ANTIBODIES AGAINST THE RGM A PROTEIN AND USES THEREOF

• US 20100028340A1
• Filed: 02/20/2009
• Published: 02/04/2010
• Est. Priority Date: 02/29/2008
• Status: Active Grant

• Current Assignee: Abbvie Deutschland GmbH & Company KG (Abbvie Incorporated)
• Original Assignee: Abbott GmbH & Company KG (Abbott Laboratories), Abbott Laboratories
• Inventors: Barlow, Eve, Leddy, Mary, Schmidt, Martin, Mueller, Bernhard K., Hsieh, Chung-Ming, Bardwell, Philip
• Granted Patent: US 8,962,803 B2
• Time in Patent Office: Days
• US Class Current: 424/133.100
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/02 for peripheral neuropathies
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/18 Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• CPC Class Codes: A61P 25/20 Hypnotics; Sedatives
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/06 Antiglaucoma agents or miotics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 2317/24 : containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/56 : variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/565 : Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/567 : Framework region [FR]
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The subject invention relates to isolated proteins, particularly monoclonal antibodies, which bind and neutralize RGM A protein. Specifically, these antibodies have the ability to inhibit the binding of RGM A to its receptor and/or coreceptors. These antibodies or portions thereof of the invention are useful for detecting RGM A and for inhibiting RGM A activity, for example in a human suffering from a disorder including but nor limited to multiple sclerosis, mammalian brain trauma, spinal cord injury, stroke, neurodegenerative diseases, and schizophrenia.

Claims

1. A binding protein that dissociates from human RGM A with a K_D Of 1×

10<sup>−

7</sup> M or less and a k_off rate constant of 1×

10<sup>−

2</sup> s<sup>−

1</sup> or less, both determined by surface plasmon resonance.

3. The binding protein of one of the preceding claims, having at least one of the following additional functional characteristics:

binding to rat RGM A,

binding to human RGM C, and

binding to rat RGM C.

4. The binding protein of one of the preceding claims, wherein said binding protein modulates the ability of RGM to bind to at least one of its receptors.

5. The binding protein of claim 4, wherein said binding protein interacts with a receptor binding domain of human RGM A.

6. The binding protein of claim 4, wherein said binding protein modulates at least one of the following interactions:

binding of human RGM A to human BMP-4.

binding of hRGM A to human Neogenin,

binding of hRGM C to human Neogenin,

binding of human RGM A to human BMP-2.

7. The binding protein according to one of the preceding claims, which is a humanized antibody.

8. The binding protein according to one of the preceding claims, comprising an antigen binding domain, said binding protein capable of binding an epitope of an RGM molecule, said antigen binding domain comprising at least one CDR comprising an amino acid sequence selected from the group consisting of

10. The binding protein according to one of the preceding claims, further comprising at least one CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

57, 58, 60, 61, 63, 64, 66, 67 and modified CDR amino acid sequences having a sequence identity of at least 50% to one of said sequences.

11. The binding protein according to any one of the preceding claims, wherein said at least one CDR comprises an amino acid sequence selected from the group consisting of:

12. The binding protein according to claim 11, comprising at least 3 CDRs which are selected from a variable domain CDR set consisting of:

13. The binding protein according to claim 12, comprising at least two variable domain CDR sets.

14. The binding protein according to claim 13, wherein said at least two variable domain CDR sets are selected from a group consisting of:

VH 5F9 set &

VL 5F9 set; and

VH 8D1 set &

VL 8D1 set

15. The binding protein according to one of the preceding claims, further comprising a human acceptor framework.

16. The binding protein according to claim 15, wherein said human acceptor framework comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO:

15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and 33.

