EA046416B1 - ANTI-BODIES AGAINST VLA-4 - Google Patents

Description

The present invention relates to alpha-4 protein binding antibodies and fragments thereof.

State of the art

Humanized antibodies can be used as therapeutic agents in place of mouse antibodies to avoid an unwanted immune response called human anti-mouse antibody (HAMA) response. Humanized antibodies are generally constructed by replacing the complementarity determining regions (CDRs) of a human antibody with CDR regions from another species, typically a region from a mouse antibody.

The VLA-4 protein (also called 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 limited to lymphoid and myeloid cells. VLA-4 binds to the endothelial cell ligand VCAM-1 (vascular endothelial adhesion molecule-1) and can mediate the attachment of T and B lymphocytes to the heparin P-binding fragment of human plasma fibronectin.

The essence of the invention

The inventors have discovered that germline variable region frameworks can be used to optimize alpha-4 binding antibodies with grafted CDR regions, such as anti-VLA-4 antibodies. Accordingly, the invention provides heavy chain variable regions (VH) and light chain variable regions (VL), and anti-VLA-4 antibody molecules containing such framework regions.

In one embodiment, the present invention provides an anti-α4 antibody VH chain comprising CDR regions from a donor anti-α4 antibody, such as the anti-α4 antibody described herein, and a VH chain framework region comprising regions 1, 2, 3 and 4 from sequence or bearing no more than 5, 10 or 15 differences from the germline variable region sequence for the VH chain. In one embodiment, variable region framework 4 (FR4) is a human consensus sequence. In one embodiment, the complete sequences of the 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.

In one embodiment, the germline sequence is IGHV1-f (SEQ ID NO: 2) shown in FIG. 1. In some embodiments, the VH chain framework sequence may differ by at least one, but not more than 2, 3, 4, 5, 10, or 15 amino acid residues from the germline sequence, e.g., SEQ ID NO: 2. B In one embodiment, the VH chain framework region further includes residues different from the corresponding residues in the human sequence. For example, the VH chain contains residues not found in humans at one or more of positions 24, 67, 76, 80, and 94 in the framework region (Kabat numbering) of SEQ ID NO: 2.

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

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

In another embodiment, at least one amino acid residue and less than 2, 3, 4, 5 or 10 amino acid residues in the FR1 region of the VH region differ from the corresponding residue found in the human germline sequence. One or more such residues may, for example, be identical to residues in the framework region of the non-human antibody from which the CDR sequences are derived. In one embodiment, the amino acid residue at position 24 (in Kabat numbering) is mutated so that it is identical to a residue in the framework region of a non-human antibody.

In another embodiment, at least one amino acid residue and less than 2, 3, 4, 5 or 10 amino acid residues in the FR2 region of the VH region differ from the corresponding residue found in the human germline sequence. One or more such residues may, for example, be identical to residues in the framework region of the non-human antibody from which the CDR sequences are derived.

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In another embodiment, at least one amino acid residue and less than 2, 3, 4, 5 or 10 amino acid residues in the FR3 region of the VH chain differ from the corresponding residue found in the human germline sequence. One or more such residues may, for example, be identical to residues in the framework region of the non-human antibody from which the CDR sequences are derived. In one embodiment, the amino acid residue at position 94 (in Kabat numbering) is identical to a residue in the framework region of a non-human antibody. In yet another embodiment, the amino acid residues at positions 67, 76, 80 and 94 (as used in Kabat numbering) are identical to residues in the framework region of the non-human antibody.

In some embodiments, the antibody VH chain has the sequence SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

In one aspect, the present invention provides an anti-VLA-4 antibody VL chain comprising CDR regions from a donor anti-VLA-4 antibody, such as the anti-VLA-4 antibody described herein, and a VL chain framework region comprising regions 1, 2, 3 and 4 from the sequence or bearing no more than 5, 10 or 15 differences (either per region or as a whole) from the germline variable region sequence for the VL chain. In one embodiment, variable region framework 4 (FR4) is a human consensus sequence. In one embodiment, the complete sequences of the framework regions FR1, FR2, FR3 and FR4 of the VL chain are present. In another embodiment, the chain is a VL region antigen binding fragment.

In yet another embodiment, the germline sequence is IGKV4-1 (SEQ ID NO: 7) shown in FIG. 2. In other embodiments, the VL chain framework sequence may differ by at least one, but not more than 2, 3, 4, 5, 10, or 15 amino acid residues from the germline framework sequence, e.g., SEQ ID NO: 7 In another embodiment, the VL chain further includes amino acid residues different from the corresponding residues in the human sequence. For example, the VL chain further contains non-human residues at one or more positions 1, 73 and 87 in the framework region (Kabat numbering) of SEQ ID NO: 7.

In one embodiment, the sequence is AAH7035.1 (SEQ ID NO: 12) or a germline engineered version thereof (SEQ ID NO: 13) shown in FIG. 2. In some embodiments, the VL chain framework sequence may differ by at least one, but not more than 5, 10, 15, 20, or 25 amino acid residues from the genetically engineered germline framework sequence, e.g., SEQ ID NO: 13. In one embodiment, the VL chain includes residues different from the corresponding residues in the human sequence. For example, the VL chain contains non-human residues at one or more positions 1 and 87 in the framework region (Kabat numbering) in SEQ ID NO: 12. In another embodiment, the VL contains amino acid substitutions in the framework regions made such that the sequence was similar to another human germline framework sequence, such as the IGKV4-1 germline sequence. In some embodiments, the VL chain framework sequence is altered to be identical to the IGKV4-1 germline sequence at positions 1-3, 5-23, 35-37, 39-42, 45-49, 57, 59- 61, 63-64, 70-72, 74-84, 86-88, 99-106 (Kabat numbering) in SEQ ID NO: 12.

In one embodiment, at least one or more complementarity determining regions (CDRs) of the variable domains are derived from a non-human α4-binding donor antibody. In another embodiment, the antigen binding regions of the heavy chain variable domain with the transplanted CDRs include CDRs corresponding to positions 24-31 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) (Kabat numbering). Thus, in one embodiment, the VL chain framework region comprises an acceptor sequence constructed from the IGKV4-1 germline sequence, the AAH70335.1 antibody, or the genetically engineered AAH70335.1 antibody.

In another embodiment, at least one amino acid residue and less than 2, 3, 4, 5, 10 or 15 amino acid residues in the FR1 region of the VL chain differ from the corresponding residue found in the human sequence. One or more such residues may, for example, be identical to residues in the framework region of the non-human antibody from which the CDR sequences are derived. In one embodiment, an amino acid residue at the N-terminal position of FR1 is mutated so that it is identical to a residue in the framework region of a non-human antibody.

In yet another embodiment, at least one amino acid residue and less than 2, 3, 4, 5, 10 or 15 amino acid residues in the FR2 region of the VL chain differ from the corresponding residue found in the human sequence. One or more such residues may, for example, be identical to residues in the framework region of the non-human antibody from which the CDR sequences are derived.

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In yet another embodiment, at least one amino acid residue and less than 2, 3, 4, 5, 10 or 15 amino acid residues in the FR3 region of the VL chain differ from the corresponding residue found in the human sequence. One or more such residues may, for example, be identical to residues in the framework region of the non-human antibody from which the CDR sequences are derived. In yet another embodiment, the amino acid residue at position 87 (in Kabat numbering) is mutated to be identical to a residue in the framework region of a non-human antibody. In yet another embodiment, the amino acid residue at positions 67 and 87 (in Kabat numbering) is mutated to be identical to a residue in the framework sequence of a non-human antibody. In yet another embodiment, the amino acid residue at positions 67, 73 and 87 (in Kabat numbering) in SEQ ID NO: 7 is mutated to be identical to a residue in the framework region of a non-human antibody.

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

In one embodiment, the CDRs of the VH and VL chain framework region acceptor sequences are selected to be similar to the CDR sequences in a non-human (e.g., mouse) antibody sequence, wherein the non-human antibody binds to alpha-4 integrin or a fragment of it. In another embodiment, the CDR sequences are selected to be similar to the CDR sequences of a non-human antibody that binds to the B1 epitope on the VLA4 α4 chain. In one embodiment, the CDRs are selected to be similar to regions of a mouse monoclonal antibody, for example, HP1/2, HP2/1, HP2/4, L25, P4C2, or 21.6 (Pulido et al., J. Biol. Chem 266:10241-10245. 1991; US Patent No. 6033665). Modifications may mean, for example, cleavage and insertion or alteration, for example, by site-directed mutagenesis.

Another feature is that the invention provides an antibody or antigen binding fragment thereof comprising:

An anti-VLA-4 antibody VL chain described herein, e.g., an anti-VLA-4 antibody VL chain comprising a CDR from a donor anti-VLA-4 antibody, e.g., an anti-VLA-4 antibody described herein, and a VL chain framework region , containing the light chain framework regions 1, 2 and 3 of the sequence or having no more than 5, 10 or 15 differences from the germline variable region sequence for the VL chain. In one embodiment, variable region 4 is a human consensus sequence; And

An anti-VLA-4 antibody VH chain described herein, e.g., an anti-VLA-4 antibody VL chain containing a CDR from a donor anti-VLA-4 antibody, e.g., an anti-VLA-4 antibody described herein, and a VL chain framework region , containing the light chain framework regions 1, 2 and 3 of the sequence or having no more than 5, 10 or 15 differences from the germline variable region sequence for the VL chain. In one embodiment, variable region 4 is a human consensus sequence.

In one embodiment, the antibody binds one or both of the α4β1 and α4β7 proteins.

In another aspect, a VL or VH chain, or an antibody or fragment thereof described herein is labeled such that detection is possible.

In yet another aspect, the invention provides a vector containing DNA encoding an antibody heavy chain or α4 binding fragment thereof described herein. In some embodiments, the vector DNA encodes a VH chain having the sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

Another aspect is that the invention provides a vector containing DNA encoding an antibody light chain or α4 binding fragment thereof described herein. In some embodiments, the vector DNA encodes a VL strand having the sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

In yet another aspect, the invention provides a vector comprising DNA encoding an antibody heavy chain or α4-binding fragment thereof described herein and an antibody light chain or α4-binding fragment thereof described herein.

Another aspect is that the invention provides a host cell containing a vector described herein, for example, a vector capable of expressing the heavy and/or light chain of an antibody or antibody fragment described herein.

In one aspect, the invention provides a method for producing a recombinant anti-α4 antibody or an α4-binding fragment thereof by obtaining a host cell transfected with (a) a DNA sequence encoding an antibody heavy chain described herein or an α4-binding fragment thereof; and (b) a DNA sequence encoding an antibody light chain described herein or an α4-binding fragment thereof, and by culturing the transfected cell to produce a recombinant anti-α4 antibody molecule or an α4-binding fragment thereof. DNA encoding

- 3 046416 the heavy and light chains of the antibody can be obtained in the same vector or on different vectors.

In one aspect, the invention provides a method for producing a recombinant anti-α4 antibody or an α4-binding fragment thereof by obtaining a host cell transfected with (a) a DNA sequence encoding an antibody heavy chain or an α4-binding fragment thereof, for example, wherein the DNA sequence may have sequences 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, wherein the DNA sequence may be SEQ ID NO: 8, 9, 10, or 11, and by culturing the transfected cell 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 within the same vector or on different vectors.

In yet another aspect, the invention provides a method of treating a disease or condition mediated by an α4 integrin, for example, α4β1 (VLA-4) or α4β7 integrin, by administering an anti-α4 antibody or antibody fragment described herein, or a pharmaceutical composition containing the antibody or fragment , a subject who requires such treatment. The subject may have or be at risk of developing, for example, inflammatory, immune or autoimmune disorders (eg, central nervous system inflammation such as multiple sclerosis, meningitis, neuromyelitis optica, neurosarcoidosis, CNS vasculitis, encephalitis and transverse myelitis), risk of tissue rejection or organ graft or graft-versus-host disease, acute CNS injury such as stroke, traumatic brain injury (TBI), or spinal cord injury (SCI); chronic kidney disease; allergies, such as allergic asthma; type 1 diabetes mellitus; inflammatory bowel disease such as Crohn's disease, ulcerative colitis; myasthenia gravis; fibromyalgia; joint disorders such as rheumatoid arthritis, psoriatic arthritis; inflammatory/immune skin disorders such as psoriasis, vitiligo, dermatitis, lichen planus; systemic lupus erythematosus; Gougerot-Sjögren syndrome; hemoblastoses, such as multiple myeloma, leukemia, lymphoma; solid cancers such as sarcomas or carcinomas, eg lung, breast, prostate, brain; and fibrotic disorders such as pulmonary fibrosis, myelofibrosis, liver cirrhosis, mesangial proliferative glomerulonephritis, crescentic glomerulonephritis, diabetic neuropathy and renal interstitial fibrosis.

In another aspect, the invention provides a method of treating a patient by administering to the patient an antibody or antibody fragment that binds to α4. In one embodiment, the patient has a cancer, such as a solid tumor or hematologic malignancy. For example, a patient receiving treatment with an antibody or antibody fragment that binds to α4 may suffer from acute myelogenous leukemia (AML) or multiple myeloma (MM).

