a patient, comprising administering the composition according to Item 1 or 2 to the patient. (Item 13) The method according to Item 12, wherein the patient has cancer. (Item 14) The method according to Item 13, wherein the patient has a solid tumor. (Item 15) The method according to Item 13, wherein the patient has a hematological malignancy. (Item 16) The method according to Item 13, wherein the patient has multiple myeloma or acute myeloid leukemia (AML). (Item 17) The method according to Item 12, wherein the patient has an inflammatory disorder. (Item 18) The method according to Item 12, wherein the patient has multiple sclerosis, asthma, rheumatoid arthritis, diabetes, optic neuritis, or Crohn's disease. (Item 19) The method according to Item 12, wherein the patient has an acute disorder. (Item 20) The method according to Item 12, wherein the patient has a spinal cord injury or a traumatic brain injury. (Item 21) The method according to Item 12, wherein the composition is administered as a dosing schedule. (Item 22) The method according to Item 12, further comprising administering a second therapeutic agent to the patient. (Item 23) The method according to Item 21, wherein the second therapeutic agent is a thrombolytic agent, a chemotherapeutic agent, a neuroprotective agent, an anti-inflammatory agent, a steroid, a cytokine, or a growth factor.

Brief Description of the Drawings

[0066]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7A

Figure 7B

Figure 7C

Figure 8A

Figure 8B

Figure 8C

Figure 9A

Figure 9B

Figure 9C

[0067] Antibodies against VLA-4 have been demonstrated to be useful in treating diseases. For example, natalizumab (Tysabri®), an anti-VLA-4 antibody, is used to treat relapsing multiple sclerosis and Crohn's disease. However, in the treatment of certain conditions, such as acute conditions like spinal cord injury (SCI) or traumatic brain injury (TBI), or in the case of treatments administered in a limited number, such as cancer treatment, it may be advantageous to treat with an anti-VLA-4 that binds with a different affinity (e.g., a higher affinity) than natalizumab. In addition, treatment with anti-VLA-4 antibodies is associated, albeit rarely, with the life-threatening disorder progressive multifocal leukoencephalopathy (PML), in which case, as part of the treatment, removal of the antibody from the treated subject is required, for example, using plasma exchange or immunoadsorption. Since antibody removal is necessary, it is also desirable to balance the advantages of an antibody having an increased affinity for VLA-4 with the disadvantages of the antibody that make removal more difficult or that give rise to risks associated with a slow turnover rate. Such antibodies may also be useful for treating conditions such as multiple sclerosis where less frequent treatment may be required or administration by means other than injection may be more effective. Enabling treatment with lower doses may also reduce the risk of adverse events such as PML. Accordingly, the present invention provides antibodies having such desirable properties.

[0068] The present invention is based at least in part on the unexpected properties of a newly designed humanized α4-binding antibody having a binding affinity for α4 that is 10-fold higher than the binding affinity of the anti-α4 antibody natalizumab.

[0069] Alpha4-binding antibodies and fragments thereof are provided, wherein the variable light chain (VL) and variable heavy chain (VH) frameworks have germline-constructed receptor sequences, such as IGKV4-1, geAAH70335.1, or IGHV1-f antibodies, or antibody sequences engineered from germline. The CDR sequences are derived from non-human anti-alpha4-binding antibodies, such as the anti-VLA-4 antibody HP1 / 2. The antibodies described herein can increase the relative affinity, for example, at least 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, relative to their murine parent. In one embodiment, the increase in affinity is at least 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, but less than 25-fold, 20-fold, or 15-fold, respectively.

[0070] Pharmaceutical composition Alpha4-binding agents, such as VLA-4-binding antibodies, can be formulated as pharmaceutical compositions. Generally, pharmaceutical compositions include a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents that are physiologically compatible.

[0071] "Pharmaceutically acceptable salts" refer to salts that retain the desired biological activity of the parent compound and do not impart any undesirable toxicological effects (see, e.g., Berge, S.M., 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, hydroiodic acid, and those derived from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine.

[0072] The antibody compositions described herein can be formulated according to methods known in the art. Pharmaceutical formulations are well-established techniques and are further described in Gennaro (ed.), Remington:The Science and Practice of Pharmacy , 20 th ed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472), Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems , 7 th Ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727), and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association , 3 rd ed. (2000) (ISBN: 091733096X).

[0073] In one embodiment, the α4 antibody can be formulated with excipient materials such as sodium chloride, disodium phosphate heptahydrate, monosodium phosphate, and polysorbate 80. In another embodiment, the α4 antibody can be formulated in citrate buffer, for example, at pH 5, 5.5, 6, 6.5, 7, or 7.5. In yet another embodiment, the α4 antibody can be formulated in a solution containing 2, 4, 5, 6, 8, 10, 12, 14, or 15% sucrose. For example, it can be provided in buffer at a concentration of about 20 mg / ml and stored at 2 - 8°C.

[0074] The pharmaceutical compositions can also be in various other forms. Such forms include, for example, liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories, among other liquid, semi-solid, and solid dosage forms. The form can depend on the intended method of administration and therapeutic use. Generally, the compositions for the agents described herein are in the form of injectable or infusible solutions.

[0075] Such compositions can be administered by parenteral modes (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular injection). As used herein, the terms "parenteral administration" and "administering parenterally" mean a mode of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal injection and infusion.

[0076] Pharmaceutical compositions generally must be sterile and stable under manufacturing and storage conditions. The pharmaceutical compositions can also be tested to ensure that they meet regulatory and industry standards for administration.

[0077] Compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high drug concentrations. Injectable sterile solutions can be prepared by incorporating the drug described herein in the required amounts in a suitable solvent with one or a combination of the above-described ingredients, and subsequently, if necessary, by sterile filtration. Generally, dispersions are prepared by incorporating the drug described herein into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for preparing injectable sterile solutions, typical methods of adjustment are vacuum drying and lyophilization, which yield the powder of the drug described herein and any additional desired ingredients from its previously sterile-filtered solution. The appropriate fluidity of a solution can be maintained, for example, by using coatings such as lecithin, and in the case of dispersions, by maintaining the required particle size and by using surfactants. Sustained absorption of injectable compositions can be accomplished by including in the composition agents that delay absorption (e.g., monostearates and gelatin).

[0078] Administration The α4-binding antibody can be administered to a subject (e.g., a human subject) by various methods. For a number of applications, the route of administration is one of intravenous injection or infusion, subcutaneous injection, or intramuscular injection. The α4-binding antibody can be administered as a fixed dose or at a dose of mg / kg. The antibody can be administered intravenously (IV) or subcutaneously (SC). For example, the antibody can be administered IV, e.g., once every 4 weeks, between about 50 and 600 mg, or SC, e.g., at least once a week (e.g., twice a week), between about 50 and 100 mg (e.g., 75 mg) as a fixed unit dose. In one embodiment, the antibody is administered IV at a fixed unit dose 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. Administration of the dose IV can be once a week, twice a week, three times a week, or more, once every 2 weeks, once every 3 weeks, once every 4 weeks, or once every 5 weeks, or at a lower frequency.

[0079] In one embodiment, the antibody is administered SC at a fixed unit dose of 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 100 mg, or 120 mg or more. Administration of the dose SC can be once a week, twice a week, three times a week, or more, once every 2 weeks, once every 3 weeks, once every 4 weeks, or once every 5 weeks, or at a lower frequency.