17. The binding protein according to claim 16, comprising a set of framework sequences selected from the group consisting of the sets:

VH3-48 set (Seq ID NO;

15, 16 and

     17)

VH3-33 set (SEQ ID NO;

21, 22 and

     23)

VH3-23 set (SEQ ID NO;

24, 25 and

     26)

each of which sets being combined with a further framework sequence, selected from

JH3 (SEQ ID NO;

18),

JH4 (SEQ ID NO;

19),

JH6 (SEQ ID NO;

20);

or

selected from the group consisting of the sets

A18 set;

(SEQ ID NO;

27, 28 and

     29)

A17 set;

(SEQ ID NO;

31, 32 and

     33)

each of which sets being combined with a further framework sequence, selected from JK2 (SEQ ID NO;

2)

18. The binding protein of any one of the preceding claims comprising at least one heavy chain variable domain selected from SEQ ID NO:

35, 36, 37, 38, 39, 40, 41, 42, and 43; and

/or at least one light chain variable domain selected from SEQ ID NO;

44, 45, and 46.

19. The binding protein of claim 18, wherein said binding protein comprises two variable domains, wherein said two variable domains have amino acid sequences selected from:

SEQ ID NOs;

35 &

44;

36 &

44;

37 &

44;

38 &

44;

39 &

44;

40 &

44;

41 &

44;

42 &

44;

43 &

44;

SEQ ID NOs;

35 &

45;

36 &

45;

37 &

45;

38 &

45;

39 &

45;

40 &

45;

41 &

45;

42 &

45;

43 &

45;

SEQ ID NOs;

35 &

46;

36 &

46;

37 &

46;

38 &

46;

39 &

46;

40 &

46;

41 &

46;

42 &

46;

43 &

46;

20. The binding protein according to any one of the claim 15 to 19, wherein said human acceptor framework comprises at least one framework region amino acid substitution at a key residue, said key residue selected from the group consisting of:

a residue adjacent to a CDR;

a glycosylation site residue;

a rare residue;

a residue capable of interacting with a RGM epitope;

a residue capable of interacting with a CDR;

a canonical residue;

a contact residue between heavy chain variable region and light chain variable region;

a residue within a Vernier zone;

an N-terminal residue capable of paraglutamate formation; and

a residue in a region that overlaps between a Chothia-defined variable heavy chain CDR1 and a Kabat-defined first heavy chain framework.

21. The binding protein according to claim 20, wherein said key residue are selected from the group consisting

(heavy chain sequence position):

1, 5, 37, 48, 49, 88, 98

(light chain sequence position);

2, 4, 41, 51

22. The binding protein of any one of the preceding claims, wherein the binding protein is a consensus human variable domain.

23. The binding protein of any one of the claims 15 to 22, wherein said human acceptor framework comprises at least one framework region amino acid substitution, wherein the amino acid sequence of the framework is at least 65% identical to the sequence of said human acceptor framework and comprises at least 70 amino acid residues identical to said human acceptor framework.

24. The binding protein of any one of the preceding claims, wherein said binding protein comprises at least one (framework mutated) variable domain having an amino acid sequence selected from the group consisting of:

SEQ ID NO;

47, 48, 49, 50;

SEQ ID NO;

51, 52, 53, and 54

25. The binding protein of claim 24, wherein said binding protein comprises two variable domains, wherein said two variable domains have amino acid sequences selected from the groups consisting of:

SEQ ID NOs;

47 &

44;

47 &

45;

47 &

46;

47 &

51;

47 &

52;

47 &

53;

47 &

54;

SEQ ID NOs;

48 &

44;

48 &

45;

48 &

46;

48 &

51;

48 &

52;

48 &

53;

48 &

54;

SEQ ID NOs;

49 &

44;

49 &

45;

49 &

46;

49 &

51;

49 &

52;

49 &

53;

49 &

54;

SEQ ID NOs;

50 &

44;

50 &

45;

50 &

46;

50 &

51;

50 &

52;

50 &

53;

50 &

54;

26. The binding protein of any one of the preceding claims, wherein said binding protein is capable of binding a target, selected from RGM molecules.

27. The binding protein of any one of the preceding claims capable of binding to human RGM A.

28. The binding protein of claim 27, having at least one of the following additional functional characteristics:

binding to rat RGM A,

binding to human RGM C,

binding to rat RGM C.

29. The binding protein of any one of the preceding claims, wherein the binding protein is capable of modulating a biological function of a target, selected from RGM molecules.

