# US 8,541,552 B2 · Antibodies to lymphotoxin-α

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- Number: US 8,541,552 B2
- Document ID: US8541552B2
- Status: Granted
- Kind: B2
- Application number: 13/026,123
- Filed: 2011-02-11
- Published: 2011-06-23
- Granted: 2013-09-24
- Earliest claimed: 2006-10-12
- Assignees: Genetech, Inc.
- Applicants: Camellia W. Adams; Jane L. Grogan; Austin L. Gurney; Krista McCutcheon
- Inventors: Camellia W. Adams; Jane L. Grogan; Austin L. Gurney; Krista McCutcheon
- Web page: https://patentgrep.com/patent/US8541552B2

## CPC

- C07K 16/242: {Lymphotoxin [LT]}
- A61P 1/00: Drugs for disorders of the alimentary tract or the digestive system
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 5/14: of the thyroid hormones, e.g. T3, T4
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- A61P 13/12: of the kidneys
- A61P 17/06: Antipsoriatics
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 21/04: for myasthenia gravis
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/04: Antibacterial agents
- A61P 31/12: Antivirals
- A61P 31/14: for RNA viruses
- A61P 37/00: Drugs for immunological or allergic disorders
- A61P 37/02: Immunomodulators
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- G06Q 99/00: Subject matter not provided for in other groups of this subclass
- A61K 2039/505: {comprising antibodies}
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/567: Framework region [FR]
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- G06Q: INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR ADMINISTRATIVE, COMMERCIAL, FINANCIAL, MANAGERIAL OR SUPERVISORY PURPOSES; SYSTEMS OR METHODS SPECIALLY ADAPTED FOR ADMINISTRATIVE, COMMERCIAL, FINANCIAL, MANAGERIAL OR SUPERVISORY PURPOSES, NOT OTHERWISE PROVIDED FOR
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The invention provides various antibodies that bind to lymphotoxin-α, methods for making such antibodies, compositions and articles incorporating such antibodies, and their uses in treating, for example, an autoimmune disorder. The antibodies include murine, chimeric, and humanized antibodies.

## Claims

### Claim 1

An isolated anti-lymphotoxin-α (LTα) antibody comprising a light chain variable region wherein the complementarity-determining region (CDR)-L1 sequence is KSSQSLLYSTXQKXFLA (SEQ ID NO:3) or KSSQSLLYSAXQKXFLA (SEQ ID NO:4) or KSSQSLLYSTXQKXALA (SEQ ID NO:6), where X is any amino acid; the CDR-L2 sequence is WASTRDS (SEQ ID NO:8); the CDR-L3 sequence is QQYYSYPRT (SEQ ID NO:13) or QQYASYPRT (SEQ ID NO:14) or QQYYAYPRT (SEQ ID NO:15), and a heavy chain variable region wherein the CDR-H1 sequence is GYTFSSYWIE (SEQ ID NO:17); the CDR-H2 sequence is EISPGSGSTXYXEEFKG (SEQ ID NO:19) or EIXPGSGSTIYXEKFKG (SEQ ID NO:110), wherein X is any amino acid; and the CDR-H3 sequence is GYHGY (SEQ ID NO:21) or GYHGA (SEQ ID NO:22).

### Claim 2 (depends on 1)

The antibody of claim 1 wherein SEQ ID NO:3 is KSSQSLLYSTAQKNFLA (SEQ ID NO:5).

### Claim 3 (depends on 1, 2)

The antibody of claim 1 or 2 wherein the CDR-L1 sequence is SEQ ID NO: 3 or 4 or 6.

### Claim 4 (depends on 1)

The antibody of claim 1 comprising either all of SEQ ID NOS:3, 8, and 13, or all of SEQ ID NOS:4, 5 or 6, 8, and 13, or all of SEQ ID NOS:3, 8, and 14 or 15, or all of SEQ ID NOS:4, 5 or 6, 8, and 14 or 15, or all of SEQ ID NOS:17, 19, and 21 or 22, or all of SEQ ID NOS:17, 110, and 21.