[0080] The anti-α4 antibody can also be administered as a bolus at a dose between about 1 and 10 mg / kg, e.g., about 6.0 mg / kg, 4.0 mg / kg, 3.0 mg / kg, 2.0 mg / kg, 1.0 mg / kg. The modified dose ranges generally include doses of less than about 600 mg / subject, about 400 mg / subject, about 300 mg / subject, about 250 mg / subject, about 200 mg / subject, or about 150 mg / subject for administration once every 4 weeks or once a month. The α4-binding antibody can be administered, for example, once every 3 to 5 weeks, e.g., once every 4 weeks, or once a month.

[0081] Dosing can be adjusted according to the clearance rate of the pre-administered anti-α4 antibody in the patient. For example, the patient does not receive a second or subsequent dose until the level of the α4 antibody in the patient's body falls below a predetermined level. In one embodiment, a patient-derived sample, such as plasma, serum, blood, urine, or cerebrospinal fluid (CSF), is assayed for the presence of the anti-α4 antibody, and if the level of the anti-α4 antibody exceeds a predetermined level, the patient does not receive a second or subsequent dose. When the level of the anti-α4 antibody in the patient's body falls below the predetermined level, the patient receives a second or subsequent dose. A patient determined to have an anti-α4 level that is too high (above the predetermined level) can be retested after 1 day, 2 days, 3 days, or 1 week, and if the level of the anti-α4 antibody in the patient's profile falls below the predetermined level, the patient may receive a second or subsequent dose of the antibody.

[0082] The dose can also be selected to reduce or avoid the production of antibodies against the α4-binding antibody, to achieve saturation exceeding 40%, 50%, 70%, 75%, or 80% of the α4 subunit, to achieve saturation less than 80%, 70%, 60%, 50%, or 40% of the α4 subunit, or to prevent an increase in circulating white blood cell levels.

[0083] In certain embodiments, the active agent may be prepared with a carrier that protects the compound from rapid release, such as a controlled-release formulation including an implant and a microencapsulated delivery system. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Numerous methods for preparing such formulations are patented or generally known. For example, Controlled Drug Delivery (Drugs and the Pharmaceutical Sciences),Second Edition,J.Robinson and V.H.L.Lee,eds. see Marcel Dekker, Inc., New York, 1987.

[0084] The pharmaceutical composition can be administered by a medical device. For example, the pharmaceutical composition can be administered by a needleless 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 include, for example, U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing drug therapy at a controlled rate; U.S. Patent No. 4,486,194, which discloses a treatment device for administering a drug through the skin; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering a drug at an accurate infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having a multi-chamber compartment; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Of course, many other such implants, delivery systems, and modules are also known.

[0085] The present disclosure also features a device for administering a first and a second agent. The device can include, for example, one or more housings for storing a pharmaceutical preparation and can be configured to deliver unit doses of the first and second agents. The first and second agents can be stored in the same or separate compartments. For example, the device can combine the agents prior to administration. It is also possible to use different devices for administering the first and second agents.

[0086] The dosing schedule is adjusted to provide a desired response, such as a therapeutic response or a combined therapeutic effect. Generally, any combination (individual or concurrent formulation) of these doses can be used to provide both the VLA-4 binder and the second agent to the subject in a biologically available amount.

[0087] As used herein, the dosage unit form, i.e., the "fixed dose", refers to physically discrete units suitable as unit dosages for the subject to be treated, each unit containing a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and, optionally, other agents.

[0088] The pharmaceutical composition may contain a "therapeutically effective amount" of the agents described herein. Such an effective amount can be determined based on the combined effects of the first and second agents administered. The therapeutically effective amount of an agent may also vary depending on factors such as the individual's disease state, age, gender, and weight, as well as the ability of the compound to elicit a desired response in the individual, such as improvement of at least one impairment parameter (e.g., a multiple sclerosis parameter), or improvement of the symptoms of at least one impairment such as myoclonus, ataxia, and tremors (e.g., the symptoms of multiple sclerosis). The therapeutically effective amount is also such that any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

[0089] Devices and Kits Formulations containing the antibodies described herein can be administered with a medical device. The device can be designed taking into account features such as portability, storage at room temperature, and ease of use so that it can be used by unskilled persons in the field or emergency responders, for example, in an emergency and transferred to a medical facility and other medical equipment. The device can include, for example, one or more housings for storing a pharmaceutical preparation containing an α4-binding antibody and can be configured to deliver one or more unit doses of the agent.

[0090] For example, the pharmaceutical composition can be administered with a transdermal 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 release control devices. The composition can be administered intravenously with standard IV equipment, such as IV tubing, regardless of the presence or absence of a series filter. In certain embodiments, the device is a syringe for use with SC or IM administration.

[0091] The pharmaceutical composition can be administered by a medical device. For example, the pharmaceutical composition can be administered by a needleless subcutaneous injection device such as the devices described 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 include, for example, U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing drug therapy at a controlled rate; U.S. Patent No. 4,486,194, which discloses a treatment device for administering a drug through the skin; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering a drug at an accurate infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having a multi-chamber compartment; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. The therapeutic composition can also be in the form of a biodegradable or non-biodegradable sustained release formulation for subcutaneous or intramuscular administration. Methods for such compositions are known in the art. Also, continuous administration can be achieved using an implantable pump or an external pump. Administration can also be intermittent, such as by once-daily injection, or continuous at a low dose in a sustained release formulation or the like. The delivery device can be modified to be optimal for the administration of the α4-binding antibody. For example, a syringe can be siliconized to an optimal degree for the storage and delivery of the antibody. Of course, numerous other such implants, delivery systems, and modules are also known.

[0092] The present disclosure also features a device for administering a first and a second agent (e.g., an antibody and a second agent). The device can include, for example, one or more housings for storing a pharmaceutical preparation and can be configured to deliver unit doses of the first and second agents. The first and second agents can be stored in the same or separate compartments. In one embodiment, the device combines the agents prior to administration. In some embodiments, the first and second agents are administered by different devices.

[0093] The α4-binding antibody can be provided in a kit. In one embodiment, the kit includes (a) a container containing a composition comprising a high concentration of a VLA-4-binding antibody, (b) a container containing a composition comprising a second agent, and optionally (c) informational material. The informational material can be a descriptive, instructional, marketing, or other material related to the methods and / or uses of the agents described herein for therapeutic effects. In one embodiment, the kit also includes the second agent. For example, the kit includes a first container containing a composition comprising the α4-binding antibody and a second container containing the second agent.

[0094] The form of the informational material of the kit is not limited. In one embodiment, the informational material can include information regarding the production, concentration, expiration date, batch, or manufacturing site information of the antibody. In one embodiment, the informational material is related to, for example, the method of administering the α4-binding antibody in a suitable dose, dosage form, or mode of administration (e.g., the dose, dosage form, or mode of administration described in the specification) for treating a subject having an acute disorder such as spinal cord injury or traumatic brain injury, or an inflammatory disease (e.g., MS), or who is at risk of experiencing symptoms associated with the inflammatory disease. The information can be provided in various forms, including printed text, computer-readable material, video or audio recordings, or information providing a link or address to a physical material.

[0095] In addition to the agent, the composition of the kit can include other components such as a solvent or buffer, stabilizer, or preservative. The agent can be provided in any form, such as liquid, dry, or lyophilized form, and in a substantially pure and / or sterile form. When the agent is provided as a liquid solution, the liquid solution is generally an aqueous solution. When the agent is provided in dry form, it is generally reconstituted by the addition of a suitable solvent. A solvent, such as sterile water or a buffer, can optionally be provided with the kit.