In another embodiment, the patient suffers from an inflammatory disorder, such as multiple sclerosis, asthma (eg, moderate to severe asthma), rheumatoid arthritis, diabetes, or Crohn's disease. In another embodiment, the composition is administered in accordance with a specific regimen. In another embodiment, the method further includes selecting a patient suitable for treatment with the composition. A patient eligible for treatment, for example, exhibited a sign or symptom indicative of disease, such as a sign or symptom indicative of multiple sclerosis.

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

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

In one embodiment, a composition comprising an α4-binding antibody is administered according to a specific schedule, for example, at regular intervals. For example, the composition may be administered once daily, weekly or monthly; once a week, twice a week, three times a week, four times a week or more often; or once every two weeks, once every three weeks, once every four weeks or more.

In one embodiment, the dosage may be adjusted according to the patient's observed rate of clearance of previously administered anti-α4 antibody. For example, in one embodiment, the patient will not be administered a second or subsequent dose until the level of anti-α4 antibodies in the patient has dropped to a level lower than a predetermined level. In one embodiment, a sample collected from a patient (eg, a plasma, serum, blood, or urine sample) is analyzed for the presence of anti-α4 antibodies, and if the level of anti-α4 antibodies is higher than a predetermined level, the patient will not be given a second or subsequent dose. If the patient's anti-α4 antibody level is below a predetermined level, the patient is given a second or subsequent dose.

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In one embodiment, the composition is administered continuously, for example, over a period of more 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 route, for example, subcutaneously, intramuscularly or intravenously.

In some embodiments, each component, the antibody and the second agent, is administered at the same dose as when each was administered as monotherapy. In other embodiments, the antibody is administered at a dosage that is equal to or less than the amount required for effective treatment when administered without an additional agent. Similarly, the second agent can be administered at a dosage that is equal to or less than the amount required for effective treatment when administered without the antibody.

Another aspect provided by the invention is a method of assessing a patient's condition by determining whether the patient meets a preselected criterion, and, if the patient meets the preselected criterion, a method of approving, providing, prescribing or administering to the patient a VLA-binding antibody composition. 4 described here. In one embodiment, the preselected criterion is the patient's failure to adequately respond to a prior alternative therapeutic treatment or regimen, such as in the case of treatment for multiple sclerosis. In another embodiment, the preselected criterion is the absence of any signs or symptoms of progressive multifocal leukoencephalopathy (PML) or the absence of any diagnosis of PML. In some cases, the selection is based on the absence of a risk factor for PML, for example, the subject does not have a positive JC virus DNA test or does not have a positive JC virus antibody test. In another embodiment, the criterion is the criterion described in PCT/US07/75577 (published as WO 2008/021954), incorporated herein by reference, which describes methods and systems for dispensing drugs and for delivering drugs to patients.

In another aspect, a method of dispensing a composition described herein is provided. The composition contains an alpha-4 binding antibody. The method includes providing to a recipient (e.g., an end user, a patient, a physician, a pharmaceutical retailer, wholesaler, distributor, or pharmaceutical department in a hospital, nursing clinic, or health care organization) a package containing a sufficient number of dosage units of a medicinal product to treat the patient in for at least 6, 12, 24, 36 or 48 months. In yet another aspect, the invention provides a method for assessing the quality of a package or series of packages (eg, to determine the potential expiration date) of a composition described herein containing an alpha-4 binding antibody. The method includes assessing whether the packaging has passed its expiration date. The shelf life is at least 6, 12, 24, 36 or 48 months, for example more than 24 or 36 months, starting from a predetermined event such as production, analysis or packaging. In some embodiments, the decision or execution of a step occurs as a result of the analysis. For example, depending on the correct analysis, the antibody in the package is used or discarded, classified, selected, released or suspended, transported, moved to another location, released for sale, sold or offered for sale, withdrawn from sale or no longer offered for sale. sales, depending on whether the product has expired.

In another aspect, the invention provides a package containing at least two unit doses of an aqueous composition containing an α4-binding antibody. In one embodiment, all unit dosages contain the same amount of antibody, and in other embodiments there are unit dosages with two or more dosage levels or with two or more different compositions, for example, having different dosages or release properties.

In another aspect, the invention includes a method of instructing a recipient to administer a composition comprising an α4-binding antibody. The method includes instructing a recipient (e.g., an end user, a patient, a physician, a pharmaceutical retailer, wholesaler, distributor, or an employee of a pharmaceutical department in a hospital, nursing clinic, or health care organization) that the antibody is to be administered to the patient as described herein regime. The method may also include instructing the recipient that the antibody must be administered before the expiration date. The shelf life is at least 6, 12, 24, 36 or 48 months, for example more than 24 or 36 months, starting from a predetermined event such as production, analysis or packaging. In one embodiment, the recipient also receives a supply of the antibody, such as a unit dose supply of the antibody.

In yet another aspect, the invention provides a method of producing an antibody that contains a CDR from a donor antibody, such as a non-human antibody, such as a mouse antibody, and framework regions of variable regions of one or both heavy and light chains derived from the framework region ( regions) variable region of the human embryonic type. The method includes one or both of items 1 and 2, where items 1 and 2 are as follows:

1) identification or selection of the framework region of the variable region of the stable acceptor heavy

- 5 046416 human chain which contains one or more of the same residues as the non-human donor heavy chain in one or more of the positions specified in points a), b) and c):

a) VH according to Kabat No. 2, 4, 24, 26, 27, 29, 36, 38, 46, 47, 48, 49, 66, 67, 69, 71, 78, 93 and 94, which are not believed to be according to some particular theory, are important for maintaining CDR conformation;

b) VH according to Kabat No. 1, 2, 27, 28, 30, 43, 66, 68, 70, 72, 73, 74 and 75, which are believed, without following any particular theory, to be able to interact with the antigen and ;

c) Kabat VH Nos. 37, 39, 44, 45, 47, 91, 93 and 103, which are believed, without following any particular theory, to be important in preserving the interaction between VH and VL; And

2) identifying or selecting a framework region of the variable region of a stable acceptor light chain that contains one or more of the same residues as the donor light chain in one or more of the positions specified in paragraphs a), b) and c):

a) Kabat VL Nos. 2, 4, 38, 43, 44, 48, 58, 64, 71 and 73, which are believed, without following any particular theory, to be important for maintaining the CDR conformation;

b) Kabat VL No. 1, 2, 49, 57, 60, 63, 65, 66, 67, 68, 69 and 70, which are believed, without following any particular theory, to potentially interact with the antigen and;

c) Kabat VL Nos. 36, 38, 43, 44, 46, 49, 87 and 98, which are believed, without following any particular theory, to be important in preserving the interaction between VH and VL;

3) creating a variable region containing donor CDRs and a selected germline framework region having matching residues specified in paragraphs. 1 and 2, such that by selecting a germline sequence and further introducing back mutations of additional residues specified in claims 1 and 2 in the germline sequence with restoration of the mouse sequence, further maximizing the match of the residues specified in claims 1 and 2; And

4) evaluation of each matching position, for example using spatial structure analysis or modeling, and in the event that the position satisfies pre-established risk standards, for example, interfering with CDR conformation, interaction with an antigen or maintaining interaction between VH and VL, - reintroduction of an equivalent residue from the mouse sequence or a standard residue for a human antibody that is compatible with the structure of the antibody.

In one embodiment, at least 3, 4 or 5 of the residues identified in (1.a) are the same. For example, in one embodiment, residues 24, 29, or 94 are the same.

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

In one embodiment, at least 3, 4 or 5 of the residues identified in (1.c) are the same. For example, in one embodiment, residues 37, 93, or 103 are the same.

In one embodiment, at least 3, 4 or 5 of the residues identified in (2.a) are the same. For example, in one embodiment, residues 2, 71 and 73 are the same.

In one embodiment, at least 3, 4 or 5 of the residues identified in (2.b) are the same. For example, in one embodiment, residues 1, 68, or 70 are the same.

In one embodiment, at least 3, 4 or 5 of the residues identified in (2.c) are the same. For example, in one embodiment, residues 46, 87, or 98 are the same.

In one embodiment, residue 6 in (1.a), residue 2 in (1.b), and residue 4 in (1.c) are the same.

In another embodiment, residue 4 in (2.a), residue 2 in (2.b), and residue 4 in (2.c) are the same.

In one embodiment, the germline heavy chain sequence belongs to the germline class VH3, VH1 and VH5. In another embodiment, the germline light chain sequence is a V-kappa or V-lambda sequence.

The term treatment refers to the administration of a therapeutic agent in an amount, manner, and/or technique effective to improve a condition, symptom, or parameter associated with a disorder, or to prevent the progression of a disorder, to a statistically significant extent or to an extent that can be determined by one skilled in the art. field of technology. The effective amount, method or technique may vary depending on the subject and may be adjusted for the subject.

The term α4-binding antibody means an antibody that binds to the α4 subunit of VLA-4 integrin (α4β 1) and at least partially inhibits VLA-4 activity, particularly VLA-4 integrin binding activity or signaling activity, e.g. VLA-4 protein-mediated signal. For example, an antibody that binds to VLA-4 may inhibit the binding of VLA-4 to a self-ligand of VLA-4, such as a cell surface protein such as VCAM-1 (vascular endothelial adhesion molecule-1), or an extracellular matrix component such like fibronectin or osteopontin. Alpha-4 binding antibody can bind to both α4β1 and α4β7. Typically, the antibody binds to the B1 epitope on α4. Antibody related

- 6046416 called α4, can bind to VLA-4 with a Kd of less than about 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 or 10 ^-11 M.

As used herein, the term antibody means a protein that contains at least one immunoglobulin variable region, for example, an amino acid sequence that contains an immunoglobulin variable domain or an immunoglobulin variable domain sequence. For example, an antibody may comprise a heavy (H) chain variable region (abbreviated herein as VH) and a light chain variable region (L) (abbreviated herein as VL). In yet another example, the antibody contains two heavy (H) chain variable regions and two light (L) chain variable regions. Immunoglobulin light chains can be of the kappa or lambda type. In one embodiment, the antibody is glycosylated. An antibody may be functionally active in antibody-mediated cytotoxicity and/or complement-mediated cytotoxicity or may be functionally inactive in one or both of these activities.

The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). The size of the FR and CDR has been precisely determined (see 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 196:901917). Definitions according to Kabat's numbering are used here. Each VH and VL region typically consists of three CDRs and four FRs, arranged from the N (amino) terminus to the C (carboxyl) terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 , FR4.

The term immunoglobulin domain means a domain from the variable or conserved domain of immunoglobulin molecules. Immunoglobulin domains typically contain two β-sheets formed from about seven β-structures and a conserved disulfide bond (see, for example, A. F. Williams and A. N. Barclay (1988) Ann. Rev. Immunol. 6:381-405).

As used herein, the term immunoglobulin variable domain sequence means an amino acid sequence that can form the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may be missing one, two or more N- or C-terminal amino acids, internal amino acids, may include one or more insertions or additional terminal amino acids, or may contain other changes. In one embodiment, a polypeptide that includes an immunoglobulin variable domain sequence can be associated with another immunoglobulin variable domain sequence to form a target binding structure (antigen binding site), for example, a structure that interacts with VLA-4.

The VH or VL chain of an antibody may further comprise all or part of a heavy or light chain conserved region and thus form an immunoglobulin heavy or light chain, respectively. In one embodiment, the antibody is a tetramer of two immunoglobulin heavy chains and two immunoglobulin light chains. The heavy and light chains of immunoglobulin can be linked by disulfide bonds. The heavy chain conserved region typically consists of three conserved domains, CH1, CH2 and CH3. The light chain conserved region typically contains a CL domain. The variable region of the heavy and light chains contains a binding domain that interacts with the antigen. Conserved regions of antibodies typically can mediate binding of the antibody to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.

The term immunoglobulin includes a wide variety of classes of polypeptides that can be distinguished from each other by their biochemical properties. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses within the classes (eg, γ1-γ4). It is the nature of this chain that 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 have functional specialization. The difference between modified versions of each of these classes and isotypes will be readily apparent to those skilled in the art in connection with the present invention, and are therefore within the scope of the present invention. All classes of immunoglobulins are clearly within the scope of the present invention. Light chains are classified as kappa or lambda (κ, λ). Each class of heavy chain can bind to either a kappa or lambda light chain.

The term antigen-binding fragment of a full-length antibody means one or more fragments of a full-length antibody that retain the ability to specifically bind to a target of interest, for example, VLA-4. Examples of binding fragments referred to as a full-length antibody antigen binding fragment include (i) Fab

- 7 046416 fragment, monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) E(ab')2 fragment. a bivalent fragment containing two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment, consisting of the VL and VH domains of a single antibody fragment, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) that retains functional activity. Additionally, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be combined using recombinant techniques through a synthetic linker, allowing them to be produced as a single protein chain in which the VL and VH regions are paired to form monovalent molecules , known as single-chain Fv (scFv). See, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

In some embodiments, the antibodies described above are pegylated.

In some embodiments, the antibodies or fragments thereof described above are multispecific. In additional embodiments, the antibodies or fragments thereof described above are monovalent or bispecific.

A detailed description of one or more embodiments of the invention is set forth in the accompanying drawings and description below. Other features, objects and advantages of the invention will be apparent from the description and drawings, as well as from the claims.

Brief description of the figures

In fig. Figure 1 shows three HP1/2 heavy chain sequence variants for the human germline heavy chain IGHV1-f. Lowercase letters above the sequence indicate insertions, according to Kabat's numbering scheme.