### Claim 5 (depends on 1)

The antibody of claim 1 comprising (i) all of the CDR-L1 to CDR-L3 amino acid sequences of SEQ ID NOS:3, 8, and 13; or of SEQ ID NOS:4, 5, and 13, or of SEQ ID NOS:6, 8, and 13, or of SEQ ID NOS:3, 8, and 14, or of SEQ ID NOS:3, 8, and 15, or of SEQ ID NOS:4, 5, and 14, or of SEQ ID NOS:4, 5, and 15 or of SEQ ID NOS:6, 8, and 14 or of SEQ ID NOS:6, 8, and 15 and (ii) all of the CDR-H1 to CDR-H3 amino acid sequences of SEQ ID NOS:17, 19, and 21, or SEQ ID NOS:17, 19, and 22, or of SEQ ID NOS:17, 110, and 21.

### Claim 6 (depends on 1)

The antibody of claim 1 comprising a human κ subgroup 1 consensus framework sequence.

### Claim 7 (depends on 6)

The antibody of claim 6 comprising a heavy-chain human subgroup III consensus framework sequence.

### Claim 8 (depends on 7)

The antibody of claim 7 wherein the framework sequence comprises a substitution at position 71, 73, or 78 of the EU index as in Kabat.

### Claim 9 (depends on 8)

The antibody of claim 8 wherein said substitution is R71A, N73T, or N78A or a combination thereof.

### Claim 10 (depends on 1)

The antibody of claim 1 that binds to lymphotoxin-α3 trimer (LTα3) and blocks the interaction of LTα3 with tumor necrosis factor receptor-I (TNFRI) and tumor necrosis factor receptor-II (TNFRII).

### Claim 11 (depends on 1)

The antibody of claim 1 that binds to a lymphotoxin-αβ heterodimer (LTαβ) complex.

### Claim 12 (depends on 1)

The antibody of claim 1 that blocks LTαβ function.

### Claim 13 (depends on 1)

The antibody of claim 1 that has an Fc region, and decreases levels of inflammatory cytokines associated with rheumatoid arthritis in an in vivo collagen-induced arthritis assay or an in vivo antibody-induced arthritis assay.

### Claim 14 (depends on 1)

The antibody of claim 1 that blocks the interaction of LTαβ with lymphotoxin-beta receptor (LTβ-R).

### Claim 15 (depends on 1)

The antibody of claim 1 that depletes LTα-positive cells.

### Claim 16 (depends on 1)

The antibody of claim 1 that modulates LTαβ-expressing cells.

### Claim 17 (depends on 1)

The antibody of claim 1 that is chimeric or humanized.

### Claim 18 (depends on 17)

The antibody of claim 17 that is humanized.

### Claim 19 (depends on 18)

The antibody of claim 18 wherein at least a portion of its framework sequence is a human consensus framework sequence.

### Claim 20 (depends on 1)

The antibody of claim 1 wherein said antibody binds LTα with an affinity of at least about 10−12 M.

### Claim 21 (depends on 1)

The antibody of claim 1 that is of the IgG isotype.

### Claim 22 (depends on 21)

The antibody of claim 21 wherein the IgG is IgG1 or IgG2a.

### Claim 23 (depends on 13)

The antibody of claim 13 wherein the IgG Fc region is a wild-type IgG Fc region.

### Claim 24 (depends on 13)

The antibody of claim 13 further comprising from one to ten amino acid substitutions selected from the group consisting of A, D, E, L, Q, T, and Y at any one or a combination of positions of the IgG Fc region selected from the group consisting of 238, 239, 246, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 309, 312, 314, 315, 320, 322, 324, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 428, 430, 434, 435, 437, 438 and 439 in its IgG Fc region that result in it having increased antibody-dependent cell-mediated cytotoxicity (ADCC) function as compared to the same antibody having a native-sequence IgG Fc region wherein the numbering is that of the EU index as in Kabat.

### Claim 25 (depends on 24)

The antibody of claim 24 comprising from one to three amino acid substitutions in its IgG Fc region.