[0096] The kit can include one or more containers for one or more compositions containing the agent. In some embodiments, the kit includes separate containers, partitions, or compartments for the composition and the information material. For example, the composition may be contained in a bottle, vial, or syringe, and the information 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 information material attached in the form of a label. In some embodiments, the kit includes a plurality of individual containers (e.g., packs), each container containing one or more unit dosage forms of the agent (e.g., the dosage forms described herein). The containers can include combined unit dosages, such as units containing an α4-binding antibody and a second agent, in the desired ratios, etc. For example, the kit can include a plurality of syringes, ampoules, foil packets, blister packs, or medical devices, each containing, for example, a single combined unit dosage. The containers of the kit can be airtight, water-resistant (e.g., impermeable to changes in humidity or evaporation), and / or light-shielding.

[0097] The kit optionally includes a device suitable for administering the composition, such as a syringe or other suitable delivery device. The device can be provided pre-filled with one or both of the agents, or can be empty but suitable for filling.

[0098] Oncology The α4-binding antibodies and methods described herein can be used to treat cancers including solid cancers and hematological malignancies. Exemplary solid cancers include sarcomas and carcinomas such as lung, breast, pancreas, colon, prostate, bladder, and brain. Hematological malignancies include cancers such as multiple myeloma, leukemia, and lymphoma.

[0099] Provided is a method for treating a patient having a hematological malignancy with a composition containing an α4-binding antibody such as an 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 leukemia (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)), lymphoma (such as Hodgkin's disease and non-Hodgkin lymphoma), multiple myeloma, Waldenström macroglobulinemia, myelodysplastic syndromes (MDS) (which can progress to AML), myeloproliferative disorders (such as polycythemia vera (PV, also called PCV or PRV), essential thrombocythemia (ET), myelofibrosis, heavy chain disease, etc.), and amyloidosis due to light chain disease.

[0100] Patients having a hematological malignancy may be identified by, for example, hematocytometry and analysis of blood smear specimens by light microscopy, which are useful for identifying malignant cells. Biopsy materials from bone marrow, etc. can also be used to identify malignant cells, and biopsy materials from lymph nodes can be useful for identifying lymphadenopathy.

[0101] α4-binding antibodies (such as humanized anti-VLA-4 antibodies like HuHP1 / 2, H1L0, H1L1, H1L2, or H1L3) are useful for the treatment of leukemia such as AML. Leukemia is a cancer that occurs in the bone marrow, and the malignant cells are white blood cells (leukocytes). AML (also called acute myeloid leukemia, acute myeloblastic leukemia, acute granulocytic leukemia, and acute non-lymphocytic leukemia) is a malignant tumor that occurs in either granulocytes or monocytes. AML is characterized by the uncontrolled overgrowth and accumulation of cells called leukemic blasts that do not function as normal blood cells, as well as the lack of red blood cells (anemia), platelets (thrombocytopenia), and normal white blood cells (especially neutrophils, i.e., neutropenia) in the blood, resulting from the disruption of the production of normal bone marrow cells.

[0102] All subtypes of AML are suitable for treatment with VLA-4 binding antibodies. Subtypes of AML are classified based on the stage of development that the myeloblasts had reached at the time of diagnosis. The categories and subsets enable physicians to determine which treatment will function optimally for that cell type and how fast the disease can progress. The subsets are M0 (myeloblastic, with special analysis), M1 (myeloblastic, without maturation), M2 (myeloblastic, with maturation), M3 (promyelocytic), M4 (myelomonocytic), M5 (monocytic), M6 (erythroleukemia), and M7 (megakaryocytic). VLA-4 antibodies can be administered together with secondary agents that are particularly suitable for the subtypes of AML. For example, acute promyelocytic leukemia (APL) and acute monocytic leukemia are subtypes of AML that require different treatments from other subtypes of AML. The second agent for treating APL can include all-trans retinoic acid (ATRA) or an antimetabolite such as cytarabine. The second agent for treating acute monocytic leukemia can include a deoxyadenosine analogue such as 2-chloro-2'-deoxyadenosine (2-CDA).

[0103] The risk factors for AML include the presence of certain genetic disorders such as Down syndrome, Fanconi anemia, Shwachman-Diamond syndrome, etc. Patients with AML and a genetic disorder can be administered a VLA-4 binding antibody and a second agent to treat the symptoms of the genetic disorder. For example, patients with AML and Fanconi anemia can be administered a VLA-4 binding antibody and an antibiotic.

[0104] Other risk factors for AML include chemotherapy or radiation therapy for treating different cancers, tobacco smoke, and exposure to large amounts of benzene.

[0105] Other cancers suitable for treatment with an α4 binding antibody include solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0106] Other disorders The formulations and methods described herein can also be used to treat inflammatory, immune, or autoimmune disorders such as inflammation of the central nervous system (e.g., in addition to multiple sclerosis, meningitis, neuromyelitis optica, neurosarcoidosis, CNS vasculitis, encephalitis, and transverse myelitis), tissue or organ transplant 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 disease (e.g., Crohn's disease, ulcerative colitis (e.g., treatment or maintenance of remission)), eosinophilic gastroenteritis, myasthenia gravis, fibromyalgia, rheumatic / immunological disorders associated with arthritis (e.g., rheumatoid arthritis, psoriatic arthritis), dermatological disorders such as inflammatory / immune skin 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., systemic progressive sclerosis (PSS), pulmonary PSS, etc.), acute or chronic eosinophilic pneumonia, Sjogren's syndrome, acute coronary syndrome (ACS), acute myocardial infarction, atherosclerosis, and fibrotic disorders (e.g., pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), pulmonary fibrosis (e.g., radiation-induced XRT-induced), myelofibrosis, cirrhosis, mesangial proliferative glomerulonephritis, crescentic glomerulonephritis, diabetic nephropathy, and interstitial renal fibrosis).

[0107] The formulations and methods described herein can also be used to treat neurological disorders such as cerebral ischemia, including prevention of transient ischemic attack and / or arterial stenosis in a patient. Other exemplary neurological disorders include chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barré syndrome (GBS), eye diseases such as macular degeneration (e.g., wet macular degeneration) and 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.

[0108] The formulations and methods described herein can also be used to treat patients who have undergone transplantation, such as kidney, heart, or bone marrow transplantation.

[0109] Multiple sclerosis Formulations containing the alpha4-binding antibodies described herein are useful for the treatment of inflammatory diseases such as multiple sclerosis (MS). Multiple sclerosis is a central nervous system disease characterized by inflammation and loss of the myelin sheath.

[0110] Patients with MS may be identified by criteria that establish a clinically definite diagnosis of MS as defined by a workshop on the diagnosis of MS (Poser et al., Ann. Neurol. 13:227, 1983). For example, an individual with clinically definite MS has two attacks and clinical evidence of any two lesions, or clinical evidence of one lesion and subclinical evidence of another separate lesion. Definite MS can also be diagnosed by two attacks and evidence of IgG oligoclonal bands in the cerebrospinal fluid, or by a combination of an attack, two lesions, and clinical evidence of IgG oligoclonal bands in the cerebrospinal fluid. The McDonald criteria can also be used to diagnose MS. (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 impairment over time in the absence of multiple clinical attacks for the diagnosis of MS. Effective treatments for multiple sclerosis may be evaluated in a number of different ways. To measure the effect of treatment, the following parameters can be used. That is, two exemplary criteria are the appearance of worsening on the EDSS (Expanded Disability Status Scale), and MRI (magnetic resonance imaging). The EDSS is a means of grading clinical disability due to MS (Kurtzke, Neurology 33:1444, 1983). Eight functional systems are evaluated for the type and severity of neuropathy. Briefly, before treatment, the patient is evaluated for impairment in the pyramidal, cerebellar, brainstem, sensory, bowel and bladder, visual, cerebral, and other systems. Follow-up examinations are performed at regular intervals. The scale ranges from 0 (normal) to 10 (death due to MS). A one-step decrease indicates that the treatment is effective (Kurtzke, Ann. Neurol. 36:573-79, 1994). Patients may also be diagnosed using other criteria used by those skilled in the art.