In fig. Figure 2 shows four variants of the HP1/2 light chain sequence for the germline antibody sequence IGKV4-1 (design L0, L1 and L2) or the genetically engineered kappa chain sequence of the human germline antibody AAH7033.1 (design L3). Lowercase letters above the sequence indicate insertions, according to Kabat's numbering scheme.

In fig. 3 is a graph depicting the results of the ELISA assay.

In fig. 4 is a graph depicting the results of the ELISA assay.

In fig. Figure 5 shows the amino acid sequence of IgG4 Fc (hinge + CH2 domain + CH3 domain). The hinge region is indicated in bold and the CH3 domain is underlined. The letter S placed in a box represents Ser228. The letter N placed in a circle represents Asn297.

In fig. 6 is a graph depicting flow cytometry data obtained from HuHP1/2 binding assays to various tumor cell lines. HP1/2 means humanized HP1/2.

In fig. 7A-7C are a panel of graphs illustrating the inhibition by HuHP1/2 molecules of binding of AML (acute myelogenous leukemia) cell lines to fibronectin or to wells coated with immobilized VCAM1-Ig. In fig. 7A shows the inhibition of binding of HL60 and KG1 cells to fibronectin-coated wells. In fig. 7B shows the inhibition of binding of KG1 cells to VCAM1-Ig-coated wells. In fig. 7C shows the inhibition of binding of HL60 cells to fibronectin- and VCAM1-Ig-coated wells when incubated with 20 μg/ml HuHP1/2 (solid bars). Open bars indicate the percentage of cell adhesion in the presence of a control with the same isotype. HP1/2 means humanized HP1/2.

In fig. 8A-8C are a panel of graphs illustrating the inhibition by HuHP1/2 molecules of binding of MM (multiple myeloma) cell lines to fibronectin or to wells coated with immobilized VCAM1-Ig. In fig. 8A shows the inhibition of binding of U266 and H929 cells to fibronectin-coated wells. In fig. 8B shows the inhibition of binding of U266 and H929 cells to VCAM1-Ig-coated wells. In fig. 8C shows the inhibition of binding of U266 cells to fibronectin- and VCAM1-Ig-coated wells when incubated with 20 μg/ml HuHP1/2 (solid bars). Open bars indicate the percentage of cell adhesion in the presence of a control with the same isotype. HP1/2 means humanized HP1/2.

In fig. 9A-9C are a panel of graphs illustrating the inhibition by HuHP1/2 molecules of binding of CLL (chronic lymphocytic leukemia) cell lines to fibronectin or to wells coated with immobilized VCAM1-Ig. In fig. 9A shows the suppression of binding of Mec1 and JM1 cells to fibronectin-coated wells. In fig. 9B shows the suppression of binding of Mec1 and JM1 cells to VCAM1-Ig-coated wells. In fig. 9C shows the inhibition of binding of Mec1 cells to fibronectin- and VCAM1-Ig-coated wells when incubated with 20 μg/ml HuHP1/2 (solid bars). Open bars indicate the percentage of cell adhesion in the presence of a control with the same isotype. HP1/2 means humanized HP1/2.

Detailed description of the invention

Antibodies against VLA-4 have been demonstrated to be useful in treating the disease. For example, natalizumab (Tysabri®), an anti-VLA-4 antibody, is used to treat recurrent MS.

- 8 046416 sclerosis and Crohn's disease. However, for the treatment of certain conditions, such as acute conditions such as spinal cord injury (SCI) or traumatic brain injury (TBI), or for treatments used in limited quantities, such as cancer treatment, it may be useful to treat with an antibody against VLA-4, which has a different binding affinity than natalizumab, for example, has a higher affinity. In addition, treatment with anti-VLA-4 antibodies is associated with a rare but sometimes fatal disorder, progressive multifocal leukoencephalopathy (PML), the treatment of which includes, among other things, removal of the antibody from the body of the treated subject, for example, using plasma replacement or immunoabsorption. Because of the need to remove the antibody, it is also desirable to balance the advantages of an antibody that has increased affinity for VLA-4 with the disadvantages of an antibody that binds so tightly that removal is difficult or poses a risk associated with a low turnover rate. Such antibodies could also be useful for treating conditions such as multiple sclerosis by allowing for less frequent drug administration or making delivery routes other than infusion more effective. The ability to treat at lower doses may also reduce the risk of adverse events such as progressive multifocal leukoencephalopathy. Accordingly, the present invention is directed to the creation of antibodies having such desirable properties.

The invention is based, at least in part, on the unexpected characteristics of novel engineered humanized α4-binding antibodies that have an α4 binding affinity that is 10 times higher than that of the anti-α4 antibody natalizumab. Antibodies that bind to alpha-4 and their fragments are proposed, in which the variable light chain (VL) and variable heavy chain (VH) framework regions contain acceptor sequences constructed from germline antibody sequences or genetically engineered germline antibody sequences , such as antibodies IGKV41 or geAAH70335.1, or IGHV1-f. The CDR sequences are derived from non-human anti-α4 antibodies, such as the anti-VLA-4 antibody called HP1/2. The antibodies described herein may have an affinity increased by at least 1.5, 2.0, 2.5, 3.0 times, for example, relative to the parent mouse antibody. In one embodiment, the increase in affinity is at least 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, but correspondingly less than 25-fold, 20-fold, or 15-fold.

Pharmaceutical compositions

An α4 binding agent, such as a VLA-4 binding antibody, can be formulated as a pharmaceutical composition. Typically, the pharmaceutical composition includes a pharmaceutically acceptable carrier. As used herein, a pharmaceutically acceptable carrier includes any and all solvents, dispersing media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

A pharmaceutically acceptable salt means a salt that retains the desired biological activity of the parent compound and does not cause any undesirable toxicological effects (see, for example, Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19) . Examples of such salts include salts formed by adding an acid and salts formed by adding a base. Salts formed by acid addition include salts derived from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, etc., as well as non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl -substituted alkanoic acids, hydroxycarboxylic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Salts formed by base addition include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., as well as non-toxic organic amines such as N,N'dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline , diethanolamine, ethylenediamine, procaine, etc.

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

In one embodiment, the anti-α4 antibody dosage form can be formulated using excipients such as sodium chloride, sodium dihydrogen phosphate heptahydrate, sodium hydrogen phosphate monobasic, and polysorbate-80. In another embodiment, the anti-α4 antibody dosage form can be formulated in a citrate buffer, for example, pH 5, 5.5, 6, 6.5, 7 or 7.5. In another embodiment, the dosage form of the anti-α4 antibody may be

- 9 046416 prepared in a solution containing 2, 4, 5, 6, 8, 10, 12, 14 or 15% sucrose. The antibody may be supplied, for example, in a buffer solution at a concentration of approximately 20 mg/ml and may be stored at 28°C.

Pharmaceutical compositions may also be in various other forms. These include, for example, liquid, semi-solid and dry dosage forms such as liquid solutions (eg injection and infusion solutions), dispersions or suspensions, tablets, dragees, powders, liposomes and suppositories. The form may depend on the intended route of administration and therapeutic use. Typically, compositions for the agents described herein are in the form of injections or infusion solutions.

Such compositions can be administered parenterally (eg, intravenous, subcutaneous, intraperitoneal or intramuscular injection). The phrases parenteral administration and parenterally administered, as used herein, mean methods of administration other than enteral and local administration, typically by injection, and including, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Pharmaceutical compositions typically must be sterile and stable under conditions of manufacture and storage. Pharmaceutical compositions may also be tested to ensure they meet regulatory and industry standards for the drugs to be administered.

The compositions may be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high drug concentrations. Sterile injectable solutions can be prepared by adding the agent described herein in the required amount to an appropriate diluent with one or a combination of the ingredients listed above, as needed, followed by sterilization by filtration. In general, dispersions are prepared by adding the agent described herein to a sterile vehicle containing a basic dispersing medium and other required ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, typical preparation methods are vacuum drying and freeze-drying, resulting in a powder of the agent described herein, mixed with any additional required ingredient, from a solution thereof previously sterilized by filtration. The required fluidity of the solution can be maintained, for example, by using a coating such as lecithin, maintaining the required particle size in the case of a dispersion and the use of surfactants. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that retards absorption, for example, monostearate and gelatin salts.

Introduction

An antibody that binds to α4 can be administered to a subject, such as a human, using a variety of routes. For many applications, the route of administration is one of the following: intravenous injection or infusion, subcutaneous injection, or intramuscular injection. The α4-binding antibody can be administered as a fixed dose or in a mg/kg dose. The antibody can be administered intravenously (IV) or subcutaneously (SC). For example, the antibody can be administered as a fixed unit dose of about 50 to 600 mg IV, for example, every 4 weeks, or about 50 to 100 mg SC (for example, 75 mg), for example, at least once per week (for example, twice a week). In one embodiment, the antibody is administered IV at a fixed unit dose of 50, 60, 80, 100, 120, 130, 140, 150, 160, 180, 200, 300, 400, 500 mg or 600 mg, or higher. IV dosing may be given once or twice or three or more times per week, or once every two, three, four or five weeks, or less frequently.

In one embodiment, the antibody is administered subcutaneously at a fixed unit dose of 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 100 mg, or 120 mg, or higher. The SC dose may be administered once or twice or three or more times per week, or once every two, three, four or five weeks, or less frequently.

The anti-α4 antibody can also be administered as a loading dose at a dosage of about 1 to 10 mg/kg, for example about 6.0, 4.0, 3.0, 2.0, 1.0 mg/kg. Modified dose ranges include a dose that is less than about 600 mg/subject, about 400 mg/subject, about 300 mg/subject, about 250 mg/subject, about 200 mg/subject, about 150 mg/subject, typically - for administration every four weeks or once a month. The α4 binding antibody may be administered, for example, every three to five weeks, for example every fourth week or monthly.

The dosage can be adjusted according to the patient's observed rate of clearance of previously administered anti-α4 antibody. For example, a patient may not receive a second or subsequent dose until the patient's anti-α4 antibody level has dropped to a level below that of

- 10 046416 not the specified level. In one embodiment, a patient sample (eg, a plasma, serum, blood, urine, or cerebrospinal fluid (CSF) sample) is analyzed for the presence of anti-α4 antibodies and, if the level of anti-α4 antibodies is above a predetermined level, the patient will not be administered second or subsequent dose. If the patient's anti-α4 antibody level is below a predetermined level, the patient is given a second or subsequent dose. A patient whose anti-α4 antibody levels are determined to be too high (above a predetermined level) may be retested after one or two or three days, or a week, and if the patient's anti-α4 antibody levels have dropped below a predetermined level, the patient can be administered a second or subsequent dose of the antibody.

The dose may also be selected to reduce or avoid the development of antibodies against the α4-binding antibody, with the goal of achieving greater than 40, 50, 70, 75, or 80% saturation of the α4 subunit, achieving less than 80, 70, 60, 50 or 40% saturation of the α4 subunit, or preventing an increase in circulating white blood cell levels.

In some embodiments, the active agent may be formulated with a carrier that will provide protection of the compound from rapid release, for example, in the form of a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Many methods for preparing such recipes are patented or well known. See, for example, Controlled Drug Delivery (Drugs and the Pharmaceutical Sciences), Second Edition, J. Robinson and V. H. L. Lee, eds., Marcel Dekker, Inc., New York, 1987.

Pharmaceutical compositions can be administered using medical devices. For example, pharmaceutical compositions can be administered using a needle-free subcutaneous administration device, such as those disclosed in US Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of well-known implants and modules are discussed, for example, in the patent. US No. 4487603, which describes an implantable microinfusion pump for dosing drugs at a controlled rate; US Pat. No. 4,486,194, which describes a therapeutic device for administering drugs through the skin; US Pat. No. 4,447,233, which describes a medical infusion pump for delivering drugs at a specific infusion rate; US Pat. No. 4,447,224, which describes implantable controlled-flow infusion devices for continuous drug delivery; US Pat. No. 4,439,196, which describes an osmotic drug delivery system with multi-chamber compartments; and US Pat. No. 4,475,196, which describes an osmotic drug delivery system. Undoubtedly, many other similar implants, delivery systems and modules are known.

The present invention also provides a device for administering the first and second agents. The device may include, for example, one or more pharmaceutical storage compartments and may be configured to release unit doses of the first and second agents. The first and second agents may be stored in the same or different compartments. For example, the device may combine the agents prior to administration. It is also possible to use different devices for administering the first and second agents.

Dosage regimens are adjusted to provide the desired response, such as a therapeutic response or a combined therapeutic effect. In general, any combination of dosages (formulated separately or together) of a VLA-4 binding agent and a second agent may be used to deliver both agents in bioavailable amounts to a subject.

Dosage unit form or fixed dose, as used herein, means physically separated units prepared as unit dosages for subjects receiving treatment; wherein each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect, in association with the desired pharmaceutical carrier and, optionally, in association with another agent.

The pharmaceutical composition may include a therapeutically effective amount of an agent described herein. Such effective amounts may be determined based on the combinatorial effect of the first and second agents administered. The therapeutically effective amount of the agent may also vary depending on factors such as the stage of the disease, age, sex and weight of the patient, and the ability of the compound to produce a desired response in the patient, such as an improvement in at least one parameter of the disorder, for example, a parameter of multiple sclerosis, or a decrease the intensity of at least one symptom of the disorder, for example, a symptom of multiple sclerosis, such as muscle atrophy, ataxia and tremor. A therapeutically effective amount is also an amount at which any toxic or harmful effects of the composition are outweighed by the therapeutically beneficial effects.