30. The binding protein of claim 29, wherein said binding protein modulates the ability of RGM to bind to at least one of its receptors.

31. The binding protein of claim 30, wherein said binding protein modulates at least one of the following interactions:

binding of human RGM A to human BMP-4.

binding of hRGM A to human Neogenin,

binding of hRGM C to human Neogenin,

binding of human RGM A to human BMP-2,

32. The binding protein of any one of the preceding claims, wherein said binding protein is capable of inhibiting an RGM biological activity.

33. The binding protein of claim 32, wherein the RGM molecule is RGM A.

34. The binding protein of claim 33, wherein the RGM A is selected from human, cynomolgus monkeys, rat, chick, frog, and fish.

35. The binding protein of any one of the preceding claims, wherein said binding protein has an on rate constant (k_on) to said target selected from the group consisting of:

at least about 10² M<sup>−

1</sup> s<sup>−

1</sup>;

at least about 10³ M<sup>−

1</sup> s<sup>−

1</sup>;

at least about 10⁴ M<sup>−

1</sup> s<sup>−

1</sup>;

at least about 10⁵ M<sup>−

1</sup> s<sup>−

1</sup>;

at least about 10⁶ M<sup>−

1</sup> s<sup>−

1</sup>, and at least about 10⁷ M<sup>−

1</sup> s<sup>−

1</sup> as measured by surface plasmon resonance.

36. The binding protein of any one of the preceding claims, wherein said binding protein has an off rate constant (k_off) to said target selected from the group consisting of:

at most about 10<sup>−

2</sup> s<sup>−

1</sup>;

at most about 10<sup>−

3</sup> s<sup>−

1</sup>;

at most about 10<sup>−

4</sup> s<sup>−

1</sup>;

at most about 10<sup>−

5</sup> s<sup>−

1</sup>; and

at most about 10<sup>−

6</sup> s<sup>−

1</sup>, as measured by surface plasmon resonance.

37. The binding protein of any one of the preceding claims, wherein said binding protein has a dissociation constant (K_D) to said target selected from the group consisting of:

at most about 10<sup>−

7</sup> M;

at most about 10<sup>−

8</sup> M;

at most about 10<sup>−

9</sup> M;

at most about 10<sup>−

10</sup> M;

at most about 10<sup>−

11</sup> M;

at most about 10<sup>−

12</sup> M; and

at most 10<sup>−

13</sup>M.

38. An antibody construct comprising a binding protein described in any one of the preceding claims, said antibody construct further comprising a linker polypeptide or an immunoglobulin constant domain.

39. The antibody construct according to claim 38, wherein said binding protein is selected from the group consisting of:

an immunoglobulin molecule,

a monoclonal antibody,

a chimeric antibody,

a CDR-grafted antibody,

a humanized antibody,

a Fab,

a Fab′

,

a F(ab′

)2,

a Fv,

a disulfide linked Fv,

a scFv,

a single domain antibody,

a diabody,

a multispecific antibody,

a dual specific antibody,

a dual variable domain immunoglobulin, and

a bispecific antibody.

40. The antibody construct according to any on of the claims 38 and 39, wherein said binding protein comprises a heavy chain immunoglobulin constant domain selected from the group consisting of;

a human IgM constant domain,

a human IgG1 constant domain,

a human IgG2 constant domain,

a human IgG3 constant domain,

a human IgG4 constant domain,

a human IgE constant domain,

a human IgD constant domain,

a human IgA1 constant domain

a human IgA2 constant domain

a human IgY constant domain and

and corresponding mutated domains.

41. The antibody construct according to any on of the claims 38 to 40, comprising an immunoglobulin constant domain having an amino acid sequence selected from the group consisting of:

SEQ ID NO;

11, 12, 13 and 14

42. An antibody conjugate comprising an antibody construct described in any one of claims 38 to 41, said antibody conjugate further comprising an agent selected from the group consisting of;

an immunoadhension molecule, an imaging agent, a therapeutic agent, and a cytotoxic agent.