[0111] Deterioration is defined as the appearance of new symptoms that are attributable to MS and are accompanied by appropriate new neurological abnormalities (IFNB MS Study Group, supra). Further, the deterioration must last at least 24 hours and must occur no sooner than after at least 30 days of stability or improvement. Briefly, the patient undergoes a standard neurological examination by a clinician. Deterioration is either mild, moderate, or severe according to changes in a neurological assessment scale (Sipe et al., Neurology 34:1368, 1984). The annual rate of deterioration and the proportion of patients without deterioration are determined.

[0112] Treatment can be considered effective if there is a statistically significant difference between the treatment group and the placebo group in the proportion or rate of patients without deterioration or without recurrence for any of these measurements. Further, the time to the first deterioration, as well as the duration and severity of the deterioration, can be measured. In this regard, a measure of effectiveness as a treatment is a statistically significant difference in the time or period to the first deterioration and in the severity of the treatment group compared to the control group. It is particularly noteworthy if the period without deterioration or without recurrence exceeds 1 year, 18 months, or 20 months. Effectiveness can also be evaluated using any method used in the art to assess symptoms of MS, including improvement in motor function using a timed walking test used alone or in combination with other criteria.

[0113] The effectiveness of administering the first agent and optionally the second agent can also be evaluated based on one or more of the following criteria. Namely, the frequency of MBP-reactive T cells determined by the limiting dilution method, the proliferative response of MBP-reactive T cell lines and clones, and the cytokine profile of T cell lines and clones against MBP established from the patient. Effectiveness is indicated by a decrease in the frequency of reactive cells, a decrease in thymidine incorporation in which modified peptides are incorporated compared to native peptides, and a decrease in TNF and IFN-α.

[0114] Clinical measurements include relapse rates at 1- and 2-year intervals, and changes in EDSS including the time from baseline to progression of 1.0 unit in EDSS that persists for 6 months. Delay in the continuous progression of disability on the Kaplan-Meier curve indicates efficacy. Other criteria include changes in the area and volume of T2 images on MRI, and changes in the number and volume of lesions determined by gadolinium-enhanced imaging.

[0115] MRI can be used to measure active lesions using gadolinium-DTPA enhanced imaging (McDonald et al. Ann. Neurol. 36:14, 1994), or to measure the location and extent of lesions using T2-weighted methods. Briefly, a baseline MRI is obtained. The same imaging plane and patient position are used for each subsequent study. The positioning and imaging sequences can be selected to maximize lesion detection and facilitate lesion follow-up. The same positioning and imaging sequences can be used for subsequent studies. The presence, location, and extent of MS lesions can be determined by a radiologist. The lesion area can be outlined and summarized for each slice of the total lesion area. Three analyses of new lesion evidence, active lesion emergence rate, and lesion area change rate may be performed (Paty et al., Neurology 43:665, 1993). Improvement with treatment can be established by statistically significant improvement in individual patients compared to baseline or between treatment and placebo groups.

[0116] Exemplary symptoms associated with multiple sclerosis that can be treated by the methods described herein include optic neuritis, diplopia, nystagmus, ocular dysmetria, internuclear ophthalmoplegia, movement and sound phosphenes, centripetal pupillary disorder, paresis, monoparesis, hemiparesis, hemiplegia, quadriparesis, paralysis, diplegia, monoplegia, tetraplegia (tetraplegia, quadraplegia), spasticity, dysarthria, muscle atrophy, spasms (spasms, cramps), hypotonia, interictal spasm, interictal myospasm, muscle undulation, restless legs syndrome, foot drop, areflexia, sensory disturbance, sensory paralysis, neuralgia, neuropathic pain and neurogenic pain, Lhermitte's sign, proprioceptive insufficiency, trigeminal neuralgia, ataxia, intention tremor, dysmetria, vestibular ataxia, dizziness, speech ataxia, dystonia, failure of reciprocal antagonistic movement, frequency of micturition, bladder spasm, flaccid bladder, detrusor-sphincter dyssynergia, erectile dysfunction, sexual dysfunction, anesthesia, constipation, tenesmus, fecal incontinence, depression, cognitive insufficiency, dementia, mood swings, emotional instability, a feeling of intoxication, bipolar syndrome, anxiety neurosis, aphasia, dysphasia, fatigue, Uhthoff's sign, gastroesophageal reflux, and sleep disorders.

[0117] Each case of MS presents one of several patterns of presentation and subsequent course. Most commonly, MS first appears as a series of attacks and then, inexplicably, remits completely or partially so that the symptoms subside, and recurs after a period of stability. This is called relapsing-remitting (RR) MS. Primary progressive (PP) MS is characterized by a progressive clinical decline with no clear remissions, although there may be temporary periods of stability or slight improvement in symptoms. Secondary progressive (SP) MS begins with a relapsing-remitting course and later follows a primary progressive course. Rarely, patients may have a progressive-relapsing (PR) course, in which the disease follows a progressive path interrupted by acute attacks. PP, SP, and PR are sometimes collectively referred to as chronic progressive MS.

[0118] A small number of patients experience malignant MS, defined as a rapid and relentless decline, which can result in significant disability or death soon after the onset of the disease. This decline can be suppressed or slowed by administering the combination therapies described herein.

[0119] Administration of an anti-α4 antibody as taken up in this specification can be effective for reducing one or more symptoms of MS, such as one or more of the aforementioned symptoms. For example, administration of an anti-α4 antibody as described in this specification can be used to treat primary or secondary progressive multiple sclerosis (PPMS or SPMS, respectively), and treatment with an anti-α4 antibody can be effective in preventing relapses.

[0120] In addition to or prior to human studies, animal models can be used to evaluate the effectiveness of using two agents. Exemplary animal models of multiple sclerosis are experimental autoimmune encephalomyelitis (EAE) mouse models such as those described in 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). In mice, the first and second agents as described in this specification can be administered prior to EAE induction. The mice are then evaluated for characteristic criteria to determine the effectiveness of using the two agents in that model.

[0121] Antibody production Recombinant antibodies that bind to alpha4 can be generated by in vivo or in vitro methods such as phage display. This method can be used to supply anti-α4 CDRs for use in the CDR-grafted antibodies described in this specification. In addition, methods such as phage display can be used to select such CDRs in the context of the germline frameworks disclosed in this specification by using libraries where the framework is a germline framework.

[0122] European Patent No. EP 239 400 (Winter et al.) describes the modification of antibodies by replacing certain complementarity determining regions (CDRs) with those of another species (within a given variable region). Since CDR-substituted antibodies contain significantly fewer non-human components, they may be less likely to induce an immune response in humans compared to true chimeric antibodies (Riechmann et al., 1988, Nature 332, 323-327; Verhoeyen et al., 1988, Science 239, 1534-1536). Generally, the CDRs of murine antibodies are replaced into the corresponding regions of human antibodies by using recombinant nucleic acid techniques to produce sequences encoding the desired substituted antibodies. Human constant region gene segments of the desired isotype (usually gamma I for CH and kappa for CL) can be added, and the heavy and light chain genes can be co-expressed in mammalian cells to produce soluble antibodies. Also, large non-immune phage display libraries may be used to isolate high affinity antibodies that can 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; U.S. 2003-0232333).