- 11 046416

Devices and kits

Compositions containing the antibody described herein can be administered via a medical device. A device may be designed with features such as portability, room temperature storage, and ease of use so that it can be used in emergency situations, such as by an untrained subject or first aid personnel in an outdoor medical setting. appointments and in the absence of other medical equipment. The device may include, for example, one or more compartments for storing pharmaceuticals that contain an α4-binding antibody and may be configured to release one or more unit doses of the agent.

For example, the pharmaceutical composition may be administered using 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 controlled release devices. The composition can be administered intravenously using standard IV administration equipment, including, for example, IV tubing with or without built-in filters. In some embodiments, the device will be a syringe for use in subcutaneous or intramuscular administration.

Pharmaceutical compositions can be administered using medical devices. For example, pharmaceutical compositions can be administered using a needle-free subcutaneous administration device, such as those disclosed in US Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of well-known implants and modules are described, for example, in Pat. US No. 4487603, which describes an implantable microinfusion pump for dosing drugs at a controlled rate; US Pat. No. 4,486,194, which describes a therapeutic device for administering drugs through the skin; US Pat. No. 4,447,233, which describes a medical infusion pump for delivering drugs at a specific infusion rate; US Pat. No. 4,447,224, which describes implantable controlled-flow infusion devices for continuous drug delivery; US Pat. No. 4,439,196, which describes an osmotic drug delivery system with multi-chamber compartments; and US Pat. No. 4,475,196, which describes an osmotic drug delivery system. The therapeutic composition may also be in the form of a biodegradable or biodegradable long-acting dosage form for subcutaneous or intramuscular administration. Methods for preparing such compositions are known to those skilled in the art. Continuous delivery can also be achieved using an implantable or external pump. Administration can also be carried out intermittently, for example, by one daily injection, or continuously at a low dose, for example, in the form of a long-acting dosage form. The delivery device can be modified to be optimally suited for the administration of α4-binding antibody. For example, a syringe can be siliconized to a degree that is optimally suited for storing and administering the antibody. Undoubtedly, many other similar implants, delivery systems and modules are known.

The present invention also provides a device for administering a first and a second agent (eg, an antibody and a second agent). The device may include, for example, one or more pharmaceutical storage compartments and may be configured to release unit doses of the first and second agents. The first and second agents may be stored in the same or different compartments. In one embodiment, the device combines the agents prior to administration. In some embodiments, the first and second agents are administered using different devices.

An antibody that binds to α4 may be supplied 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 ) information material. Information material may be description, instruction, advertising or other material that relates to the methods described herein and/or to the use of agents for therapeutic purposes. In one embodiment, the kit also includes a second agent. For example, the kit includes a first container containing a composition including an α4 binding antibody and a second container containing a second agent.

The information material in the set is not limited in form. In one embodiment, the information material may include information about the antibody's receipt, concentration, expiration date, batch or manufacturing location information, and the like. In one embodiment, the information material relates to methods of administering an α4-binding antibody, e.g., in a suitable dosage, dosage form, or route of administration (e.g., the dosage, dosage form, or route of administration described herein) for the purpose of treating a subject. suffering from an acute disorder such as spinal cord injury or traumatic injury

- 12 046416 tion of the brain, or from an inflammatory disease (eg, multiple sclerosis), or a subject who is at risk of experiencing an episode associated with an inflammatory disease. The information may be presented in a variety of formats, including printed text, computer reading material, video or audio recording, or information that provides a link or reference to material material.

In addition to the agent, the composition in the kit may include other ingredients such as a solvent or buffer solution, a stabilizing agent or a preservative. The agent may be in any form, for example, liquid, dry or lyophilized form, and substantially pure and/or sterile. If the agents are presented in the form of a liquid solution, the liquid solution is typically an aqueous solution. If the agents are in dry form, reconstitution is generally accomplished by adding a suitable solvent. A solvent, such as sterile water or a buffer solution, may optionally be provided in the kit.

The kit may include one or more containers for a composition or compositions containing agents. In some embodiments, the kit includes separate containers, dividers, or compartments for composition and 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 pocket or bag. In other embodiments, the individual elements of the set are contained in a single, non-divided container. For example, the composition is in a bottle, vial or syringe, to which is attached information material in the form of a label. In some embodiments, the kit includes a plurality (eg, a package) of unit containers, each of which contains one or more unit dosage forms (eg, the dosage form described herein) of the agents. The container may include a combination dosage unit, for example, a unit that includes both the α4-binding antibody and the second agent, for example, in a desired ratio. For example, a kit may include a plurality of syringes, ampoules, foil pouches, blister packs, or medical devices, each containing, for example, a single combined unit dose. Kit containers may be airtight, watertight (eg, impervious to changes in humidity or evaporation), and/or lighttight.

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

Oncology

Antibodies that bind to α4 and the methods described herein can be used to treat cancer, including solid cancers and hematologic malignancies. Examples of solid cancers include sarcomas and carcinomas, such as those of the lung, breast, pancreas, colon, prostate, bladder, and brain. Hemoblastoses include cancers such as multiple myeloma, leukemia and lymphoma.

Methods are provided for treating patients with hematologic malignancies using a composition containing an α4-binding antibody, such as the anti-VLA-4 antibody described herein. Hemoblastoses are oncological diseases of the hematopoietic and immune systems of the body. This type of cancer affects the blood, bone marrow and/or lymph nodes. Hemoblastoses include leukemias such as acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia, acute erythromyelosis and hairy cell leukemia (HCL); lymphomas such as Hodgkin's lymphoma and non-Hodgkin's lymphoma; and multiple myelomas; Waldenström's macroglobulinemia; myelodysplastic syndrome (MDS) (which can lead to AML); myeloproliferative syndrome such as polycythemia vera (also known as primary polycythemia, erythremia, Vaquez disease), idiopathic thrombocytosis (IT), myelofibrosis, heavy chain disease; and amyloidosis caused by light chain disease.

Patients with hematologic malignancies can be identified by blood count and blood smear analysis, such as light microscopy, which is useful for identifying malignant cells. A biopsy, such as bone marrow, may also be used to identify malignant cells, and a lymph node biopsy may be useful to detect lymphadenopathy.

An antibody that binds to a4 (eg, a humanized anti-VLA-4 antibody such as HuHP1/2, H1L0, H1L1, H1L2 or H1L3) is useful for treating leukemia such as AML. Leukemias are cancers that begin in the bone marrow, with the malignant cells being white blood cells (leukocytes). AML (also called acute myelocytic leukemia, acute myeloblastic leukemia, acute granulocytic leukemia, and acute nonlymphoblastic leukemia) is a malignant disease that arises in either granulocytes or monocytes. AML is characterized by uncontrolled, excessive growth and accumulation of cells called

- 13 046416 caused by leukoblasts that are unable to function as normal blood cells, and blockade of the formation of normal bone marrow cells, which leads to a deficiency of red cells (anemia) and platelets (thrombocytopenia), and normal white cells (especially neutrophils, i.e. to neutropenia) in the blood.

All AML subtypes are suitable for treatment with an antibody that binds to VLA-4. AML subtypes are classified based on the stage of development that the myeloblasts have reached at the time of diagnosis. The categories and subtypes allow the doctor to decide which treatment will be most effective for a particular cell type and how quickly the disease may progress. The following subtypes are distinguished: M0 - myeloblastic, according to the results of a special analysis; M1 myeloblastic, without signs of maturation; M2 - myeloblastic, with signs of maturation; M3 - promyelocytic; M4 myelomonocytic; M5 - monocytic; M6 - erythroleukemia and M7 - megacryoblastic. The anti-VLA-4 antibody can be administered together with a second agent that is appropriate for the specific AML subtype. For example, acute promyelocytic leukemia (APL) and acute monocytic leukemia are subtypes of AML that require different treatments than other AML subtypes. A second agent for the treatment of ALI may include all-trans retinoic acid (ATRA) or an antimetabolite such as cytarabine. The second agent for treating acute monocytic leukemia may include a deoxyadenosine analog such as 2-chloro-2'-deoxyadenosine (2-CDA).

Risk factors for developing AML include the presence of certain genetic disorders, such as Down syndrome, Fanconi anemia, Shwachman-Diamond syndrome, etc. A patient with AML and a genetic disorder may be given an antibody that binds to VLA-4 and a second agent to treat the symptom of the genetic disorder. For example, a patient with AML and Fanconi anemia may be given an antibody that binds to VLA-4 and an antibiotic.

Other risk factors for developing AML include chemotherapy or radiation therapy for various types of cancer, tobacco smoking, and exposure to large amounts of benzene.

Other cancers suitable for treatment with α4-binding antibody include solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, ovarian cancer, squamous cell carcinoma, basal cell epithelioma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, carcinoma kidney cell cinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oli Godendroglioma , schwannoma, meningioma, melanoma, neuroblastoma and retinoblastoma).

Other violations

The compositions and methods described herein may also be used to treat other inflammatory, immune or autoimmune disorders, such as inflammation of the central nervous system (eg, in addition to multiple sclerosis, meningitis, neuromyelitis optica, neurosarcoidosis, CNS vasculitis, encephalitis and transverse myelitis); tissue or organ graft rejection or graft-versus-host disease, acute central nervous system injury such as stroke or spinal cord injury (SCI); chronic kidney disease; allergies, such as allergic asthma, moderate to severe allergic rhinitis, allergic eye diseases; type 1 diabetes mellitus; inflammatory bowel diseases, for example, Crohn's disease, ulcerative colitis (for example, for treatment or maintenance of remission); eosinophilic gastroenteritis; myasthenia gravis; fibromyalgia; rheumatoid/immunological disorders such as joint disorders, eg rheumatoid arthritis, psoriatic arthritis; dermatological disorders such as inflammatory/immune skin disorders, eg psoriasis, vitiligo, dermatitis (eg diffuse neurodermatitis), lichen planus, moderate to severe chronic urticaria; systemic lupus erythematosus (SLE; eg, lupus nephritis); scleroderma (eg, progressive systemic sclerosis (PSS), such as PSS of the lung); acute or chronic primary eosinophilic pneumonia; Gougerot-Sjögren syndrome; acute coronary syndrome (ACS); acute myocardial infarction; atherosclerosis; and fibrotic disorders, for example, pulmonary fibrosis (for example, idiopathic pulmonary fibrosis), pulmonary fibrosis, for example, induced by radiation therapy), myelofibrosis, liver cirrhosis, mesangial proliferative glomerulonephritis, crescentic glomerulonephritis, diabetic neuropathy and interstitial fibrosis of the kidney.

The compositions and methods described herein can also be used for the treatment of neurological disorders such as cerebral ischemia, including prophylaxis in patients with transient ischemic attacks and/or arterial stenosis. Other examples of neurological disorders include chronic inflammatory demyelinating polyneuropathy (CIDP); Guillain-Barre syndrome

- 14 046416 (SGB); eye diseases such as macular degeneration (eg, wet macular degeneration) and anterior ischemic optic neuropathy; neuropathic pain (eg, symptomatic neuropathic pain); Alzheimer's disease; amyotrophic lateral sclerosis (ALS) (eg disease-modifying ALS) and Parkinson's disease.

The compositions and methods described herein can also be used to treat patients who have undergone a transplant, such as a kidney, heart, or bone marrow transplant.

Multiple sclerosis

Compositions containing the alpha-4 binding antibody described herein are useful for the treatment of inflammatory diseases such as multiple sclerosis (MS). Multiple sclerosis is a disease of the central nervous system that is characterized by inflammation and loss of myelin sheaths.

Patients with MS can be identified by criteria for the diagnosis of clinically significant MS, as outlined in the workshop on the diagnosis of MS (Poser et al., Ann. Neurol. 13:227, 1983). For example, a patient with clinically significant MS has had two exacerbations, and there is either clinical evidence of two lesions, or clinical evidence of one lesion and paraclinical evidence of a second lesion different from the first. Full-blown MS can also be diagnosed by evidence of two flares and the presence of an IgG oligoclonal band in the cerebrospinal fluid, or by the combination of a flare, clinical evidence of two flares and the presence of an IgG oligoclonal band in the cerebrospinal fluid. The MacDonald criteria can also be used to diagnose PC. (McDonald et al., 2001, Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the Diagnosis of Multiple Sclerosis, Ann. Neurol. 50:121127). The McDonald criteria include the use of MRI evidence of CNS deterioration over time, which can be used to make a diagnosis of MS in the absence of multiple clinical exacerbations. The effectiveness of treatment for multiple sclerosis can be assessed in several different ways. The following parameters can be used to measure the effectiveness of treatment. Two examples of criteria include: EDSS (Extended Disability Score) and MRI (Magnetic Resonance Imaging) detection of exacerbations. The Expanded Disability Scale (EDSS) is a way of measuring clinical impairment caused by MS (Kurtzke, Neurology 33:1444, 1983). Eight functional systems are assessed according to the type and severity of neurological deterioration. Briefly, before starting treatment, the patient is assessed for deterioration in the following systems: pyramidal, cerebellar, brain stem, sensory, bowel and bladder, visual, cerebral, etc. Follow-up is carried out at specified intervals. The scale ranges from 0 (normal) to 10 (death as a result of PC). A decrease of one full scale step indicates the effectiveness of treatment (Kurtzke, Ann. Neurol. 36:573-79, 1994). It is also possible to diagnose patients using other criteria known to those skilled in the art.