### Claim 26 (depends on 24)

The antibody of claim 24 wherein the IgG Fc region has the amino acid substitutions at any one or any combination of positions that are 268D, or 298A, or 326D, or 333A, or 334A, or 298A together with 333A, or 298A together with 334A, or 239D together with 332E, or 239D together with 298A and 332E, or 239D together with 268D and 298A and 332E, or 239D together with 268D and 298A and 326A and 332A, or 239D together with 268D and 298A and 326A and 332E, or 239D together with 268D and 283L and 298A and 332E, or 239D together with 268D and 283L and 298A and 326A and 332E, or 239D together with 330L and 332E and 272Y and 254T and 256E, or 250Q together with 428L, or 265A, or 297A, wherein the 265A substitution is in the absence of 297A and the 297A substitution is in the absence of 265A, wherein the numbering is that of the EU index as in Kabat.

### Claim 27 (depends on 18)

The antibody of claim 18 having a monovalent affinity to human LTα that is about the same as or greater than the monovalent affinity to human LTα of a murine antibody produced by a hybridoma cell line deposited under American Type Culture Collection Accession Number PTA-7538 (hybridoma murine Lymphotoxin alpha2 beta1 s5H3.2.2).

### Claim 28 (depends on 27)

The humanized antibody of claim 27 wherein the monovalent affinity is expressed as a Kd value.

### Claim 29 (depends on 28)

The humanized antibody of claim 28 wherein the monovalent affinity is measured by optical biosensor or radioimmunoassay.

### Claim 30 (depends on 1)

The antibody of claim 1 having a light-chain variable domain comprising SEQ ID NO:108, or a heavy-chain variable domain comprising SEQ ID NO:109, or having light-chain and heavy-chain variable domains comprising both SEQ ID NOS:108 and 109.

### Claim 31 (depends on 1)

An antibody of claim 1 having reduced fusose relative to the amount of fucose on the same antibody produced in a Chinese hamster ovary cell that has not been modified to reduce fucosylation.

### Claim 32 (depends on 1)

The antibody of claim 1 having no fucose.

### Claim 33 (depends on 1)

An antibody composition comprising the antibodies of claim 1 having an IgG Fc region, wherein about 20-100% of the antibodies in the composition comprise a mature core carbohydrate structure in the Fc region that lacks a fucose.

### Claim 34 (depends on 33)

The composition of claim 33 wherein the antibody comprises an IgG Fc region comprising from one to ten amino acid substitutions selected from the group consisting of A, D, E, L, Q, T, and Y at any one or any combination of positions of the IgG Fc region selected from the group consisting of: 238, 239, 246, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 309, 312, 314, 315, 320, 322, 324, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 428, 430, 434, 435, 437, 438 and 439 in its IgG Fc region that result in it having increased antibody-dependent cell-mediated cytotoxicity (ADCC) function as compared to the same antibody having a native-sequence IgG Fc region wherein the numbering is that of EU index as in Kabat.

### Claim 35 (depends on 33)

The composition of claim 33 wherein the antibody comprises an Fc substitution that is 268D or 326D or 333A together with 334A, or 298A together with 333A, or 298A together with 334A, or 239D together with 332E, or 239D together with 298A and 332E, or 239D together with 268D and 298A and 332E, or 239D together with 268D and 298A and 326A and 332A, or 239D together with 268D and 298A and 326A and 332E, or 239D together with 268D and 283L and 298A and 332E, or 239D together with 268D and 283L and 298A and 326A and 332E, or 239D together with 330L and 332E, wherein the numbering is that of EU index as in Kabat.

### Claim 36 (depends on 33)

The composition of claim 33 wherein the antibody binds an FcγRIII.

### Claim 37 (depends on 33)

The composition of claim 33 wherein the antibody binds the FcγRIII with better affinity, or mediates antibody-dependent cell-mediated cytotoxicity (ADCC) more effectively, than a glycoprotein with a mature core carbohydrate structure including fucose attached to the Fc region of the glycoprotein.

### Claim 38 (depends on 33)

The composition of claim 33 wherein the antibody has been produced by a Chinese hamster ovary (CHO) cell.

### Claim 39 (depends on 38)

The composition of claim 38 wherein the CHO cell is a Lec13 cell.