[0123] The anti-α4 or antibody fragments described herein can recognize epitopes of the α4 subunit that are involved in binding to cognate ligands, such as VCAM-1 or fibronectin. The antibodies described herein can inhibit binding to one or more cognate ligands, such as VCAM-1 and fibronectin.

[0124] In some embodiments, the antibodies discussed herein can interact with VLA-4 on cells, such as lymphocytes, but do not cause cell aggregation.

[0125] Exemplary α4-binding antibodies have one or more CDRs, such as all three heavy chain (HC) CDRs and / or all three light chain (LC) CDRs of the specific antibodies disclosed herein, or, in summary, have CDRs that are at least 80, 85, 90, 92, 94, 95, 96, 97, 98, 99% identical to such antibodies. In one embodiment, the H1 and H2 hypervariable loops have the same canonical structure as those of the antibodies described herein. In one embodiment, the L1 and L2 hypervariable loops have the same canonical structure as those of the antibodies described herein.

[0126] In one embodiment, the amino acid sequences of the HC and / or LC variable domain sequences are at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to the amino acid sequences of the HC and / or LC variable domains of the antibodies described herein. The amino acid sequences of the HC and / or LC variable domain sequences may differ from the corresponding sequences of the antibodies described herein by at least one amino acid, but not more than 10, 8, 6, 5, 4, 3, or 2. For example, this difference may be primarily or entirely within the framework regions.

[0127] The amino acid sequences of the HC and LC variable domain sequences can be encoded by nucleic acid sequences that hybridize under high stringency conditions to the nucleic acid sequences described herein, or by nucleic acid 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 antibodies described herein. In one embodiment, one or more heavy or light chain framework regions (e.g., HC FR1, 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 from human germline antibodies.

[0128] The calculation of "homology" or "sequence identity" (these terms are used interchangeably herein) between two sequences is performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). The optimal alignment is determined as the best score using the GAP program of the GCG software package with a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Next, the amino acid residues or nucleotides are compared at the corresponding amino acid or nucleotide positions. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (the "identity" of an amino acid or nucleic acid as used herein corresponds to the "homology" of an amino acid or nucleic acid). The percent identity between two sequences is a function of the number of identical positions shared by the sequences.

[0129] The term "hybridizes under high stringency conditions" as used herein describes the conditions for hybridization and washing. Guidance for performing the hybridization reaction can be found in Current Protocols in Molecular Biology , John Wiley & Sons N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and non-aqueous methods are described in that reference and either can be used. High stringency hybridization conditions include hybridization in 6X SSC at about 45° C. followed by one or more washes at 65° C. in 0.2X SSC, 0.1% SDS, or substantially similar conditions.

[0130] Antibody production Antibodies can be produced in prokaryotic and eukaryotic cells. In one embodiment, antibodies (e.g., scFvs) are expressed in yeast cells such as Pichia spp. (see, e.g., Powers et al. (2001) J Immunol Methods. 251:123-35), Hansenula spp., or Saccharomyces spp.

[0131] In one embodiment, antibodies, particularly full-length antibodies (e.g., IgG), are produced in mammalian cells. Exemplary mammalian host cells for recombinant expression include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220 and used with a DHFR selectable marker as described in, e.g., Kaufman and Sharp (1982) Mol. Biol. 159:601-621), lymphocyte cell lines such as NS0 myeloma cells and SP2 cells, COS cells, K562, and transformed animals, e.g., cells derived from transformed mammals. For example, the cells are mammary epithelial cells.

[0132] In addition to the nucleic acid sequence encoding the immunoglobulin domain, the recombinant expression vector may carry additional nucleic acid sequences such as sequences that regulate the replication of the vector in the host cell (e.g., origin of replication) and a selectable marker gene. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). Exemplary selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0133] In an exemplary system for the recombinant expression of an antibody (e.g., a full-length antibody or an antigen-binding portion thereof), a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy chain gene and the antibody light chain gene are each operably linked to an enhancer / promoter regulatory element (e.g., a CMV enhancer / AdMLP promoter regulatory element or an SV40 enhancer / AdMLP promoter regulatory element, etc. derived from SV40, CMV, adenovirus, etc.) to drive high-level gene transcription. The recombinant expression vector also retains the DHFR gene, enabling the selection of CHO cells transfected with the vector using methotrexate selection / amplification. The selected transformed host cells are cultured to enable the expression of the antibody heavy chain and the antibody light chain, and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select the transformants, culture the host cells, and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with protein A or protein G. For example, a purified α4-binding antibody can be concentrated to about 100 mg / mL to about 200 mg / mL using protein concentration techniques known in the art.

[0134] The antibody may also include modifications, e.g., modifications that alter Fc function, to reduce or eliminate interaction with, for example, Fc receptors or C1q or both. For example, the human IgG4 constant region can have a mutation from Ser to Pro at residue 228 to lock the hinge region. The amino acid sequence of IgG4 Fc (hinge + CH2 + CH3 domains) is provided in FIG. 5.

[0135] In another embodiment, the human IgG1 constant region can be mutated at residues, e.g., one or more of residues 234 and 237, according to, for example, U.S. Patent No. 5,648,260. Other exemplary modifications include those described in U.S. Patent No. 5,648,260.

[0136] For some antibodies that include an Fc domain, the antibody production system may be designed to synthesize antibodies in which the Fc region is glycosylated. In another embodiment, the Fc domain of an IgG molecule is glycosylated at asparagine 297 of the CH2 domain (see Figure 5). This asparagine is the site for modification by a biantennary oligosaccharide. This glycosylation is involved in effector functions mediated by Fcγ receptors 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 a mammalian expression system that appropriately glycosylates the residue corresponding to asparagine 297. The Fc domain can also include other eukaryotic post-translational modifications.

[0137] Other suitable Fc domain modifications include those described in International Publication No. WO2004 / 029207. For example, the Fc domain can be XmAb® Fc (Xencor, Monrovia, CA). The Fc domain or a fragment thereof can have substitutions in the Fcγ receptor (FcγR) binding region, such as the domains and fragments described in International Publication No. WO05 / 063815. In some embodiments, the Fc domain or a fragment thereof can have substitutions in the neonatal Fc receptor (FcRn) binding region, such as the domains and fragments described in International Publication No. WO05047327. In other embodiments, the Fc domain is single-chain, a fragment thereof, or a modified version thereof, such as those described in International Publication No. WO2008143954. Other suitable Fc modifications are known and described in the art.

[0138] Antibodies can also be produced by transgenic animals. For example, U.S. Patent No. 5,849,992 describes a method for expressing antibodies in the mammary gland of transgenic mammals. The transgene is constructed to include a milk-specific promoter and nucleic acid sequence encoding the antibody of interest, such as the antibodies described herein, and a signal sequence for secretion. Milk produced by female such transgenic mammals contains the antibody of interest secreted therein, such as the antibodies described herein. The antibodies can be purified from the milk or, in some applications, used directly.

[0139] Antibodies can be modified, for example, in a portion that improves their stability and / or residence in circulation, such as in blood, serum, lymph, bronchoalveolar lavage fluid, or other tissues, by at least 1.5, 2, 5, 10, or 50-fold.

[0140] For example, VLA-4 binding antibodies can be associated with a polymer, such as a polyalkylene oxide or polyethylene oxide, such as a substantially non-antigenic polymer. Suitable polymers vary substantially in weight. Polymers having a number average molecular weight in the range of about 200 to about 35,000 (or about 1,000 to about 15,000, and 2,000 to about 12,500) daltons can be used.