43. The antibody conjugate according to claim 42, wherein said agent is an imaging agent selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin.

44. The antibody conjugate according to claim 43, wherein said imaging agent is a radiolabel selected from the group consisting of:

³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, and ¹⁵³Sm.

45. The antibody conjugate according to claim 43, wherein said agent is a therapeutic or cytotoxic agent selected from the group consisting of;

an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, toxin, and an apoptotic agent.

46. The antibody construct according to any on of the claims 38 to 41, wherein said binding protein possesses a human glycosylation pattern.

47. The antibody conjugate according to any on of the claims 42 to 45, wherein said binding protein possesses a human glycosylation pattern.

48. The binding protein according to any one of the claims 1 to 37, wherein said binding protein exists as a crystal.

49. The antibody construct according to any one of the claims 38 to 41, wherein said antibody construct exists as a crystal.

50. The antibody conjugate according to any one of the claims 42 to 45, wherein said antibody construct exists as a crystal.

51. The binding protein according to claim 48, wherein said crystal is a carrier-free pharmaceutical controlled release crystal.

52. The antibody construct according to claim 49, wherein said crystal is a carrier-free pharmaceutical controlled release crystal.

53. The antibody conjugate according to claim 50, wherein said crystal is a carrier-free pharmaceutical controlled release crystal.

54. The binding protein according to claim 48, wherein said binding protein has a greater half life in vivo than the soluble counterpart of said binding protein.

55. The antibody construct according to claim 49, wherein said antibody construct has a greater half life in vivo than the soluble counterpart of said antibody construct.

56. The antibody conjugate according to claim 50, wherein said antibody conjugate has a greater half life in vivo than the soluble counterpart of said antibody conjugate.

57. The binding protein according to claim 48, wherein said binding protein retains biological activity.

58. The antibody construct according to claim 49, wherein said antibody construct retains biological activity.

59. The antibody conjugate according to claim 50, wherein said antibody conjugate retains biological activity.

60. An isolated nucleic acid encoding a binding protein amino acid sequence of any one of claims 1-37.

61. An isolated nucleic acid encoding an antibody construct amino acid sequence of any one of claims 38-41.

62. An isolated nucleic acid encoding an antibody conjugate amino acid sequence of any one of claims 42-45.

63. A vector comprising an isolated nucleic acid according to any one of claims 60 to 62.

64. The vector of claim 63 wherein said vector is selected from the group consisting of pcDNA, pTT, pTT3, PEFBOS, pBV, pJV, pHybE, and pBJ.

65. A host cell comprising a vector according to any one of claims 63 and 64.

66. The host cell according to claim 65, wherein said host cell is a prokaryotic cell.

67. The host cell according to claim 66, wherein said host cell is E. coli.

68. The host cell according to claim 67, wherein said host cell is a eukaryotic cell.

69. The host cell according to claim 68, wherein said eukaryotic cell is selected from the group consisting of protist cell, animal cell, plant cell and fungal cell.

70. The host cell according to claim 69, wherein said eukaryotic cell is an animal cell selected from the group consisting of;

a mammalian cell, an avian cell, and an insect cell.

71. The host cell according to claim 69, wherein said host cell is selected from HEK Cells, CHO cells COS cells and yeast cells.

72. The host cell according to claim 71, wherein said yeast cell is Saccharomyces cerevisiae.

73. The host cell according to claim 70, wherein said host cell is an insect Sf9 cell.

74. A method of producing a protein capable of binding RGM, comprising culturing a host cell of any one of claims 65 to 73 in culture medium under conditions sufficient to produce a binding protein capable of binding RGM.

75. A protein produced according to the method of claim 74.

76. A composition for the release of a binding protein said composition comprising

(a) a formulation, wherein said formulation comprises a crystallized product protein according to any one of claims 48 to 50, and an ingredient;

and

(b) at least one polymeric carrier.