Exacerbations are defined as the appearance of a new symptom that is characteristic of MS and is accompanied by a corresponding new neurological disorder (IFNB MS Study Group, see earlier). In addition, the exacerbation must last for at least 24 hours and must be preceded by at least 30 days of stability or improvement. Briefly, patients undergo a standard neurological examination by a clinician. Exacerbations may be mild, moderate or severe, according to changes in Neurological Status Rating Scale scores (Sipe et al., Neurology 34:1368, 1984). The annual frequency of exacerbations and the proportion of patients who did not experience exacerbations are determined.

A treatment can be considered effective if there is a statistically significant difference in the frequency or proportion of patients without exacerbations or patients without relapses between the treatment group and the placebo group for any of these indicators. In addition, the time to first exacerbation and the duration and severity of the exacerbation can also be measured. In this case, the measure of treatment effectiveness is the statistically significant difference in time to first exacerbation or duration and severity of exacerbation between the treatment group and the control group. Particular attention should be paid to a period without exacerbation or without relapse of more than one year, 18 months or 20 months. Efficacy can also be assessed using any method known to those skilled in the art, for example, by assessing symptoms of MS, including improvement in mobility, using a timed walking test, alone or in combination with other criteria.

The effectiveness of administration of the first agent, and optionally the second agent, may also be assessed based on one or more of the following criteria: frequency of MBP-reactive T cells as determined by limited dilution, proliferative response of MBP-reactive T cell lines and clones, cytokine profile of the lines, and clones of T cells in response to MBP obtained from patients. Efficacy is evidenced by a decrease in the frequency of reactive cells, a decrease in thymidine incorporation with a change in peptide compared to the native one, and a decrease in the levels of TNF and IFN-α.

Clinical indicators include relapse rates at one- and two-year intervals and infidelity

- 15 046416 EDSS score, including time to disease progression from baseline to 1.0 EDSS over six months. According to the Kaplan-Meier survival curve, effectiveness is indicated by a delay in the continuous progression of disability. Other criteria include changes in T2-weighted MRI area and volume and the number and volume of lesions identified on gadolinium-enhanced images.

MRI can be used to measure active lesions using gadolinium-DTPA contrast-enhanced imaging (McDonald et al. Ann. Neurol. 36:14, 1994) or to determine the location and extent of lesions using T ₂ -weighting techniques. Briefly, a baseline MR image is obtained. For each subsequent examination, the same imaging plane and patient position are used. Image acquisition positions and sequences can be selected to maximize the likelihood of lesion detection and facilitate lesion tracking. The same imaging position and sequence can be used for each subsequent examination. The presence, location and degree of development of PC lesions can be determined by radiologists. The area of the lesions can be delineated and summed, layer by layer, to determine the total area of the lesions. Three analyzes can be performed: evidence of a new lesion, frequency of active lesions, and percentage change in lesion area (Paty et al., Neurology 43:665, 1993). Improvement resulting from therapy can be determined by statistically significant improvement in an individual patient compared to baseline or in the treated group compared to the placebo group.

Examples of symptoms associated with multiple sclerosis that can be treated with the methods described here include: optic neuritis, diplopia, nystagmus, ocular dysmetria, internal ophthalmoplegia, motion and sound phosphenes, afferent pupillary defect, paresis, monoparesis, paraparesis, hemiparesis, quadraparesis, plegia, paraplegia, hemiplegia, tetraplegia, quadraplegia, spasticity, dysarthria, muscle atrophy, spasms, cramps, hypotension, muscle twitching, myoclonus, myokymia, restless leg syndrome, drop foot, reflex disorders, paresthesia, anesthesia, neuralgia , neuropathic and neurogenic pain, Lhermitte's symptom, proprioceptive dysfunction, trigeminal neuralgia, ataxia, tremor of intention, dysmetria, vestibular ataxia, dizziness, speech ataxia, dystonia, dysdiadochokinesia, frequent urination, bladder spasticity, decreased bladder tone, detrusor-sphincteric dyssynergia, erectile dysfunction, anorgasmia, frigidity, constipation, defecation urgency, fecal incontinence, depression, cognitive disorders, dementia, mood swings, emotional lability, euphoria, bipolar syndrome, anxiety, aphasia, dysplasia, fatigue, Uthoff's sign, gastroesophageal reflux and sleep disorders.

Each case of PC represents one of several patterns of manifestations and subsequent course of the disease. Most often, MS first manifests as a series of exacerbations, followed by complete or partial remissions, in which the severity of symptoms inexplicably decreases, only to return after a period of stability. This form is called relapsing-remitting (RR) MS. Primary progressive (IP) MS is characterized by gradual clinical deterioration without significant remissions, although temporary stabilization or a slight decrease in the severity of symptoms may be observed. Secondary progressive (SP) MS begins with a relapsing-remitting course, followed by a later transition to a primary progressive course. A progressive-relapsing (IR) course is rarely observed, in which the disease has a progressive development path, interrupted by acute exacerbations. AI, VI and IR forms are sometimes combined and called chronic progressive MS.

Several patients have malignant MS, defined as rapid and unrelenting deterioration leading to significant disability or even death soon after disease onset. This deterioration can be stopped or slowed by administration of the combination therapeutic agent described herein.

Administration of the anti-α4 antibody provided herein may be effective in reducing one or more symptoms of MS, such as one or more of the symptoms described above. For example, administration of the anti-α4 antibody described herein can be used to treat primary or secondary progressive multiple sclerosis (PPMS or PVMS, respectively), and treatment with the anti-α4 antibody may be effective in preventing relapse.

In addition to or prior to human studies, an animal model may be used to evaluate the efficacy of the two agents. An example of an animal model of multiple sclerosis is the mouse model of experimental autoimmune encephalitis (EAE), for example, as 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). Mice can be administered the first and second agents described herein before inducing EAE. The mice are then assessed against characteristic criteria to determining the effectiveness of using two agents in the model.

- 16 046416

Creation of Antibodies

Recombinant antibodies that bind to alpha-4 can be generated using in vivo or in vitro methods such as phage display. The methods can be used to obtain anti-α4 CDRs for use in the CDR-grafted antibodies described herein. Additionally, methods such as phage display can be used to select such CDRs in the context of the germline framework regions disclosed herein, for example, using a library in which the framework region is a germline framework region.

Application EP 239400 (Winter et al.) describes a method for modifying antibodies by replacing (within a given variable region) their complementarity determining regions (CDRs) belonging to one biological species with regions belonging to another species. Antibodies with substituted CDRs are less likely to elicit an immune response in humans compared to true chimeric antibodies because antibodies with substituted CDRs contain substantially fewer non-human components. (Riechmann et al., 1988, Nature 332, 323-327; Verhoeyen et al., 1988, Science 239, 1534-1536). Typically, mouse CDR antibodies replace corresponding regions in a human antibody using recombinant nucleic acid technology to produce sequences encoding the desired substituted antibody. Gene segments of the conserved regions of a human antibody of the desired isotype (typically 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 a soluble antibody. Large unimmunized libraries in phage display format can also be used to isolate high-affinity antibodies that can be used to develop human therapeutics using standard phage technology (see, for example, Hoogenboom et al. (1998) Immunotechnology 4:1-20, and Hoogenboom et al (2000) Immunol Today 2:371-8; U.S. 20030232333).

The anti-a4 antibody or fragment of such an antibody described herein can recognize epitopes of the α4 subunit that are involved in binding to its own ligand, for example, VCAM-1 or fibronectin. The antibodies described herein can inhibit one or more of these self-ligands (eg, VCAM-1 and fibronectin).

In some embodiments, the antibodies provided herein can interact with VLA-4 on the surface of cells, such as lymphocytes, but do not cause cell aggregation.

An example of an α4-binding antibody contains one or more CDRs, e.g., all three heavy chain (HC) CDRs and/or all three light chain (LC) CDRs of a particular antibody disclosed herein, or CDRs that add up to at least 80, 85, 90, 92, 94, 95, 96, 97, 98, 99% identical to such an antibody. In one embodiment, the hypervariable loops H1 and H2 have the same canonical structure as the corresponding loops of the antibody described herein. In one embodiment, the hypervariable loops L1 and L2 have the same canonical structure as the corresponding loops of the antibody described herein.

In one embodiment, the amino acid sequence of the variable domain sequence in the HC and/or LC is at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to the amino acid sequence of the variable domain in the HC and /or LC antibody described herein. The amino acid sequence of the variable domain sequence within the HC and/or LC may differ in at least one amino acid, but not more than ten, eight, six, five, four, three or two amino acids, from the corresponding sequence of the antibody described herein. For example, the differences may be located primarily or entirely in wireframe regions.

The amino acid sequences of the HC and LC variable domain sequences may be encoded by a nucleic acid sequence that hybridizes under highly stringent conditions to a nucleic acid sequence described herein or to one encoding a variable domain or amino acid sequence 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 variable domain within the HC and/or LC are at least 70, 80, 85, 90, 92, 95 , 97, 98, 99 or 100% identical to the corresponding framework regions of the variable domains of the HC and LC antibodies described herein. In one embodiment, one or more heavy or light chain framework regions (e.g., FR1, FR2, and FR3 HCs) are at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 100% sequence identical to the corresponding framework regions. regions from human germline antibodies.

The calculation of homology or sequence identity between two sequences (these terms are used interchangeably herein) is carried out as follows. For purposes of optimal comparison, the sequences are aligned (eg, gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be omitted from the comparison). Optimal alignment is defined as obtaining the best

- 17 046416 estimates using the GAP program from the GCG software package with a Blossum 62 substitution matrix and a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The amino acids are then compared residues or nucleotides at corresponding amino acid positions or nucleotide positions. If a position in the first sequence is occupied by the same amino acid residue or nucleotide that is at the corresponding position in the second sequence, then the molecules are identical at that position (as used here, the term amino acid or nucleic acid identity is equivalent to the term amino acid or nucleic acid homology). The percentage identity between two sequences is a function of the number of identical positions that share the sequences.

When used here, the term hybridizes under highly stringent conditions describes the hybridization and washing conditions. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, the contents of which are incorporated herein by reference. This publication describes both aqueous and non-aqueous methods, and any of them can be used. Highly stringent hybridization conditions include hybridization in 6xSSC at approximately 45°C, followed by one or more washes in 0.2xSSC with 0.1% SDS at 65°C or substantially similar conditions.

Obtaining antibodies

Antibodies can be produced in prokaryotic and eukaryotic cells. In one embodiment, the antibodies (eg, scFvs) are expressed in a yeast cell such as Pichia (see, for example, Powers et al. (2001) J. Immunol. Methods 251:123-35), Hanseula or Saccharomyces.

In one embodiment, the antibodies, in particular full-length antibodies, such as IgG, are produced in mammalian cells. Examples of mammalian host cells for expression of recombinant proteins include Chinese hamster ovary cells (CHO cells) (including dhfr-CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77 :42164220 used with a DHFR selectable marker, e.g. as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621), lymphocyte cell lines, e.g. NS0 myeloma cells and SP2 cells, COS cells, K562 cells and cells derived from transgenic animals, such as transgenic mammals. For example, the cell is a mammalian epithelial cell.

In addition to the nucleic acid sequence encoding the immunoglobulin domain, recombinant expression vectors may carry additional nucleic acid sequences, such as sequences that regulate the replication of the vector in host cells (eg, origins of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, for example, US patent numbers 4399216, 4634665 and 5179017). Examples of selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with selection/amplification in the presence of methotrexate) and the peo gene (for selection in the presence of G418).

For example, in a system for expressing a recombinant antibody (eg, a full-length antibody or an antigen-binding portion thereof), a recombinant expression vector encoding both the heavy chain and the light chain of the antibody is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, each of the antibody heavy and light chain genes is operably linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus, etc., such as the CMV enhancer/AdMLP promoter regulatory element or the SV40 enhancer/ promoter regulatory element AdMLP), in order to obtain high levels of gene transcription. The recombinant expression vector also carries the DHFR gene, which allows selection of CHO cells that have been transfected with the vector using methotrexate-dependent selection/amplification. Selected transformed host cells are cultured to express the antibody heavy and light chains, and the intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect host cells, select transformants, cultivate host cells, and extract the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography using Protein A or Protein G. For example, purified α4-binding antibodies can be concentrated to a concentration of from about 100 mg/ml to about 200 mg/ml using protein concentration techniques known in the art. specialists in this field of technology.

Antibodies may also include modifications, for example, modifications that alter Fc functions, for example, to reduce or eliminate interaction with the Fc receptor or with C1q, or with both. For example, the conserved region of human IgG4 may contain a Ser to Pro mutation at residue position 228 to anchor the hinge region. The amino acid sequence of IgG4 Fc (hinge + CH2 domain + CH3 domain) is shown in FIG. 5.

In yet another example, a conserved region of human IgG1 may be mutated one at a time

- 18 046416 or more residues, for example, one or more of residues 234 and 237, for example, according to the numbering adopted in US patent No. 5648260. Other examples of modifications include the modifications described in US patent No. 5648260.