[0141] For example, a VLA-4 binding antibody can be conjugated to a water-soluble polymer, such as a hydrophilic vinyl polymer (e.g., polyvinyl alcohol or polyvinyl pyrrolidone). A non-limiting list of such polymers includes polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylenated polyols, copolymers thereof, and block copolymers thereof, provided that the water solubility of the block copolymer is maintained. Further useful polymers include polyoxyalkylene (such as polyoxyethylene, polyoxypropylene, copolymers of polyoxyethylene, and polyoxypropylene (Pluronic), etc.); polymethacrylate; carbomer; saccharide monomers D-mannose, D-galactose, 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 homopolysaccharides and heteropolysaccharides (such as lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextran sulfate, dextran, dextrin, glycogen, or acidic mucopolysaccharides (e.g., hyaluronic acid), etc.) containing neuraminic acid, branched or unbranched polysaccharides; polymers of sugar alcohols such as polysorbitol and polymannitol; heparin or heparan.

[0142] Exemplary second agent In some cases, the formulations described herein, such as those containing an alpha4 binding antibody, are administered either in combination with a formulation containing a second agent or in combination with a second agent.

[0143] In one implementation example, the α4-binding antibody and the second agent are provided as a co-formulation, and this co-formulation is administered to the subject. Further, for example, at least 24 hours before or after administering the co-formulation, a single dose of the α4-binding antibody is administered, and then a single dose formulation containing the second agent is administered separately. In another implementation example, the antibody and the second agent are provided as separate formulations, and the administration step includes sequentially administering the antibody and the second agent. The sequential administration can be provided on the same day (e.g., within 1 hour of each other, or at least 3, 6, or 12 hour intervals), or on different days.

[0144] Generally, the antibody and the second agent are each administered as multiple doses at intervals of time. The antibody and the second agent are generally each administered according to a dosing schedule. One or both of the dosing schedules may have a certain periodicity. The dosing schedule of the antibody can have a different periodicity from the dosing schedule of the second agent, for example, one can be administered more frequently than the other. In one implementation example, one of the antibody and the second agent is administered once a week and the other is administered once a month. In another implementation example, one of the antibody and the second agent is administered continuously, for example, over a period exceeding 30 minutes but less than 1, 2, 4, or 12 hours, and the other is administered as a bolus. The antibody and the second agent can be administered by any suitable method, for example, subcutaneously, intramuscularly, or intravenously.

[0145] In some embodiments, each of the antibody and the second agent is administered at the same dose as the dose prescribed for each for monotherapy. In other embodiments, the antibody is administered at a dose that is less than the amount required for efficacy when administered alone. Similarly, the second agent can be administered at a dose that is less than the amount required for efficacy when administered alone.

[0146] Non-limiting examples of a second agent for treating multiple sclerosis in combination with an α4-binding antibody include · Interferons, for example, human interferon beta 1a (e.g., AVONEX® or Rebif®) and interferon beta 1b (BETASERON™, human interferon beta substituted at position 17, Berlex / Chiron), · Glatiramer acetate (also known as copolymer 1 (Cop-1), COPAXONE®, Teva Pharmaceutical Industries, Inc.), · Rituxan® (rituximab) or another anti-CD20 antibody (e.g., one that competes with or binds to overlapping epitopes with rituximab), · Mitoxantrone (NOVANTRONE®, Lederle), · Chemotherapeutic agents, for example, cladribine (LEUSTATIN®), azathioprine (IMURAN®), cyclophosphamide (CYTOXAN®), cyclosporin A, methotrexate, 4-aminopyridine, and tizanidine, · Corticosteroids (e.g., methylprednisolone (MEDRONE®, Pfizer), prednisone), · Immunoglobulins, for example, Rituxan® (rituximab), CTLA4Ig, alemtuzumab (MabCAMPATH®), or daclizumab (an antibody that binds CD25), · Statins, and · TNF antagonists, are included.

[0147] Glatiramer acetate is a protein formed by a random chain of the amino acids - glutamic acid, lysine, alanine, and tyrosine (hence, GLATiramer). Glatiramer acetate can be synthesized in solution from these amino acids at a ratio of approximately 5 parts alanine to 3 parts lysine, 1.5 parts glutamic acid, and 1 part tyrosine using N-carboxyamino acid anhydrides.

[0148] Additional 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-II, GM-CSF, FGF, and PDGF. Further exemplary second agents include antibodies to cell surface molecules, such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90, or their ligands. For example, daclizumab is an anti-CD25 antibody that can improve multiple sclerosis.

[0149] Still other exemplary antibodies include antibodies that provide the activity of the agents described herein, such as antibodies that engage an interferon receptor, e.g., an antibody that engages the interferon beta receptor. Generally, in embodiments where the second agent comprises an antibody, it binds to a target protein other than VLA-4 or alpha4 integrin, or to at least an epitope on VLA-4 other than that recognized by the first agent.

[0150] Still other additional exemplary second agents include FK506, rapamycin, mycophenolate mofetil, leflunomide, non-steroidal anti-inflammatory drugs (NSAIDs), e.g., phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents that interfere with signaling by pro-inflammatory cytokines as described herein, IL-1β converting enzyme inhibitors (e.g., Vx740), anti-P7, PSGL, TACE inhibitors, T cell signaling inhibitors, e.g., kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurine, angiotensin converting enzyme inhibitors, soluble cytokine receptors and their derivatives as described herein, anti-inflammatory cytokines (e.g., IL-4, IL-10, IL-13, and TGF).

[0151] In some embodiments, the second agent can 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, docusate, pemoline, dantrolene, desmopressin, dexamethasone, tolterodine, phenytoin, oxybutynin, bisacodyl, venlafaxine, amitriptyline, methenamine, clonazepam, isoniazid, vardenafil, nitrofurantoin, psyllium hydrophilic mucilloid, alprostadil, gabapentin, nortriptyline, paroxetine, propantheline bromide, modafinil, fluoxetine, phenazopyridine, methylprednisolone, carbamazepine, imipramine, diazepam, sildenafil, bupropion, and sertraline. A number of second agents that are small molecules have a molecular weight between 150 and 5000 daltons.

[0152] Examples of TNF antagonists include chimeric antibodies, humanized antibodies, human antibodies, or antibodies generated in vitro (or antigen-binding fragments thereof) against TNF (e.g., human TNFα), (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), etc.); anti-TNF antibody fragments (e.g., CPD870); soluble fragments of TNF receptors, e.g., p55 or p75 human TNF receptors or derivatives thereof, e.g., 75kdTNFR-IgG (75kD TNF receptor-IgG fusion protein, ENBREL™); Immunex (e.g., Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A), see also p55kdTNFR-IgG (55kD TNF receptor-IgG fusion protein (LENERCEPT™))); enzyme antagonists (e.g., TNFα converting enzyme (TACE) inhibitors (e.g., alpha sulfonyl hydroxamic acid derivatives, International Publication No. WO01 / 55112, and N-hydroxyformamide TACE inhibitors GW 3333, -005, or -022)); and TNF-bp / s-TNFR (soluble TNF binding proteins, e.g., see Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S284, Amer. J. Physiol.-Heart and Circulatory Physiology (1995) Vol. 268, pp. 37-42).