### Claim 40 (depends on 33)

The composition of claim 33 wherein the antibody has been produced by a mammalian cell lacking a fucosyltransferase gene.

### Claim 41 (depends on 40)

The composition of claim 40 wherein the fucosyltransferase gene is the FUT8 gene.

### Claim 42 (depends on 33)

The composition of claim 33 wherein the antibody is free of bisecting N-acetylglucosamine (GlcNAc) attached to its mature core carbohydrate structure.

### Claim 43 (depends on 33)

The composition of claim 33 wherein the antibody has bisecting N-acetylglucosamine (GlcNAc) attached to its mature core carbohydrate structure.

### Claim 44 (depends on 33)

The composition of claim 33 wherein the antibody has one or more galactose residues attached to its mature core carbohydrate structure.

### Claim 45 (depends on 33)

The composition of claim 33 wherein the antibody is free of one or more galactose residues attached to its mature core carbohydrate structure.

### Claim 46 (depends on 33)

The composition of claim 33 wherein the antibody has one or more sialic acid residues attached to its mature core carbohydrate structure.

### Claim 47 (depends on 33)

The composition of claim 33 wherein the antibody is free of one or more sialic acid residues attached to its mature core carbohydrate structure.

### Claim 48 (depends on 33)

The composition of claim 33 that comprises at least about 2% afucosylated antibodies.

### Claim 49 (depends on 33)

The composition of claim 33 that comprises at least about 4% afucosylated antibodies.

### Claim 50 (depends on 33)

The composition of claim 33 that comprises at least about 10% afucosylated antibodies.

### Claim 51 (depends on 33)

The composition of claim 33 that comprises at least about 19% afucosylated antibodies.

### Claim 52 (depends on 33)

The composition of claim 33 that comprises about 100% afucosylated antibodies.

### Claim 53 (depends on 1)

A composition comprising the antibody of claim 1 and a carrier.

### Claim 54 (depends on 53)

The composition of claim 53 further comprising a second medicament, wherein the anti-LTα antibody is a first medicament.

### Claim 55 (depends on 54)

The composition of claim 54 wherein the second medicament is a an immunosuppressive agent, an antagonist that binds a B-cell surface marker, a B-cell Activating Factor (BAFF) antagonist, a disease-modifying anti-rheumatic drug (DMARD), an integrin antagonist, a non-steroidal anti-inflammatory drug (NSAID), a cytokine antagonist, or a combination thereof.

### Claim 56

An isolated anti-lymphotoxin-α (LTα) antibody comprising a light chain variable region wherein complementarity-determining region (CDR)-L1 comprises amino acids 24-40 of SEQ ID NO: 108, CDR-L2 comprises amino acids 56-62 of SEQ ID NO: 108, and CDR-L3 comprises amino acids 95-103 of SEQ ID NO: 108, and a heavy chain variable region wherein CDR-H1 comprises amino acids 26-35 of SEQ ID NO: 109, CDR-H2 comprises amino acids 50-66 of SEQ ID NO: 109, and CDR-H3 comprises amino acids 99-103 of SEQ ID NO: 109.

### Claim 57 (depends on 56)

The antibody of claim 56 that is chimeric or humanized.

### Claim 58 (depends on 57)

The antibody of claim 57 that is humanized.

### Claim 59 (depends on 56)

The antibody of claim 56 that is of the IgG Isotype.

### Claim 60 (depends on 56)

The antibody of claim 56 wherein the IgG is IgG1 or IgG2a.

### Claim 61 (depends on 56)

An antibody of claim 56 having reduced fucose relative to the amount of fucose on the same antibody produced in a Chinese hamster ovary cell that has not been modified to reduce fucosylation.

### Claim 62 (depends on 61)

The antibody of claim 61 having no fucose.

### Claim 63 (depends on 56)

An antibody composition comprising antibodies of claim 56 having an Fc region, wherein about 20-100% of the antibodies in the composition comprise a mature core carbohydrate structure in the Fc region that lacks a fucose.

### Claim 64 (depends on 56)

A composition comprising the antibody of claim 56 and a carrier.