For some antibodies that contain an Fc domain, the antibody production system can be designed to produce antibodies in which the Fc region is glycosylated. In yet another example, the Fc domain of IgG molecules is glycosylated at asparagine residue 297 in the CH2 domain (see FIG. 5). This asparagine residue is a site for modification by two-arm oligosaccharides. This glycosylation is involved in effector functions mediated by receptors for Fcy and the complement component 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 cell-based expression system that ensures correct glycosylation of the residue corresponding to asparagine residue 297. The Fc domain can also contain other post-translational modifications characteristic of eukaryotes.

Other suitable modifications to the Fc domain include those described in WO 2004/029207. For example, the Fc domain may be XmAb® Fc (Xencor, Monrovia, CA, USA). The Fc domain or fragment thereof may contain a substitution in the FcY receptor (FcyR) binding region, such as in the domains and fragments described in WO 05/063815. In some embodiments, the Fc domain or fragment thereof contains a substitution in the neonatal Fc receptor (FcRn) binding region, such as in the domains and fragments described in WO 05047327. In other embodiments, the Fc domain is a single chain or fragment, or a modified version thereof, such as those described in WO 2008143954. Other suitable modifications of Fc are known and described in the art.

Antibodies can also be produced by transgenic animals. For example, US Pat. No. 5,849,992 describes a method for expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that contains a promoter specific for milk protein genes and nucleic acid sequences encoding an antibody of interest, such as the antibody described herein, as well as a signal sequence for secretion. The milk produced by females of such transgenic mammals contains an antibody of interest secreted therein, such as the antibody described herein. The antibody can be purified from milk or, for some applications, applied directly.

Antibodies can be modified, for example, with a moiety that improves their stability and/or retention in the circulation, for example, in blood, serum, lymph, bronchoalveolar secretions or other tissues, for example in at least 1.5, 2, 5. 10 or 50 times.

For example, an antibody that binds to VLA-4 may be associated with a polymer, such as a substantially non-immunogenic polymer such as polyalkylene oxide or polypropylene oxide. Suitable polymers will vary significantly in weight. Polymers with number average molecular weights ranging from about 200 to about 35,000 daltons (or from about 1,000 to about 15,000 and from about 2,000 to about 12,500) can be used.

For example, an antibody that binds to VLA-4 may be conjugated to a water-soluble polymer, such as a hydrophilic polyvinyl polymer, such as polyvinyl alcohol or polyvinylpyrrolidone. A non-limiting list of such polymers includes homopolymers of polyalkylene oxides such as polyethylene glycol (PEG) or polypropylene glycols, polyethylene polyols, copolymers thereof, and block copolymers thereof, provided that the water solubility of the block copolymers is maintained. Additional suitable polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and polyoxyethylene-polyoxypropylene block copolymers (Pluronics); polymethacrylates; carbomers; branched and unbranched polysaccharides containing monomers of the saccharides D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid (for example , polymannuronic acid or alginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminic acid, including homopolysaccharides and heteropolysaccharides such as lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextran sulfate, dextran, dextrins, glycogen, or acid polysaccharide subunit mucopolysaccharides, for example, hyaluronic acid; sugar alcohol polymers such as polysorbitol and polymannitol; heparin or heparon.

Examples of second agents

In some cases, the compositions described herein, for example, compositions containing an alpha-4 binding antibody, include a second agent or are administered in combination with a composition comprising a second agent.

In one embodiment, the α4-binding antibody and the second agent are provided as a combination composition, and the combination composition is administered to the subject. Additionally, you can, for example, at least 24 hours before or 24 hours after administration of the combination composition, separately administer one dose of the composition containing the α4-binding antibody, followed by one dose

- 19 046416 composition containing a second agent. In yet another embodiment, the antibody and the second agent are provided as separate compositions, and the administration step includes sequential administration of the antibody and the second agent. Sequential administration can be performed on the same day (eg, with an interval of one hour between administrations or at least 3, 6 or 12 hours between administrations), or on different days.

In general, each component, the antibody and the second agent, is administered in multiple doses separated over time. Each component, the antibody and the second agent, is generally administered according to a regimen. The regime for one of them or for both may be characterized by regular periodicity. The antibody regimen may have a different frequency than the second agent regimen, for example, one component may be administered more frequently than another. In one embodiment, either the antibody or the second agent is administered once a week and the other once a month. In yet another embodiment, either the antibody or the second agent is administered continuously, for example, over a period of more than 30 minutes but less than 1, 2, 4, or 12 hours, and the second is administered as a loading dose. The antibody and second agent can be administered by any suitable route, for example, subcutaneously, intramuscularly or intravenously.

In some embodiments, each component, the antibody and the second agent, is administered at the same dose as when each was administered as monotherapy. In other embodiments, the antibody is administered at a dosage that is equal to or less than the amount required for effective treatment when administered without an additional agent. Similarly, the second agent can be administered at a dosage that is equal to or less than the amount required for effective treatment when administered without the antibody.

Non-limiting examples of second agents for the treatment of multiple sclerosis in combination with an anti-a4 antibody include:

interferons, for example, human interferon beta-1a (eg, Avonex® or Rebif®) and human interferon-^ (BETASERON™);

human interferon beta, substituted at position 17; Berlex/Chiron company);

glatiramer acetate (also called copolymer 1, Cop-1; Copaxone™);

Teva Pharmaceutical Industries, Inc.);

Rituxan® (rituximab) or another anti-CD20 antibody, such as an antibody that competes with rituximab or binds to an epitope that overlaps with the epitope of rituximab;

mitoxantrone (NOVANTRONE®, Lederle);

a chemotherapeutic agent, for example, cladribine (LEUSTATIN®), azathioprine (IMURAN®), cyclophosphamide (CYTOXAN®), cyclosporine-A, methotrexate, 4-aminopyridine and tizanidine;

corticosteroid, eg methylprednisolone (MEDRONE®, Pfizer), prednisone;

immunoglobulin, such as Rituxan® (rituximab);

CTLA4 Ig; alemtuzumab (MabCAMPATH®) or daclizumab (an antibody that binds to CD25);

statins; and TNF antagonists.

Glatiramer acetate is a protein formed by the random alternation of glutamic acid, lysine, alanine and tyrosine in a chain of amino acids (hence, GLATuramer). Glatiramer acetate can be synthesized in solution from these amino acids in 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.

Additional second agents include antibodies or antagonists for other human cytokines or growth factors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-12 IL-15, IL- 16, IL-18, EMAP-11, GM-CSF, FGF and PDGF. Other examples of second agents include antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 and other ligands. For example, daclizumab is an anti-CD25 antibody that can reduce the severity of multiple sclerosis.

Additional examples of antibodies include antibodies that promote the activity of an agent described herein, such as antibodies that contact an interferon receptor, such as interferon receptor beta. In a typical application in which the second agent includes an antibody, the antibody binds to a target protein other than VLA-4 or other than α4 integrin, or at least an epitope on VLA-4 different from the epitope recognized by the first agent.

Additional examples of second agents include: FK506, rapamycin, mycophenolate mofetil, leflunomide, nonsteroidal anti-inflammatory drugs (NSAIDs), e.g., phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents that interfere with proinflammatory cytokine signaling, as described

- 20 046416 here, IL-converting enzyme inhibitors (eg Vx740), anti-P7 antibodies, P-selectin glycoprotein ligand (PSGL), TACE inhibitors, T cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine , azatloprine, 6-mercaptopurines, angiotensin-converting enzyme inhibitors, soluble cytokine receptors and their derivatives as described herein, anti-inflammatory cytokines (eg, IL-4, IL-10, IL-13 and TGF).

In some embodiments, the second agent may be used to treat one or more symptoms or side effects of MS. Such agents include, for example, amantadine, baclofen, papaverine, meclizine, hydroxyzine, sulfamethoxazole, ciprofloxacin, docusate, pemoline, dantrolene, desmopressin, dexamethasone, tolterodine, phenytoin, oxybutynin, bisacodyl, venlafaxine, amitriptyline, methenamine, clonazepam, isoniazid, var denafil, nitrofurantoin, hydrophilic psyllium husk fibers, alprostadil, gabapentin, nortriptyline, paroxetine, propantheline bromide, modafinil, fluoxetine, phenazopyridine, methylprednisolone, carbamazepine, imipramine, diazepam, sildenafil, bupropion and sertraline. Many second agents, which are small molecules, have a molecular weight ranging from 150 to 5000 daltons.

Examples of TNF antagonists include chimeric, humanized, human or in vitro antibodies (or antigen binding fragments thereof) to TNF (e.g., human TNFa), such as D2E7, (anti-human TNFa antibody, US Pat. No. 6,258,562; BASF), CDP571/CDP-870/BAY-10-3356 (humanized anti-TNFa antibody; Celltech/Pharmacia), cA2 (chimeric anti-TNFa antibody; REMICADE™, Centocor); anti-TNF antibody fragments (eg CPD870); soluble TNF receptor fragments, e.g. human p55 or p75 TNF receptors or derivatives thereof, e.g. 75 kdTNFR-IgG (75 kDa TNF receptor chimeric protein and IgG, Enbrel™; Immunex; see e.g. Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A), p55 kdTNFR-IgG (chimeric protein receptor for TNF 55 kDa and IgG, Lenercept (LENERCEPT™)); enzyme antagonists, for example, TNFa converting enzyme (TACE) inhibitors (for example, alpha-sulfonylhydroxamic acid derivative, WO 01/55112, and Nhydroxyformamide TACE inhibitor - GW 3333, -005, or -022); and TNF-bp/s-TNFR (soluble protein that binds to TNF; see, for example, Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S284; Amer. J. Physiol. - Heart and Circulatory Physiology (1995) Vol. 268, pp. 37-42).

In addition to the second agent, other agents may also be administered to the subject. However, in some embodiments, proteins or biologics other than the a4-binding antibody and the second agent are not administered to the subject as a pharmaceutical composition. The α4-binding antibody and the second agent may be the only agents that are injected. In embodiments in which the second agent is a recombinant protein, the a4-binding antibody and the second agent may be the only recombinant agents that are administered to the subject, or at least the only recombinant agents that modulate immune or inflammatory responses. In further embodiments, only the α4 binding antibody is the only recombinant agent or the only biologic administered to the subject.

Unless otherwise specified, all technical and scientific terms used in this document have the same meanings as commonly accepted in the field to which the present invention relates. Although it is possible to use methods and materials similar or equivalent to those described herein in practicing or testing the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other reference information mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present description of the invention, including definitions, will serve as control. Moreover, the materials, methods, and examples are illustrative only and are not limiting.

Example

Example 1 Anti-VLA-4 antibody variants are more potent than humanized HP1/2.

Anti-VLA-4 antibodies were constructed using the IGKV4-1 germline framework (for designs L1 and L2) or the genetically engineered germline sequence AAH7033.1 (for L3 design) for the VL chain and the IGHV1-f germline framework for VH. These antibodies contain fewer back mutations than the humanized HP1/2 antibody described in US Pat. No. 6,602,503.

Heavy chain changes

The sequences of the three heavy chain variants are shown in FIG. 1 and are designated as design H0, design H1 and design H2. Each design contains a mouse HP1/2 antibody CDR transplanted into the IGHV1-f framework. The HO design contains no back mutations in the framework regions, whereas the H1 and H2 designs contain varying numbers of back mutations in the framework sequences to optimize the affinity of the humanized antibody.

- 21 046416

Light chain changes

The sequences of the four light chain variants are shown in FIG. 2 and are designated as L0 design, L1 design, L2 design and L3 design (also called L0, L1, L2, L3). Each design contains a mouse HP1/2 antibody CDR grafted into a germline scaffold. The IGKV4-1 germline framework was used for the L0, L1 and L2 designs, and the genetically engineered AAH70335 germline framework was used for the L3 design. Design L0 contains no back mutations in the framework regions, while designs L1, L2 and L3 contain varying numbers of back mutations in the framework regions to optimize the affinity of the humanized antibody.

The results of the competitive analysis using ELISA are presented in table. 1 and Fig. 3. In this experiment, α4β1 was preincubated with the test monoclonal antibodies (mAbs), and then mouse HP1/2 antibodies were used as a competition reagent. The results of this experiment indicate that antibodies containing L2 or L3 light chains were more active than the humanized HuHP1/2 antibody described in US Pat. No. 6,602,503. The results are presented in table. 1 below and in FIG. 3. The heavy chain (H1) of the antibodies used for this assay had the sequence according to the H1 design shown in FIG. 1, while L1 refers to the design L1 in FIG. 2.

Table 1

Competitive analysis using ELISA

Monoclonal antibody IC50, nM Chimeric HP 1/2 1.06 H1L0 1.87 HILI 1.67 H1L2 0.9 H1L3 0.49 HuHPl/2 1.05

In table 1 chimeric monoclonal antibody is a chimeric HP1/2 antibody in which the murine heavy and light chain variable regions are genetically fused to the conserved regions of human IgG1. This antibody is substantially identical in binding affinity to the parent mouse HP1/2 antibody (Sanchez-Madrid et al., Eur. J. Immunol. 16:1343-1349, 1996). The results of the experiment indicate that it is possible to improve the affinity of a monoclonal antibody relative to the sequence of the original mouse antibody used to create it by humanization based on a germline acceptor framework region created by genetic engineering methods.

Another competition assay experiment compared the binding affinity of the new antibodies to the humanized anti-a4 antibody 21.6 (Tysabri® (natalizumab)), described in US Pat. No. 5,840,299. This experiment examined the binding of a mixture of mouse HP1/2 antibody and a test monoclonal antibody to the protein α4β1. The results of this experiment are shown in Fig. 4 and in table. 2 below and indicate that the new engineered antibodies are approximately 10 times more effective than natalizumab.

table 2

Competitive analysis using ELISA

Monoclonal antibody IC50, nM Chimeric HP 1/2 1.64 H1L0 4.46 HILI 4.55 H1L2 1.34 10 HuHPl/2 1.41 Tysabri® 10.9

Example 2 Humanized HP1/2 antibody (HuHP1/2) binds to VLA-4 on the surface of tumor cells.

The binding of the anti-VLA-4 antibody, HuHP1/2, to various cell lines was examined using flow cytometry. Binding was studied using CLL cell lines

- 22 046416 (chronic lymphocytic leukemia) Mec1 and JM1; MM (multiple myeloma) cell lines U266 and H929; and AML (acute myelogenous leukemia) cell lines HL60 and KG1. HuHP1/2 antibodies bound to cells of all tumor cell lines tested (Fig. 6). Flow cytometry data were used to calculate _EC50 values for antibody binding to each cell line. This information is presented in the table below. 3.

HuHP1/2 was also found to block the attachment of AML cell lines to fibronectin and the VCAM1-Ig chimeric protein. To test whether the antibody was able to block attachment, AML cell lines - HL60 or KG1 - were allowed to adhere to fibronectin-coated wells (Fig. 7A) or VCAM1-Ig-coated wells (Fig. 7B) in the presence of increasing concentrations of HP1/2 or antibody serving as isotype control. The HuHP1/2 antibody blocked the attachment of both cell types to fibronectin-coated wells and to VCAM1-Ig-coated wells. Maximal inhibition of HL60 cell binding to both ligands was achieved at 20 μg/ml HuHP1/2 (Fig. 1C).

HuHP1/2 was also found to block the attachment of MM cell lines to fibronectin and the VCAM1-Ig chimeric protein. The MM cell lines U266 and H929 were allowed to adhere to fibronectin-coated wells (Fig. 8A) or VCAM1-Ig-coated wells (Fig. 8B) in the presence of increasing concentrations of HP1/2 or isotype control antibody. The HuHP1/2 antibody blocked the attachment of both cell lines to wells coated with fibronectin and VCAM1-Ig. Maximal inhibition of U266 cell binding to both ligands was achieved at 20 μg/ml HuHP1/2 (Fig. 8C).

HuHP1/2 was also found to block the attachment of CLL cell lines to fibronectin and the VCAM1-Ig chimeric protein. The CLL cell lines Mec1 and JM1 were allowed to adhere to fibronectin-coated wells (Fig. 9A) or VCAM1-Ig-coated wells (Fig. 9B) in the presence of increasing concentrations of HP1/2 or isotype control antibody. The HuHP1/2 antibody blocked the attachment of both cell lines to wells coated with fibronectin and VCAM1-Ig. Maximal inhibition of Mec1 cell binding to both ligands was achieved at 20 μg/ml HuHP1/2 (Fig. 9C).

IC50 values for HuHP1/2 antibody binding to tumor cell lines were calculated from the data presented in FIG. 7-9. These data are presented in table. 3.

Table 3

Calculations for HuHP1/2 Binding to Tumor Cell Lines

- 23 046416

Sequence listing <110> BIOGEN IDEC MA INC.

<120> ANTI-VLA-4 ANTIBODIES <130> B2047-7046WO <140> PCT/US11/32641 <141> 2011-04-15 <150> 61/324,944 <151> 2010-04-16 <160> 15 <170 > Patent version 3.5 <210> 1 <211> 121 <212> protein <213> Mus sp.

<400> 1

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 15 1015

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn lie Lys Asp Thr 20 2530

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp lie 35 4045

Gly Arg lie Asp Pro Ala Ser Gly Asp Thr Lys Tyr Asp Pro Lys Phe 50 5560

Gln Val Lys Ala Thr lie Thr Ala Asp Thr Ser Ser Asn Thr Ala Trp 65 70 7580

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 9095

Ala Asp Gly Met Trp Val Ser Thr Gly Tyr Ala Leu Asp Phe Trp Gly 100 105110

Gln Gly Thr Thr Val Thr Val Ser Ser

115120 <210> 2 <211> 98 <212> protein <213> Homo sapiens <400> 2

- 24 046416

Glu Vai Gin Leu Vai Gin Ser Gly Ala Glu Vai Lys Lys Pro Gly Ala 15 1015

Thr Vai Lys lie Ser Cys Lys Vai Ser Gly Tyr Thr Phe Thr Asp Tyr 20 2530

Tyr Met His Trp Vai Gin Gin Ala Pro Gly Lys Gly Leu Glu Trp Met 35 4045

Gly Leu Vai Asp Pro Glu Asp Gly Glu Thr lie Tyr Ala Glu Lys Phe 50 5560

Gin Gly Arg Vai Thr lie Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 7580

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Vai Tyr Tyr Cys 85 9095

Ala Thr <210> 3 <211> 121 <212> protein <213> artificial sequence <220>

<221> source <223> /Note =0writing of an artificial sequence: synthetic polypeptide <400> 3

Glu Vai Gin Leu Vai Gin Ser Gly Ala Glu Vai Lys Lys Pro Gly Ala

1015

Thr Vai Lys lie Ser Cys Lys Vai Ser Gly Phe Asn lie Lys Asp Thr 20 2530

Tyr Met His Trp Vai Gin Gin Ala Pro Gly Lys Gly Leu Glu Trp Met 35 4045

Gly Arg lie Asp Pro Ala Ser Gly Asp Thr Lys Tyr Asp Pro Lys Phe 50 5560

Gin Vai Arg Vai Thr lie Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 7580

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Vai Tyr Tyr Cys 85 9095

- 25 046416

Ala Thr Gly Met Trp Vai Ser Thr Gly Tyr Ala Leu Asp Phe Trp Gly

100 105 110

Gin Gly Thr Leu Vai Thr Vai Ser Ser

115 120 <210> 4 <211> 121 <212> protein <213> artificial sequence <220>

<221> source <223> /Note =Description of artificial sequence: synthetic polypeptide <400> 4

Glu Vai Gin Leu Vai Gin Ser Gly Ala Glu Vai Lys Lys Pro Gly Ala

1015

Thr Vai Lys lie Ser Cys Lys Ala Ser Gly Phe Asn lie Lys Asp Thr 20 2530

Tyr Met His Trp Vai Gin Gin Ala Pro Gly Lys Gly Leu Glu Trp Met 35 4045

Gly Arg lie Asp Pro Ala Ser Gly Asp Thr Lys Tyr Asp Pro Lys Phe 50 5560

Gin Vai Arg Vai Thr lie Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 7580

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Vai Tyr Tyr Cys 85 9095

Ala Asp Gly Met Trp Vai Ser Thr Gly Tyr Ala Leu Asp Phe Trp Gly 100 105110

Gin Gly Thr Leu Vai Thr Vai Ser Ser

115120 <210> 5 <211> 121 <212> protein <213> artificial sequence <220>

<221> source <223> /Note =0writing of an artificial sequence: synthetic polypeptide <400> 5

- 26 046416

Glu Vai Gin Leu Vai Gin Ser Gly Ala Glu Vai Lys Lys Pro Gly Ala 15 1015

Thr Vai Lys lie Ser Cys Lys Ala Ser Gly Phe Asn lie Lys Asp Thr 20 2530

Tyr Met His Trp Vai Gin Gin Ala Pro Gly Lys Gly Leu Glu Trp lie 35 4045

Gly Arg lie Asp Pro Ala Ser Gly Asp Thr Lys Tyr Asp Pro Lys Phe 50 5560

Gin Vai Arg Ala Thr lie Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 7580

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Vai Tyr Tyr Cys 85 9095

Ala Asp Gly Met Trp Vai Ser Thr Gly Tyr Ala Leu Asp Phe Trp Gly 100 105110

Gin Gly Thr Leu Vai Thr Vai Ser Ser

115120 <210> 6 <211> 107 <212> protein <213> Mus sp.

<400> 6

Ser lie Vai Met Thr Gin Thr Pro Lys Phe Leu Leu Vai Ser Ala Gly 15 1015

Asp Arg Vai Thr lie Thr Cys Lys Ala Ser Gin Ser Vai Thr Asn Asp 20 2530

Vai Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu lie 35 4045

Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Vai Pro Asp Arg Phe Thr Gly 50 5560

Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr He Ser Thr Vai Gin Ala 65 70 7580

Glu Asp Leu Ala Vai Tyr Phe Cys Gin Gin Asp Tyr Ser Ser Pro Tyr 85 9095

Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys 100105 <210> 7 <211> 101 <212> protein <213> Homo sapiens <400> 7

Asp lie Vai Met Thr Gin Ser Pro Asp Ser Leu Ala Vai Ser Leu Gly

1015

Glu Arg Ala Thr lie Asn Cys Lys Ser Ser Gin Ser Vai Leu Tyr Ser 20 2530

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 4045

Pro Pro Lys Leu Leu lie Tyr Trp Ala Ser Thr Arg Glu Ser Gly Vai 50 5560

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 7580 lie Ser Ser Leu Gin Ala Glu Asp Vai Ala Vai Tyr Tyr Cys Gin Gin 85 9095

Tyr Tyr Ser Thr Pro 100 <210> 8 <211> 107 <212> protein <213> artificial sequence <220>

<221> source <223> /Note =0writing of an artificial sequence: synthetic polypeptide <400> 8

Asp lie Vai Met Thr Gin Ser Pro Asp Ser Leu Ala Vai Ser Leu Gly

1015

Glu Arg Ala Thr He Asn Cys Lys Ala Ser Gin Ser Vai Thr Asn Asp 20 2530

Vai Ala Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu He 35 4045

Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Vai Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Leu Gin Ala 65 70 75 80

- 28 046416

Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin Asp Tyr Ser Ser Pro Tyr 85 90 95

Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys 100 105 <210> 9 <211> 107 <212> protein <213> artificial sequence <220>

<221> source <223> /Note =0writing of an artificial sequence: synthetic polypeptide <400> 9

Ser lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1015

Glu Arg Ala Thr lie Asn Cys Lys Ala Ser Gin Ser Val Thr Asn Asp 20 2530

Val Ala Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu lie 35 4045

Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Ala 65 70 7580

Glu Asp Val Ala Val Tyr Phe Cys Gin Gin Asp Tyr Ser Ser Pro Tyr 85 9095

Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys 100105 <210> 10 <211> 107 <212> protein <213> artificial sequence <220>

<221> source <223> /Note =0writing of an artificial sequence: synthetic polypeptide <400> 10

Ser Not Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

10 15

- 29 046416

Glu Arg Ala Thr lie Asn Cys Lys Ala Ser Gln Ser Val Thr Asn Asp 20 2530

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu lie 35 4045

Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 5560

Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gln Ala 65 70 7580

Glu Asp Val Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr 85 9095

Thr Phe Gly Gln Gly Thr Lys Val Glu lie Lys 100105 <210> 11 <211> 107 <212> protein <213> artificial sequence <220>

<221> source <223> /Note =0writing of an artificial sequence: synthetic polypeptide <400> 11

Ser lie Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1015

Glu Arg Ala Thr lie Asn Cys Lys Ala Ser Gln Ser Val Thr Asn Asp 20 2530

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu lie 35 4045

Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 5560

Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Gln Ala 65 70 7580

Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr 85 9095

Thr Phe Gly Gln Gly Thr Lys Val Glu lie Lys 100105 <210> 12

- 30 046416 <211> 107 <212> protein <213> Homo sapiens <400> 12

Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai Gly

1015

Asp Arg Vai Thr He Thr Cys Gin Ala Ser Gin Asp He Lys Thr Tyr 20 2530

Leu Ser Trp Phe Gin Gin Lys Pro Gly Lys Pro Pro Lys Leu Leu He 35 4045

Ser Asp Ala Ser Gly Phe Gin Pro Gly Vai Pro Ser Arg Phe Ser Gly 50 5560

Ser Gly Tyr Gly Thr Asp Phe Ser Phe Thr He Thr Ser Leu Arg Pro 65 70 7580

Asp Asp Thr Ala Thr Tyr Tyr Cys Gin Gin Tyr Glu Lys Vai Pro Phe 85 9095

Thr Phe Gly Pro Gly Lys Vai Gly Phe Asn Arg 100105 <210> 13 <2H> 107 <212> protein <213> artificial sequence <220>

<221> source <223> /Note =0writing of an artificial sequence: synthetic polypeptide <400> 13

Asp Not Vai Met Thr Gin Ser Pro Asp Ser Leu Ala Vai Ser Leu Gly

1015

Glu Arg Ala Thr He Asn Cys Gin Ala Ser Gin Asp He Lys Thr Tyr 20 2530

Leu Ser Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu He 35 4045

Tyr Asp Ala Ser Gly Phe Gin Pro Gly Vai Pro Asp Arg Phe Ser Gly 50 5560

Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr He Ser Ser Leu Gin Ala 65 70 7580

Glu Asp Vai Ala Thr Tyr Tyr Cys Gin Gin Tyr Glu Lys Vai Pro Tyr 85 9095

- 31 046416

Thr Phe Gly Gln Gly Thr Lys Val Glu lie Lys 100 105 <210> 14 <211> 228 <212> protein <213> artificial sequence <220>

<221> source <223> /Note =Description of artificial sequence: synthetic polypeptide <400> 14

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe

1015

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 2530

Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 4045

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 5560

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 7580

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 9095

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105110

Ser lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala

145 150 155160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185190

Thr Vai Asp Lys Ser Arg Trp Gin Glu Gly Asn Vai Phe Ser Cys Ser

195 200 205

Vai Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser 210 215 220

Leu Ser Leu Gly 225 <210> 15 <211> 236 <212> protein <213> Homo sapiens <400> 15

Met Asp Met Arg Vai Pro Ala Gin Leu Leu Gly Leu Leu Leu Leu Trp

1015

Leu Ser Gly Ala Arg Cys Asp lie Gin Met Thr Gin Ser Pro Ser Ser 20 2530

Leu Ser Ala Ser Vai Gly Asp Arg Vai Thr lie Thr Cys Gin Ala Ser 35 4045

Gin Asp lie Lys Thr Tyr Leu Ser Trp Phe Gin Gin Lys Pro Gly Lys 50 5560

Pro Pro Lys Leu Leu lie Ser Asp Ala Ser Gly Phe Gin Pro Gly Vai 65 70 7580

Pro Ser Arg Phe Ser Gly Ser Gly Tyr Gly Thr Asp Phe Ser Phe Thr 85 9095 lie Thr Ser Leu Arg Pro Asp Asp Thr Ala Thr Tyr Tyr Cys Gin Gin 100 105110

Tyr Glu Lys Vai Pro Phe Thr Phe Gly Pro Gly Thr Lys Vai Gly Phe 115 120125

Asn Arg Thr Vai Ala Ala Pro Ser Vai Phe lie Phe Pro Pro Ser Asp

130 135140

Glu Gin Leu Lys Ser Gly Thr Ala Ser Vai Vai Cys Leu Leu Asn Asn

145 150 155160

Phe Tyr Pro Arg Glu Ala Lys Vai Gin Trp Lys Vai Asp Asn Ala Leu

165 170175

Gin Ser Gly Asn Ser Gin Glu Ser Vai Thr Glu Gin Asp Ser Lys Asp 180 185190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200205

Glu Lys His Lys Vai Tyr Ala Cys Glu Vai Thr His Gin Gly Leu Ser

210 215220

Ser Pro Vai Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230235

Claims (27)

1. A recombinant antibody molecule or its antigen-binding fragment containing a light chain variable domain and a heavy chain variable domain, where: (a) the light chain variable domain contains the light chain variable domain framework sequence from IGKV4-1 (SEQ ID NO: 7) and the CDR from the VL chain of the mouse antibody HP1/2 (SEQ ID NO: 6), wherein CDR1 contains the sequence KASQSVTNDVA , CDR2 contains the sequence YASNRYT and CDR3 contains the sequence QQDYSSPYT, and the light chain variable domain framework sequence contains amino acid substitutions at one or more of positions 1, 67, 73, 85 and 87 of the framework according to the Kabat numbering scheme, wherein the replaced amino acid at position 1 framework region is serine (S), the replaced amino acid at position 67 of the framework region is tyrosine (Y), the replaced amino acid at position 73 is phenylalanine (F), the replaced amino acid at position 85 is threonine (T), and the replaced the amino acid at position 87 is phenylalanine (F); and (b) the heavy chain variable domain comprises a heavy chain variable domain framework sequence from IGHV1-f (SEQ ID NO: 2) and a CDR from the VH chain of the mouse HP1/2 antibody (SEQ ID NO: 1), wherein CDR1 contains the sequence NIKDTYM, CDR2 contains the sequence IDPASGDTKYDPKFQ and CDR3 contains the sequence WVSTGYALDF, and the heavy chain variable domain framework sequence contains amino acid substitutions at one or more of positions 24 and 94 of the framework according to the Kabat numbering scheme, wherein in the case of a substitution, the replaced amino acid is at position 24 of the framework region is alanine (A), the replaced amino acid at position 94 of the framework region is aspartic acid (D); where the recombinant antibody molecule or its antigen-binding fragment binds the α4 chain of the VLA-4 protein. 2. A recombinant antibody molecule or its antigen-binding fragment containing a light chain variable domain and a heavy chain variable domain, where: (a) the light chain variable domain contains the light chain variable domain framework sequence from IGKV4-1 (SEQ ID NO: 7) and the CDR from the VL chain of the mouse antibody HP1/2 (SEQ ID NO: 6), wherein CDR1 contains the sequence KASQSVTNDVA , CDR2 contains the sequence YASNRYT and CDR3 contains the sequence QQDYSSPYT, and the light chain variable domain framework sequence contains amino acid substitutions at one or more of positions 1, 67, 73, 85 and 87 of the framework according to the Kabat numbering scheme, wherein the replaced amino acid at position 1 framework region is serine (S), the replaced amino acid at position 67 of the framework region is tyrosine (Y), the replaced amino acid at position 73 is phenylalanine (F), the replaced amino acid at position 85 is threonine (T), and the replaced the amino acid at position 87 is phenylalanine (F); and (b) the heavy chain variable domain comprises a heavy chain variable domain framework sequence from design H0 (SEQ ID NO: 3) and a CDR from the VH chain of the mouse antibody HP1/2 (SEQ ID NO: 1), wherein CDR1 contains the sequence NIKDTYM , CDR2 contains the sequence IDPASGDTKYDPKFQ and CDR3 contains the sequence WVSTGYALDF, and the heavy chain variable domain framework sequence contains amino acid substitutions at one or more of positions 24 and 94 of the framework according to the Kabat numbering scheme, wherein in the case of a substitution, the replaced amino acid is at position 24 of the framework is alanine (A), the amino acid replaced at position 94 of the framework region is aspartic acid (D); where the recombinant antibody molecule or its antigen-binding fragment binds the α4 chain of the VLA-4 protein. 3. A recombinant antibody molecule or its antigen-binding fragment containing a light chain variable domain and a heavy chain variable domain, where: (a) the light chain variable domain contains a light chain variable domain framework region sequence from the genetically engineered germline sequence AAH70335.1 (SEQ ID NO: 12) and a CDR from the VL chain of the mouse antibody HP1/2 (SEQ ID NO : 6), where CDR1 contains the sequence KASQSVTNDVA, CDR2 contains the sequence YASNRYT and CDR3 contains the sequence QQDYSSPYT; and the light chain variable domain framework sequence comprises amino acid substitutions at one or more of framework positions 1 and 87 according to the Kabat numbering scheme, wherein the replaced amino acid at position 1 of the framework is serine (S) and the replaced amino acid at position 87 is phenylalanine (F); and (b) the heavy chain variable domain comprises the heavy chain variable domain framework sequence from IGHV1-f (SEQ ID NO: 2) and the CDR from the mouse HP1/2 antibody VH chain (SEQ ID - 34 046416 NO: 1), wherein CDR1 contains the sequence NIKDTYM, CDR2 contains the sequence IDPASGDTKYDPKFQ and CDR3 contains the sequence WVSTGYALDF, and the heavy chain variable domain framework sequence contains amino acid substitutions at one or more of positions 24 and 94 of the framework, according to the Kabat numbering scheme, wherein, in the case of substitution, the replaced amino acid at position 24 of the framework region is alanine (A), the replaced amino acid at position 94 of the framework region is aspartic acid (D); where the recombinant antibody molecule or its antigen-binding fragment binds the α4 chain of the VLA-4 protein. 4. A recombinant antibody molecule or its antigen-binding fragment containing a light chain variable domain and a heavy chain variable domain, where: (a) the light chain variable domain contains a light chain variable domain framework region sequence from the genetically engineered germline sequence AAH70335.1 (SEQ ID NO: 12) and a CDR from the VL chain of the mouse antibody HP1/2 (SEQ ID NO : 6), where CDR1 contains the sequence KASQSVTNDVA, CDR2 contains the sequence YASNRYT and CDR3 contains the sequence QQDYSSPYT; and the light chain variable domain framework sequence comprises amino acid substitutions at one or more of framework positions 1 and 87 according to the Kabat numbering scheme, wherein the replaced amino acid at position 1 of the framework is serine (S) and the replaced amino acid at position 87 is phenylalanine (F); and (b) the heavy chain variable domain comprises a heavy chain variable domain framework sequence from design H0 (SEQ ID NO: 3) and a CDR from the VH chain of the mouse antibody HP1/2 (SEQ ID NO: 1), wherein CDR1 contains the sequence NIKDTYM , CDR2 contains the sequence IDPASGDTKYDPKFQ and CDR3 contains the sequence WVSTGYALDF, and the heavy chain variable domain framework sequence contains amino acid substitutions at one or more of positions 24 and 94 of the framework, according to the Kabat numbering scheme, wherein in the case of a substitution, the replaced amino acid is at position 24 of the framework region is alanine (A), the replaced amino acid at position 94 of the framework region is aspartic acid (D); where the recombinant antibody molecule or its antigen-binding fragment binds the α4 chain of the VLA-4 protein. 5. The recombinant antibody molecule or antigen binding fragment thereof according to any one of claims 1, 2, 3 or 4, wherein the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. 6. The recombinant antibody molecule or antigen binding fragment thereof according to any one of claims 1, 2, 3 or 4, wherein the light chain variable region consists of the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. 7. A recombinant antibody molecule or antigen-binding fragment thereof according to any one of claims 1, 2, 3 or 4, characterized in that the heavy chain variable region consists of the amino acid sequence SEQ ID NO: 4. 8. A recombinant antibody molecule or antigen-binding fragment thereof according to any one of claims 1, 2, 3 or 4, characterized in that the light chain variable region consists of the amino acid sequence SEQ ID NO: 11. 9. A recombinant antibody molecule or antigen-binding fragment thereof according to any one of claims 1, 2, 3 or 4, characterized in that the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 4, and the light chain variable region consists of the amino acid sequence of SEQ ID NO: 11. 10. A vector containing DNA encoding the variable region of the heavy chain of a recombinant antibody molecule or an antigen-binding fragment thereof according to any one of claims 1, 2, 3 or 4. 11. A vector containing DNA encoding the variable region of the light chain of a recombinant antibody molecule or an antigen-binding fragment thereof according to any one of claims 1, 2, 3 or 4. 12. A vector containing DNA encoding a light chain variable region of a recombinant antibody molecule or an antigen-binding fragment thereof according to any one of claims 1, 2, 3 or 4, wherein the light chain variable region contains the amino acid sequence of SEQ ID NO: 4. 13. A vector containing DNA encoding the light chain variable region of a recombinant antibody molecule or antigen binding fragment thereof according to any one of claims 1, 2, 3, or 4, wherein the light chain variable region contains SEQ ID NO: 11. 14. A vector containing DNA encoding a heavy chain variable region and a light chain variable region of a recombinant antibody molecule or an antigen-binding fragment thereof according to claim 1, 2, 3 or 4, where the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 4 and variable the light chain region contains the amino acid sequence SEQ ID - 35 046416 NO: 11. 15. A method for producing a recombinant antibody molecule or its antigen-binding fragment that binds the VLA-4 protein chain, comprising: a) obtaining a host cell containing (i) a DNA sequence encoding a recombinant antibody molecule or an antigen-binding fragment thereof that binds to oc4 at any one of p. 1, 2, 3 or 4; And b) culturing the cell to obtain a recombinant antibody molecule or its antigen-binding fragment. 16. A method of treating a patient, including administering to said patient a recombinant antibody molecule or its antigen-binding fragment according to any of the following. 1, 2, 3 or 4, where the patient suffers from a disease or disorder selected from cancer, an inflammatory disease, a neurological disease or an acute disorder. 17. The method according to claim 16, characterized in that the cancer includes a solid tumor. 18. The method according to claim 16, characterized in that the cancer includes hematological malignancy. 19. The method of claim 16, wherein the cancer includes multiple myeloma or acute myelogenous leukemia (AML). 20. The method of claim 16, wherein the inflammatory disorder includes multiple sclerosis, asthma, rheumatoid arthritis, diabetes, optic neuritis or Crohn's disease. 21. The method of claim 16, wherein the inflammatory disorder includes multiple sclerosis or Crohn's disease. 22. The method of claim 16, wherein the acute disorder includes spinal cord injury or traumatic brain injury. 23. The method according to claim 16, characterized in that the recombinant antibody molecule or its fragment that binds to oc4 is administered in accordance with a specific regimen selected from once a day, once a month, once a week, twice a week a week, three times a week, four times a week, once every two weeks, or once every three weeks. 24. The method of claim 16, further comprising administering to the patient a second therapeutic agent selected from a thrombolytic agent, a chemotherapeutic agent, a neuroprotective agent, an anti-inflammatory agent, a steroid, a cytokine, or a growth factor. 25. Recombinant antibody molecule or its antigen-binding fragment according to any of the pi. 1-9 or 15-24, where the recombinant antibody molecule or its antigen-binding fragment binds to VLA-4 with a K _d of less than ΙΟ' ⁶ , ΙΟ' ⁷ , ΙΟ' ⁸ , ΙΟ' ⁹ , 10 ¹⁰ or 10 ¹¹ M. 26. The vector of claim 10, wherein the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 3, 4 or 5. 27. The vector of claim 11, wherein the light chain variable region consists of the amino acid sequence of SEQ ID NO: 8, 9, 10 or 11.