[0153] In addition to the second agent, it is also possible to deliver other agents to the subject. However, in some embodiments, no protein other than the α4-binding antibody and the second agent, nor any biologic, is administered to the subject as a pharmaceutical composition. The α4-binding antibody and the second agent may be the only agents delivered by injection. In embodiments where the second agent is a recombinant protein, the α4-binding antibody and the second agent may be the only recombinant agents administered to the subject, or the only recombinant agents that modulate an immune or inflammatory response. In still other embodiments, the α4-binding antibody alone may be the only recombinant agent or the only biologic agent administered to the subject.

[0154] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, and the appropriate methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Examples

[0155] Example 1. The variable anti-VLA-4 antibody is more potent than humanized HP1 / 2. Anti-VLA-4 antibodies were constructed using the germline framework IGKV4-1 (designs L1 and L2), or AAH7033.1 (design L3) with the germline manipulated for the VL chain and the germline framework IGHV1-f for the VH chain. These antibodies had fewer revertant mutations than the humanized HP1 / 2 antibody described in U.S. Patent No. 6,602,503.

[0156] Heavy chain mutations The arrays of three mutations of the heavy chain are shown in FIG. 1 as Design H0, Design H1, and Design H2. Each design has the CDRs of murine HP1 / 2 transplanted into the IGHV1-f framework. Design H0 contains no revertant mutations in the framework region, while Design H1 and Design H2 have various degrees of revertant mutations in the framework region sequence to optimize the affinity of the humanized antibody.

[0157] Light chain mutations The arrays of four mutations of the light chain are shown in FIG. 2 as Design L0, Design L1, Design L2, and Design L3 (also referred to as L0, L1, L2, L3). Each design has the CDRs of murine HP1 / 2 transplanted into the germline framework. The IGKV4-1 germline framework was used for Design L0, Design L1, and Design L2, and the engineered framework of AAH70335 germline was used for Design L3. Design L0 contains no revertant mutations in the framework region, while Design L1, Design L2, and Design L3 have various degrees of revertant mutations in the framework region to optimize the affinity of the humanized antibody.

[0158] The results of the competitive ELISA assay are shown in Table 1 and FIG. 3. In this experiment, α4β1 was pre-incubated with the test mAb, and then murine HP1 / 2 was used as the competitor. The results of this experiment showed that the antibodies having light chain L2 or L3 were more potent than the humanized antibody HuHP1 / 2 described in U.S. Patent No. 6,602,503. The results are shown in Table 1 below and in FIG. 3. The heavy chain (H1) of the antibody for this assay has the "Design H1" sequence shown in FIG. 1, while L1 refers to Design L1 shown in FIG. 2.

Table 1

[0159] In Table 1, the chimeric mAb is the chimerized HP1 / 2 antibody, and the murine 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 murine HP1 / 2 antibody (Sanchez-Madrid et al., Eur. J. Immunol. 16:1343-1349, 1996). The results of the experiments indicate that it is possible to improve the affinity of monoclonal antibodies for their murine parent sequences through humanization of the receptor framework engineered in the germline.

[0160] Another competitive assay compares the binding affinity of the new antibody to the humanized 21.6 anti-α4 antibody (Tysabri® (natalizumab)) described in U.S. Patent No. 5,840,299. In this experiment, the binding of a mixture of murine HP1 / 2 with the test mAb to α4β1 was assayed. The results of this experiment are shown in Figure 4 and Table 2 below, indicating that the newly designed antibody is approximately 10-fold more potent than natalizumab.

Table 2

[0161] Example 2. Humanized HP1 / 2 (HuHP1 / 2) binds to VLA-4 on 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 with the CLL (chronic lymphocytic leukemia patient) cell lines Mec1 and JM1, the MM (multiple myeloma) cell lines U266 and H929, and the AML (acute myeloid leukemia patient) cell lines HL60 and KG1. HuHP1 / 2 bound to all of the tumor cell lines tested (Figure 6). The flow cytometry data were used to calculate EC50 values for the antibodies binding to each of the different cell lines. This information is shown in Table 3 below.

[0162] HuHP1 / 2 was also found to block the adhesion of AML cell lines to fibronectin (FN) and VCAM1-Ig fusion protein. To test whether the antibody could block adhesion, the AML cell lines HL60 or KG1 were allowed to adhere to FN-coated wells (Figure 7A) or VCAM1-Ig-coated wells (Figure 7B) in the presence of increasing concentrations of HP1 / 2 or isotype control antibody. HuHP1 / 2 blocked the adhesion of both cell types to FN-coated wells and VCAM1-Ig-coated wells. The maximum inhibition of HL60 cell binding to both ligands was achieved with 20 μg / mL of HuHP1 / 2 (Figure 7C).

[0163] HuHP1 / 2 was also found to block the adhesion of MM cell lines to FN and VCAM1-Ig fusion protein. The MM cell lines U266 and H929 were allowed to adhere to FN-coated wells (Figure 8A) or VCAM1-Ig-coated wells (Figure 8B) in the presence of increasing concentrations of HP1 / 2 or isotype control antibody. HuHP1 / 2 blocked the adhesion of both cell line types to FN-coated wells and VCAM1-Ig-coated wells. The maximum inhibition of U266 cell binding to both ligands was achieved with 20 μg / mL of HuHP1 / 2 (Figure 8C).

[0164] HuHP1 / 2 was also found to block the adhesion of CLL cell lines to FN and VCAM1-Ig fusion protein. The CLL cell lines Mec1 and JM1 were allowed to adhere to FN-coated wells (Figure 9A) or VCAM1-Ig-coated wells (Figure 9B) in the presence of increasing concentrations of HP1 / 2 or isotype control antibody. HuHP1 / 2 blocked the adhesion of both cell line types to FN-coated wells and VCAM1-Ig-coated wells. The maximum inhibition of Mec1 cell binding to both ligands was achieved with 20 μg / mL of HuHP1 / 2 (Figure 9C).

[0165] The IC50 values for the binding of HuHP1 / 2 to tumor cell lines were calculated from the data shown in Figures 7 - 9. These data are shown in Table 3.

Table 3

[0166] Other embodiments are within the scope of the claims.

Claims

1. A recombinant antibody molecule or its α4 binding fragment having heavy and light chain CDRs derived from the mouse antibody HP1 / 2, comprising a variable heavy chain framework sequence derived from IGHV1-f (SEQ ID NO: 2) and a variable light chain framework sequence derived from IGKV4-1 (SEQ ID NO: 7) or AAH70335.1 (SEQ ID NO: 13) with manipulated germline, wherein: (a) the variable heavy chain of the recombinant antibody molecule comprises CDRs derived from the VH chain of the mouse antibody HP1 / 2, CDR1 comprises the sequence GFNIKDTYM (SEQ ID NO: 16), CDR2 comprises the sequence RIDPASGDTKYDPKFQV (SEQ ID NO: 17), and CDR3 comprises the sequence GMWVSTGYALDF (SEQ ID NO: 18); (b) the variable light chain of the recombinant antibody molecule comprises CDRs derived from the VL chain of the mouse antibody HP1 / 2, CDR1 comprises the sequence KASQSVTNDVA (SEQ ID NO: 19), CDR2 comprises the sequence YASNRYT (SEQ ID NO: 20), and CDR3 comprises the sequence QQDYSSPYT (SEQ ID NO: 21); (c) the variable heavy chain of the recombinant antibody molecule comprises substitutions at framework positions 24 and 94 according to the Kabat numbering scheme, the residue substituted at framework position 24 is alanine (A), and the residue substituted at framework position 94 is aspartic acid (D); the recombinant antibody molecule or its α4 binding fragment binds to VLA-4 with a higher binding affinity than that of its mouse parent. Recombinant antibody molecule or its α4 binding fragment.

2. A recombinant antibody molecule or its α4 binding fragment capable of binding to α4, comprising heavy and light chain CDR1-3 derived from the mouse antibody HP1 / 2, a variable light chain framework comprising a receptor sequence derived from IGKV4-1 (SEQ ID NO: 7), and a variable heavy chain framework comprising a receptor sequence derived from IGHV1-f (SEQ ID NO: 2), wherein: The variable light chain framework contains substitutions at framework positions 1, 67, 73, 85, and 87 according to the Kabat numbering scheme, the residue substituted at framework position 1 is serine (S), the residue substituted at framework position 67 is tyrosine (Y), the residue substituted at framework position 73 is phenylalanine (F), the residue substituted at framework position 85 is threonine (T), and the residue substituted at framework position 87 is phenylalanine (F), The recombinant antibody molecule or its α4 binding fragment binds to VLA-4 with a higher binding affinity than that of its mouse parent, The heavy chain CDR1-3 derived from the mouse antibody HP1 / 2 includes CDR1 containing the sequence GFNIKDTYM (SEQ ID NO: 16), CDR2 containing the sequence RIDPASGDTKYDPKFQV (SEQ ID NO: 17), and CDR3 containing the sequence GMWVSTGYALDF (SEQ ID NO: 18), The light chain CDR1-3 derived from the mouse antibody HP1 / 2 includes CDR1 containing the sequence KASQSVTNDVA (SEQ ID NO: 19), CDR2 containing the sequence YASNRYT (SEQ ID NO: 20), and CDR3 containing the sequence QQDYSSPYT (SEQ ID NO: 21), A recombinant antibody molecule or its α4 binding fragment.

3. The recombinant antibody molecule or its α4 binding fragment according to claim 2, wherein the light chain variable region contains the sequence of SEQ ID NO:

11.

4. a) DNA encoding an anti-α4 antibody heavy chain or its heavy chain variable region containing SEQ ID NO: 4 or 5; b) DNA encoding an anti-α4 antibody light chain or its light chain variable region containing SEQ ID NO: 9, 10, or 11 A vector comprising wherein the anti-α4 antibody heavy chain or its heavy chain variable region, and the anti-α4 antibody light chain or its light chain variable region form an antibody or a fragment thereof that specifically binds to α4.

5. A combination comprising a vector containing DNA encoding an anti-α4 antibody heavy chain or a heavy chain variable region thereof, which contains SEQ ID NO: 4 or 5, and a vector containing DNA encoding an anti-α4 antibody light chain or a light chain variable region thereof, which contains SEQ ID NO: 9, 10 or 11, wherein the anti-α4 antibody heavy chain or its heavy chain variable region, and the anti-α4 antibody light chain or its light chain variable region form an antibody or a fragment thereof that specifically binds to α4.

6. A method for producing a recombinant anti-α4 antibody molecule or an α4-binding fragment thereof, comprising: (a) providing a host cell comprising (i) a DNA sequence encoding an anti-α4 antibody heavy chain or a heavy chain variable region thereof, which contains SEQ ID NO: 4 or 5, and (ii) a DNA sequence encoding an anti-α4 antibody light chain or a light chain variable region thereof, which contains SEQ ID NO: 9, 10 or 11; (b) culturing the cells to produce the recombinant anti-α4 antibody molecule or an α4-binding fragment thereof that specifically binds to α4. A method comprising the above steps.

7. A composition comprising the recombinant antibody molecule or an α4-binding fragment thereof according to claim 1 for treating a patient.

8. The composition according to claim 7, wherein the patient has cancer.

9. The composition according to claim 8, wherein the patient has a solid tumor, a hematological malignancy, multiple myeloma or acute myeloid leukemia (AML).

10. The composition according to claim 7, wherein the patient has an inflammatory disorder, multiple sclerosis, asthma, rheumatoid arthritis, diabetes, optic neuritis, Crohn's disease, acute injury, spinal cord injury or traumatic brain injury.

11. The composition according to claim 7, wherein the composition is administered according to a dosing schedule at a regular interval selected from once a day, once a week or once a month; 1 to 4 times a week; once every two weeks; once every three weeks; or once every four weeks.

12. The composition according to claim 7, wherein the composition is administered to the patient in combination with a second therapeutic agent.

13. The composition according to claim 12, wherein the second therapeutic agent is a thrombolytic agent, a chemotherapeutic agent, a neuroprotective agent, an anti-inflammatory agent, a steroid, a cytokine, or a growth factor.

14. The recombinant antibody molecule according to claim 2 or an α4 binding fragment thereof, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 4 or SEQ ID NO:

5.

15. The antibody according to claim 2, wherein the antibody binds to VLA-4.

16. A composition comprising the recombinant antibody molecule according to claim 2 or an α4 binding fragment thereof for treating a patient.

17. The composition according to claim 16, wherein the patient has cancer.

18. The composition according to claim 17, wherein the patient has a solid tumor, a hematological malignancy, multiple myeloma or acute myeloid leukemia (AML).

19. The composition according to claim 16, wherein the patient has an inflammatory disorder, multiple sclerosis, asthma, rheumatoid arthritis, diabetes, optic neuritis, Crohn's disease, an acute disorder, a spinal cord injury or a traumatic brain injury.

20. The composition according to claim 16, wherein the composition is administered according to a dosing schedule at regular intervals selected from once a day, once a week or once a month; 1 to 4 times a week; once every two weeks; once every three weeks; or once every four weeks.

21. The composition according to claim 16, wherein the composition is administered to the patient in combination with a second therapeutic agent.

22. The composition according to claim 21, wherein the second therapeutic agent is a thrombolytic agent, a chemotherapeutic agent, a neuroprotective agent, an anti-inflammatory agent, a steroid, a cytokine, or a growth factor.

23. A composition for treating a patient suffering from a disease or disorder mediated by α4 integrin, comprising a recombinant anti-α4 antibody molecule having heavy and light chain CDRs derived from the mouse antibody HP1 / 2, comprising a variable heavy chain framework sequence derived from IGHV1-f (SEQ ID NO: 2) and a variable light chain framework sequence derived from IGKV4-1 (SEQ ID NO: 7) or AAH70335.1 (SEQ ID NO: 13) with engineered germline, or an α4 binding fragment thereof, (a) the variable heavy chain of the recombinant anti-α4 antibody molecule comprises CDRs derived from the VH chain of the mouse antibody HP1 / 2, CDR1 comprises the sequence GFNIKDTYM (SEQ ID NO: 16), CDR2 comprises the sequence RIDPASGDTKYDPKFQV (SEQ ID NO: 17), and CDR3 comprises the sequence GMWVSTGYALDF (SEQ ID NO: 18); (b) the variable light chain of the recombinant anti-α4 antibody molecule comprises CDRs derived from the VL chain of the mouse antibody HP1 / 2, CDR1 comprises the sequence KASQSVTNDVA (SEQ ID NO: 19), CDR2 comprises the sequence YASNRYT (SEQ ID NO: 20), and CDR3 comprises the sequence QQDYSSPYT (SEQ ID NO: 21), (c) the variable heavy chain of the recombinant anti-α4 antibody molecule comprises substitutions at framework positions 24 and 94 according to the Kabat numbering scheme, the residue substituted at framework position 24 is alanine (A), and the residue substituted at framework position 94 is aspartic acid (D), wherein the recombinant anti-α4 antibody molecule or its α4 binding fragment binds to VLA-4 with a higher binding affinity than that of its mouse parental counterpart, Composition.

Images