77. The composition according to claim 76, wherein said polymeric carrier is a polymer selected from one or more of the group consisting of:

poly(acrylic acid), poly(cyanoacrylates), poly(amino acids), poly(anhydrides), poly (depsipeptide), poly(esters), poly(lactic acid), poly(lactic-co-glycolic acid) or PLGA, poly(b-hydroxybutryate), poly(caprolactone), poly(dioxanone);

poly (ethylene glycol), poly((hydroxypropyl)methacrylamide, poly [(organo) phosphazene], poly(ortho esters), poly(vinyl alcohol), poly(vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polyeaccharides, blends and copolymers thereof.

78. The composition according to claim 76, wherein said ingredient is selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β

-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol.

79. A method for treating a mammal comprising the step of administering to the mammal an effective amount of the composition according to any one of the claims 77 and 78.

80. A pharmaceutical composition comprising the product of any one of claims 1 to 59, and a pharmaceutically acceptable carrier.

81. The pharmaceutical composition of claim 80, wherein said pharmaceutically acceptable carrier functions as adjuvant useful to increase the absorption, or dispersion of said binding protein.

82. The pharmaceutical composition of claim 81, wherein said adjuvant is hyaluronidase.

83. The pharmaceutical composition of claim 82 further comprising at least one additional therapeutic agent for treating a disorder in which RGM activity is detrimental.

84. The pharmaceutical composition of claim 83, wherein said additional agent is selected from the group consisting of:

Therapeutic agent, imaging agent, cytotoxic agent, angiogenesis inhibitors;

kinase inhibitors;

co-stimulation molecule blockers;

adhesion molecule blockers;

anti-cytokine antibody or functional fragment thereof;

methotrexate;

cyclosporin;

rapamycin;

FK506;

detectable label or reporter;

a TNF antagonist;

an antirheumatic;

a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressive, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an epinephrine or analog, a cytokine, and a cytokine antagonist.

85. A method for reducing human RGM A activity comprising contacting human RGM A with the product of any one of claims 1 to 59 such that human RGM A activity is reduced.

86. A method for decreasing hRGM A binding to Neogenin receptor in a subject in need thereof, comprising the step of administering to the subject a product of any one of claims 1 to 59.

87. A method for decreasing hRGM A binding to bone morphogenetic protein-2 and bone morphogenetic protein-4 (BMP-2 and BMP-4) in a subject in need thereof, comprising the step of administering to the subject a product of any one of claims 1 to 59.

88. A method of treating a subject for a disorder associated with RGM A activity comprising the step of administering alone or in combination with other therapeutic agents a product of any one of claims 1 to 59.

89. A method for reducing RGM A activity in a subject suffering from a disorder in which RGM A activity is detrimental, comprising administering to the subject a product of any one of claims 1 to 59, alone or in combination with other therapeutic agents.

90. The method of claim 89, wherein the disorder comprises neurological diseases selected from the group comprising Amyotrophic Lateral Sclerosis, Brachial Plexus Injury, Brain Injury, including traumatic brain injury, Cerebral Palsy, Guillain Barre, Leukodystrophies, Multiple Sclerosis, Post Polio, Spina Bifida, Spinal Cord Injury, Spinal Muscle Atrophy, Spinal Tumors, Stroke, Transverse Myelitits;

dementia, senile dementia, mild cognitive impairment, Alzheimer-related dementia, Huntington'"'"'s chorea, tardive dyskinesia, hyperkinesias, manias, Morbus Parkinson, steel-Richard syndrome, Down'"'"'s syndrome, myasthenia gravis, nerve trauma, vascular amyloidosis, cerebral hemorrhage I with amyloidosis, brain inflammation, acute confusion disorder, amyotrophic lateral sclerosis, glaucoma and Alzheimer'"'"'s disease.

91. An isolated CDR of a binding protein as defined in any one of the claims 1 to 51.

2. The binding protein that binds to human RGM A and neutralizes the neurite outgrowth inhibitory activity of human RGM A as determined in a standard in vitro assay.

9. A binding protein comprising an antigen binding domain, said binding protein capable of binding an epitope of an RGM molecule, said antigen binding domain comprising at least one CDR comprising an amino acid sequence selected from the group consisting of: