WO2010045388A2 - Use of mmp-9 and mmp-12 binding proteins for the treatment and prevention of systemic sclerosis - Google Patents

Use of mmp-9 and mmp-12 binding proteins for the treatment and prevention of systemic sclerosis Download PDF

Info

Publication number
WO2010045388A2
WO2010045388A2 PCT/US2009/060716 US2009060716W WO2010045388A2 WO 2010045388 A2 WO2010045388 A2 WO 2010045388A2 US 2009060716 W US2009060716 W US 2009060716W WO 2010045388 A2 WO2010045388 A2 WO 2010045388A2
Authority
WO
WIPO (PCT)
Prior art keywords
mmp
protein
antibody
binding
variable domain
Prior art date
Application number
PCT/US2009/060716
Other languages
French (fr)
Other versions
WO2010045388A3 (en
Inventor
Simone Nicholson
Clive Wood
Laetitia Devy
Original Assignee
Dyax Corp.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dyax Corp. filed Critical Dyax Corp.
Publication of WO2010045388A2 publication Critical patent/WO2010045388A2/en
Publication of WO2010045388A3 publication Critical patent/WO2010045388A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • MMPs Matrix Metalloproteinases
  • the MMP family consists of at least 26 members, all of which share a common catalytic core with a zinc molecule in the active site.
  • MMP-9 binding proteins proteins that bind MMP-9 or MMP-12, herein referred to as "MMP-9 binding proteins” or “MMP-12 binding proteins,” respectively, and methods of using such proteins to treat systemic sclerosis.
  • Systemic sclerosis is a clinically heterogeneous, systemic disorder which affects the connective tissue of the skin, internal organs and the walls of blood vessels. It is characterized by alterations of the microvasculature, disturbances of the immune system and by massive deposition of collagen and other matrix substances in the connective tissue. Sjogren's syndrome can also be associated with systemic sclerosis.
  • this disclosure relates, inter alia, to a method of treating or preventing systemic sclerosis in a subject, the method comprising: administering an isolated protein (e.g., antibody, e.g., human antibody) that binds MMP-9 to the subject, wherein the antibody binds the same epitope or competes for binding with an antibody selected from the group consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
  • the antibody competes with or binds the same epitope as DX- 2802.
  • the protein may also compete with another MMP-9 binding protein described herein.
  • the disclosure provides a method of treating or preventing systemic sclerosis in a subject, the method comprising: administering an isolated protein (e.g., antibody, e.g., human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence to the subject, wherein: the heavy chain immunoglobulin variable domain sequence comprises one, two, or three (e.g., three) CDR regions from the heavy chain variable domain of DX-2802, 539 A- M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166- FlO, M0279-A03, M0279-B02, M0288-C08, and M0281-F06, and/or the light chain immunoglobulin variable domain sequence comprises one, two, or three (e.g., three) CDR regions from the light chain variable domain of DX-2802, 539A- M0
  • the one, two, or three (e.g., three) CDR regions from the heavy chain variable domain are from DX-2802 and/or the one, two, or three (e.g., three) CDR regions from the light chain variable domain are from DX-2802.
  • the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of DX-2802, 539A-M0240-B03, M0078-G07,
  • the light chain immunoglobulin variable domain sequence comprises the light chain variable domain of DX-2802, 539A-M0240-B03, M0078- G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06 (respectively).
  • the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of DX-2802, and/or the the light chain immunoglobulin variable domain sequence comprises the light chain variable domain of DX- 2802.
  • the protein comprises the heavy chain of DX-2802, 539 A-M0240-
  • the protein comprises the heavy chain of DX-2802, and/or the light chain of DX-2802.
  • the protein may contain one or more (e.g., 1, 2, or 3) heavy chain and/or light chain CDR regions from another MMP-9 binding protein described herein. In some embodiments, the protein may contain the heavy chain and/or light chain variable domain from another MMP-9 binding protein described herein. In some embodiments, the protein may contain the heavy chain and/or light chain from another MMP-9 binding protein described herein.
  • this disclosure relates to a method of treating or preventing systemic sclerosis in a subject, the method comprising: administering an isolated protein that binds MMP-9 (e.g., an MMP-9 binding protein described herein) to the subject.
  • MMP-9 e.g., an MMP-9 binding protein described herein
  • the MMP-9 binding protein is used to treat the diffuse form of systemic sclerosis. In some embodiments, the MMP-9 binding protein is used to treat the limited form of systemic sclerosis.
  • the MMP-9 binding protein is used in combination with a second therapeutic agent.
  • the second therapeutic agent is another MMP-9 binding protein, e.g., another MMP-9 binding protein described herein.
  • the second therapeutic agent is another inhibitor described herein, e.g., a small molecule inhibitor of MMP-9.
  • the second therapeutic agent is an MMP- 12 binding protein, e.g., an MMP- 12 binding protein described herein.
  • the second therapeutic agent is e.g., cyclosporin, an NSAID (e.g., naproxen), a calcium channel blocker (e.g., nifedipine), an angiotensin receptor blocker (e.g., losartan), epoprostenol (prostacyclin), a prostacyclin analogue (e.g., iloprost, e.g., aerolized iloprost), a dual endothelin-receptor antagonist (e.g., bosentan), prostaglandin El (e.g., alprostadil), a proton pump inhibitor, an antibiotic (e.g., tetracycline), an immunosuppressant (e.g., cyclophosphamide, methotrexate or azathioprine), an ACE inhibitor, a sympathetic blocker, or surgery (e.g., lung transplantation).
  • an NSAID e.g., naproxen
  • These proteins include antibodies (including antibody fragments) (e.g., primate antibodies and Fabs, especially human antibodies and Fabs) that bind to MMP-9 (e.g., human MMP-9).
  • these proteins include antibodies (including antibody fragments) (e.g., primate antibodies and Fabs, especially human antibodies and Fabs) that inhibit MMP-9 (e.g., human MMP-9) (e.g., inhibit the catalytic activity of MMP-9).
  • the MMP-9 binding proteins can be used in the treatment of systemic sclerosis (scleroderma), in which excess or inappropriate activity of MMP-9 features. In many cases, the proteins have tolerable low or no toxicity.
  • the method uses a protein (e.g., an antibody, peptide or Kunitz domain protein) that binds MMP-9, in particular, a protein (e.g., an antibody (e.g., human antibody), peptide or Kunitz domain protein) that binds and inhibits MMP-9.
  • a protein e.g., an antibody (e.g., human antibody), peptide or Kunitz domain protein
  • the method uses an antibody (e.g., a human antibody) that binds to human MMP-9.
  • the human antibody is an inhibitor of the catalytic activity of MMP-9.
  • the antibody can be, e.g., an IgGl, IgG2, IgG3, IgG4, Fab, Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen combining site of one of the antibodies herein listed.
  • the antibody is used to guide a nano-particle or toxin to a cell expressing MMP-9 on the cell surface.
  • the antibody causes effector functions (CDC or ADCC) to kill the cell which expresses MMP-9.
  • the VH and VL regions of the binding proteins can be provided as IgG, Fab, Fab2, Fab2', scFv, PEGylated Fab, PEGylated scFv, PEGylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA + VH::CH1, HSA: :LC + VH::CH1, or other appropriate construct.
  • the binding protein comprises a Kunitz domain protein or modified version (e.g., HSA fusion) or peptide-based MMP-9 binding protein that can inhibit MMP-9 activity.
  • the method uses a protein (e.g., an isolated protein) that binds to MMP-9 (e.g., human MMP-9) and includes at least one immunoglobulin variable region.
  • MMP-9 e.g., human MMP-9
  • the protein includes a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence.
  • the protein binds to and inhibits MMP-9 (e.g., inhibits MMP-9 catalytic activity), e.g., human MMP- 9.
  • the protein binds to human MMP-9 specifically, and not to MMP- 9 from another species (e.g., the protein does not bind to MMP-9 from another species with greater than background levels of binding).
  • the protein binds MMP-9 specifically, and not to another matrix metalloproteinase (e.g., the protein does not bind to any other matrix metalloproteinase with greater than background levels of binding).
  • binding proteins can be conjugated to a drug (e.g., to form a MMP-9 binding protein-drug conjugate) and used therapeutically.
  • a drug e.g., to form a MMP-9 binding protein-drug conjugate
  • This disclosure relates, in part, to MMP-9 binding protein-drug conjugates, the preparation of these conjugates, and uses thereof.
  • the protein can include one or more of the following characteristics: (a) a human CDR or human framework region; (b) the HC immunoglobulin variable domain sequence comprises one or more (e.g., 1, 2, or 3) CDRs that are at least 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a HC variable domain described herein; (c) the LC immunoglobulin variable domain sequence comprises one or more (e.g., 1, 2, or 3) CDRs that are at least 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a LC variable domain described herein; (d) the LC immunoglobulin variable domain sequence is at least 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a LC variable
  • the protein can bind to MMP-9, e.g., human MMP-9, with a binding affinity of at least 10 5 , 10 6 , 10 7 ,10 8 , 10 9 , 10 10 and 10 11 M “1 . In one embodiment, the protein binds to MMP-9 with a K off slower than 1 X 10 ⁇ 3 , 5 X 10 "4 s "1 , or 1 X 10 ⁇ 4 s "1 . In one embodiment, the protein binds to
  • the protein inhibits human MMP-9 activity, e.g., with a Ki of less than 10 ⁇ 5 , 10 ⁇ 6 , 10 ⁇ 7 , 10 ⁇ 8 , 10 ⁇ 9 , and 10 ⁇ 10 M.
  • the protein can have, for example, an IC50 of less than 100 nM, 10 nM or 1 nM. In some embodiments, the protein has an IC50 of about 1.8 nM.
  • the affinity of the protein for MMP-9 can be characterized by a K D of less than 100 nm, less than 10 nM, or about 3 nM (e.g., 3.1 nM), about 5 nM (e.g., 5 nM), about 6 nm (e.g., 5.9 nM), about 7 nM (e.g., 7.1 nM), or about 10 nM (e.g., 9.6 nM).
  • K D K D of less than 100 nm, less than 10 nM, or about 3 nM (e.g., 3.1 nM), about 5 nM (e.g., 5 nM), about 6 nm (e.g., 5.9 nM), about 7 nM (e.g., 7.1 nM), or about 10 nM (e.g., 9.6 nM).
  • the protein has a K D ⁇ 200 nM.
  • the protein has a tl/2 of at least about 10 minutes (e.g., 11 minutes), at least about 20 minutes (e.g., 18 minutes), at least about 25 minutes(e.g., 25 minutes), at least about 35 minutes (e.g., 33 minutes), or at least about 60 minutes (e.g., 57 minutes).
  • the protein binds the catalytic domain of human MMP-9, e.g., the protein contacts residues in or near the active site of MMP-9.
  • the protein does not contact residues in or near the active site of MMP-9 but instead binds elsewhere on MMP-9 and causes a steric change in MMP-9 that affects (e.g., inhibits) its activity.
  • the protein also binds to MMP-16 and/or MMP-24, e.g., with a binding affinity of at least 10 5 , 10 6 , 10 7 ,10 8 , 10 9 , 10 10 and 10 11 M "1 .
  • the protein binds to both MMP-9 and to MMP-16 or MMP-24 with a binding affinity of at least 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 and 10 11 M "1 .
  • the protein is an antibody (e.g., a human antibody) having the light and heavy chains of antibodies selected from the group consisting of DX-2802, 539A- M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
  • an antibody e.g., a human antibody having the light and heavy chains of antibodies selected from the group consisting of DX-2802, 539A- M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
  • the protein is an antibody (e.g., a human antibody) having its heavy selected from the group consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081- D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288- C08, and M0281-F06.
  • an antibody e.g., a human antibody having its heavy selected from the group consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081- D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288- C08, and M0281-F06.
  • the protein is an antibody (e.g., a human antibody) having its light chain selected from the group consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
  • an antibody e.g., a human antibody having its light chain selected from the group consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs selected from the corresponding CDRs of the group of heavy chains consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
  • an antibody e.g., a human antibody having one or more (e.g., 1, 2, or 3) heavy chain CDRs selected from the corresponding CDRs of the group of heavy chains consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs selected from the corresponding CDRs of the group of light chains consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
  • an antibody e.g., a human antibody having one or more (e.g., 1, 2, or 3) light chain CDRs selected from the corresponding CDRs of the group of light chains consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs selected from the corresponding CDRs of the group of light chains consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06and one or more (e.g., 1, 2, or 3) heavy chain CDRs selected from the corresponding CDRs of the group of heavy chains consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M
  • the protein is an antibody (e.g., a human antibody) having light and heavy antibody variable regions of an antibody selected from the group consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075- D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
  • an antibody e.g., a human antibody having light and heavy antibody variable regions of an antibody selected from the group consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075- D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
  • the protein is an antibody (e.g., a human antibody) having a heavy chain antibody variable region of an antibody selected from the group consisting of: DX- 2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
  • an antibody e.g., a human antibody having a heavy chain antibody variable region of an antibody selected from the group consisting of: DX- 2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
  • the protein is an antibody (e.g., a human antibody) having a light chain antibody variable region of an antibody selected from the group consisting of: DX- 2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
  • an antibody e.g., a human antibody having a light chain antibody variable region of an antibody selected from the group consisting of: DX- 2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
  • the protein is an antibody (e.g., a human antibody) having the light and heavy chains of antibodies from M0166-F10. In another preferred embodiment, the protein is an antibody (e.g., a human antibody) having its heavy chain from M0166-F10. In yet another preferred embodiment, the protein is an antibody (e.g., a human antibody) having its light chain from M0166-F10.
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain of M0166-F10.
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chain of M0166-F10.
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain of M0166-F10 and one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains of M0166- FlO.
  • an antibody e.g., a human antibody having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain of M0166-F10 and one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains of M0166- FlO.
  • the protein is an antibody (e.g., a human antibody) having light and heavy antibody variable regions of M0166-F10.
  • the protein is an antibody (e.g., a human antibody) having the heavy chain antibody variable region of M0166-F10.
  • the protein is an antibody (e.g., a human antibody) having the light chain antibody variable region of M0166-F10.
  • the protein is an antibody (e.g., a human antibody) having the light and heavy chains of antibodies from 539A-M0240-B03.
  • the protein is an antibody (e.g., a human antibody) having its heavy chain from 539A-M0240-B03.
  • the protein is an antibody (e.g., a human antibody) having its light chain from 539A-M0240-B03.
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain of 539A-M0240-B03.
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains of 539A-M0240-B03.
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain of 539A- M0240-B03 and one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains of 539A-M0240-B03.
  • the protein is an antibody (e.g., a human antibody) having light and heavy antibody variable regions of 539A-M0240-B03.
  • the protein is an antibody (e.g., a human antibody) having the heavy chain antibody variable region of 539A-M0240-B03.
  • the protein is an antibody (e.g., a human antibody) having the light chain antibody variable region of 539A- M0240-B03.
  • the protein is an antibody (e.g., a human antibody) having the light and heavy chains of antibodies from X0034-C02 (DX-2802).
  • the protein is an antibody (e.g., a human antibody) having its heavy chain from X0034-C02.
  • the protein is an antibody (e.g., a human antibody) having its light chain from X0034-C02.
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain of X0034-C02.
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains of X0034-C02.
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain of X0034-C02 and one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains of X0034- C02.
  • the protein is an antibody (e.g., a human antibody) having light and heavy antibody variable regions of X0034-C02.
  • the protein is an antibody (e.g., a human antibody) having the heavy chain antibody variable region of X0034-C02.
  • the protein is an antibody (e.g., a human antibody) having the light chain antibody variable region of X0034-C02.
  • the protein may contain one or more (e.g., 1, 2, or 3) heavy chain and/or light chain CDR regions from another MMP-9 binding protein described herein. In some embodiments, the protein may contain the heavy chain and/or light chain variable domain from another MMP-9 binding protein described herein. In some embodiments, the protein may contain the heavy chain and/or light chain from another MMP-9 binding protein described herein.
  • the HC and LC variable domain sequences are components of the same polypeptide chain. In another, the HC and LC variable domain sequences are components of different polypeptide chains.
  • the protein is an IgG, e.g., IgGl, IgG2, IgG3, or IgG4.
  • the protein can be a soluble Fab (sFab).
  • the protein includes a Fab2', scFv, minibody, scFv::Fc fusion, Fab: :HS A fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen combining site of one of the binding proteins herein.
  • VH and VL regions of these Fabs can be provided as IgG, Fab, Fab2, Fab2', scFv, PEGylated Fab, PEGylated scFv, PEGylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA + VH::CH1, HSA: :LC + VH::CH1, or other appropriate construction.
  • the protein is a human or humanized antibody or is non- immunogenic in a human.
  • the protein includes one or more human antibody framework regions, e.g., all human framework regions.
  • the protein includes a human Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or 99% identical to a human Fc domain.
  • the protein is a primate or primatized antibody or is non- immunogenic in a human.
  • the protein includes one or more primate antibody framework regions, e.g., all primate framework regions.
  • the protein includes a primate Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or 99% identical to a primate Fc domain.
  • "Primate” includes humans ⁇ Homo sapiens), chimpanzees ⁇ Pan troglodytes and Pan paniscus (bonobos)), gorillas ⁇ Gorilla gorilla), gibons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and tarsiers.
  • the protein includes no sequences from mice or rabbits (e.g., is not a murine or rabbit antibody).
  • the protein is capable of binding to tumor cells expressing MMP-9, e.g., to Colo205 (a human colorectal carcinoma cell line), or MCF-7 (a human breast adenocarcinoma cell line) cells.
  • protein is physically associated with a nanoparticle, and can be used to guide a nanoparticle to a cell expressing MMP-9 on the cell surface.
  • the protein causes effector cells (CDC or ADCC) to kill a cell which expresses MMP-9.
  • the MMP-9 binding protein is administered (alone or in combination with a second agent (e.g., a second agent described herein)) to a subject to treat a symptom of systemic sclerosis, e.g., Raynaud's syndrome, swelling (eventually skin tightening and contractures of the fingers), polyarthralgia, dysphagia, dyspnea, lung fibrosis, acute alveolitis, pulmonary hypertension, heartburn, cardiac arrhythmias or renal crisis, or other symptom described herein.
  • a symptom of systemic sclerosis e.g., Raynaud's syndrome, swelling (eventually skin tightening and contractures of the fingers), polyarthralgia, dysphagia, dyspnea, lung fibrosis, acute alveolitis, pulmonary hypertension, heartburn, cardiac arrhythmias or renal crisis, or other symptom described herein.
  • the more evident symptom is usually the hardening of the skin and
  • the MMP-9 binding protein is administered (alone or in combination with a second agent (e.g., a second agent described herein)) to a subject to treat Sjogren's syndrome associated with SSc.
  • a second agent e.g., a second agent described herein
  • the MMP-9 binding protein is administered (alone or in combination with a second agent (e.g., a second agent described herein)) to a subject at risk of developing systemic sclerosis, e.g., with a familial predisposition for the disease (e.g., a polymorphism in COL1A2 or TGF- ⁇ l), or with another risk factor, e.g., a prior cytomegalovirus (CMV) infection, or exposure to organic solvents or other chemical agents (e.g., vinyl chloride, bleomycin, pentazocine, epoxy and/or aromatic hydrocarbons, contaminated rapeseed oil or L-tryptophan).
  • a familial predisposition for the disease e.g., a polymorphism in COL1A2 or TGF- ⁇ l
  • CMV cytomegalovirus
  • organic solvents or other chemical agents e.g., vinyl chloride, bleomycin, pentazocine, epoxy and/or aromatic
  • the disclosure features an MMP-9 binding protein that is a competitive inhibitor of MMP-9.
  • the binding protein competes with an MMP-9 substrate (e.g., collagen), e.g., binds to the same epitope as the substrate, e.g., and prevents substrate binding.
  • the method includes inhibiting an interaction between MMP-9 and an
  • MMP-9 substrate e.g., collagen
  • the method includes contacting an MMP-9 binding protein (e.g., an MMP-9 binding protein described herein) with MMP-9 (e.g., in vitro or in vivo), wherein the binding protein binds to MMP-9 and thereby prevents the binding of an MMP-9 substrate to MMP-9.
  • the binding protein binds to the same epitope on MMP-9 as the substrate, e.g., the binding protein is a competitive inhibitor.
  • the binding protein does not bind the same epitope as the substrate but causes a steric change in MMP-9 that decreases or inhibits the ability of the substrate to bind.
  • the method features a MMP-9 binding protein-drug conjugate that includes a MMP-9 binding protein (e.g., an MMP-9 binding protein described herein) and a drug.
  • a MMP-9 binding protein e.g., an MMP-9 binding protein described herein
  • the binding protein comprises at least one immunoglobulin variable region, and/or the protein binds to and/or inhibits MMP-9, e.g., inhibits MMP-9 catalytic activity.
  • the drug is a cytotoxic or cytostatic agent.
  • the cytotoxic agent can be, e.g., selected from the group consisting of an auristatin, a DNA minor groove binding agent, a DNA minor groove alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a podophyllotoxin, a baccatin derivative, a cryptophysin, a combretastatin, a maytansinoid, and a vinca alkaloid.
  • the cytotoxic agent is an auristatin and, e.g., the auristatin is selected from AFP, MMAF, MMAE, AEB, AEVB and auristatin E. In one embodiment, the auristatin is AFP or MMAF. In another embodiment, the cytotoxic agent is a maytansinoid and, e.g., the maytansinoid is selected from a maytansinol, maytansine, DMl, DM2, DM3 and DM4. In one embodiment, the maytansinoid is DMl.
  • the cytotoxic agent is selected from paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatstatin, calicheamicin, and netropsin.
  • the cytotoxin is an auri statin, a maytansinoid, or calicheamicin.
  • the cytotoxic agent is an antitubulin agent and, e.g., the antitubulin agent is selected from AFP, MMAP, MMAE, AEB, AEVB, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicines, colcimid, estramustine, cemadotin, discodermolide, maytansinol, maytansine, DMl, DM2, DM3, DM4 and eleutherobin.
  • the antitubulin agent is selected from AFP, MMAP, MMAE, AEB, AEVB, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothil
  • the MMP-9 binding protein (e.g., antibody) is conjugated to the cytotoxic agent via a linker.
  • the linker is cleavable under intracellular conditions, e.g., the cleavable linker is a peptide linker cleavable by an intracellular protease.
  • the linker is a peptide linker, e.g., a dipeptide linker, e.g., a val-cit linker or a phe-lys linker.
  • the cleavable linker is hydrolyzable at a pH of less than 5.5, e.g., the hydrolyzable linker is a hydrazone linker. In another embodiment, the cleavable linker is a disulfide linker.
  • a binding protein described herein can be provided as a pharmaceutical composition, e.g., including a pharmaceutically acceptable carrier.
  • the composition can be at least 10, 20, 30, 50, 75, 85, 90, 95, 98, 99, or 99.9% free of other protein species.
  • the binding protein can be produced under GMP (good manufacturing practices).
  • the binding protein is provided in pharmaceutically acceptable carriers, e.g., suitable buffers or excipients.
  • the dose of a binding protein is sufficient to block about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% of the activity of MMP-9 in the patient, e.g., at the site of disease.
  • this may require a dose, e.g., of between about 0.01 mg/Kg to about 100 mg/Kg, e.g., between about 0.1 and about 10 mg/Kg.
  • the dose can be a dose of about 0.1, about 1, about 3, about 6, or about 10 mg/Kg.
  • these doses correspond to approximately 18 nM, 180 nM, 540 nM, 1.08 microM, and 1.8 microM, respectively, of binding sites for a 5 L blood volume.
  • the optimal dose will be established by clinical trials, but will most likely lie in this range.
  • the disclosure features a method of detecting an MMP-9 in a sample, e.g., a sample from a patient (e.g., tissue biopsy or blood sample).
  • the method includes: contacting the sample with an MMP-9 binding protein (e.g., an MMP-9 binding protein described herein); and detecting an interaction between the protein and the MMP-9, if present.
  • the protein includes a detectable label.
  • An MMP-9 binding protein can be used to detect MMP-9 in a subject.
  • the method includes: administering an MMP-9 binding protein (e.g., an MMP-9 binding protein described herein) to a subject; and detecting the protein in the subject, e.g., detecting an interaction between the protein and the MMP-9, if present.
  • the protein further includes a detectable label.
  • the disclosure features a method of modulating MMP-9 activity.
  • the method includes: contacting an MMP-9 with an MMP-9 binding protein (e.g., an MMP-9 binding protein described herein) (e.g., in a human subject), thereby modulating MMP-9 activity.
  • an MMP-9 binding protein e.g., an MMP-9 binding protein described herein
  • the binding protein inhibits MMP-9 activity (e.g., inhibits MMP-9 catalytic activity).
  • the disclosure features a method of treating systemic sclerosis.
  • the method includes: administering, to a subject, an MMP-9 binding protein (e.g., an MMP-9 binding protein described herein) in an amount sufficient to treat systemic sclerosis in the subject.
  • an MMP-9 binding protein e.g., an MMP-9 binding protein described herein
  • MMP-9 binding proteins can be useful for modulating systemic sclerosis in a subject.
  • the protein can be administered, to the subject, in an amount effective to modulate systemic sclerosis.
  • the disclosure features a method of treating Sjogren's syndrome associated with SSc. The method includes: administering, to a subject, an MMP-9 binding protein (e.g., an MMP-9 binding protein described herein) in an amount sufficient to treat Sjogren's syndrome associated with SSc in the subject.
  • an MMP-9 binding protein e.g., an MMP-9 binding protein described herein
  • An MMP-9 binding protein described herein can be administered in combination with one or more other MMP inhibitors, e.g., small molecule inhibitors, e.g., broad specificity inhibitors.
  • the small molecule inhibitors are one or more of neovastat, marimastat, BAY 12-9566, or prinomastat.
  • the one or more MMP inhibitors include another MMP-9 binding protein (e.g., an MMP-9 binding protein described herein), or an MMP-12 binding protein, e.g., an MMP-12 binding protein described herein.
  • the disclosure features the use of an MMP-9 binding protein described herein for the manufacture of a medicament for the treatment of a disorder described herein, e.g., systemic sclerosis.
  • the disclosure features a method of treating or preventing systemic sclerosis in a subject, the method comprising: administering an isolated protein (e.g., antibody, e.g., human antibody) that binds MMP-
  • an isolated protein e.g., antibody, e.g., human antibody
  • the antibody binds the same epitope or competes for binding with an antibody selected from the group consisting of DX-2712, a mutant or variant of DX-2712, 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03
  • the antibody competes with or binds the same epitope as DX- 2712.
  • the antibody competes with or binds the same epitope as 539B- X0041-D02.
  • the disclosure features a method of treating or preventing systemic sclerosis in a subject, the method comprising: administering an isolated protein (e.g., antibody, e.g., human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence to the subject, wherein: the heavy chain immunoglobulin variable domain sequence comprises one, two, or three (e.g., three) CDR regions from the heavy chain variable domain of DX-2712, a mutant or variant of DX-2712, 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01,
  • an isolated protein e.g., antibody, e.g., human antibody
  • the heavy chain immunoglobulin variable domain sequence comprises one, two, or
  • the light chain immunoglobulin variable domain sequence comprises one, two, or three (e.g., three) CDR regions from the light chain variable domain of DX-2712, a mutant or variant of DX-2712, 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09,
  • the one, two, or three (e.g., three) CDR regions from the heavy chain variable domain are from DX-2712 and/or the one, two, or three (e.g., three) CDR regions from the light chain variable domain are from DX-2712.
  • the one, two, or three (e.g., three) CDR regions from the heavy chain variable domain are from 539B-X0041-D02and/or the one, two, or three (e.g., three) CDR regions from the light chain variable domain are from 539B-X0041-D02.
  • the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of DX-2712, a mutant or variant of DX-2712, 539B- X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134- BOl, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134- C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134- E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05,
  • the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of DX-2712, and/or the the light chain immunoglobulin variable domain sequence comprises the light chain variable domain of DX- 2712.
  • the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of 539B-X0041-D02, and/or the the light chain immunoglobulin variable domain sequence comprises the light chain variable domain of 539B- X0041-D02.
  • the protein comprises the heavy chain of DX-2712, a mutant or variant of DX-2712, 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A
  • the protein comprises the heavy chain of DX-2712, and/or the light chain of DX-2712. In some embodiments, the protein comprises the heavy chain of 539B-X0041-D02, and/or the light chain of 539B-X0041-D02.
  • This disclosure also relates to a method of treating or preventing systemic sclerosis in a subject, the method comprising: administering an isolated protein that binds MMP- 12 (e.g., an MMP- 12 binding protein described herein) to the subject.
  • an isolated protein that binds MMP- 12 e.g., an MMP- 12 binding protein described herein
  • the MMP- 12 binding protein is used to treat the diffuse form of systemic sclerosis.
  • the MMP- 12 binding protein is used to treat the limited form of systemic sclerosis.
  • the MMP- 12 binding protein is used in combination with a second therapeutic agent.
  • the second therapeutic agent is another MMP-12 binding protein, e.g., another MMP-12 binding protein described herein.
  • the second therapeutic agent is another inhibitor described herein, e.g., a small molecule inhibitor of MMP-12.
  • the second therapeutic agent is an MMP-9 binding protein, e.g., an MMP-9 binding protein described herein.
  • the second therapeutic agent is e.g., cyclosporin, an NSAID (e.g., naproxen), a calcium channel blocker (e.g., nifedipine), an angiotensin receptor blocker (e.g., losartan), epoprostenol (prostacyclin), a prostacyclin analogue (e.g., iloprost, e.g., aerolized iloprost), a dual endothelin-receptor antagonist (e.g., bosentan), prostaglandin El (e.g., alprostadil), a proton pump inhibitor, an antibiotic (e.g., tetracycline), an immunosuppressant (e.g., cyclophosphamide, methotrexate or azathioprine), an ACE inhibitor, a sympathetic blocker, or surgery (e.g., lung transplantation).
  • an NSAID e.g., naproxen
  • These proteins include antibodies (including antibody fragments) (e.g., primate antibodies and Fabs, especially human antibodies and Fabs) that bind to MMP-12 (e.g., human MMP-12).
  • these proteins include antibodies (including antibody fragments) (e.g., primate antibodies and Fabs, especially human antibodies and Fabs) that inhibit MMP-12 (e.g., human MMP-12) (e.g., inhibit the catalytic activity of MMP-12).
  • the MMP-12 binding proteins can be used in the treatment of systemic sclerosis (scleroderma), in which excess or inappropriate activity of MMP-12 features. In many cases, the proteins have tolerable low or no toxicity.
  • the method uses a protein (e.g., an antibody, peptide or Kunitz domain protein) that binds MMP-12, in particular, a protein (e.g., an antibody (e.g., human antibody), peptide or Kunitz domain protein) that binds and inhibits MMP-12.
  • a protein e.g., an antibody (e.g., human antibody), peptide or Kunitz domain protein
  • the method uses an antibody (e.g., a human antibody) that binds to human MMP-12.
  • the human antibody is an inhibitor of the catalytic activity of MMP-12.
  • the antibody can be, e.g., an IgGl, IgG2, IgG3, IgG4, Fab, Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen combining site of one of the antibodies herein listed.
  • the antibody is used to guide a nano-particle or toxin to a cell expressing MMP-12 on the cell surface.
  • the antibody causes effector functions (CDC or ADCC) to kill the cell which expresses MMP-12.
  • the VH and VL regions of the binding proteins can be provided as IgG, Fab, Fab2, Fab2', scFv, PEGylated Fab, PEGylated scFv, PEGylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA + VH::CH1, HSA: :LC + VH::CH1, or other appropriate construct.
  • the binding protein comprises a Kunitz domain protein or modified version (e.g., HSA fusion) or peptide-based MMP-12 binding protein that can inhibit MMP-12 activity.
  • the method uses a protein (e.g., an isolated protein) that binds to MMP-12 (e.g., human MMP-12) and includes at least one immunoglobulin variable region.
  • MMP-12 e.g., human MMP-12
  • the protein includes a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence.
  • the protein binds to and inhibits MMP-12 (e.g., inhibits MMP-12 catalytic activity), e.g., human MMP-12.
  • the protein binds to human MMP-12 specifically, and not to MMP-12 from another species (e.g., the protein does not bind to MMP-12 from another species with greater than background levels of binding).
  • the protein binds MMP-12 specifically, and not to another matrix metalloproteinase (e.g., the protein does not bind to any other matrix metalloproteinase with greater than background levels of binding).
  • binding proteins can be conjugated to a drug (e.g., to form a MMP-12 binding protein-drug conjugate) and used therapeutically.
  • a drug e.g., to form a MMP-12 binding protein-drug conjugate
  • This disclosure relates, in part, to MMP-12 binding protein-drug conjugates, the preparation of these conjugates, and uses thereof.
  • the conjugates can be used, e.g., in the treatment of disorders, e.g., for the treatment of systemic sclerosis.
  • the protein can include one or more of the following characteristics: (a) a human CDR or human framework region; (b) the HC immunoglobulin variable domain sequence comprises one or more (e.g., 1, 2, or 3) CDRs that are at least 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a HC variable domain described herein; (c) the LC immunoglobulin variable domain sequence comprises one or more (e.g., 1, 2, or 3) CDRs that are at least 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a LC variable domain described herein; (d) the LC immunoglobulin variable domain sequence is at least 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a LC variable
  • the protein can bind to MMP-12, e.g., human MMP-12, with a binding affinity of at least 10 5 , 10 6 , 10 7 ,10 8 , 10 9 , 10 10 and 10 11 M “1 .
  • the protein binds to MMP-12 with a K off slower than 1 X 10 "3 , 5 X 10 ⁇ 4 s "1 , or 1 X 10 ⁇ 4 s "1 .
  • the protein binds to MMP-12 with a K 0n faster than 1 X 10 2 , 1 X 10 3 , or 5 X 10 3 M 1 S 1 .
  • the protein inhibits human MMP-12 activity, e.g., with a Ki of less than 10 "5 , 10 "6 , 10 “7 , 10 “8 , 10 “9 , and 10 "10 M.
  • the protein can have, for example, an IC50 of less than 100 nM, 10 nM or 1 nM. In some embodiments, the protein has an IC50 of about 1.8 nM.
  • the affinity of the protein for MMP-12 can be characterized by a K D of less than 100 nm, less than 10 nM, or about 3 nM (e.g., 3.1 nM), about 5 nM (e.g., 5 nM), about 6 nm (e.g., 5.9 nM), about 7 nM (e.g., 7.1 nM), or about 10 nM (e.g., 9.6 nM).
  • K D K D of less than 100 nm, less than 10 nM, or about 3 nM (e.g., 3.1 nM), about 5 nM (e.g., 5 nM), about 6 nm (e.g., 5.9 nM), about 7 nM (e.g., 7.1 nM), or about 10 nM (e.g., 9.6 nM).
  • the protein has a K D ⁇ 200 nM.
  • the protein has a tl/2 of at least about 10 minutes (e.g., 11 minutes), at least about 20 minutes (e.g., 18 minutes), at least about 25 minutes(e.g., 25 minutes), at least about 35 minutes (e.g., 33 minutes), or at least about 60 minutes (e.g., 57 minutes).
  • the protein binds the catalytic domain of human MMP-12, e.g., the protein contacts residues in or near the active site of MMP-12.
  • the protein does not contact residues in or near the active site of MMP-12 but instead binds elsewhere on MMP-12 and causes a steric change in MMP-12 that affects (e.g., inhibits) its activity.
  • the protein is an antibody (e.g., a human antibody) having the light and heavy chains of antibodies selected from the group consisting of DX-2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134- BI l, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134- C12, M0134-D
  • the protein is an antibody (e.g., a human antibody) having its heavy chain selected from the group consisting of DX-2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M01
  • the protein is an antibody (e.g., a human antibody) having its light chain selected from the group consisting of DX-2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M01
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs selected from the corresponding CDRs of the group of heavy chains consisting of DX-2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134--
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs selected from the corresponding CDRs of the group of light chains consisting of DX-2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134--
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs selected from the corresponding CDRs of the group of light chains consisting of DX-2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134--
  • the protein is an antibody (e.g., a human antibody) having light and heavy antibody variable regions of an antibody selected from the group consisting of DX-2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134- A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134
  • the protein is an antibody (e.g., a human antibody) having a heavy chain antibody variable region of an antibody selected from the group consisting of: DX- 2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134- A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M
  • the protein is an antibody (e.g., a human antibody) having a light chain antibody variable region of an antibody selected from the group consisting of: DX- 2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134- A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M
  • the protein is an antibody (e.g., a human antibody) having the light and heavy chains of antibody M0008-H09, M0131-A06 or M0121-E07.
  • the protein is an antibody (e.g., a human antibody) having its heavy chain from M0008-H09, M0131-A06 or M0121-E07.
  • the protein is an antibody (e.g., a human antibody) having its light chain from M0008-H09, M0131-A06 or M0121-E07.
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain of M0008-H09, M0131-A06 or M0121-E07.
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains of M0008-H09, M0131- A06 or M0121-E07.
  • the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs and one or more (e.g., 1, 2, or3 ) light chain CDRs from the corresponding CDRs of the heavy chain and light chain of M0008-H09, M0131-A06 or M0121-E07 (respectively).
  • the protein is an antibody (e.g., a human antibody) having the light and heavy chain variable regions of antibody M0008-H09, M0131-A06 or M0121-E07.
  • the protein is an antibody (e.g., a human antibody) having its heavy chain variable region from M0008-H09, M0131-A06 or M0121-E07.
  • the protein is an antibody (e.g., a human antibody) having its light chain variable region from M0008-H09, M0131-A06 or M0121-E07.
  • the protein is an antibody (e.g., human antibody) having the light and heavy chains of antibody DX-2712 (also referred to as M0131-A06-GA-S).
  • the protein is a human antibody having its heavy chain from antibody DX-2712.
  • the protein is a human antibody having its light chain from antibody DX-2712.
  • the protein is a human antibody having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain from antibody DX-2712. In another even more preferred embodiment, the protein is a human antibody having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains from antibody DX-2712.
  • the protein is a human antibody having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains from antibody DX-2712 and having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain from antibody DX- 2712.
  • the protein is an antibody (e.g., human antibody) having the light and heavy chain variable regions of antibody DX-2712 (also referred to as M0131-A06- GA-S).
  • the protein is a human antibody having its heavy chain variable region from antibody DX-2712.
  • the protein is a human antibody having its light chain variable region from antibody DX-2712.
  • the protein is a human antibody having the light and heavy chains of a mutant or variant of DX-2712, e.g., a mutant or variant described herein.
  • the protein is a human antibody having its heavy chain from a mutant or variant of DX-2712, e.g., a mutant or variant described herein.
  • the protein is a human antibody having its light chain from a variant of DX-2712, e.g., a mutant or variant described herein.
  • the protein is a human antibody having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain from a mutant or variant of DX-2712, e.g., a mutant or variant described herein.
  • the protein is a human antibody having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains from a mutant or variant of DX- 2712, e.g., a mutant or variant described herein.
  • the protein is a human antibody having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains from a mutant or variant of DX-2712, e.g., a mutant or variant described herein and having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain from a mutant or variant of DX-2712, e.g., a mutant or variant described herein.
  • one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains from a mutant or variant of DX-2712, e.g., a mutant or variant described herein having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain from a mutant or variant of DX-2712, e.g., a mutant or variant described herein.
  • the protein is an antibody (e.g., human antibody) having the light and heavy chain variable regions of a mutant or variant of DX-2712, e.g., a mutant or variant described herein.
  • the protein is a human antibody having its heavy chain variable region from a mutant or variant of DX-2712, e.g., a mutant or variant described herein.
  • the protein is a human antibody having its light chain variable region from a mutant or variant of DX-2712, e.g., a mutant or variant described herein.
  • the protein is a human antibody having the light and heavy chains of antibody 539B-X0041-D02. In another preferred embodiment, the protein is a human antibody having its heavy chain from antibody 539B-X0041-D02. In yet another preferred embodiment, the protein is a human antibody having its light chain from antibody 539B-X0041-D02.
  • the protein is a human antibody having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain from antibody 539B-X0041-D02.
  • the protein is a human antibody having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains from antibody 539B-X0041-D02.
  • the protein is a human antibody having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains from antibody 539B-X0041-D02 and having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain from antibody 539B-X0041-D02.
  • the protein is an antibody (e.g., human antibody) having the light and heavy chain variable regions of antibody 539B-X0041-D02.
  • the protein is a human antibody having its heavy chain variable region from antibody 539B-X0041-D02.
  • the protein is a human antibody having its light chain variable region from antibody 539B-X0041-D02.
  • the protein is a human antibody having the light and heavy chains of a variant of 539B-X0041-D02, e.g., a variant described herein.
  • the protein is a human antibody having its heavy chain from a variant of 539B- X0041-D02, e.g., a variant described herein.
  • the protein is a human antibody having its light chain from a variant of 539B-X0041-D02, e.g., a variant described herein.
  • the protein is a human antibody having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain from a variant of 539B-X0041-D02, e.g., a variant described herein.
  • the protein is a human antibody having one or more (e.g., 1, 2, or 3) light chain
  • the protein is a human antibody having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains from a variant of 539B-X0041-D02, e.g., a variant described herein and having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain from a variant of 539B-X0041-D02, e.g., a variant described herein.
  • the protein is an antibody (e.g., human antibody) having the light and heavy chain variable regions of a variant of 539B-X0041-D02, e.g., a variant described herein.
  • the protein is a human antibody having its heavy chain variable region from a variant of 539B-X0041-D02, e.g., a variant described herein.
  • the protein is a human antibody having its light chain variable region from a variant of 539B-X0041-D02, e.g., a variant described herein.
  • the HC and LC variable domain sequences are components of the same polypeptide chain. In another, the HC and LC variable domain sequences are components of different polypeptide chains.
  • the protein is an IgG, e.g., IgGl, IgG2, IgG3, or IgG4.
  • the protein can be a soluble Fab (sFab).
  • the protein includes a Fab2', scFv, minibody, scFv::Fc fusion, Fab: :HS A fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen combining site of one of the binding proteins herein.
  • VH and VL regions of these Fabs can be provided as IgG, Fab, Fab2, Fab2', scFv, PEGylated Fab, PEGylated scFv, PEGylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA + VH::CH1, HSA: :LC + VH::CH1, or other appropriate construction.
  • the protein is a human or humanized antibody or is non- immunogenic in a human.
  • the protein includes one or more human antibody framework regions, e.g., all human framework regions.
  • the protein includes a human Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or 99% identical to a human Fc domain.
  • the protein is a primate or primatized antibody or is non- immunogenic in a human.
  • the protein includes one or more primate antibody framework regions, e.g., all primate framework regions.
  • the protein includes a primate Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or 99% identical to a primate Fc domain.
  • "Primate” includes humans ⁇ Homo sapiens), chimpanzees ⁇ Pan troglodytes and Pan paniscus (bonobos)), gorillas ⁇ Gorilla gorilla), gibons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and tarsiers.
  • the protein includes no sequences from mice or rabbits (e.g., is not a murine or rabbit antibody).
  • protein is physically associated with a nanoparticle, and can be used to guide a nanoparticle to a cell expressing MMP- 12 on the cell surface.
  • the protein causes effector cells (CDC or ADCC) to kill a cell which expresses MMP- 12.
  • the MMP-12 binding protein is administered (alone or in combination with a second agent described herein) to a subject to treat a symptom of systemic sclerosis, e.g., Raynaud's syndrome, swelling (eventually skin tightening and contractures of the fingers), polyarthralgia, dysphagia, dyspnea, lung fibrosis, acute alveolitis, pulmonary hypertension, heartburn, cardiac arrhythmias or renal crisis, or other symptom described herein.
  • a symptom of systemic sclerosis e.g., Raynaud's syndrome, swelling (eventually skin tightening and contractures of the fingers), polyarthralgia, dysphagia, dyspnea, lung fibrosis, acute alveolitis, pulmonary hypertension, heartburn, cardiac arrhythmias or renal crisis, or other symptom described herein.
  • the more evident symptom is usually the hardening of the skin and associated scarring. Blood vessels may also be more visible
  • the MMP-12 binding protein is administered (alone or in combination with a second agent (e.g., a second agent described herein)) to a subject to treat Sjogren's syndrome associated with SSc.
  • a second agent e.g., a second agent described herein
  • the MMP-12 binding protein is administered (alone or in combination with a second agent (e.g., a second agent described herein)) to a subject at risk of developing systemic sclerosis, e.g., with a familial predisposition for the disease (e.g., a polymorphism in COLl A2 or TGF- ⁇ l), or with another risk factor, e.g., a prior cytomegalovirus (CMV) infection, or exposure to organic solvents or other chemical agents (e.g., vinyl chloride, bleomycin, pentazocine, epoxy and/or aromatic hydrocarbons, contaminated rapeseed oil or L-tryptophan).
  • a familial predisposition for the disease e.g., a polymorphism in COLl A2 or TGF- ⁇ l
  • CMV cytomegalovirus
  • organic solvents or other chemical agents e.g., vinyl chloride, bleomycin, pentazocine, epoxy and/or aromatic
  • the method uses an MMP-12 binding protein that is a competitive inhibitor of MMP-12.
  • the binding protein competes with an MMP-12 substrate (e.g., lung extracellular matrix, elastin, gelatin, fibronectin, apo[a], apoB-100, collagen, osteonectin, TFPI, alpha 1-protease inhibitor, uPAR and CD14), e.g., binds to the same epitope as the substrate, e.g., and prevents substrate binding.
  • an MMP-12 substrate e.g., lung extracellular matrix, elastin, gelatin, fibronectin, apo[a], apoB-100, collagen, osteonectin, TFPI, alpha 1-protease inhibitor, uPAR and CD14
  • the method includes inhibiting an interaction between MMP-12 and an
  • MMP-12 substrate e.g., lung extracellular matrix, elastin, gelatin, fibronectin, apo[a], apoB-100, collagen, osteonectin, TFPI, alpha 1-protease inhibitor, uPAR and CD14.
  • the method includes contacting an MMP- 12 binding protein described herein with MMP- 12 (e.g., in vitro or in vivo), wherein the binding protein binds to MMP- 12 and thereby prevents the binding of an MMP- 12 substrate to MMP- 12.
  • the binding protein binds to the same epitope on MMP- 12 as the substrate, e.g., the binding protein is a competitive inhibitor.
  • the binding protein does not bind the same epitope as the substrate but causes a steric change in MMP- 12 that decreases or inhibits the ability of the substrate to bind.
  • the disclosure features an MMP-12 binding protein-drug conjugate that includes a MMP-12 binding protein (e.g., an MMP-12 binding protein described herein) and a drug.
  • the binding protein comprises at least one immunoglobulin variable region, and/or the protein binds to and/or inhibits MMP-12, e.g., inhibits MMP-12 catalytic activity.
  • the drug is a cytotoxic or cytostatic agent.
  • the cytotoxic agent can be, e.g., selected from the group consisting of an auristatin, a DNA minor groove binding agent, a DNA minor groove alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a podophyllotoxin, a baccatin derivative, a cryptophysin, a combretastatin, a maytansinoid, and a vinca alkaloid.
  • an auristatin e.g., selected from the group consisting of an auristatin, a DNA minor groove binding agent, a DNA minor groove alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a podophyllotoxin, a baccatin derivative, a crypto
  • the cytotoxic agent is an auristatin and, e.g., the auristatin is selected from AFP, MMAF, MMAE, AEB, AEVB and auristatin E. In one embodiment, the auristatin is AFP or MMAF. In another embodiment, the cytotoxic agent is a maytansinoid and, e.g., the maytansinoid is selected from a maytansinol, maytansine, DMl, DM2, DM3 and DM4. In one embodiment, the maytansinoid is DMl.
  • the cytotoxic agent is selected from paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin- 10, echinomycin, combretatstatin, calicheamicin, and netropsin.
  • the cytotoxin is an auristatin, a maytansinoid, or calicheamicin.
  • the cytotoxic agent is an antitubulin agent and, e.g., the antitubulin agent is selected from AFP, MMAP, MMAE, AEB, AEVB, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicines, colcimid, estramustine, cemadotin, discodermolide, maytansinol, maytansine, DMl, DM2, DM3, DM4 and eleutherobin.
  • the antitubulin agent is selected from AFP, MMAP, MMAE, AEB, AEVB, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothil
  • the MMP-12 binding protein (e.g., antibody) is conjugated to the cytotoxic agent via a linker.
  • the linker is cleavable under intracellular conditions, e.g., the cleavable linker is a peptide linker cleavable by an intracellular protease.
  • the linker is a peptide linker, e.g., a dipeptide linker, e.g., a val-cit linker or a phe-lys linker.
  • the cleavable linker is hydrolyzable at a pH of less than 5.5, e.g., the hydrolyzable linker is a hydrazone linker. In another embodiment, the cleavable linker is a disulfide linker.
  • a binding protein described herein can be provided as a pharmaceutical composition, e.g., including a pharmaceutically acceptable carrier.
  • the composition can be at least 10, 20, 30, 50, 75, 85, 90, 95, 98, 99, or 99.9% free of other protein species.
  • the binding protein can be produced under GMP (good manufacturing practices).
  • the binding protein is provided in pharmaceutically acceptable carriers, e.g., suitable buffers or excipients.
  • the dose of a binding protein is sufficient to block about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% of the activity of MMP-12 in the patient, e.g., at the site of disease.
  • this may require a dose, e.g., of between about 0.01 mg/Kg to about 100 mg/Kg, e.g., between about 0.1 and about 10 mg/Kg.
  • the dose can be a dose of about 0.1, about 1, about 3, about 6, or about 10 mg/Kg.
  • these doses correspond to approximately 18 nM, 180 nM, 540 nM, 1.08 microM, and 1.8 microM, respectively, of binding sites for a 5 L blood volume.
  • the optimal dose will be established by clinical trials, but will most likely lie in this range.
  • An MMP-12 binding protein can be used to detect MMP-12 in a subject.
  • the method includes: administering an MMP-12 binding protein (e.g., an MMP-12 binding protein described herein) to a subject; and detecting the protein in the subject, e.g., detecting an interaction between the protein and the MMP- 12, if present.
  • the protein further includes a detectable label.
  • the disclosure features a method of modulating MMP- 12 activity.
  • the method includes: contacting an MMP-12 with an MMP-12 binding protein (e.g., an MMP-12 binding protein described herein) (e.g., in a human subject), thereby modulating MMP-12 activity.
  • the binding protein inhibits MMP-12 activity (e.g., inhibits MMP-12 catalytic activity).
  • the disclosure features a method of treating systemic sclerosis.
  • the method includes: administering, to a subject, an MMP-12 binding protein (e.g., an MMP-12 binding protein described herein) in an amount sufficient to treat systemic sclerosis in the subject.
  • an MMP-12 binding protein e.g., an MMP-12 binding protein described herein
  • the disclosure features a method of treating Sjogren's syndrome associated with SSc.
  • the method includes: administering, to a subject, an MMP-9 binding protein (e.g., an MMP-12 binding protein described herein) in an amount sufficient to treat Sjogren's syndrome associated with SSc in the subject.
  • an MMP-9 binding protein e.g., an MMP-12 binding protein described herein
  • An MMP-12 binding protein described herein can be administered in combination with one or more other MMP inhibitors, e.g., small molecule inhibitors, e.g., broad specificity inhibitors.
  • the small molecule inhibitors are one or more of the small molecule inhibitors described herein.
  • the one or more MMP inhibitors include another MMP-12 binding protein, e.g., another MMP-12 binding protein described herein, or an MMP-9 binding protein, e.g., an MMP-9 binding protein described herein.
  • the disclosure features the use of an MMP-12 binding protein described herein for the manufacture of a medicament for the treatment of a disorder described herein, e.g., systemic sclerosis.
  • a disorder described herein e.g., systemic sclerosis.
  • FIGURE 1 is a graph depicting inhibition assays of MMP- 12 using non-inhibitor binding proteins.
  • FIGURE 2A is a line graph showing human MMP-12 activity (Fluo/min) in the presence of increasing concentrations (nM) of an MMP-12 binding protein (539B-M08H09).
  • FIGURE 2B is a line graph showing the binding rate of elastin (dF/min) in the presence of increasing concentrations (nM) of an MMP-12 binding protein (539B-M08H09).
  • FIGURE 2C is a line graph showing murine MMP-12 activity (Fluo/sec) in the presence of increasing concentrations (nM) of an MMP-12 binding protein (539B-M08H09).
  • FIGURE 3 A is a line graph showing measured IC 50 (nM) of an MMP-12 binding protein (M08H09) versus substrate concentration ( ⁇ M) of human MMP-12.
  • FIGURE 3B is a line graph showing measured IC 50 (nM) of an MMP-12 binding protein (M08H09) versus substrate concentration ( ⁇ M) of murine MMP-12.
  • FIGURES 4 A and 4B are line graphs showing murine MMP-12 activity (Fluo/sec) in the presence of increasing concentrations (nM) of an MMP-12 binding protein (539B-M11H11).
  • FIGURE 4A Fab
  • FIGURE 4B IgG
  • FIGURE 5 is a bar graph showing ELISA competition assays with four different MMP-
  • FIGURE 6 is a bar graph showing the effect of various doses of MMP-12 binding proteins M08-H09 and Mil -HIl on peribronchial inflammation score in OVA-challenged mouse model of airway inflammation.
  • FIGURES 7A and 7B are bar graphs showing eosinophil percentages (FIGURE 7A) and counts (FIGURE 7B) in OVA-challenged mouse model of airway inflammation administered various doses of MMP-12 binding proteins M08-H09 and Mil -HIl.
  • FIGURE 8 is a bar graph showing the effect of an MMP-12 binding protein (M08-H09) on inflammatory cell infiltration into a carrageenan-stimulated mouse air pouch.
  • FIGURE 9 is a line graph showing human MMP- 12 activity (Fluo/sec) in the presence of increasing concentrations (nM) of an MMP-12 binding protein (539B-M131 A06) and an MMP- 12 binding protein (539B-M131A06-GA-S) which is a version of 539B-M131A06 that has been modified to remove a glycosylation site.
  • FIGURES 1OA abd 1OB summarize the identification of amino acid changes in affinity matured variant HV-CDRs (cycles 1 and 2) that contribute to improvement in affinity and inhibition properties.
  • FIGURE 11 A is a line graph showing human MMP-9 activity (Fluo/sec) in the presence of increasing concentrations (nM) of an MMP-9 binding protein (539A-M0166-F10).
  • FIGUREIlB is a table showing that an MMP-9 binding protein (539A- M0166-F10) is specific for human MMP-9.
  • FIGURE 12 is a line graph showing measured IC 50 (nM) versus substrate concentration ( ⁇ M).
  • FIGURE 13 is a series of line graphs showing IC 50 measurements at various concentrations of substrate.
  • SSc systemic sclerosis
  • MMP-9 and TIMP-I serum concentrations of MMP-9 and TIMP-I
  • the serum concentrations of MMP-9 correlated well with the degree of skin involvement, as determined by the Rodnan score (thickness).
  • Circulating TGF- ⁇ strongly correlated with MMP-9 concentrations.
  • Dermal fibroblasts from patients with SSc produced more MMP-9 than those from healthy controls upon stimulation with IL-Ib, TNF- ⁇ , or TGF- ⁇ .
  • the overproduced MMP-9 may induce microvascular damage and leakage of substances that further augment endothelial cell damage or fibroblast activation in SSc.
  • MMP-12 Matrix metalloproteinase-12 blocks angiogenesis by cleavage of the endothelial urokinase- type plasminogen activator receptor (uPAR).
  • uPAR endothelial urokinase- type plasminogen activator receptor
  • SSc MVEC-conditioned medium impaired uPA-dependent proliferation and invasion as well as capillary morphogenesis in normal microvascular endothelial cells (MVECs) in vitro.
  • An anti-MMP-12 antibody can restore SSc MVEC function.
  • SSc systemic sclerosis
  • MMPs matrix metalloproteinases
  • TIMP-I levels are significantly raised in the serum of diffuse cutaneous SSc and limited cutaneous SSc patients compared with the normal controls. Serum MMP-I levels do not differ significantly between either of the SSc groups or between SSc groups and normal controls.
  • Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are 72- and 92-kD, respectively, type IV collagenases that are members of a group of secreted zinc metalloproteases which, in mammals, degrade the collagens of the extracellular matrix.
  • Other members of this group include interstitial collagenase (MMP-I) and stromelysin (MMP-3).
  • MMP-2 the 72-kD type IV collagenase (also known as CLG4A)
  • MMP-9 the 92-kD collagenase (also known as CLG4B)
  • CLG4B the 92-kD collagenase
  • the present disclosure provides proteins that bind to MMP-9 and, in some instances, inhibit MMP-9 activity.
  • Matrix metalloproteinase-12 (MMP- 12) is a type IV collagenase that is a member of a group of secreted zinc metalloproteases which, in mammals, degrade various proteins of the extracellular matrix.
  • MMP-I interstitial collagenase
  • MMP-3 stromelysin
  • MMP- 12 a.k.a. macrophage elastase, macrophage metalloelastase, or matrix metalloproteinase 12
  • MMP-12 is thought to be involved in many disease states.
  • Many small molecule inhibitors of MMP- 12 have been tested for safety and efficacy in cancer and other diseases. So far, all have lacked either sufficient potency or sufficient specificity or both.
  • the present disclosure provides proteins that bind MMP-12, and in some instances, inhibit MMP-12 activity. In many instances, the disclosed MMP-12 binding proteins bind and inhibit human and mouse MMP12 enzyme activity.
  • MMP-12 binding proteins are disclosed that bind MMP-12 but do not inhibit MMP-12 activity. Such MMP-12 binding proteins are useful, e.g., to determine the presence of MMP-12.
  • binding protein refers to a protein that can interact with a target molecule. This term is used interchangeably with "ligand.”
  • An "MMP-9 binding protein” refers to a protein that can interact with MMP-9, and includes, in particular, proteins that preferentially interact with and/or inhibit MMP-9.
  • the MMP-9 binding protein is an antibody.
  • An “MMP-12 binding protein” refers to a protein that can interact with MMP-12, and includes, in particular, proteins that preferentially interact with and/or inhibit MMP-12.
  • the MMP-12 binding protein is an antibody.
  • an antibody refers to a protein that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence.
  • an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL).
  • VH heavy chain variable region
  • L light chain variable region
  • an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions.
  • antibody encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab') 2 , Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (de Wildt et al., Eur J Immunol. 1996; 26(3):629-39.)) as well as complete antibodies.
  • An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof).
  • Antibodies may be from any source, but primate (human and non-human primate) and primatized are preferred.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” ("CDR"), interspersed with regions that are more conserved, termed “framework regions” ("FR").
  • CDR complementarity determining regions
  • FR framework regions
  • the extent of the framework region and CDRs has been precisely defined (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. MoI. Biol.
  • Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.
  • an "immunoglobulin variable domain sequence” refers to an amino acid sequence which can form the structure of an immunoglobulin variable domain such that one or more (e.g., 1, 2, or 3) CDR regions are positioned in a conformation suitable for an antigen binding site.
  • the sequence may include all or part of the amino acid sequence of a naturally-occurring variable domain.
  • the sequence may omit 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 include other alterations.
  • a polypeptide that includes immunoglobulin variable domain sequence can associate with another immunoglobulin variable domain sequence to form an antigen binding site, e.g., a structure that preferentially interacts with an MMP-9 protein, e.g., the MMP-9 catalytic domain.
  • the VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain, respectively.
  • the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds.
  • the heavy chain constant region includes three immunoglobulin domains, CHl, CH2 and CH3.
  • the light chain constant region includes a CL domain.
  • the variable region of the heavy and light chains contains a binding domain that interacts with an antigen.
  • the constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system.
  • the light chains of the immunoglobulin may be of types kappa or lambda.
  • the antibody is glycosylated.
  • An antibody can be functional for antibody-dependent cytotoxicity and/or complement-mediated cytotoxicity.
  • One or more regions of an antibody can be human or effectively human.
  • one or more of the variable regions can be human or effectively human.
  • one or more of the CDRs can be human, e.g., HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3.
  • Each of the light chain CDRs can be human.
  • HC CDR3 can be human.
  • One or more of the framework regions can be human, e.g., FRl, FR2, FR3, and FR4 of the HC or LC.
  • the Fc region can be human.
  • all the framework regions are human, e.g., derived from a human somatic cell, e.g., a hematopoietic cell that produces immunoglobulins or a non-hematopoietic cell.
  • the human sequences are germline sequences, e.g., encoded by a germline nucleic acid.
  • the framework (FR) residues of a selected Fab can be convertered to the amino-acid type of the corresponding residue in the most similar primate germline gene, especially the human germline gene.
  • One or more of the constant regions can be human or effectively human.
  • an immunoglobulin variable domain the constant region, the constant domains (CHl, CH2, CH3, CLl), or the entire antibody can be human or effectively human. All or part of an antibody can be encoded by an immunoglobulin gene or a segment thereof.
  • Exemplary human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the many immunoglobulin variable region genes.
  • Full-length immunoglobulin "light chains” (about 25 KDa or about 214 amino acids) are encoded by a variable region gene at the NH2- terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH- terminus.
  • Full-length immunoglobulin "heavy chains” (about 50 KDa or about 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).
  • the length of human HC varies considerably because HC CDR3 varies from about 3 amino-acid residues to over 35 amino-acid residues.
  • antigen-binding fragment of a full length antibody refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to a target of interest.
  • binding fragments encompassed within the term "antigen-binding fragment” of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab') 2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (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 functionality.
  • CDR
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv).
  • scFv single chain Fv
  • Antibody fragments can be obtained using any appropriate technique including conventional techniques known to those with skill in the art.
  • the term “monospecific antibody” refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes a "monoclonal antibody” or “monoclonal antibody composition,” which as used herein refer to a preparation of antibodies or fragments thereof of single molecular composition, irrespective of how the antibody was generated.
  • an “effectively human” immunoglobulin variable region is an immunoglobulin variable region that includes a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human.
  • An “effectively human” antibody is an antibody that includes a sufficient number of human amino acid positions such that the antibody does not elicit an immunogenic response in a normal human.
  • a “humanized” immunoglobulin variable region is an immunoglobulin variable region that is modified to include a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human.
  • Descriptions of "humanized” immunoglobulins include, for example, US 6,407,213 and US 5,693,762.
  • binding affinity refers to the apparent association constant or K 3 .
  • the K 3 is the reciprocal of the dissociation constant (K d ).
  • a binding protein may, for example, have a binding affinity of at least 10 5 , 10 6 , 10 7 ,10 8 , 10 9 , 10 10 and 10 11 M "1 for a particular target molecule, e.g., MMP-9, MMP-16, or MMP-24.
  • Higher affinity binding of a binding protein to a first target relative to a second target can be indicated by a higher K 3 (or a smaller numerical value K d ) for binding the first target than the K 3 (or numerical value K d ) for binding the second target.
  • the binding protein has specificity for the first target (e.g., a protein in a first conformation or mimic thereof) relative to the second target (e.g., the same protein in a second conformation or mimic thereof; or a second protein).
  • Differences in binding affinity can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, or 10 5 fold.
  • Binding affinity can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay).
  • Exemplary conditions for evaluating binding affinity are in TRIS-buffer (5OmM TRIS, 15OmM NaCl, 5mM CaCl 2 at pH7.5). These techniques can be used to measure the concentration of bound and free binding protein as a function of binding protein (or target) concentration.
  • the concentration of bound binding protein ([Bound]) is related to the concentration of free binding protein ([Free]) and the concentration of binding sites for the binding protein on the target where (N) is the number of binding sites per target molecule by the following equation:
  • K 3 it is not always necessary to make an exact determination of K 3 , though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to K 3 , and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.
  • a functional assay e.g., an in vitro or in vivo assay.
  • compositions refers to a composition that is removed from at least 90% of at least one component of a natural sample from which the isolated composition can be obtained.
  • compositions produced artificially or naturally can be "compositions of at least" a certain degree of purity if the species or population of species of interests is at least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure on a weight-weight basis.
  • an “epitope” refers to the site on a target compound that is bound by a binding protein (e.g., an antibody such as a Fab or full length antibody).
  • a binding protein e.g., an antibody such as a Fab or full length antibody.
  • the site can be entirely composed of amino acid components, entirely composed of chemical modifications of amino acids of the protein (e.g., glycosyl moieties), or composed of combinations thereof.
  • Overlapping epitopes include at least one common amino acid residue, glycosyl group, phosphate group, sulfate group, or other molecular feature.
  • An (first) antibody "binds to the same epitope” as another (second) antibody if the antibody binds to the same site on a target compound that the second antibody binds, or binds to a site that overlaps (e.g., 50%, 60%, 70%, 80%, 90%, or 100% overlap, e.g., in terms of amino acid sequence or other molecular feature (e.g., glycosyl group, phosphate group, or sulfate group) with the site that the second antibody binds.
  • overlaps e.g., 50%, 60%, 70%, 80%, 90%, or 100% overlap, e.g., in terms of amino acid sequence or other molecular feature (e.g., glycosyl group, phosphate group, or sulfate group) with the site that the second antibody binds.
  • An (first) antibody "competes for binding” with another (second) antibody if the binding of the first antibody to its epitope decreases (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) the amount of the second antibody that binds to its epitope.
  • the competition can be direct (e.g., the first antibody binds to an epitope that is the same as, or overlaps with, the epitope bound by the second antibody), or indirect (e.g., the binding of the first antibody to its epitope causes a steric change in the target compound that decreases the ability of the second antibody to bind to its epitope).
  • sequence identity is calculated as follows.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences.
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence.
  • the reference sequence may be the length of the immunoglobulin variable domain sequence.
  • the term "substantially identical” is used herein to refer to a first amino acid or nucleic acid sequence that contains a sufficient number of identical or equivalent (e.g., with a similar side chain, e.g., conserved amino acid substitutions) amino acid residues or nucleotides to a second amino acid or nucleic acid sequence such that the first and second amino acid or nucleic acid sequences have (or encode proteins having) similar activities, e.g., a binding activity, a binding preference, or a biological activity.
  • the second antibody has the same specificity and has at least 50%, at least 25%, or at least 10% of the affinity relative to the same antigen.
  • sequences similar or homologous e.g., at least about 85% sequence identity
  • sequence identity can be about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.
  • substantial identity exists when the nucleic acid segments hybridize under selective hybridization conditions (e.g., highly stringent hybridization conditions), to the complement of the strand.
  • the nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions describes conditions for hybridization and washing.
  • 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. Aqueous and nonaqueous methods are described in that reference and either can be used.
  • Specific hybridization conditions referred to herein are as follows: (1) low stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1% SDS at least at 50 0 C (the temperature of the washes can be increased to 55°C for low stringency conditions); (2) medium stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60 0 C; (3) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and (4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C.
  • SSC sodium chloride/sodium citrate
  • Very high stringency conditions are the preferred conditions and the ones that should be used unless otherwise specified.
  • the disclosure includes nucleic acids that hybridize with low, medium, high, or very high stringency to a nucleic acid described herein or to a complement thereof, e.g., nucleic acids encoding a binding protein described herein.
  • the nucleic acids can be the same length or within 30, 20, or 10% of the length of the reference nucleic acid.
  • the nucleic acid can correspond to a region encoding an immunoglobulin variable domain sequence described herein.
  • An MMP-9 binding protein may have mutations (e.g., at least one, two, or four, and/or less than 15, 10, 5, or 3) relative to a binding protein described herein (e.g., conservative or nonessential amino acid substitutions), which do not have a substantial effect on protein function. Whether or not a particular substitution will be tolerated, i.e., will not adversely affect biological properties, such as binding activity can be predicted, e.g., by evaluating whether the mutation is conservative or by the method of Bowie, et al. (1990) Science 247:1306-1310.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). It is
  • Motif sequences for biopolymers can include positions which can be varied amino acids.
  • the symbol "X” in such a context generally refers to any amino acid (e.g., any of the twenty natural amino acids or any of the nineteen non-cysteine amino acids).
  • Other allowed amino acids can also be indicated for example, using parentheses and slashes.
  • “(A/W/F/N/Q)" means that alanine, tryptophan, phenylalanine, asparagine, and glutamine are allowed at that particular position.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of the binding agent, e.g., the antibody, without abolishing or more preferably, without substantially altering a biological activity, whereas changing an "essential" amino acid residue results in a substantial loss of activity.
  • cognate ligand refers to a naturally occurring ligand of an MMP-9, including naturally occurring variants thereof (e.g., splice variants, naturally occurring mutants, and isoforms).
  • Statistical significance can be determined by any art known method. Exemplary statistical tests include: the Students T-test, Mann Whitney U non-parametric test, and Wilcoxon non-parametric statistical test. Some statistically significant relationships have a P value of less than 0.05 or 0.02. Particular binding proteins may show a difference, e.g., in specificity or binding, that are statistically significant (e.g., P value ⁇ 0.05 or 0.02).
  • agonist is meant to refer to an agent that mimics or up- regulates (e.g., potentiates or supplements) the bioactivity of a protein.
  • An agonist can be a wild- type protein or derivative thereof having at least one bioactivity of the wild-type protein.
  • An agonist can also be a compound that upregulates expression of a gene or which increases at least one bioactivity of a protein.
  • An agonist can also be a compound which increases the interaction of a polypeptide with another molecule, e.g., a target peptide or nucleic acid.
  • Antagonist as used herein is meant to refer to an agent that downregulates (e.g., suppresses or inhibits) at least one bioactivity of a protein.
  • An antagonist can be a compound which inhibits or decreases the interaction between a protein and another molecule, e.g., a target peptide or enzyme substrate.
  • An antagonist can also be a compound that downregulates expression of a gene or which reduces the amount of expressed protein present.
  • a "patient”, “subject” or “host” to be treated by the subject method may mean either a human or non-human animal.
  • preventing or to "prevent” a disease in a subject refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease is prevented, that is, administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) so that it protects the host against developing the unwanted condition.
  • a pharmaceutical treatment e.g., the administration of a drug
  • Preventing a disease may also be referred to as “prophylaxis” or “prophylactic treatment.” Sequences similar or homologous (e.g., at least about 85% sequence identity) to the sequences disclosed herein are also part of this application.
  • the sequence identity can be about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.
  • substantial identity exists when the nucleic acid segments hybridize under selective hybridization conditions (e.g., highly stringent hybridization conditions), to the complement of the strand.
  • the nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • treat refers to the application or administration of an agent, alone or in combination with one or more other agents (e.g., a second agent) to a subject, e.g., a patient, e.g., a patient who has a disorder (e.g., a disorder as described herein), a symptom of a disorder or a predisposition for a disorder, e.g., to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder.
  • Treating a cell refers to a reduction in an activity of a cell. A reduction does not necessarily require a total elimination of activity, but a reduction, e.g., a statistically significant reduction, in the activity or the number of cells.
  • MMP-9 Binding Proteins The disclosure provides proteins that bind to MMP-9 (e.g., human MMP-9) and include at least one immunoglobin variable region.
  • the MMP-9 binding protein includes a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence.
  • HC heavy chain
  • LC light chain
  • a number of exemplary MMP-9 binding proteins are described herein.
  • the MMP-9 binding protein may be an isolated protein (e.g., at least 70, 80, 85, 90, 91,
  • the MMP-9 binding protein may additionally inhibit MMP-9, e.g., human MMP-9.
  • the binding protein can inhibit the catalytic activity of MMP-9 (e.g., human MMP-9).
  • the protein binds the catalytic domain of human MMP-9, e.g., the protein contacts residues in or near the active site of MMP-9.
  • the protein does not contact residues in or near the active site of MMP-9 but instead binds elsewhere on MMP-9 and causes a steric change in MMP-9 that affects (e.g., inhibits) its activity.
  • Exemplary MMP-9 binding proteins include DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06, or proteins that comprise the HC and/or LC CDRs of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, and M0166-F10, or proteins that comprise the HC and/or LC variable regions of DX-2802, 539A- M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-
  • MMP-9 binding antibodies may have their HC and LC variable domain sequences included in a single polypeptide (e.g., scFv), or on different polypeptides (e.g., IgG or Fab).
  • MMP-9/MMP-2 dual binding prioteins e.g., antibodies, e.g., human antibodies
  • MMP-9 Matrix Metalloproteinase 9 (MMP-9) MMP-9 Sequences.
  • MMP-9 is encoded by a gene designated as MMP9 with full name
  • Matrix metalloproteinase-9 precursor Synonyms for MMP-9 include matrix metalloproteinase 9, gelatinase B (GELB), 92kDa gelatinase (CLG4B), 92kDa type IV collagenase (EC 3.4.24.35).
  • the DNA sequence is known for Homo sapiens and Mus musculus.
  • An exemplary cDNA sequence encoding human MMP9 and the amino acid sequence are shown below.
  • Exemplary cDNA sequences encoding murine MMP9 and amino acid sequences are also shown below.
  • An exemplary MMP-9 protein can include the human or mouse MMP-9 amino acid sequence, a sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to one of these sequences, or a fragment thereof, e.g., a fragment without the signal sequence or prodomain.
  • Table A shows the similar genes in other organisms and the percentage of similarity with human MMP-9.
  • gosspyii yeast (Ashbya gossypii), K. lactis yeast (Kluyveromyces lactis), medicago trunc (Medicago truncatula), malaria parasite ⁇ Plasmodium falciparum), schistosome parasite ⁇ Schistosoma mansoni), sorghum ⁇ Sorghum bicolor), toxoplasmosis ⁇ Toxoplasma gondii).
  • MMP-9 belongs to the peptidase MlOA family. MMP-9 consists of five domains; the amino-terminal and zinc-binding domains shared by all members of the secreted metalloprotease gene family, the collagen-binding fibronectin-like domain also present in the 72-kDa type IV collagenase, a carboxyl-terminal hemopexin-like domain shared by all known enzymes of this family with the exception of PUMP-I, and a unique 54-amino-acid- long proline-rich domain homologous to the alpha 2 chain of type V collagen (Wilhelm et al. (1989) /. Biol. Chem. 264, 17213-17221) (Table B).
  • MMP-9 Factors that regulate MMP-9.
  • the catalytic activity of MMP-9 is inhibited by histatin-3 1/24 (histatin-5).
  • MMP-9 is activated by urokinase-type plasminogen activator; plasminogen; IL-lbeta, 4-aminophenylmercuric acetate and phorbol ester.
  • MMP-9 exists as monomer, disulfide-linked homodimer, and as a heterodimer with a 25 kDa protein. Macrophages and transformed cell lines produce only the monomeric MMP-9, the hetrodimeric form is produced by normal alveolar macrophages and granulocytes.
  • the processing of the precursor yields different active forms of 64, 67 and 82 kDa. Sequentially processing by MMP-3 yields the 82 kDa matrix metalloproteinase-9. In arthritis patients, this enzyme can contribute to the pathogenesis of joint destruction and can be a useful marker of disease status.
  • MMP-9 Endogenous inhibitors of MMP-9.
  • MMP-9 has a number of endogenous inhibitors. Like other MMPs, MMP-9 is inhibited by TIMPs (Murphy, G., and Willenbrock, F. (1995) Methods Enzymol. 248, 496-510).
  • a characteristic of MMP-9 (and MMP-2) is the ability of their zymogens to form tight non-covalent and stable complexes with TIMPs. It has been shown that pro-MMP-2 binds TIMP-2 (Goldberg et al. (1989) Proc. Natl. Acad. ScL U. S. A. 86, 8207- 8211), whereas pro-MMP-9 binds TIMP-I (Wilhelm et al.
  • TIMPs typically are slow, tight binding inhibitors.
  • a MMP-9 binding protein e.g., antibody
  • recombinant TIMP-I can be administered to inhibit MMP-9, e.g., in combination with a MMP-9 binding protein decscribed herein.
  • Small molecule inhibitors of MMP-9 Small molecule inhibitors of MMP-9. Skiles et al. (2004, Curr Med Chem, 11:2911-77) reported that first generation small-molecule MMP inhibitors had poor bioavailability and the second generation had caused musculoskeletal pain and inflammation. Most small-molecule MMP inhibitors interact with the catalytic zinc but have fairly low affinity. Thus, a higher concentration is needed to have effect. The interaction with the catalytic zinc leads to inhibition of other MMPs and toxic side effects.
  • a MMP-9 binding protein described herein can be used in combination with a small molecule inhibitor. For example, because the inhibitors are used in combination, the dose of the small molecule used can be decreased and therefore result in fewer side effects.
  • small molecule MMP-9 inhibitors include small synthetic anthranilic acid-based inhibitors (see, e.g., Calbiochem Inhibitor-I, catalogue #444278 and Levin et al., 2001, Bioorg. Med. Chem. Lett. 11:2975-2978).
  • Small interfering RNA inhibitors of MMP-9 can be inhibited by small interfering RNA (siRNA).
  • siRNA small interfering RNA
  • Examples of siRNA that can be used include:
  • the siRNA can be administered to inhibit MMP-9, e.g., in combination with a MMP-9 binding protein decscribed herein.
  • the disclosure provides proteins that bind to MMP-12 (e.g., human MMP-12 and/or murine MMP-12) and include at least one immunoglobin variable region.
  • MMP-12 e.g., human MMP-12 and/or murine MMP-12
  • the MMP-12 binding protein includes a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence.
  • HC heavy chain
  • LC light chain
  • the MMP- 12 binding protein may be an isolated protein (e.g., at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% free of other proteins).
  • the MMP-12 binding protein may additionally inhibit MMP-12, e.g., human and/or murine MMP-12.
  • the binding protein can inhibit the catalytic activity of MMP-12 (e.g., human MMP-12).
  • the protein binds the catalytic domain of human MMP-12, e.g., the protein contacts residues in or near the active site of MMP-12.
  • the protein does not contact residues in or near the active site of MMP-12 but instead binds elsewhere on MMP-12 and causes a steric change in MMP-12 that affects (e.g., inhibits) its activity.
  • Exemplary MMP-12 binding proteins include M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135- A06M0135-A07M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-
  • the MMP-12 binding protein is M0030-A10, M0032-H09, M0038-A03, M0038-H04, M0039-B02, M0040-B05, M0041-A05, R011-B11, M0007-A10 (also referred to as M7A10) or M0008-E08 (also referred to as M8E8).
  • Others include DX-2712 (also referred to as M0131-A06-GA-S), a mutant or variant of DX-2712 (e.g., as described herein), and 539B- X0041-D02.
  • Others include proteins that comprise the HC and/or LC CDRs of these antibodies, or proteins that comprise the HC and/or LC variable regions of any of these antibodies.
  • MMP-12 binding proteins may be antibodies.
  • MMP-12 binding antibodies may have their HC and LC variable domain sequences included in a single polypeptide (e.g., scFv), or on different polypeptides (e.g., IgG or Fab).
  • MMP-12 Matrix Metalloproteinase 12
  • MMP-12 is encoded by a gene designated as MMP 12 with full name Matrix metalloproteinase- 12 precursor. Synonyms for MMP-12 include matrix metalloproteinase 12, macrophage elastase, macrophage metalloelastase.
  • the DNA sequence is known for Homo sapiens and Mus musculus.
  • An exemplary cDNA sequence encoding human MMP 12 and the amino acid sequence are shown below.
  • Exemplary cDNA sequences encoding murine MMP 12 and amino acid sequences are also shown below.
  • An exemplary MMP-12 protein can include the human or mouse MMP-12 amino acid sequence, a sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to one of these sequences, or a fragment thereof, e.g., a fragment without the signal sequence or prodomain.
  • MMP- 12 Factors that regulate MMP- 12. Expression of MMP- 12 is regulated by many factors. Reports of upregulation include: Oncogene. 2004 Jan 22;23(3):845-51. (recurrence in stage I lung cancer, 2/10 cases), Ann Neurol. 2003 Jun;53(6):731-42. (collagenase-induced rat model of intracerebral hemorrhage), Cancer Res. 2005 May 15;65(10):4261-72. (protein kinase C/p53 resistant cells), Br J Dermatol. 2005 Apr;152(4):720-6. (Samples from nine patients with squamous cell carcinoma), Cardiovasc Res. 2005 May l;66(2):410-9. (Aging), J Immunol.
  • MMP-12 has a number of endogenous inhibitors. Like other MMPs, MMP-12 is inhibited by TIMPs (Murphy, G., and Willenbrock, F. (1995) Methods Enzymol 248, 496-510).
  • Small molecule inhibitors of MMP-12 Small molecule inhibitors of MMP-12. Small molecule inhibitors of MMP-12 have been synthesized and tested. Most of these have either insufficient potency or insufficient specificity, or both. The reports include: Proc Natl Acad Sci USA. 2005 Apr 12;102(15):5334-9. (acetohydroxamic acid and N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid); Arthritis Rheum. 2004 Oct;50(10):3275-85. (a general hydroxamate inhibitor of MMP activity); Arch Biochem Biophys. 2003 Jan 15;409(2):335-40. (peptide Iin24); J MoI Biol.
  • the small molecule can be administered to inhibit MMP-12, e.g., in combination with a MMP-12 binding protein described herein.
  • MMP- 12 can be inhibited by small interfering RNA (siRNA). Examples of siRNA that can be used are described in US Patent Publication No.: 20040087533 and PCT Publication No.: WO 200409098.
  • the siRNA can be administered to inhibit MMP-12, e.g., in combination with a MMP-12 binding protein described herein.
  • the MMP-9 binding proteins described herein can be conjugated to a drug (e.g., a cytotoxic, cytostatic, or immunomodulatory agent).
  • a drug e.g., a cytotoxic, cytostatic, or immunomodulatory agent.
  • the conjugates can be used therapeutically or prophylactically, e.g., the binding protein can target the drug, e.g., in vivo, e.g., to a site of disease (e.g., a tumor or site of inflammation), e.g., such that the drug affects the site of disease (e.g., causes a cytostatic or cytotoxic effect on targeted cells).
  • the binding protein itself has therapeutic or prophylactic efficacy (e.g., the protein can modulate (e.g., antagonize) MMP-9 or -12, or cause a cytostatic or cytotoxic effect on a cell that expresses MMP-9 or -12 (e.g., an endothelial cell or tumor cell)).
  • the protein can modulate (e.g., antagonize) MMP-9 or -12, or cause a cytostatic or cytotoxic effect on a cell that expresses MMP-9 or -12 (e.g., an endothelial cell or tumor cell)).
  • the binding protein-drug conjugate can be used such that the binding protein and drug both contribute (e.g., additively or synergistically) to an effect on MMP-9 or -12 (e.g., a therapeutic effect, e.g., in vivo, e.g., to a site of disease (e.g., a tumor or site of undesired angiogenesis or vascularization).
  • the drug and/or binding protein can be, for example, cytotoxic, cytostatic or otherwise prevent or reduce the ability of a targeted cell to divide and/or survive (e.g., when the drug is taken up or internalized by the targeted cell and/or upon binding of the binding protein to MMP-9 or -12).
  • the drug and/or binding protein can prevent or reduce the ability of the cell to divide and/or metastasize.
  • useful classes of drugs that can be used in the binding protein-drug conjugates described herein include cytotoxic or immunomodulatory agents such as, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, pre-forming compounds, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxa
  • cytotoxic or immunomodulatory agents such as
  • cytotoxic or immunomodulatory agents include, for example, an androgen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (actinomycin), daunorubicin, decarbazine, docetaxel, doxorubicin, an estrogen, 5- fluorodeoxyuridine, 5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), mechlorethamine, melphalan
  • the drug comprises a cytotoxic agent.
  • Suitable cytotoxic agents include, for example, dolastatins (e.g., auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (e.g., enediynes and lexitropsins), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide, eleutherobin, and mitoxantrone.
  • the drug is a cytotoxic agent such as AFP
  • AEVB auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatstatin, chalicheamicin, maytansine, DM-I, or netropsin.
  • the drug is a cytotoxic agent that comprises a conventional chemotherapeutic such as, for example, doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C or etoposide.
  • the drug can be a combined therapy, such as CHOP (Cyclophosphamide, Doxorubicin, Prednisolone and Vincristine), CHOP-R (Cyclophosphamide, Doxorubicin Vincristine, Prednisolone, and rituximab) or ABVD (Doxorubicin, Bleomycin, Vinblastine and dacarbazine).
  • CC-1065 analogues e.g., DCl
  • calicheamicin maytansine
  • analogues of dolastatin 10 rhizoxin
  • palytoxin can also be used.
  • the drug can be a cytotoxic or cytostatic agent that comprises auristatin E (also known in the art as dolastatin-10) or a derivative thereof.
  • the auristatin E derivative is, e.g., an ester formed between auristatin E and a keto acid.
  • auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively.
  • auristatin derivatives include AFP, MMAF, and MMAE.
  • the synthesis and structure of auristatin E and its derivatives are described in US 20030083263 and US 20050009751, and U.S. Pat. Nos. 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414.
  • MMAF or AFP is used.
  • the drug is a cytotoxic agent that comprises a DNA minor groove binding agent.
  • a cytotoxic agent that comprises a DNA minor groove binding agent.
  • the minor groove binding agent is a CBI compound.
  • the minor groove binding agent is an enediyne (e.g., calicheamicin).
  • anti-tubulin agents examples include, but are not limited to, taxanes (e.g., TAXOL® (paclitaxel), TAXOTERE® (docetaxel)), T67 (Tularik), vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), and dolastatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB).
  • taxanes e.g., TAXOL® (paclitaxel), TAXOTERE® (docetaxel)
  • T67 Tularik
  • vinca alkyloids e.g., vincristine, vinblastine, vindesine, and vinorelbine
  • dolastatins e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB
  • antitubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine, cryptophysins, cemadotin, maytansinoids, combretastatins, discodermolide, eleutherobin, rhizoxin/maytansine, auristatin dolastatin 10 MMAE, and peloruside A.
  • taxane analogs e.g., epothilone A and B
  • nocodazole e.g., colchicine and colcimid
  • estramustine e.g., epothilone A and B
  • cryptophysins e.g., cemadotin
  • maytansinoids combretastatins
  • discodermolide e.g., eleutherobin
  • the drug is a cytotoxic agent such as an anti-tubulin agent.
  • the anti-tubulin agent is an auristatin, a vinca alkaloid, a podophyllotoxin, a taxane, a baccatin derivative, a cryptophysin, a maytansinoid, a combretastatin, or a dolastatin.
  • the antitubulin agent is AFP, MMAP, MMAE, AEB, AEVB, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP- 16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicines, colcimid, estramustine, cemadotin, discodermolide, maytansine, DMl, DM2, DM3, DM4, or eleutherobin.
  • the cytotoxic agent comprises a maytansinoid, another group of anti-tubulin agents.
  • the maytansinoid is maytansine or DM-I (ImmunoGen, Inc.; see also Chari et al. Cancer Res. 52:127-131 (1992)).
  • sterically hindered thiol and disulfide-containing maytansinoids in which the alpha-carbon atom bearing the sulfur atom bears one or two alkyl substituents are used in the binding protein-drug conjugate, e.g., US 2007-0292422; US 2007-0264266.
  • the drug comprises an agent that acts to disrupt DNA.
  • the drug may be selected from enediynes (e.g., calicheamicin and esperamicin) and non-enediyne small molecule agents (e.g., bleomycin, methidiumpropyl-EDTA-Fe(II)).
  • enediynes e.g., calicheamicin and esperamicin
  • non-enediyne small molecule agents e.g., bleomycin, methidiumpropyl-EDTA-Fe(II)
  • Other useful drugs include daunorubicin, doxorubicin, distamycin A, cisplatin, mitomycin C, ecteinascidins, duocarmycin/CC-1065, and bleomycin/pepleomycin.
  • the drug can comprise an alkylating agent such as Asaley NSC
  • the drug can comprise an antimitotic agent such as allocolchicine NSC 406042, Halichondrin B NSC 609395, colchicine NSC 757, colchicine derivative NSC 33410, dolastatin 10 NSC 376128 (NG-auristatin derived), maytansine NSC 153858, rhizoxin NSC 332598, taxol NSC 125973, taxol derivative NSC 608832, thiocolchicine NSC 361792, trityl cysteine NSC 83265, vinblastine sulfate NSC 49842, or vincristine sulfate NSC 67574.
  • an antimitotic agent such as allocolchicine NSC 406042, Halichondrin B NSC 609395, colchicine NSC 757, colchicine derivative NSC 33410, dolastatin 10 NSC 376128 (NG-auristatin derived), maytansine NSC 153858, rhizoxin NSC 332598
  • the drug can comprise an topoisomerase I inhibitor such as camptothecin NSC 94600, camptothecin, Na salt NSC 100880, aminocamptothecin NSC 603071, camptothecin derivative NSC 95382, camptothecin derivative NSC 107124, camptothecin derivative NSC 643833, camptothecin derivative NSC 629971, camptothecin derivative NSC 295500, camptothecin derivative NSC 249910, camptothecin derivative NSC 606985, camptothecin derivative NSC 374028, camptothecin derivative NSC 176323, camptothecin derivative NSC 295501, camptothecin derivative NSC 606172, camptothecin derivative NSC 606173, camptothecin derivative NSC 610458, camptothecin derivative NSC 618939, camptothecin derivative NSC 610457, camptothecin derivative NSC 610459, camptothecin derivative NSC
  • the drug can comprise an topoisomerase II inhibitor such as doxorubicin NSC 123127, amonafide NSC 308847, m-AMSA NSC 249992, anthrapyrazole derivative NSC 355644, pyrazoloacridine NSC 366140, bisantrene HCL NSC 337766, daunorubicin NSC 82151, deoxydoxorubicin NSC 267469, mitoxantrone NSC 301739, menogaril NSC 269148, N,N-dibenzyl daunomycin NSC 268242, oxanthrazole NSC 349174, rubidazone NSC 164011, VM-26 NSC 122819, or VP-16 NSC 141540.
  • an topoisomerase II inhibitor such as doxorubicin NSC 123127, amonafide NSC 308847, m-AMSA NSC 249992, anthrapyrazole derivative NSC 35
  • the drug can comprise an RNA or DNA antimetabolite such as L- alanosine NSC 153353, 5-azacytidine NSC 102816, 5-fluorouracil NSC 19893, acivicin NSC 163501, aminopterin derivative NSC 132483, aminopterin derivative NSC 184692, aminopterin derivative NSC 134033, an antifol NSC 633713, an antifol NSC 623017, Baker's soluble antifol NSC 139105, dichlorallyl lawsone NSC 126771, brequinar NSC 368390, ftorafur (pro-drug) NSC 148958, 5,6-dihydro-5-azacytidine NSC 264880, methotrexate NSC 740, methotrexate derivative NSC 174121, N-(phosphonoacetyl)-L-aspartate (PALA) NSC 224131, pyrazofurin NSC 143095
  • AFP refers to dimethylvaline-valine-dolaisoleuine-dolaproine- phenylalanine-p-phenylened-iamine (e.g., see Formula XVI in US 2006-0233794).
  • the abbreviation “MAE” refers to monomethyl auristatin E (see Formula XI in US 2006- 0233794).
  • the abbreviation “AEB” refers to an ester produced by reacting auristatin E with paraacetyl benzoic acid (e.g., see Formula XX in US 2006-0233794)
  • the abbreviation “AEVB” refers to an ester produced by reacting auristatin E with benzoylvaleric acid (e.g., see Formula XXI in US 2006-0233794).
  • MMAF dovaline-valine-dolaisoleunine-dolaproine- phenylalanine (e.g., see Formula IVIV in US 2006-0233794).
  • fk and phe-lys refer to the linker phenylalanine-lysine.
  • the drug is a cytotoxic agent selected from the group consisting of an auristatin, a DNA minor groove binding agent, a DNA minor groove alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid.
  • a cytotoxic agent selected from the group consisting of an auristatin, a DNA minor groove binding agent, a DNA minor groove alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid.
  • the drug is a cytotoxic agent such as AFP or MMAF.
  • the drug is an immunosuppressive agent such as gancyclovir, etanercept, cyclosporine, tacrolimus, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, Cortisol, aldosterone, dexamethasone, a cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, or a leukotriene receptor antagonist.
  • an immunosuppressive agent such as gancyclovir, etanercept, cyclosporine, tacrolimus, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, Cortisol, aldosterone, dexamethasone, a cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, or a leukotriene receptor antagonist.
  • the binding proteins described herein can be associated with a drug to form a binding protein-drug conjugate by being linked to the drug directly.
  • the binding protein is directly conjugated to the drug.
  • the binding proteins described herein can be associated with a drug to form a binding protein-drug conjugate by use of a linker region between the drug and the binding protein.
  • the binding protein is conjugated to the drug via a linker.
  • the linker can be cleavable under intracellular conditions, e.g., such that cleavage of the linker releases the drug from the binding protein in the intracellular environment.
  • the cleavable linker is a peptide linker cleavable by an intracellular protease.
  • the peptide linker is a dipeptide linker.
  • the dipeptide linker is a val-cit (vc) linker or a phe-lys (fk) linker.
  • the cleavable linker is hydrolyzable at a pH of less than 5.5. In some embodiments, the hydrolyzable linker is a hydrazone linker. In some embodiments, the cleavable linker is a disulfide linker.
  • the linker is cleavable by a cleaving agent that is present in the intracellular environment (e.g., within a lysosome or endosome or caveolea).
  • the linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease.
  • the peptidyl linker is at least two amino acids long or at least three amino acids long.
  • Cleaving agents can include cathepsins B and D and plasmin, which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside target cells (see, e.g., Dubowchik and Walker Pharm. Therapeutics 83:67-123 (1999)).
  • peptidyl linkers are cleavable by enzymes that are present in targeted cells (e.g., cancer cells).
  • a peptidyl linker that is cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancerous tissue can be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Gly linker).
  • the peptidyl linker cleavable by an intracellular protease is a Val-Cit (vc) linker or a Phe-Lys linker (fk) (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the val-cit linker).
  • vc Val-Cit
  • fk Phe-Lys linker
  • One advantage of using intracellular proteolytic release of the drug is that the drug can be attenuated when conjugated and the serum stabilities of the conjugates are typically high.
  • a vc linker is used in the binding protein-drug conjugates described herein.
  • a binding protein- vcAFP or a binding protein- vcMMAF conjugate e.g., a MMP-9 or -12 binding protein-vcAFP or a MMP-9 or -12 binding protein- vcMMAF conjugate
  • a binding protein- vcAFP or a binding protein- vcMMAF conjugate is prepared.
  • the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values.
  • the pH-senstive linker is hydrolyzable under acidic conditions.
  • an acid-labile linker that is hydrolyzable in the lysosome e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal., ketal., or the like
  • an acid-labile linker that is hydrolyzable in the lysosome e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal., ketal., or the like
  • the hydrolyzable linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929)).
  • the linker is cleavable under reducing conditions (e.g., a disulfide linker).
  • a disulfide linker e.g., a disulfide linker.
  • disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N- succinimidyl-3-(2-pyridyldithio propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)- , SPDB and SMPT (See, e.g., Thorpe et al.
  • the linker is a malonate linker (Johnson et al. Anticancer Res. 15:1387-93 (1995)), a maleimidobenzoyl linker (Lau et al. Bioorg-Med-Chem. 3(10):1299-1304 (1995), or a 3'-N-amide analog (Lau et al. Bioorg-Med-Chem. 3(10):1305-12 (1995)).
  • the linker is not substantially sensitive to the extracellular environment.
  • "not substantially sensitive to the extracellular environment" in the context of a linker means that no more than about 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linkers, in a sample of a binding protein- drug conjugate, are cleaved when the binding protein-drug conjugate is present in an extracellular environment (e.g., in plasma).
  • Whether a linker is not substantially sensitive to the extracellular environment can be determined, for example, by incubating independently with plasma both (a) the binding protein-drug conjugate (the "conjugate sample”) and (b) an equal molar amount of unconjugated binding protein or drug (the “control sample”) for a predetermined time period (e.g., 2, 4, 8, 16, or 24 hours) and then comparing the amount of unconjugated binding protein or drug present in the conjugate sample with that present in control sample, as measured, for example, by high performance liquid chromatography.
  • a predetermined time period e.g., 2, 4, 8, 16, or 24 hours
  • the linker promotes cellular internalization. In certain embodiments, the linker promotes cellular internalization when conjugated to the drug (i.e., in the milieu of the linker-drug moiety of the binding protein-drug conjugate described herein). In yet other embodiments, the linker promotes cellular internalization when conjugated to both the drug and the binding protein.
  • linkers that can be used with the present compositions and methods are described in WO 2004010957.
  • the binding protein-drug conjugates described herein are used therapeutically in the treatment of a disorder (e.g., cancer or inflammation).
  • a disorder e.g., cancer or inflammation.
  • it is desirable to only target a binding protein-drug conjugate to a cell that expresses the target to which the binding protein binds e.g., to only target a MMP-9 expressing cell to which a MMP-9 binding protein binds, and not target a nearby "bystander" cell
  • it is desirable to target a binding protein-drug conjugate to a cell expressing the target to which the binding protein binds and also to bystander cells e.g., to elicit a "bystander effect").
  • a binding protein-drug conjugate e.g., a MMP-9 binding protein-drug conjugate can be engineered to exert a precise killing of only antigen-presenting cells without damaging proximal antigen-negative tissues, e.g., by preparing thioether-linked conjugates.
  • it can be engineered to produce a bystander effect, e.g., by preparing disulfide-linked conjugates.
  • targets e.g., antigens
  • the bystander cytotoxicity associated with disulfide linker-containing conjugates provides a rationale for treatment of sites of a disorder (e.g., tumors) with binding protein-drug conjugates even if the sites exhibit heterogeneous target expression.
  • the bystander effect adds a degree of nonselective killing activity. Potentially, this could be a drawback if normal cells in tissues surrounding the site of disorder (e.g., tumor) are affected.
  • the bystander cytotoxicity may damage tissues intricately involved in supporting the disorder, such as endothelial cells and pericytes of tumor neovasculature, or tumor stromal cells, resulting, for example, in enhanced antitumor activity of the binding protein-drug conjugate against tumors expressing the antigen either homogeneously or heterogeneously. See also Kovtum et al. Cancer Res. 66:3214 (2006).
  • a display library is a collection of entities; each entity includes an accessible polypeptide component and a recoverable component that encodes or identifies the polypeptide component.
  • the polypeptide component is varied so that different amino acid sequences are represented.
  • the polypeptide component can be of any length, e.g. from three amino acids to over 300 amino acids.
  • a display library entity can include more than one polypeptide component, for example, the two polypeptide chains of an sFab.
  • a display library can be used to identify proteins that bind to MMP-9 or -12. In a selection, the polypeptide component of each member of the library is probed with MMP-9 or -12 (e.g., the catalytic domain of MMP-9 or -12 or other fragment) and if the polypeptide component binds to the MMP-9, the display library member is identified, typically by retention on a support.
  • Retained display library members are recovered from the support and analyzed.
  • the analysis can include amplification and a subsequent selection under similar or dissimilar conditions. For example, positive and negative selections can be alternated.
  • the analysis can also include determining the amino acid sequence of the polypeptide component and purification of the polypeptide component for detailed characterization.
  • Phage Display The protein component is typically covalently linked to a bacteriophage coat protein. The linkage results from translation of a nucleic acid encoding the protein component fused to the coat protein.
  • the linkage can include a flexible peptide linker, a protease site, or an amino acid incorporated as a result of suppression of a stop codon. Phage display is described, for example, in U.S.
  • Bacteriophage displaying the protein component can be grown and harvested using standard phage preparatory methods, e.g. PEG precipitation from growth media. After selection of individual display phages, the nucleic acid encoding the selected protein components can be isolated from cells infected with the selected phages or from the phage themselves, after amplification. Individual colonies or plaques can be picked, the nucleic acid isolated and sequenced.
  • Display Formats include cell based display (see, e.g., WO 03/029456), protein-nucleic acid fusions (see, e.g., US 6,207,446), ribosome display (See, e.g., Mattheakis et al. (1994) P roc. Natl. Acad. Sci. USA 91 : 9022 and Hanes et al. (2000) Nat
  • Scaffolds useful for display include: antibodies (e.g., Fab fragments, single chain Fv molecules (scFV), single domain antibodies, camelid antibodies, and camelized antibodies); T-cell receptors; MHC proteins; extracellular domains (e.g., fibronectin Type III repeats, EGF repeats); protease inhibitors (e.g., Kunitz domains, ecotin, BPTI, and so forth); TPR repeats; trifoil structures; zinc finger domains; DNA-binding proteins; particularly monomeric DNA binding proteins; RNA binding proteins; enzymes, e.g., proteases (particularly inactivated proteases), RNase; chaperones, e.g., thioredoxin and heat shock proteins; intracellular signaling domains (such as SH2 and SH3 domains); linear and constrained peptides; and linear peptide substrates.
  • Display libraries can include synthetic and/or natural diversity. See, e.g., US 2004-0005709.
  • Display technology can also be used to obtain binding proteins (e.g., antibodies) that bind particular epitopes of a target. This can be done, for example, by using competing non-target molecules that lack the particular epitope or are mutated within the epitope, e.g., with alanine. Such non-target molecules can be used in a negative selection procedure as described below, as competing molecules when binding a display library to the target, or as a pre-elution agent, e.g., to capture in a wash solution dissociating display library members that are not specific to the target. Iterative Selection.
  • display library technology is used in an iterative mode. A first display library is used to identify one or more binding proteins for a target. These identified binding proteins are then varied using a mutagenesis method to form a second display library. Higher affinity binding proteins are then selected from the second library, e.g., by using higher stringency or more competitive binding and washing conditions.
  • the mutagenesis is targeted to regions at the binding interface. If, for example, the identified binding proteins are antibodies, then mutagenesis can be directed to the CDR regions of the heavy or light chains as described herein. Further, mutagenesis can be directed to framework regions near or adjacent to the CDRs. In the case of antibodies, mutagenesis can also be limited to one or a few of the CDRs, e.g., to make precise step-wise improvements. Exemplary mutagenesis techniques include: error-prone PCR, recombination, DNA shuffling, site-directed mutagenesis and cassette mutagenesis.
  • the methods described herein are used to first identify a protein from a display library that binds MMP-9 with at least a minimal binding specificity for a target or a minimal activity, e.g., an equilibrium dissociation constant for binding of less than 1 nM, 10 nM, or 100 nM.
  • the nucleic acid sequence encoding the initial identified proteins are used as a template nucleic acid for the introduction of variations, e.g., to identify a second protein that has enhanced properties (e.g., binding affinity, kinetics, or stability) relative to the initial protein.
  • the methods described herein can be used to isolate binding proteins with a desired (e.g., reduced) kinetic dissociation rate for a binding interaction to a target.
  • the library is contacted to an immobilized target.
  • the immobilized target is then washed with a first solution that removes non-specifically or weakly bound biomolecules.
  • the bound binding proteins are eluted with a second solution that includes a saturating amount of free target or a target specific high-affinity competing monoclonal antibody, i.e., replicates of the target that are not attached to the particle.
  • the free target binds to biomolecules that dissociate from the target. Rebinding is effectively prevented by the saturating amount of free target relative to the much lower concentration of immobilized target.
  • the second solution can have solution conditions that are substantially physiological or that are stringent.
  • the solution conditions of the second solution are identical to the solution conditions of the first solution. Fractions of the second solution are collected in temporal order to distinguish early from late fractions. Later fractions include biomolecules that dissociate at a slower rate from the target than biomolecules in the early fractions.
  • display library screening methods described herein can include a selection or screening process that discards display library members that bind to a non-target molecule.
  • non-target molecules include streptavidin on magnetic beads, blocking agents such as bovine serum albumin, non-fat bovine milk, any capturing or target immobilizing monoclonal antibody, or non-transfected cells which do not express the human MMP-9 or -12 target.
  • a so-called "negative selection” step is used to discriminate between the target and related non-target molecule and a related, but distinct non-target molecules.
  • the display library or a pool thereof is contacted to the non-target molecule.
  • Members of the sample that do not bind the non-target are collected and used in subsequent selections for binding to the target molecule or even for subsequent negative selections.
  • the negative selection step can be prior to or after selecting library members that bind to the target molecule.
  • a screening step is used. After display library members are isolated for binding to the target molecule, each isolated library member is tested for its ability to bind to a non-target molecule (e.g., a non-target listed above). For example, a high-throughput ELISA screen can be used to obtain this data. The ELISA screen can also be used to obtain quantitative data for binding of each library member to the target as well as for cross species reactivity to related targets or subunits of the target (e.g., mouse MMP-9) and also under different condition such as pH6 or pH 7.5. The non-target and target binding data are compared (e.g., using a computer and software) to identify library members that specifically bind to the target.
  • a non-target molecule e.g., a non-target listed above.
  • a high-throughput ELISA screen can be used to obtain this data.
  • the ELISA screen can also be used to obtain quantitative data for binding of each library member to the target as well as for cross species reactivity
  • proteins with a particular property e.g., ability to bind MMP-9 or -12 and/or ability to modulate MMP-9 or -12
  • proteins arrays of antibodies see, e.g., De Wildt et al. (2000) Nat. Biotechnol. 18:989-994
  • lambda gtll libraries two-hybrid libraries and so forth.
  • PRIMATIZED antibodies genetically engineered from cynomolgus macaque monkey and human components, are structurally indistinguishable from human antibodies. They may, therefore, be less likely to cause adverse reactions in humans, making them potentially suited for long-term, chronic treatment " Curr Opin Investig Drugs. (2001) 2(5):635-8.
  • One exemplary type of library presents a diverse pool of polypeptides, each of which includes an immunoglobulin domain, e.g., an immunoglobulin variable domain.
  • an immunoglobulin domain e.g., an immunoglobulin variable domain.
  • display libraries where the members of the library include primate or "primatized” (e.g., such as human, non-human primate or “humanized”) immunoglobin domains (e.g., immunoglobin variable domains) or chimeric primatized Fabs with human constant regions.
  • Human or humanized immunoglobin domain libraries may be used to identify human or "humanized” antibodies that, for example, recognize human antigens. Because the constant and framework regions of the antibody are human, these antibodies may avoid themselves being recognized and targeted as antigens when administered to humans.
  • the constant regions may also be optimized to recruit effector functions of the human immune system.
  • the in vitro display selection process surmounts the inability of a normal human immune system to generate antibodies against self- antigens.
  • a typical antibody display library displays a polypeptide that includes a VH domain and a VL domain.
  • An "immunoglobulin domain” refers to a domain from the variable or constant domain of immunoglobulin molecules. Immunoglobulin domains typically contain two ⁇ -sheets formed of about seven ⁇ -strands, and a conserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay, 1988, Ann. Rev. Immunol. 6:381-405).
  • the display library can display the antibody as a Fab fragment (e.g., using two polypeptide chains) or a single chain Fv (e.g., using a single polypeptide chain). Other formats can also be used.
  • the displayed antibody can include one or more constant regions as part of a light and/or heavy chain.
  • each chain includes one constant region, e.g., as in the case of a Fab.
  • additional constant regions are displayed.
  • Antibody libraries can be constructed by a number of processes (see, e.g., de Haard et al., 1999, /. Biol. Chem. 274:18218-30; Hoogenboom et al., 1998, Immunotechnology 4:1-20; Hoogenboom et al., 2000, Immunol. Today 21:371-378, and Hoet et al. (2005) Nat Biotechnol. 23(3)344-8. Further, elements of each process can be combined with those of other processes. The processes can be used such that variation is introduced into a single immunoglobulin domain (e.g., VH or VL) or into multiple immunoglobulin domains (e.g., VH and VL).
  • a single immunoglobulin domain e.g., VH or VL
  • multiple immunoglobulin domains e.g., VH and VL
  • the variation can be introduced into an immunoglobulin variable domain, e.g., in the region of one or more of CDRl, CDR2, CDR3, FRl, FR2, FR3, and FR4, referring to such regions of either and both of heavy and light chain variable domains.
  • the variation(s) may be introduced into all three CDRs of a given variable domain, or into CDRl and CDR2, e.g., of a heavy chain variable domain. Any combination is feasible.
  • antibody libraries are constructed by inserting diverse oligonucleotides that encode CDRs into the corresponding regions of the nucleic acid.
  • the oligonucleotides can be synthesized using monomeric nucleotides or trinucleotides.
  • Knappik et al., 2000, J. MoI. Biol. 296:57-86 describe a method for constructing CDR encoding oligonucleotides using trinucleotide synthesis and a template with engineered restriction sites for accepting the oligonucleotides.
  • an animal e.g., a rodent
  • the animal is immunized with MMP-9 or -12.
  • the animal is optionally boosted with the antigen to further stimulate the response.
  • spleen cells are isolated from the animal, and nucleic acid encoding VH and/or VL domains is amplified and cloned for expression in the display library.
  • antibody libraries are constructed from nucleic acid amplified from na ⁇ ve germline immunoglobulin genes.
  • the amplified nucleic acid includes nucleic acid encoding the VH and/or VL domain. Sources of immunoglobulin-encoding nucleic acids are described below.
  • Amplification can include PCR, e.g., with primers that anneal to the conserved constant region, or another amplification method.
  • Nucleic acid encoding immunoglobulin domains can be obtained from the immune cells of, e.g., a primate (e.g., a human), mouse, rabbit, camel, or rodent.
  • the cells are selected for a particular property. B cells at various stages of maturity can be selected.
  • the B cells are na ⁇ ve.
  • fluorescent-activated cell sorting FACS is used to sort B cells that express surface-bound IgM, IgD, or IgG molecules. Further, B cells expressing different isotypes of IgG can be isolated.
  • the B or T cell is cultured in vitro.
  • the cells can be stimulated in vitro, e.g., by culturing with feeder cells or by adding mitogens or other modulatory reagents, such as antibodies to CD40, CD40 ligand or CD20, phorbol myristate acetate, bacterial lipopolysaccharide, concanavalin A, phytohemagglutinin, or pokeweed mitogen.
  • mitogens or other modulatory reagents such as antibodies to CD40, CD40 ligand or CD20, phorbol myristate acetate, bacterial lipopolysaccharide, concanavalin A, phytohemagglutinin, or pokeweed mitogen.
  • the cells are isolated from a subject that has a disease of condition described herein, e.g., a cancer (e.g., metastatic cancer, e.g., metastatic breast cancer), an inflammatory disease (e.g., synovitis, atherosclerosis), rheumatoid arthritis, osteoarthritis, an ocular condition (e.g., macular degeneration), diabetes, Alzheimer's Disease, cerebral ischemia, endometriosis, fibrin-invasive activity, angiogenesis, or capillary tube formation
  • a cancer e.g., metastatic cancer, e.g., metastatic breast cancer
  • an inflammatory disease e.g., synovitis, atherosclerosis
  • rheumatoid arthritis e.g., osteoarthritis
  • an ocular condition e.g., macular degeneration
  • diabetes Alzheimer's Disease, cerebral ischemia, endometriosis, fibrin-invasive activity, angiogenesis, or capillary tube
  • the cells have activated a program of somatic hypermutation.
  • Cells can be stimulated to undergo somatic mutagenesis of immunoglobulin genes, for example, by treatment with antiimmunoglobulin, anti-CD40, and anti-CD38 antibodies (see, e.g., Bergthorsdottir et al., 2001, /. Immunol. 166:2228).
  • the cells are na ⁇ ve.
  • the nucleic acid encoding an immunoglobulin variable domain can be isolated from a natural repertoire by the following exemplary method.
  • the reverse transcription of the first (antisense) strand can be done in any manner with any suitable primer. See, e.g., de Haard et al., 1999, /. Biol. Chem. 274:18218-30.
  • the primer binding region can be constant among different immunoglobulins, e.g., in order to reverse transcribe different isotypes of immunoglobulin.
  • the primer binding region can also be specific to a particular isotype of immunoglobulin.
  • the primer is specific for a region that is 3' to a sequence encoding at least one CDR.
  • poly-dT primers may be used (and may be preferred for the heavy-chain genes).
  • a synthetic sequence can be ligated to the 3' end of the reverse transcribed strand.
  • the synthetic sequence can be used as a primer binding site for binding of the forward primer during PCR amplification after reverse transcription.
  • the use of the synthetic sequence can obviate the need to use a pool of different forward primers to fully capture the available diversity.
  • variable domain-encoding gene is then amplified, e.g., using one or more rounds. If multiple rounds are used, nested primers can be used for increased fidelity.
  • the amplified nucleic acid is then cloned into a display library vector.
  • each candidate library member can be further analyzed, e.g., to further characterize its binding properties for the target, e.g., MMP-9 or -12, or for binding to other protein, e.g., another metalloproteinase.
  • Each candidate library member can be subjected to one or more secondary screening assays.
  • the assay can be for a binding property, a catalytic property, an inhibitory property, a physiological property (e.g., cytotoxicity, renal clearance, immunogenicity), a structural property (e.g., stability, conformation, oligomerization state) or another functional property.
  • the same assay can be used repeatedly, but with varying conditions, e.g., to determine pH, ionic, or thermal sensitivities.
  • the assays can use a display library member directly, a recombinant polypeptide produced from the nucleic acid encoding the selected polypeptide, or a synthetic peptide synthesized based on the sequence of the selected polypeptide.
  • the Fabs can be evaluated or can be modified and produced as intact IgG proteins.
  • Exemplary assays for binding properties include the following.
  • Binding proteins can be evaluated using an ELISA assay. For example, each protein is contacted to a microtitre plate whose bottom surface has been coated with the target, e.g., a limiting amount of the target. The plate is washed with buffer to remove non- specifically bound polypeptides. Then the amount of the binding protein bound to the target on the plate is determined by probing the plate with an antibody that can recognize the binding protein, e.g., a tag or constant portion of the binding protein. The antibody is linked to a detection system (e.g. , an enzyme such as alkaline phosphatase or horse radish peroxidase (HRP) which produces a colorimetric product when appropriate substrates are provided).
  • a detection system e.g. , an enzyme such as alkaline phosphatase or horse radish peroxidase (HRP) which produces a colorimetric product when appropriate substrates are provided.
  • FRET fluorescence resonance energy transfer
  • a fluorophore label on the first molecule (e.g., the molecule identified in the fraction) is selected such that its emitted fluorescent energy can be absorbed by a fluorescent label on a second molecule (e.g., the target) if the second molecule is in proximity to the first molecule.
  • the fluorescent label on the second molecule fluoresces when it absorbs to the transferred energy. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal.
  • a binding event that is configured for monitoring by FRET can be conveniently measured through standard fluorometric detection means, e.g., using a fluorimeter. By titrating the amount of the first or second binding molecule, a binding curve can be generated to estimate the equilibrium binding constant.
  • ALPHASCREENTM Packard Bioscience, Meriden CT
  • ALPHASCREEN TM uses two labeled beads. One bead generates singlet oxygen when excited by a laser. The other bead generates a light signal when singlet oxygen diffuses from the first bead and collides with it. The signal is only generated when the two beads are in proximity.
  • One bead can be attached to the display library member, the other to the target. Signals are measured to determine the extent of binding.
  • SPR Surface Plasmon Resonance
  • the interaction of binding protein and a target can be analyzed using SPR.
  • SPR or Biomolecular Interaction Analysis (BIA) detects biospecific interactions in real time, without labeling any of the interactants. Changes in the mass at the binding surface (indicative of a binding event) of the BIA chip result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)). The changes in the refractivity generate a detectable signal, which are measured as an indication of real-time reactions between biological molecules.
  • Methods for using SPR are described, for example, in U.S. Patent No.
  • BIAcore Flexchip can be used to compare and rank interactions in real time, in terms of kinetics, affinity or specificity without the use of labels.
  • Information from SPR can be used to provide an accurate and quantitative measure of the equilibrium dissociation constant (K d ), and kinetic parameters, including K 0n and K Off , for the binding of a binding protein to a target.
  • Such data can be used to compare different biomolecules. For example, selected proteins from an expression library can be compared to identify proteins that have high affinity for the target or that have a slow K Off .
  • This information can also be used to develop structure- activity relationships (SAR). For example, the kinetic and equilibrium binding parameters of matured versions of a parent protein can be compared to the parameters of the parent protein. Variant amino acids at given positions can be identified that correlate with particular binding parameters, e.g., high affinity and slow K Off .
  • This information can be combined with structural modeling (e.g., using homology modeling, energy minimization, or structure determination by x-ray crystallography or NMR). As a result, an understanding of the physical interaction between the protein and its target can be formulated and used to guide other design processes.
  • Binding proteins can be screened for ability to bind to cells which transiently or stably express and display the target of interest on the cell surface.
  • MMP-9 binding proteins can be fluorescently labeled and binding to MMP-9 in the presence of absence of antagonistic antibody can be detected by a change in fluorescence intensity using flow cytometry e.g., a FACS machine.
  • MMP-9 or -12 binding antibody In addition to the use of display libraries, other methods can be used to obtain a MMP-9 or -12 binding antibody.
  • MMP-9 or -12 protein or a region thereof can be used as an antigen in a non-human animal, e.g., a rodent.
  • the non-human animal includes at least a part of a human immunoglobulin gene.
  • a human immunoglobulin gene For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci.
  • antigen-specific monoclonal antibodies (Mabs) derived from the genes with the desired specificity may be produced and selected. See, e.g., XENOMOUSETM, Green et al., 1994, Nat. Gen. 7:13-21; U.S. 2003-0070185, WO 96/34096, published Oct. 31, 1996, and PCT Application No. PCT/US96/05928, filed Apr. 29, 1996.
  • a monoclonal antibody is obtained from the non-human animal, and then modified, e.g., humanized or deimmunized.
  • Winter describes a CDR-grafting method that may be used to prepare the humanized antibodies (UK Patent Application GB 2188638A, filed on March 26, 1987; US Patent No. 5,225,539. All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen.
  • Humanized antibodies can be generated by replacing sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions.
  • General methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. US Patent Nos. 5,585,089, US 5,693,761 and US 5,693,762.
  • Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Numerous sources of such nucleic acid are available.
  • nucleic acids may be obtained from a hybridoma producing an antibody against a predetermined target, as described above.
  • the recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.
  • Immunoglobin MMP-9 or -12 binding proteins may be modified to reduce immunogenicity. Reduced immunogenicity is desirable in MMP-9 or -12 binding proteins intended for use as therapeutics, as it reduces the chance that the subject will develop an immune response against the therapeutic molecule. Techniques useful for reducing immunogenicity of MMP-9 binding proteins include deletion/modification of potential human T cell epitopes and 'germlining' of sequences outside of the CDRs (e.g., framework and Fc).
  • An MMP-9 or -12 -binding antibody may be modified by specific deletion of human T cell epitopes or "deimmunization" by the methods disclosed in WO 98/52976 and WO 00/34317. Briefly, the heavy and light chain variable regions of an antibody are analyzed for peptides that bind to MHC Class II; these peptides represent potential T-cell epitopes (as defined in WO 98/52976 and WO 00/34317).
  • peptide threading For detection of potential T-cell epitopes, a computer modeling approach termed "peptide threading" can be applied, and in addition a database of human MHC class II binding peptides can be searched for motifs present in the VH and VL sequences, as described in WO 98/52976 and WO 00/34317. These motifs bind to any of the 18 major MHC class II DR allotypes, and thus constitute potential T cell epitopes.
  • Potential T-cell epitopes detected can be eliminated by substituting small numbers of amino acid residues in the variable regions, or preferably, by single amino acid substitutions. As far as possible conservative substitutions are made, often but not exclusively, an amino acid common at this position in human germline antibody sequences may be used.
  • nucleic acids encoding V H and V L can be constructed by mutagenesis or other synthetic methods (e.g., de novo synthesis, cassette replacement, and so forth).
  • Mutagenized variable sequence can, optionally, be fused to a human constant region, e.g., human IgGl or K constant regions.
  • a potential T cell epitope will include residues which are known or predicted to be important for antibody function. For example, potential T cell epitopes are usually biased towards the CDRs. In addition, potential T cell epitopes can occur in framework residues important for antibody structure and binding. Changes to eliminate these potential epitopes will in some cases require more scrutiny, e.g., by making and testing chains with and without the change. Where possible, potential T cell epitopes that overlap the CDRs were eliminated by substitutions outside the CDRs. In some cases, an alteration within a CDR is the only option, and thus variants with and without this substitution should be tested.
  • the substitution required to remove a potential T cell epitope is at a residue position within the framework that might be critical for antibody binding.
  • variants with and without this substitution should be tested.
  • several variant deimmunized heavy and light chain variable regions were designed and various heavy/light chain combinations tested in order to identify the optimal deimmunized antibody.
  • the choice of the final deimmunized antibody can then be made by considering the binding affinity of the different variants in conjunction with the extent of deimmunization, i.e., the number of potential T cell epitopes remaining in the variable region.
  • Deimmunization can be used to modify any antibody, e.g., an antibody that includes a non-human sequence, e.g., a synthetic antibody, a murine antibody other non-human monoclonal antibody, or an antibody isolated from a display library.
  • a non-human sequence e.g., a synthetic antibody, a murine antibody other non-human monoclonal antibody, or an antibody isolated from a display library.
  • MMP-9 or -12 binding antibodies are "germlined" by reverting one or more non-germline amino acids in framework regions to corresponding germline amino acids of the antibody, so long as binding properties are substantially retained. Similar methods can also be used in the constant region, e.g., in constant immunoglobulin domains.
  • Antibodies that bind to MMP-9, or -12 e.g., an antibody described herein may be modified in order to make the variable regions of the antibody more similar to one or more germline sequences.
  • an antibody can include one, two, three, or more amino acid substitutions, e.g., in a framework, CDR, or constant region, to make it more similar to a reference germline sequence.
  • One exemplary germlining method can include identifying one or more germline sequences that are similar (e.g., most similar in a particular database) to the sequence of the isolated antibody. Mutations (at the amino acid level) are then made in the isolated antibody, either incrementally or in combination with other mutations. For example, a nucleic acid library that includes sequences encoding some or all possible germline mutations is made. The mutated antibodies are then evaluated, e.g., to identify an antibody that has one or more additional germline residues relative to the isolated antibody and that is still useful (e.g., has a functional activity). In one embodiment, as many germline residues are introduced into an isolated antibody as possible.
  • mutagenesis is used to substitute or insert one or more germline residues into a framework and/or constant region.
  • a germline framework and/or constant region residue can be from a germline sequence that is similar (e.g., most similar) to the non-variable region being modified.
  • activity e.g., binding or other functional activity
  • Similar mutagenesis can be performed in the framework regions.
  • Selecting a germline sequence can be performed in different ways. For example, a germline sequence can be selected if it meets a predetermined criteria for selectivity or similarity, e.g., at least a certain percentage identity, e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity. The selection can be performed using at least 2, 3, 5, or 10 germline sequences. In the case of CDRl and CDR2, identifying a similar germline sequence can include selecting one such sequence.
  • a predetermined criteria for selectivity or similarity e.g., at least a certain percentage identity, e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity.
  • the selection can be performed using at least 2, 3, 5, or 10 germline sequences.
  • identifying a similar germline sequence can include selecting one
  • identifying a similar germline sequence can include selecting one such sequence, but may including using two germline sequences that separately contribute to the amino-terminal portion and the carboxy-terminal portion. In other implementations more than one or two germline sequences are used, e.g., to form a consensus sequence.
  • a related variable domain sequence has at least 30, 40, 50, 60, 70, 80, 90, 95 or 100% of the CDR amino acid positions that are not identical to residues in the reference CDR sequences, residues that are identical to residues at corresponding positions in a human germline sequence (i.e., an amino acid sequence encoded by a human germline nucleic acid).
  • a related variable domain sequence has at least 30, 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of the FR regions identical to FR sequence from a human germline sequence, e.g., a germline sequence related to the reference variable domain sequence.
  • an antibody which has similar activity to a given antibody of interest, but is more similar to one or more germline sequences, particularly one or more human germline sequences.
  • an antibody can be at least 90, 91, 92, 93, 94, 95,
  • an antibody can include at least 1, 2, 3, 4, or 5 germline residues in a CDR region, the germline residue being from a germline sequence of similar (e.g., most similar) to the variable region being modified.
  • Germline sequences of primary interest are human germline sequences.
  • the activity of the antibody e.g., the binding activity as measured by K A
  • Germline sequences of human immunoglobin genes have been determined and are available from a number of sources, including the international ImMunoGeneTics information system® (IMGT), available via the world wide web at imgt.cines.fr, and the V BASE directory
  • IMGT international ImMunoGeneTics information system
  • Exemplary germline reference sequences for Vkappa include: 012/02, 018/08, A20,
  • a germline reference sequence for the HC variable domain can be based on a sequence that has particular canonical structures, e.g., 1-3 structures in the Hl and H2 hypervariable loops.
  • hypervariable loops of an immunoglobulin variable domain can be inferred from its sequence, as described in Chothia et al., 1992, /. MoI. Biol. 227:799-817;
  • Exemplary sequences with a 1-3 structure include: DP-I, DP-8, DP-12, DP-2,
  • Standard recombinant nucleic acid methods can be used to express a protein that binds to MMP-9 or -12.
  • a nucleic acid sequence encoding the protein is cloned into a nucleic acid expression vector.
  • each chain can be cloned into an expression vector, e.g., the same or different vectors, that are expressed in the same or different cells.
  • Some antibodies can be produced in bacterial cells, e.g., E. coli cells.
  • the Fab is encoded by sequences in a phage display vector that includes a suppressible stop codon between the display entity and a bacteriophage protein (or fragment thereof)
  • the vector nucleic acid can be transferred into a bacterial cell that cannot suppress a stop codon.
  • the Fab is not fused to the gene III protein and is secreted into the periplasm and/or media.
  • Antibodies can also be produced in eukaryotic cells.
  • the antibodies e.g., scFv's
  • the antibodies are expressed in a yeast cell such as Pichia (see, e.g., Powers et al., 2001, /. Immunol. Methods. 251:123-35), Hanseula, or Saccharomyces.
  • antibodies are produced in mammalian cells.
  • Preferred mammalian host cells for expressing the clone antibodies or antigen-binding fragments thereof include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, 1980, Proc. Natl. Acad. ScL USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, 1982, MoI. Biol. 159:601 621), lymphocytic cell lines, e.g., NSO myeloma cells and SP2 cells, COS cells, HEK293T cells (J. Immunol. Methods (2004) 289(l-2):65-80.), and a cell from a transgenic animal, e.g., a transgenic mammal.
  • the cell is a mammary epithelial cell.
  • the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017).
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhff host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhff CHO cells by calcium phosphate-mediated transfection.
  • the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/ AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element) to drive high levels of transcription of the genes.
  • enhancer/promoter regulatory elements e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/ AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element
  • the recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification.
  • the selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium.
  • Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.
  • the antibody production system may produce antibodies in which the Fc region is glycosylated.
  • the Fc domain of IgG molecules is glycosylated at asparagine 297 in the CH2 domain.
  • This asparagine is the site for modification with biantennary-type oligosaccharides. It has been demonstrated that this glycosylation is required for effector functions mediated by Fcg receptors and complement CIq (Burton and Woof, 1992, Adv. Immunol. 51:1-84; Jefferis et al., 1998, Immunol. Rev. 163:59-76).
  • the Fc domain is produced in a mammalian expression system that appropriately glycosylates the residue corresponding to asparagine 297.
  • the Fc domain can also include other eukaryotic post-translational modifications.
  • Antibodies can also be produced by a transgenic animal.
  • U.S. Patent No. 5,849,992 describes a method of expressing an antibody in the mammary gland of a transgenic mammal.
  • a transgene is constructed that includes a milk-specific promoter and nucleic acids encoding the antibody of interest and a signal sequence for secretion.
  • the milk produced by females of such transgenic mammals includes, secreted-therein, the antibody of interest.
  • the antibody can be purified from the milk, or for some applications, used directly.
  • MMP-9 Binding of MMP-9 binding proteins to cells expressing MMP-9 can be characterized in a number assays known in the art, including FACS (Fluorescence Activated Cell Sorting), immunofluorescence, and immunocytochemistry.
  • MMP-9 binding protein is contacted with cells and/or tissues which express or contain MMP-9 and binding is detected in accordance with the method being used.
  • a fluorescent detection system e.g., fluorescent- labeled secondary antibody
  • immunofluorescence analysis e.g., a fluorescent- labeled secondary antibody
  • a enzymatic system is used for immunocytochemistry are generally used in these assayscan be performed on non-perm.
  • MMP-9 binding proteins can be characterized as to cellular binding by FACS (Fluorescence Activated Cell Sorting) using cells expressing MMP-9.
  • FACS Fluorescence Activated Cell Sorting
  • Individual cells held in a thin stream of fluid are passed through one or more laser beams cause light to scatter and fluorescent dyes to emit light at various frequencies.
  • Photomultiplier tubes (PMT) convert light to electrical signals and cell data is collected.
  • Forward and side scatter are used for preliminary identification of cells. Forward and side scatter are used to exclude debris and dead cells.
  • Fluorescent labeling allows investigation of cell structure and function.
  • Cell autofluorescence is generated by labeling cell structures with fluorescent dyes.
  • FACS collects fluorescence signals in one to several channels corresponding to different laser excitation and fluorescence emission wavelength.
  • Immunofluorescence involves the staining of cells with antibodies conjugated to fluorescent dyes such as fluorescein and phycoerythrin (PE).
  • fluorescent dyes such as fluorescein and phycoerythrin (PE).
  • PE phycoerythrin
  • This method can be used to label MMP-9 on the cell surface of MDA- MB-231 cells using biotinylated MMP-9 binding proteins.
  • Biotin is used in this two-step detection systems in concert with conjugated steptavidin. Biotin is typically conjugated to proteins via primary amines (i.e., lysines). Usually, between 1.5 and 3 biotin molecules are conjugated to each antibody.
  • a second fluorescently conjugated antibody streptavidin/PE is added which is specific for biotin.
  • MMP-9 binding proteins can be characterized in cultured cells expressing the MMP-9 antigen.
  • the method generally used is immunocytochemistry. Immunocytochemistry involves the use of antibodies that recognize parts of the receptor that are exposed to the outside environment when expressed at the cell surface (the 'primary antibody'). If the experiment is carried out in intact cells, such an antibody will only bind to surface expressed receptors. Biotinylated or non-biotinylated MMP-9 binding proteins can be used.
  • the secondary antibody is then either a streptavidin/HRP antibody (for biotinylated MMP-9 binding protein) or an anti- human IgG/HRP (for non-biotinylated MMP-9 binding protein).
  • the staining can then be detected using an inverted microscope.
  • the assay can be performed in the absence of MMP-9 binding protein and in presence of lO ⁇ g/mL of MMP-9 binding protein.
  • MMP-9 binding proteins can be characterized in assays that measure their modulatory activity toward MMP-9 or fragments thereof in vitro or in vivo.
  • MMP-9 can be combined with a substrate such as Mca-Pro-Leu-Ala-Cys(Mob)-Trp-Ala-Arg-Dap(Dnp)-NH? under assay conditions permitting cleavage by MMP-9.
  • the assay is performed in the absence of the MMP-9 binding protein, and in the presence of increasing concentrations of the MMP-9 binding protein.
  • the concentration of binding protein at which 50% of the MMP-9 activity (e.g., binding to the substrate) is inhibited is the IC 50 value (Inhibitory Concentration 50%) or EC 50 (Effective Concentration 50%) value for that binding protein.
  • IC 50 value Inhibitory Concentration 50%
  • EC 50 Effective Concentration 50%
  • Exemplary binding proteins have an IC 50 value of less than 800 nM, 400 nM, 100 nM, 25 nM, 5 nM, or 1 nM, e.g., as measured in an in vitro assay for inhibition of MMP-9 activity when the MMP-9 is at 2 pM.
  • MMP-9 binding proteins may also be characterized with reference to the activity of
  • MMP-9 on substrates e.g., collagen, gelatin.
  • substrates e.g., collagen, gelatin.
  • cleavage of gelatin by MMP-9 can be detected in zymography.
  • the method is based on a SDS gel impregnated with a substrate, which is degraded by the proteases resolved during the incubation period. Coomassie blue staining of the gels reveals proteolytic fragments as white bands on a dark blue background. Within a certain range, the band intensity can be related linearly to the amount of the protease loaded.
  • Cells expressing MMP-9 are used in this assay. The assay is performed in the absence of the MMP-9 binding protein, and in the presence of increasing concentrations of the MMP-9 binding protein.
  • the concentration of binding protein at which 50% of the MMP-9 activity (e.g., binding to the substrate) is inhibited is the IC 50 value (Inhibitory Concentration 50%) or EC 50 (Effective Concentration 50%) value for that binding protein.
  • IC 50 value Inhibitory Concentration 50%
  • EC 50 Effective Concentration 50%
  • those having lower IC 50 or EC 50 values are considered more potent inhibitors of MMP-9 than those binding proteins having higher IC50 or EC50 values.
  • Exemplary binding proteins have an IC 50 value of less than 800 nM, 400 nM, 100 nM, 25 nM, 5 nM, or 1 nM, e.g., as measured in an in vitro assay for inhibition of MMP-9 activity.
  • the binding proteins can also be evaluated for selectivity toward MMP-9.
  • a MMP-9 binding protein can be assayed for its potency toward MMP-9 and a panel of MMPs and other enzymes, e.g., human and/or mouse enzymes, e.g., MMP-I, -2, -3, -7, -8, -12, -13, -14, -16, -17, -24, and TACE, and an IC 50 value or EC 50 value can be determined for each MMP.
  • a compound that demonstrates a low IC50 value or EC50 value for the MMP-9, and a higher IC 50 value or EC 50 value, e.g., at least 2-, 5-, or 10- fold higher, for another MMP within the test panel (e.g., MMP-I, -10) is considered to be selective toward MMP-9.
  • MMP-9 binding proteins can be evaluated for their ability to inhibit MMP-9 in a cell based assay, e.g., in situ zymography, e.g., in Colo205 cells or MCF-7 cells.
  • a pharmacokinetics study in rat, mice, or monkey can be performed with MMP-9 binding proteins for determining MMP-9 half-life in the serum.
  • the effect of the binding protein can be assessed in vivo, e.g., in an animal model for a disease, for use as a therapeutic, for example, to treat a disease or condition described herein, e.g., systemic sclerosis, a cancer (e.g., metastatic cancer, e.g., metastatic breast cancer), an inflammatory disease (e.g., chronic obstructive pulmonary disease (COPD), asthma, rhinitis (e.g., allergic rhinitis), inflammatory bowel disease, synovitis, rheumatoid arthritis), heart failure, septic shock, neuropathic pain, inflammatory pain, osteoarthritis, or an ocular condition (e.g., macular degeneration).
  • COPD chronic obstructive pulmonary disease
  • COPD chronic obstructive pulmonary
  • MMP-12 Binding of MMP- 12 binding proteins to cells expressing MMP- 12 can be characterized in a number assays known in the art, including FACS (Fluorescence Activated Cell Sorting), immunofluorescence, and immunocytochemistry.
  • MMP-12 binding protein is contacted with cells and/or tissues which express or contain MMP-12, and binding is detected in accordance with the method being used.
  • a fluorescent detection system e.g., fluorescent-labeled secondary antibody
  • FACS Fluorescence Activated Cell Sorting
  • This method can be used to label MMP- 12 on the cell surface of MDA- MB-231 cells using biotinylated MMP- 12 binding proteins.
  • Biotin is used in this two-step detection systems in concert with conjugated steptavidin.
  • Biotin is typically conjugated to proteins via primary amines (i.e., lysines).
  • primary amines i.e., lysines
  • streptavidin/PE is added which is specific for biotin.
  • MMP- 12 binding proteins can be characterized in cultured cells expressing the MMP- 12 antigen. The method generally used is immunocytochemistry.
  • Immunocytochemistry involves the use of antibodies that recognize parts of the receptor that are exposed to the outside environment when expressed at the cell surface (the 'primary antibody'). If the experiment is carried out in intact cells, such an antibody will only bind to surface expressed receptors.
  • Biotinylated or non-biotinylated MMP- 12 binding proteins can be used.
  • the secondary antibody is then either a streptavidin/HRP antibody (for biotinylated MMP- 12 binding protein) or an anti- human IgG/HRP (for non-biotinylated MMP- 12 binding protein).
  • the staining can then be detected using an inverted microscope.
  • the assay can be performed in the absence of MMP-12 binding protein and in presence of lO ⁇ g/mL of MMP-12 binding protein.
  • MMP-12 binding proteins can be characterized in assays that measure their modulatory activity toward MMP-12 or fragments thereof in vitro or in vivo.
  • MMP-12 can be combined with a substrate such as Mca-Pro-Leu-Ala-Cys(Mob)-Trp-Ala-Arg-Dap(Dnp)-NH? under assay conditions permitting cleavage by MMP-12.
  • the assay is performed in the absence of the MMP-12 binding protein, and in the presence of increasing concentrations of the MMP-12 binding protein.
  • the concentration of binding protein at which 50% of the MMP-12 activity (e.g., binding to the substrate) is inhibited is the IC 50 value (Inhibitory Concentration 50%) or EC 50 (Effective Concentration 50%) value for that binding protein.
  • IC 50 value Inhibitory Concentration 50%
  • EC 50 Effective Concentration 50%
  • those having lower IC 50 or EC 50 values are considered more potent inhibitors of MMP- 12 than those binding proteins having higher IC 50 or EC 50 values.
  • Exemplary binding proteins have an IC 50 value of less than 800 nM, 400 nM, 100 nM, 25 nM, 5 nM, or 1 nM, e.g., as measured in an in vitro assay for inhibition of MMP- 12 activity when the MMP- 12 is at 2 pM.
  • MMP- 12 binding proteins may also be characterized with reference to the activity of MMP-12 on substrates (e.g., lung extracellular matrix, elastin, gelatin, etc.). For example, cleavage of gelatin by MMP-12 can be detected in zymography. The method is based on a SDS gel impregnated with a substrate, which is degraded by the proteases resolved during the incubation period. Coomassie blue staining of the gels reveals proteolytic fragments as white bands on a dark blue background. Within a certain range, the band intensity can be related linearly to the amount of the protease loaded. Cells expressing MMP-12 are used in this assay.
  • substrates e.g., lung extracellular matrix, elastin, gelatin, etc.
  • cleavage of gelatin by MMP-12 can be detected in zymography. The method is based on a SDS gel impregnated with a substrate, which is degraded by the proteases resolved during the
  • the assay is performed in the absence of the MMP-12 binding protein, and in the presence of increasing concentrations of the MMP-12 binding protein.
  • concentration of binding protein at which 50% of the MMP-12 activity (e.g., binding to the substrate) is inhibited is the IC 50 value (Inhibitory Concentration 50%) or EC 50 (Effective Concentration 50%) value for that binding protein.
  • IC 50 value Inhibitory Concentration 50%
  • EC 50 Effective Concentration 50%
  • Exemplary binding proteins have an IC50 value of less than 800 nM, 400 nM, 100 nM, 25 nM, 5 nM, or 1 nM, e.g., as measured in an in vitro assay for inhibition of MMP-12 activity.
  • the binding proteins can also be evaluated for selectivity toward MMP-12.
  • a MMP-12 binding protein can be assayed for its potency toward MMP-12 and a panel of MMPs and other enzymes, e.g., human and/or mouse enzymes, e.g., MMP-I, -2, -3, -7, -8, -9, -13, -14, - 16, -17, -24, and TACE, and an IC 50 value or EC 50 value can be determined for each MMP.
  • a compound that demonstrates a low IC 50 value or EC 50 value for the MMP-12, and a higher IC 50 value or EC 50 value, e.g., at least 2-, 5-, or 10- fold higher, for another MMP within the test panel (e.g., MMP-I, -10) is considered to be selective toward MMP-12.
  • MMP-12 binding proteins can be evaluated for their ability to inhibit MMP-12 in a cell based assay, e.g., in situ zymography, e.g., in Colo205 cells or MCF-7 cells.
  • a pharmacokinetics study in rat, mice, or monkey can be performed with MMP- 12 binding proteins for determining MMP- 12 half-life in the serum.
  • the effect of the binding protein can be assessed in vivo, e.g., in an animal model for a disease, for use as a therapeutic, for example, to treat a disease or condition described herein, e.g., systemic sclerosis, a cancer (e.g., metastatic cancer, e.g., metastatic colorectal, lung, or hepatocellular cancer), an inflammatory disease (e.g., chronic obstructive pulmonary disease (COPD), asthma, rhinitis (e.g., allergic rhinitis), atherosclerosis, multiple sclerosis, rheumatoid arthritis), cardiovascular disease, aneurysym, wound healing, aging and nerve damage associated with excess or inappropriate activity of MMP- 12.
  • a disease or condition described herein e.g., systemic sclerosis, a cancer (e.g., metastatic cancer, e.g., metastatic colorectal, lung, or hepatocellular cancer), an inflammatory disease (e
  • compositions e.g., pharmaceutically acceptable compositions or pharmaceutical compositions, which include an MMP-9 or -12-binding protein, e.g., an antibody molecule, other polypeptide or peptide identified as binding to MMP-9 or -12 described herein.
  • MMP-9 or -12 binding protein can be formulated together with a pharmaceutically acceptable carrier.
  • Pharmaceutical compositions include therapeutic compositions and diagnostic compositions, e.g., compositions that include labeled MMP-9 or -12 binding proteins for in vivo imaging.
  • a pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion), although carriers suitable for inhalation and intranasal administration are also contemplated.
  • the MMP-9 or -12 binding protein may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
  • a pharmaceutically acceptable salt is a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al., 1977, /. Pharm. Sci. 66:1-19).
  • Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like
  • nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like.
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.
  • compositions may be in a variety of forms. These include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • liquid solutions e.g., injectable and infusible solutions
  • dispersions or suspensions tablets, pills, powders, liposomes and suppositories.
  • the form can depend on the intended mode of administration and therapeutic application.
  • Many compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for administration of humans with antibodies.
  • An exemplary mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • the MMP- 9 binding protein is administered by intravenous infusion or injection.
  • the MMP-9 binding protein is administered by intramuscular or subcutaneous injection.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration.
  • Sterile injectable solutions can be prepared by incorporating the binding protein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • an MMP-9 or -12 binding protein can be administered by a variety of methods, although for many applications, the preferred route/mode of administration is intravenous injection or infusion.
  • the MMP-9 or -12 binding protein can be administered by intravenous infusion at a rate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m 2 or 7 to 25 mg/m 2 .
  • the route and/or mode of administration will vary depending upon the desired results.
  • the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are available. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., 1978, Marcel Dekker, Inc., New York.
  • compositions can be administered with medical devices.
  • a pharmaceutical composition disclosed herein can be administered with a device, e.g., a needleless hypodermic injection device, a pump, or implant.
  • an MMP-9 or -12 binding protein can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier excludes many highly hydrophilic compounds.
  • the therapeutic compounds disclosed herein cross the BBB (if desired) they can be formulated, for example, in liposomes.
  • liposomes For methods of manufacturing liposomes, see, e.g., U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399,331.
  • the liposomes may comprise one or more moieties that are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade, 1989, /. Clin. Pharmacol. 29:685).
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • an exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody disclosed herein is 0.1-20 mg/kg, more preferably 1-10 mg/kg.
  • An anti- MMP-9 or -12 antibody can be administered, e.g., by intravenous infusion, e.g., at a rate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m or about 5 to 30 mg/m .
  • Dosage values may vary with the type and severity of the condition to be alleviated. For a particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
  • compositions disclosed herein may include a "therapeutically effective amount” or a “prophylactically effective amount” of an MMP-9 or -12 binding protein disclosed herein.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the protein to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the composition is outweighed by the therapeutically beneficial effects.
  • a "therapeutically effective dosage” preferably modulates a measurable parameter, e.g., levels of circulating IgG antibodies or enzymatic activity, by a statistically significant degree or at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects.
  • a measurable parameter e.g., levels of circulating IgG antibodies or enzymatic activity
  • the ability of a compound to modulate a measurable parameter e.g., a disease-associated parameter
  • this property of a composition can be evaluated by examining the ability of the compound to modulate a parameter in vitro.
  • prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, because a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • an MMP-9 or -12 binding protein is physically associated with a moiety that improves its stabilization and/or retention in circulation, e.g., in blood, serum, lymph, or other tissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold.
  • an MMP-9 or -12 binding protein can be associated with a polymer, e.g., a substantially non-antigenic polymer, such as polyalkylene oxides or polyethylene oxides. Suitable polymers will vary substantially by weight. Polymers having molecular number average weights ranging from about 200 to about 35,000 (or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used.
  • an MMP-9 or -12 binding protein can be conjugated to a water soluble polymer, e.g., hydrophilic polyvinyl polymers, e.g. polyvinylalcohol and polyvinylpyrrolidone.
  • a water soluble polymer e.g., hydrophilic polyvinyl polymers, e.g. polyvinylalcohol and polyvinylpyrrolidone.
  • a non-limiting list of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained.
  • An MMP-9 or -12 binding protein can also be associated with a carrier protein, e.g., a serum albumin, such as a human serum albumin.
  • a carrier protein e.g., a serum albumin, such as a human serum albumin.
  • a translational fusion can be used to associate the carrier protein with the MMP-9 or -12 binding protein.
  • kits An MMP-9 or -12 binding protein described herein can be provided in a kit, e.g., as a component of a kit.
  • the kit includes (a) an MMP-9 or -12 binding protein, e.g., a composition (e.g., a pharmaceutical composition) that includes an MMP-9 or -12 binding protein, and, optionally (b) informational material.
  • the informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of an MMP-9 or -12 binding protein for the methods described herein.
  • the informational material of the kits is not limited in its form.
  • the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to using the binding protein to treat, prevent, or diagnosis of disorders and conditions, e.g., systemic sclerosis.
  • the informational material can include instructions to administer an MMP-9 or -12 binding protein in a suitable manner to perform the methods described herein, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein).
  • the informational material can include instructions to administer an MMP-9 or -12 binding protein to a suitable subject, e.g., a human, e.g., a human having, or at risk for, a disorder or condition described herein, e.g., systemic sclerosis.
  • the material can include instructions to administer an MMP-9 or -12 binding protein to a patient with a disorder or condition described herein, e.g., systemic sclerosis.
  • the informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in print but may also be in other formats, such as computer readable material.
  • An MMP-9 or -12 binding protein can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that an MMP-9 or -12 binding protein be substantially pure and/or sterile.
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred.
  • reconstitution generally is by the addition of a suitable solvent.
  • the solvent e.g., sterile water or buffer, can optionally be provided in the kit.
  • the kit can include one or more containers for the composition containing an MMP-9 or - 12 binding protein.
  • the kit contains separate containers, dividers or compartments for the composition and informational material.
  • the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained association with the container.
  • the separate elements of the kit are contained within a single, undivided container.
  • the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label.
  • the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of an MMP-9 or -12 binding protein.
  • the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of an MMP-9 or -12 binding protein.
  • the containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.
  • the kit optionally includes a device suitable for administration of the composition, e.g., a syringe, inhalant, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device.
  • a device suitable for administration of the composition e.g., a syringe, inhalant, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device.
  • the device is an implantable device that dispenses metered doses of the binding protein.
  • the disclosure also features a method of providing a kit, e.g., by combining components described herein.
  • Proteins that bind to MMP-9 or -12 and identified by the method described herein and/or detailed herein have therapeutic and prophylactic utilities, particularly in human subjects. These binding proteins are administered to a subject to treat, prevent, and/or diagnose systemic sclerosis. Treating includes administering an amount effective to alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder. The treatment may also delay onset, e.g., prevent onset, or prevent deterioration of a disease or condition.
  • an amount of an target-binding agent effective to prevent a disorder, or a prophylactically effective amount of the binding agent refers to an amount of a target binding agent, e.g., an MMP-9 or -12 binding protein, e.g., an anti-MMP-9 or -12 antibody described herein, which is effective, upon single- or multiple-dose administration to the subject, for preventing or delaying the occurrence of the onset or recurrence of a disorder, e.g., a disorder described herein, e.g., systemic sclerosis.
  • a binding agent described herein can be used to reduce angiogenesis in a subject, e.g., to treat systemic sclerosis.
  • the method includes administering the binding protein (e.g., an MMP-9 or -12 binding protein, e.g., an anti-MMP-9 or -12 binding protein described herein) to the subject, e.g., in an amount effective to modulate systemic sclerosis, a symptom of the disorder, or progression of the disorder.
  • the agent e.g., an MMP-9 or -12 binding protein, e.g., an anti- MMP-9 or -12 antibody
  • MMP-9 or -12 binding proteins and other agents are also described in "Pharmaceutical Compositions.” Suitable dosages of the molecules used can depend on the age and weight of the subject and the particular drug used.
  • the binding proteins can be used as competitive agents to inhibit, reduce an undesirable interaction, e.g., between a natural or pathological agent and the MMP-9 or -12.
  • the dose of the MMP-9 or -12 binding protein can be the amount sufficient to block 90%, 95%, 99%, or 99.9% of the activity of MMP- 9 or -12 in the patient, especially at the site of disease. Depending on the disease, this may require 0.1, 1.0, 3.0, 6.0, or 10.0 mg/Kg.
  • these doses correspond to approximately 18 nM, 180 nM, 540 nM, 1.08 ⁇ M, and 1.8 ⁇ M of binding sites for a 5 L blood volume.
  • the MMP-9 or -12 binding proteins are used to inhibit an activity of a cell, e.g., a fibroblast or endothelial cell in vivo.
  • the binding proteins can be used by themselves or conjugated to an agent, e.g., a cytotoxic drug, cytotoxin enzyme, or radioisotope.
  • This method includes: administering the binding protein alone or attached to an agent (e.g., a cytotoxic drug), to a subject requiring such treatment.
  • MMP-9 or -12 binding proteins that do not substantially inhibit MMP-9 or -12 may be used to deliver nanoparticles containing agents, such as toxins, to MMP-9 or -12 associated cells or tissues, e.g., fibroblasts or endothelial cells.
  • agents such as toxins
  • the binding proteins may be used to deliver an agent (e.g., any of a variety of cytotoxic and therapeutic drugs) to cells and tissues where MMP-9 or -12 is present.
  • agents include a compound emitting radiation, molecules of plants, fungal, or bacterial origin, biological proteins, and mixtures thereof.
  • the cytotoxic drugs can be intracellularly acting cytotoxic drugs, such as toxins short range radiation emitters, e.g., short range, high energy ⁇ -emitters.
  • a prodrug system can be used.
  • a first binding protein is conjugated with a prodrug which is activated only when in close proximity with a prodrug activator.
  • the prodrug activator is conjugated with a second binding protein, preferably one which binds to a non competing site on the target molecule. Whether two binding proteins bind to competing or non competing binding sites can be determined by conventional competitive binding assays. Exemplary drug prodrug pairs are described in Blakely et al., (1996) Cancer Research, 56:3287 3292.
  • the MMP-9 or -12 binding proteins can be used directly in vivo to eliminate antigen- expressing cells via natural complement-dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC).
  • the binding proteins described herein can include complement binding effector domain, such as the Fc portions from IgGl, -2, or -3 or corresponding portions of IgM which bind complement.
  • a population of target cells is ex vivo treated with a binding agent described herein and appropriate effector cells. The treatment can be supplemented by the addition of complement or serum containing complement.
  • phagocytosis of target cells coated with a binding protein described herein can be improved by binding of complement proteins.
  • cells coated with the binding protein which includes a complement binding effector domain are lysed by complement.
  • MMP-9 or -12 binding proteins Methods of administering MMP-9 or -12 binding proteins are described in "Pharmaceutical Compositions.” Suitable dosages of the molecules used will depend on the age and weight of the subject and the particular drug used.
  • the binding proteins can be used as competitive agents to inhibit or reduce an undesirable interaction, e.g., between a natural or pathological agent and the MMP-9 or -12.
  • the MMP-9 or -12 binding protein can be used to deliver macro and micromolecules, e.g., a gene into the cell for gene therapy purposes into the endothelium or epithelium and target only those tissues expressing the MMP-9 or -12.
  • the binding proteins may be used to deliver a variety of cytotoxic drugs including therapeutic drugs, a compound emitting radiation, molecules of plants, fungal, or bacterial origin, biological proteins, and mixtures thereof.
  • the cytotoxic drugs can be intracellularly acting cytotoxic drugs, such as short range radiation emitters, including, for example, short range, high energy ⁇ emitters, as described herein.
  • recombinant nucleic acid techniques can be used to construct a nucleic acid that encodes the binding protein (e.g., antibody or antigen-binding fragment thereof) and the cytotoxin (or a polypeptide component thereof) as translational fusions.
  • the recombinant nucleic acid is then expressed, e.g., in cells and the encoded fusion polypeptide isolated.
  • the MMP-9 or -12 binding protein can be coupled to high energy radiation emitters, for example, a radioisotope, such as I, a ⁇ -emitter, which, when localized at a site, results in a killing of several cell diameters. See, e.g., S.E.
  • Radiolabeled Antibody in Cancer Therapy Monoclonal Antibodies for Cancer Detection and Therapy, R.W. Baldwin et al. (eds.), pp 303 316 (Academic Press 1985).
  • Other suitable radioisotopes include a emitters, such as 212 Bi, 213 Bi, and 211 At, and b emitters, such as 186 Re and 90 Y.
  • 177 Lu may also be used as both an imaging and cytotoxic agent.
  • Radioimmunotherapy (RIT) using antibodies labeled with 131 1 , 90 Y, and 177 Lu is under intense clinical investigation. There are significant differences in the physical characteristics of these three nuclides and as a result, the choice of radionuclide is very critical in order to deliver maximum radiation dose to a tissue of interest.
  • the higher beta energy particles of 90 Y may be good for bulky tumors.
  • the relatively low energy beta particles of I are ideal, but in vivo dehalogenation of radioiodinated molecules is a major disadvantage for internalizing antibody.
  • Lu has low energy beta particle with only 0.2-0.3 mm range and delivers much lower radiation dose to bone marrow compared to Y. In addition, due to longer physical half- life (compared to Y), the residence times are higher.
  • Lu labeled agents As a result, higher activities (more mCi amounts) of Lu labeled agents can be administered with comparatively less radiation dose to marrow.
  • Meredith RF Meredith RF, et al., 1996, /. Nucl. Med. 37: 1491-1496
  • Alvarez RD et al., 1997, Gynecol. Oncol. 65: 94-101.
  • the MMP-9 or -12 binding proteins described herein are useful to treat diseases or conditions in which MMP-9 or -12 is implicated (respectively), e.g., a disease or condition described herein, or to treat one or more symptoms associated therewith.
  • the MMP-9 or -12 binding protein e.g., MMP-9 or -12 binding IgG or Fab
  • diseases and conditions include systemic sclerosis.
  • a therapeutically effective amount of a MMP-9 or -12 binding protein is administered to a subject having or suspected of having a disorder in which MMP-9 or -12 (respectively) is implicated, thereby treating (e.g., ameliorating or improving a symptom or feature of a disorder, slowing, stabilizing or halting disease progression) the disorder.
  • the MMP-9 or -12 binding protein is administered in a therapeutically effective amount.
  • a therapeutically effective amount of an MMP-9 binding protein or an MMP- 12 binding protein is the amount which is effective, upon single or multiple dose administration to a subject, in treating a subject, e.g., curing, alleviating, relieving or improving at least one symptom of a disorder in a subject to a degree beyond that expected in the absence of such treatment.
  • a therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the composition is outweighed by the therapeutically beneficial effects.
  • a therapeutically effective amount can be administered, typically an amount of the compound which is effective, upon single or multiple dose administration to a subject, in treating a subject, e.g., curing, alleviating, relieving or improving at least one symptom of a disorder in a subject to a degree beyond that expected in the absence of such treatment.
  • a therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the composition is outweighed by the therapeutically beneficial effects.
  • a therapeutically effective dosage preferably modulates a measurable parameter, favorably, relative to untreated subjects. The ability of a compound to inhibit a measurable parameter can be evaluated in an animal model system predictive of efficacy in a human disorder.
  • Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • SSc Systemic Sclerosis
  • scleroderma is the generalized type of scleroderma, which is a chronic disease characterized by excessive deposits of collagen in the skin or other organs such as joints, esophagus, lower gastrointestinal (GI) tract, lung, heart and kidney.
  • GI gastrointestinal
  • SSc can be fatal as a result of heart, kidney, lung, or intestinal damage.
  • scleroderma diffuse, limited and morphea/linear. Diffuse and limited sclerodermas are both systemic diseases. There is also a subset of the systemic form known as "systemic scleroderma sine scleroderma", in which the usual skin involvement is not present.
  • Calcinosis is the formation of calcium deposits in the connective tissues, which can be detected by x ray. They are typically found on the fingers, hands, face, and trunk and on the skin above elbows and knees. When the deposits break through the skin, painful ulcers can result.
  • Raynaud's phenomenon is a condition in which the small blood vessels of the hands and/or feet contract in response to cold or anxiety. As the vessels contract, the hands or feet turn white and cold, then blue. As blood flow returns, they become red. Fingertip tissues may suffer damage, leading to ulcers, scars, or gangrene.
  • Esophageal dysfunction impaired function of the esophagus that occurs when smooth muscles in the esophagus lose normal movement. In the upper esophagus, the result can be swallowing difficulties; in the lower esophagus, the problem can cause chronic heartburn or inflammation.
  • An MMP-9 and/or -12 binding protein e.g., an anti-MMP-9 or -12 binding protein described herein can be used to treat or prevent one or more of these symptoms.
  • Limited scleroderma typically comes on gradually and affects the skin only in certain areas: the fingers, hands, face, lower arms, and legs. Many people with limited disease have Raynaud's phenomenon for years before skin thickening starts. Others start out with skin problems over much of the body, which improves over time, leaving only the face and hands with tight, thickened skin. Telangiectasias and calcinosis often follow. Because of the predominance of CREST in people with limited disease, some doctors refer to limited disease as the CREST syndrome. Diffuse scleroderma typically comes on suddenly.
  • Skin thickening occurs quickly and over much of the body, affecting the hands, face, upper arms, upper legs, chest, and stomach in a symmetrical fashion (for example, if one arm or one side of the trunk is affected, the other is also affected). Some people may have more area of their skin affected than others. Internally, it can damage key organs such as the heart, lungs, and kidneys. Scleroderma affects the skin, and in more serious cases it can affect the blood vessels and internal organs.
  • Common symptoms include, e.g., Raynaud's syndrome, swelling (eventually skin tightening and contractures of the fingers), polyarthralgia, dysphagia, dyspnea, lung fibrosis, acute alveolitis, pulmonary hypertension, heartburn, cardiac arrhythmias and renal crisis.
  • the more evident symptom is usually the hardening of the skin and associated scarring. Blood vessels may also be more visible.
  • Many SSc patients (over 80%) have vascular symptoms and Raynaud's phenomenon. During an attack, there is discoloration of the hands and feet in response to cold. Raynaud's normally affects the fingers and toes.
  • SSc and Raynaud's can cause painful ulcers on the fingers or toes which are known as digital ulcers.
  • Calcinosis is also common in SSc, and is often seen near the elbows, knees or other joints.
  • Diffuse scleroderma can cause musculoskeletal, pulmonary, gastrointestinal, renal and other complications. Patients with larger amounts of cutaneous involvement are more likely to have involvement of the internal tissues and organs.
  • CMV cytomegalovirus
  • Treatments for some of the symptoms of scleroderma include drugs that soften the skin and reduce inflammation.
  • Topical treatment for the skin changes of scleroderma do not alter the disease course, but may improve pain and ulceration.
  • the skin tightness may be treated systemically with methotrexate and cyclosporin.
  • a range of NSAIDs nonsteroidal anti- inflammatory drugs
  • naproxen can be used to ease painful symptoms.
  • Episodes of Raynaud's phenomenon can respond to calcium channel blockers, e.g., nifedipine; angiotensin receptor blockers, e.g., losartan; or dual endothelin-receptor antagonists, e.g., bosentan.
  • Severe digital ulceration may respond to epoprostenol (prostacyclin or its analogue iloprost), prostaglandin El (alprostadil) or sympathetic blockers.
  • Reflux esophagitis can be treated with proton pump inhibitors.
  • Broad-spectrum antibiotics e.g., tetracycline can suppress overgrowth of intestinal flora and alleviate malabsorption symptoms.
  • Scleroderma renal crisis the occurrence of acute renal failure and malignant hypertension (very high blood pressure with evidence of organ damage) in people with scleroderma, is effectively treated with drugs from the class of the ACE (Angiotensin Converting Enzyme) inhibitors.
  • ACE Angiotensin Converting Enzyme
  • Active alveolitis is often treated with pulses of immunosuppressants, e.g., cyclophosphamide, methotrexate and azathioprine often together with a small dose of steroids.
  • Surgery e.g., lung transplantation can also be performed.
  • Pulmonary hypertension may be treated, e.g., with epoprostenol (prostacyclin), bosentan and possibly aerolized iloprost.
  • the disclosure provides methods of treating or preventing SSc (e.g., ameliorating symptoms or the worsening of SSc) by administering a therapeutically effective amount of a MMP-9 and/or MMP-12 binding protein (e.g., an MMP-9 or MMP-12 binding proteindescribed herein; an inhibitory MMP-9 or MMP-12 binding protein, e.g., an anti-MMP-9 or anti-MMP-12 IgG or Fab) to a subject having or suspected of having SSc.
  • a MMP-9 and/or MMP-12 binding protein e.g., an MMP-9 or MMP-12 binding proteindescribed herein; an inhibitory MMP-9 or MMP-12 binding protein, e.g., an anti-MMP-9 or anti-MMP-12 IgG or Fab
  • MMP-9 and/or MMP-12 binding protein e.g., an MMP-9 or MMP-12 binding proteindescribed herein
  • another SSc treatment e.g., cyclosporin, NSAIDs (e.g., naproxen), calcium channel blockers (e.g., nifedipine), angiotensin receptor blockers (e.g., losartan), epoprostenol (prostacyclin), prostacyclin analogues (e.g., iloprost), dual endothelin-receptor antagonists (e.g., bosentan), prostaglandin El, proton pump inhibitors, antibiotics (e.g., tetracycline), immunosuppressants (e.g., cyclophosphamide, methotrexate or azathioprine), ACE inhibitors, and surgery (e.g., lung transplantation).
  • a MMP-9 and/or MMP-12 binding protein e.g., an M
  • Sjogren's syndrome can be associated with SSc.
  • Sjogren's syndrome is an autoimmune disorder in which immune cells attack and destroy the exocrine glands that produce tears and saliva.
  • Sjogren's syndrome can exist as a disorder in its own right
  • Sjogren's syndrome may cause skin, nose, and vaginal dryness, and may affect other organs of the body, including the kidneys, blood vessels, lungs, liver, pancreas, and brain.
  • An MMP-9 and/or -12 binding protein e.g., an anti-MMP-9 or -12 binding protein described herein
  • a second agent e.g., a second agent dercribed herein or a second treatment for Sjorgen's syndrome
  • the MMP-9 binding proteins described herein can be administered in combination with one or more of the other therapies for treating a disease or condition associated with MMP-9 activity, e.g., a disease or condition described herein, e.g., systemic sclerosis.
  • a disease or condition associated with MMP-9 activity e.g., a disease or condition described herein, e.g., systemic sclerosis.
  • an MMP-9 binding protein can be used therapeutically or prophylactically with surgery, another MMP-9 inhibitor, e.g., a small molecule inhibitor, another anti-MMP-9 Fab or IgG (e.g., another Fab or IgG described herein), peptide inhibitor, or small molecule inhibitor.
  • MMP-9 inhibitors that can be used in combination therapy with an MMP-9 binding protein are described herein.
  • One or more small-molecule MMP inhibitors can be used in combination with one or more MMP-9 binding proteins described herein.
  • the combination can result in a lower dose of the small-molecule inhibitor being needed, such that side effects are reduced.
  • the MMP-12 binding proteins described herein can be administered in combination with one or more of the other therapies for treating a disease or condition associated with MMP-12 activity, e.g., a disease or condition described herein, e.g., systemic sclerosis.
  • a disease or condition associated with MMP-12 activity e.g., a disease or condition described herein, e.g., systemic sclerosis.
  • an MMP-12 binding protein can be used therapeutically or prophylactically with surgery, another MMP-12 inhibitor, e.g., a small molecule inhibitor, another anti-MMP-12 Fab or IgG (e.g., another Fab or IgG described herein), peptide inhibitor, or small molecule inhibitor.
  • MMP-12 inhibitors that can be used in combination therapy with an MMP-12 binding protein are described herein.
  • One or more small-molecule MMP inhibitors can be used in combination with one or more MMP-12 binding proteins described herein.
  • the combination can result in a lower dose of the small-molecule inhibitor being needed, such that side effects are reduced.
  • the MMP-9 or -12 binding proteins described herein can be administered in combination with one or more current therapies for treating systemic sclerosis.
  • proteins that inhibit IGF-II or that inhibit a downstream event of IGF-II/IGF-IIE activity can also be used in combination with another treatment for SSc-associated pulmonary fibrosis, such as surgery or administration of a second agent.
  • the second agent can include certain anti- inflammatory drugs (e.g., steroids), cytotoxic drugs, immunosuppressive agents, collagen synthesis inhibitors, endothelin receptor antagonist or surgery.
  • certain anti- inflammatory drugs e.g., steroids
  • cytotoxic drugs e.g., cytotoxic drugs
  • immunosuppressive agents e.g., collagen synthesis inhibitors, endothelin receptor antagonist or surgery.
  • high doses of oral corticosteroids e.g., prednisone, 40 to 80 mg daily
  • Cytotoxic drugs such as cyclophosphamide and immunosuppressants such as azathioprine (cyclophosphamide is also an immunosuppressant) have also been used.
  • Collagen synthesis inhibitors such as Pirfenidone and endothelin receptor antagonists such as Bosentan may also be effective. Lung transplantation for highly selected patients with end-stage pulmonary fibrosis has been reported.
  • cyclophosphamide or azathioprine can be used to treat SSc-associated pulmonary fibrosis.
  • pulses of cyclophosphamide often together with a small dose of steroids; epoprostenol, bosentan or iloprost (e.g, aerolized iloprost) can be used.
  • the term "combination" refers to the use of the two or more agents or therapies to treat the same patient, wherein the use or action of the agents or therapies overlap in time.
  • the agents or therapies can be administered at the same time (e.g., as a single formulation that is administered to a patient or as two separate formulations administered concurrently) or sequentially in any order. Sequential administrations are administrations that are given at different times.
  • the time between administration of the one agent and another agent can be minutes, hours, days, or weeks.
  • the use of an MMP-9 or -12 binding protein described herein can also be used to reduce the dosage of another therapy, e.g., to reduce the side-effects associated with another agent that is being administered, e.g., to reduce the side-effects of the second therapy.
  • a combination can include administering a second agent at a dosage at least 10, 20, 30, or 50% lower than would be used in the absence of the MMP-9 or -12 binding protein.
  • the second agent or therapy can also be another agent for systemic sclerosis, e.g., cyclosporin, an NSAID (e.g., naproxen), a calcium channel blocker (e.g., nifedipine), an angiotensin receptor blocker (e.g., losartan), epoprostenol (prostacyclin), a prostacyclin analogue (e.g., iloprost, e.g., aerolized iloprost), a dual endothelin-receptor antagonist (e.g., bosentan), prostaglandin El (e.g., alprostadil), a proton pump inhibitor, an antibiotic (e.g., tetrocycline), an immunosuppressant (e.g., cyclophosphamide, methotrexate or azathioprine), an ACE inhibitor, a sympathetic blocker, or surgery (e.g., lung transplantation).
  • NSAID
  • 88/518 unique sFab were identified by ELISA and sequencing (campaign A) and 168 by sequencing (campaign B). Their ability to inhibit human MMP-12, murine MMP-12 and other MMPs (1, 2, 3, 7, 8, 9, 10, 13, 14, 16, 17 and 24) was determined by usual means. The sFabs were converted to IgGIs.
  • Table III Ki values of four of the Campaign A MMP- 12 binding proteins that act as inhibitors:
  • 539B-M0008-H09 is cross reactive with human MMP-12 and murine MMP-12.
  • 539B-M0008-H09 showed a linear relationship between IC 50 and concentration ( ⁇ M) for both human MMP-12 and murione MMP-12, see FIGURE 3.
  • the Ki of 539B-M0008-H09 for human MMP-12 is 2.8 + 0.8 nM and the Km is 16 ⁇ 6 ⁇ M.
  • the Ki of 539B-M0008-H09 for murine MMP-12 is 2.2 ⁇ 0.6 nM and the Km is 42 ⁇ 17 ⁇ M.
  • 539B-M0011-Hl 1 was found to be an inhibitor of murine MMP-12 but not human MMP-12. This is shown in FIGURE 4.
  • mice were sensitized by IP administration of OVA/ Alum on day 0 and day 7.
  • M08-H09 was administered IP to the mice.
  • the mice were challenged by pulmonary administration of OVA.
  • metacholine induction and airway hyperresponsiveness was measured.
  • Six different groups were tested. Group 1 was a control group that received PBS. Grous 2, 3, 4 and 5 were administered M08-H09 at doses of 1 mg/kg, 5 mg/kg, 10 mg/kg and 25 mg/kg.
  • Group 6 was administered 25 mg/kg of Mll-Hll.
  • the OVA challenged mice were assessed for BAL inflammation based upon differential cell counts, lung histology by quantifying global inflammation, measurement of airway responsiveness to metacholine challenge, IL-4, IL-5 and IL- 13 ELISA measurement of BAL or protein lung extracts, serum specific IgE measurement, lung histology using congo red staining to determine eosinophil counts around bronchi and measurement of MMP-12 activity in the lungs using a fluorogenic substrate.
  • M08-H09 at all doses resulted in a decrease in the peribronchial inflammation score. A significant decrease was seen at 10 mg/kg and 25 mg/kg doses of M08- H09.
  • the differential cell counts showed that M08-H09 results in eosinophil percentages that are decreased.
  • M08-H09 significantly decreased total white blood cell infiltration and specifically neutrophil and lymphocyte infiltration.
  • Example 3 Affinity Matured Variants of M08-H09 Table IX summarizes cycle 1 of the affinity maturation of M08-H09.
  • 539B-M131A06 was cross reactive with human and murine MMP-12. It has a IC50 or Ki of ⁇ 0.3 nM and a KD of 180 pM for human MMP-12 and 73-150 pM for murine MMP-12.
  • Example 4 Modification of 539B-M131-A06 (M131A06) to remove a glycosylation site in CDRl of the variable light chain.
  • CDR 1 of the variable light chain of M131 A06 was modified to remove a glycosylation site. Removal of the glycosylation site in CDRl did not effect binding of M131A06 to MMP-12.
  • the MMP-12 antibody with the glycosylation site removed is referred to as 539B-M131A06-GA-S.
  • FIGURES 1OA and 1OB summarize the identification of amino acid changes in affinity matured variant HV-CDRs (cycles 1 and 2) that contribute to improvement in affinity and inhibition properties.
  • Example 6 Additional MMP- 12 Binding Proteins Twenty-one additional mutants were prepared: Eighteen mutants of DX-2712 were made by changing residues in the CDRl and 2 of the HC of DX-2712. A residue in the light chain of each of DX-2712, M0121-E07, and M0008-H09 (the parental antibody) was glycosylated.
  • MMP-9 (PMA activated) in solution. Phage, suitably depleted (e.g., previous contact with streptavidin) were allowed to interact with the target, unbound phage washed away and the output sampled and/or amplified for the next round of selection. This was repeated until the output phage in ELISA analysis indicate a high percentage of binders. 128/2076 unique sFabs were identified by ELISA and sequencing.
  • the phage display were converted into sFabs and then into IgGIs. Their ability to inhibit MMP-9 and other MMPs (1, 3, 7, 8, 9, 10, 12, 13, 14) was determined by usual means. Compounds were initially screened at 1 ⁇ M against MMP-9 and those compounds that inhibited MMP-9 >80% were subjected to additional screens against purified recombinant human MMPs. For these additional screens, an IC 50 value was determined.
  • antibody 539A-M0166-F10 has an IC50 of 4.3 ⁇ 1.9 nM on human MMP-9 activity.
  • the IC50 is ⁇ 33nM for the 539A-M0166-F10 Fab.
  • FIGURE HB shows that 539A-M0166-F10 is specific for human MMP-9 (hMMP-9) as compared to the other human (h) and murine (m) MMPs tested.
  • the residual enzyme activity was measured in the presence of 1 ⁇ M antibody (Fab or hlgG-l, as indicated in FIG. 1 IB).
  • the human MMP-I, -2, -3, -7, -8, -9, -10, -12, -13, and -14 were obtained from BIOMOL (Human MMP-9: SE-244, BIOMOL; Human MMP-14: SE-259, BIOMOL; Human MMP-I, -2, -8, -13: MMP MultiPack-1 from BIOMOL; Human MMP-3, -7, -10, -12: MMP MultiPack-2 from BIOMOL).
  • the mouse MMP-2 and -9 were from R&D (Mouse MMP-9: 909-MM, R&D Mouse MMP-2: 924-MP, R&D).
  • the substrate was Mca-Pro-Lys-Pro-Leu-Ala-Leu-Dap(Dnp)- Ala-Arg-NH2 (M-2225, Bachem) for human MMP-3, and Mca-Lys-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala- Arg-NH2 (M-2350, Bachem) for all the other enzymes.
  • the substrate concentration in the assay was 10 ⁇ M.
  • 539A-M0166-F10 also decreases MMP-9 activity in MCF-7 and Colo205 tumors, as measured by in situ zymography (data not shown).
  • Example 16 The DNA and amino acid sequences of variable regions of 539A-M0166-F10 sFAB are as follows:
  • the IC 50 values were then plotted against the substrate concentration.
  • the plot of the measured IC 50 (nM) vs. the substrate concentration ( ⁇ M) is shown in FIGURE 12.
  • the IC 50 increases linearly with the substrate concentration, which indicates that M0166-F10 behaves as a competitive inhibitor of the human MMP-9.
  • the following equation applies: and therefore the value of the K 1 can be calculated from the intercept.
  • K 1 0.3 + 0.5 nM.
  • the IC 50 measurements at various concentrations of substrate (3 ⁇ M, 5 ⁇ M, 7.5 ⁇ M, 10 ⁇ M, 12.5 ⁇ M, and 15 ⁇ M) are shown in FIGURE 13.
  • 539A-M0240-B03 is a selective inhibitor of MMP-9. 539A-M0240-B03 can decrease or inhibit the activity of human and mouse MMP-9.
  • CDRs complememtarity determining regions
  • a protein containing the HC CDR sequences of 539A-M0240-B03 (M0240-B03) and the light chain sequence shown below can be used in the methods described herein.
  • a protein containing the LC CDRs shown below and the HC CDRs of 539A-M0240-B03, or a protein containing the LC variable region (light V gene) shown below and the 539A-M0240-B03 HC CDRs can also be used in the methods described herein.
  • the protein can include a constant region sequence, such as the constant region (LC- lambda 1) shown below.
  • amino acid and nucleic acid sequences for another exemplary protein that can be used in the methods described herein are provided below.
  • a protein containing the LC and HC CDRs shown below, or a protein containing the light chain and heavy chain variable regions (LV and HV, respectively) shown below can also be used in the methods described herein.
  • 539A-M0240-B03 Parental isolate (sFab; IgG-pBhl(f)).
  • 539A-X0034-C02 (GS clone) (X0034-C02): DX-2802: Germlined, sequence optimized. The entire antibody fragment, containing the signal sequence, variable region and constant region of both the light and heavy chains were sequenced. The sequence data is available in 539A-R0108-A01 (539A-X0034-C02).
  • amino acid and nucleic acid sequences for another exemplary protein that can be used in the methods described herein are provided below.
  • a protein containing the LC and HC CDRs shown below, or a protein containing the light chain and heavy chain variable regions (LV and HV, respectively) shown below can also be used in the methods described herein.
  • a protein containing the light chain and heavy chain (designated as LV+ LC and HV + HC, respectively, below) sequences can also be used.
  • Table 80 gives the LV and HV CDR sequences of M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
  • M237-D02 (also referred to herein as 539A-M0237-D02) was used as the parent antibody for affinity maturation.
  • Two libraries were built and Fabs that bind all of the targets (hMMP9, hMMP2, mMMP9, and mMMP2) were selected.
  • One library allows the selected LC of M237-D02 to be replaced with any LC of the FAB -310 library (Hoet et al., Nat Biotechnol. 2005 23:344-348).
  • the other library allowed HC CDRl-2 to be replaced by any HC CDRl-2 of the FAB-310 library.
  • Table 50 gives the LV and HV CDR sequences of the affinity matured variants of M237-D02.
  • Table 60 shows the selected FABs from the HC-CDRl-2 library at positions 25- 66. Non-standard position 58a was allowed so that sequences having an insert could be displayed.
  • "-" means that the sequence is identical to M237-D02; "#” means there is a deletion.
  • Table 70 shows the VLs of M0237-D02 and M0275-D02 compared to germline.
  • "-" means that the sequence is identical to germline L6::JK5.
  • Antibodies having the sequences described herein can be expressed by joining DNA encoding a signal sequence to DNA encoding the sequences given as is known to persons with skill in the art.
  • VH CDRl contains residues 31-35 and only residues 31, 33, and 35 were allowed to vary. All but one of the isolates have W35, suggesting the importance for binding all four targets.
  • the P33 of the parental M0237-D02 is preferentially replaced with D, evidently preferred for binding all four targets. At position 31, there is no strong preference although four isolates have H. Antibodies that are more likely to have suitable solubility properties could be picked.
  • VH CDR2 contains residues 50, 51, 52, 52a, and 53-65. Positions 50, 52, 52a, 56, and 58 were varied. At position 50, the parental Y has mostly changed to V (only VGYRSW were allowed). At position 52, the parental V has mostly changed to Y (only VGYRSW were allowed). At 52a, only P and S were allowed and both seem to be accepted. M0275-D03 lacks an amino acid at position 55. At position 56, all amino acids except C were allowed. The parental R has been rejected but there is no clear preference; there are 4 Ys, 3 Ps, 3 As, 2 Ts, 1 F, and 1 G. At position 58, all amino acids except C were allowed. Two of the isolates retain the parental Y, 5 have F, 2 have G, 2 have S, and one has A.
  • VLs there are only two VLs in this collection of isolates (SEQ ID NO: 1-30).
  • Antibodies X0106-A01, X0106-B02, X0106-C04, X0106-E4, and X0106-F05 were produced as IgGIs and tested for inhibition of human and mouse MMP-9 and MMP-2.
  • X0106-A01, X0106-B02, X0106-C03, X0106-E4, and X0106-F05 are the germlined, optimized version of M0256-D11, M0276-F11, M0274-G08, M0275-D03, and M0307-F04, respectively.
  • Table 110 gives the LV and HV CDR sequences of the germlined, optimized MMP-9/MMP-2 specific antibodis (X0106-A01, X0106-B02, X0106-C04, X0106-E4, and X0106-F05). Following Table 10 is a listing of the sequences of VL and VH plus part of the constant regions for these antibodies.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Methods of using proteins that bind to MMP-9 or MMP- 12 for the treatment or prevention of systemic sclerosis are described.

Description

USE OF MMP-9 AND MMP- 12 BINDING PROTEINS FOR THE TREATMENT AND PREVENTION OF SYSTEMIC SCLEROSIS
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Application Serial No. 61/105,373, filed on October 14, 2008. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.
BACKGROUND
Matrix Metalloproteinases (MMPs) are a family of zinc metalloendopeptidases secreted by cells, and are responsible for much of the turnover of matrix components. The MMP family consists of at least 26 members, all of which share a common catalytic core with a zinc molecule in the active site.
SUMMARY
This disclosure relates, inter alia, to proteins that bind MMP-9 or MMP-12, herein referred to as "MMP-9 binding proteins" or "MMP-12 binding proteins," respectively, and methods of using such proteins to treat systemic sclerosis.
Systemic sclerosis is a clinically heterogeneous, systemic disorder which affects the connective tissue of the skin, internal organs and the walls of blood vessels. It is characterized by alterations of the microvasculature, disturbances of the immune system and by massive deposition of collagen and other matrix substances in the connective tissue. Sjogren's syndrome can also be associated with systemic sclerosis.
In one aspect, this disclosure relates, inter alia, to a method of treating or preventing systemic sclerosis in a subject, the method comprising: administering an isolated protein (e.g., antibody, e.g., human antibody) that binds MMP-9 to the subject, wherein the antibody binds the same epitope or competes for binding with an antibody selected from the group consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06. In some embodiments, the antibody competes with or binds the same epitope as DX- 2802.
In some embodiments, the protein may also compete with another MMP-9 binding protein described herein.
In another aspect, the disclosure provides a method of treating or preventing systemic sclerosis in a subject, the method comprising: administering an isolated protein (e.g., antibody, e.g., human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence to the subject, wherein: the heavy chain immunoglobulin variable domain sequence comprises one, two, or three (e.g., three) CDR regions from the heavy chain variable domain of DX-2802, 539 A- M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166- FlO, M0279-A03, M0279-B02, M0288-C08, and M0281-F06, and/or the light chain immunoglobulin variable domain sequence comprises one, two, or three (e.g., three) CDR regions from the light chain variable domain of DX-2802, 539A- M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166- FlO, M0279-A03, M0279-B02, M0288-C08, and M0281-F06 (respectively), and the protein binds to (e.g., and inhibits) MMP-9.
In some embodiments, the one, two, or three (e.g., three) CDR regions from the heavy chain variable domain are from DX-2802 and/or the one, two, or three (e.g., three) CDR regions from the light chain variable domain are from DX-2802.
In some embodiments, the the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of DX-2802, 539A-M0240-B03, M0078-G07,
M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06, and/or the the light chain immunoglobulin variable domain sequence comprises the light chain variable domain of DX-2802, 539A-M0240-B03, M0078- G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279- B02, M0288-C08, and M0281-F06 (respectively). In some embodiments, the the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of DX-2802, and/or the the light chain immunoglobulin variable domain sequence comprises the light chain variable domain of DX- 2802. In some embodiments, the protein comprises the heavy chain of DX-2802, 539 A-M0240-
B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279- A03, M0279-B02, M0288-C08, and M0281-F06, and/or the light chain of DX-2802, 539A- M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06 (respectively). In some embodiments, the protein comprises the heavy chain of DX-2802, and/or the light chain of DX-2802.
In some embodiments, the protein may contain one or more (e.g., 1, 2, or 3) heavy chain and/or light chain CDR regions from another MMP-9 binding protein described herein. In some embodiments, the protein may contain the heavy chain and/or light chain variable domain from another MMP-9 binding protein described herein. In some embodiments, the protein may contain the heavy chain and/or light chain from another MMP-9 binding protein described herein.
In one aspect, this disclosure relates to a method of treating or preventing systemic sclerosis in a subject, the method comprising: administering an isolated protein that binds MMP-9 (e.g., an MMP-9 binding protein described herein) to the subject.
In some embodiments, the MMP-9 binding protein is used to treat the diffuse form of systemic sclerosis. In some embodiments, the MMP-9 binding protein is used to treat the limited form of systemic sclerosis.
In some embodiments, the MMP-9 binding protein is used in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is another MMP-9 binding protein, e.g., another MMP-9 binding protein described herein. In some embodiments, the second therapeutic agent is another inhibitor described herein, e.g., a small molecule inhibitor of MMP-9. In some embodiments, the second therapeutic agent is an MMP- 12 binding protein, e.g., an MMP- 12 binding protein described herein.
In some embodiments, the second therapeutic agent is e.g., cyclosporin, an NSAID (e.g., naproxen), a calcium channel blocker (e.g., nifedipine), an angiotensin receptor blocker (e.g., losartan), epoprostenol (prostacyclin), a prostacyclin analogue (e.g., iloprost, e.g., aerolized iloprost), a dual endothelin-receptor antagonist (e.g., bosentan), prostaglandin El (e.g., alprostadil), a proton pump inhibitor, an antibiotic (e.g., tetracycline), an immunosuppressant (e.g., cyclophosphamide, methotrexate or azathioprine), an ACE inhibitor, a sympathetic blocker, or surgery (e.g., lung transplantation). These proteins include antibodies (including antibody fragments) (e.g., primate antibodies and Fabs, especially human antibodies and Fabs) that bind to MMP-9 (e.g., human MMP-9). In some embodiments, these proteins include antibodies (including antibody fragments) (e.g., primate antibodies and Fabs, especially human antibodies and Fabs) that inhibit MMP-9 (e.g., human MMP-9) (e.g., inhibit the catalytic activity of MMP-9). The MMP-9 binding proteins can be used in the treatment of systemic sclerosis (scleroderma), in which excess or inappropriate activity of MMP-9 features. In many cases, the proteins have tolerable low or no toxicity.
In some embodiments, the method uses a protein (e.g., an antibody, peptide or Kunitz domain protein) that binds MMP-9, in particular, a protein (e.g., an antibody (e.g., human antibody), peptide or Kunitz domain protein) that binds and inhibits MMP-9.
In one embodiment, the method uses an antibody (e.g., a human antibody) that binds to human MMP-9. In one embodiment, the human antibody is an inhibitor of the catalytic activity of MMP-9. The antibody can be, e.g., an IgGl, IgG2, IgG3, IgG4, Fab, Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen combining site of one of the antibodies herein listed. In one embodiment, the antibody is used to guide a nano-particle or toxin to a cell expressing MMP-9 on the cell surface. In one embodiment, the antibody causes effector functions (CDC or ADCC) to kill the cell which expresses MMP-9.
In some embodiments, the VH and VL regions of the binding proteins (e.g., Fabs) can be provided as IgG, Fab, Fab2, Fab2', scFv, PEGylated Fab, PEGylated scFv, PEGylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA + VH::CH1, HSA: :LC + VH::CH1, or other appropriate construct.
In another embodiment, the binding protein comprises a Kunitz domain protein or modified version (e.g., HSA fusion) or peptide-based MMP-9 binding protein that can inhibit MMP-9 activity.
In one embodiment, the method uses a protein (e.g., an isolated protein) that binds to MMP-9 (e.g., human MMP-9) and includes at least one immunoglobulin variable region. For example, the protein includes a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence. In one embodiment, the protein binds to and inhibits MMP-9 (e.g., inhibits MMP-9 catalytic activity), e.g., human MMP- 9.
In some embodiments, the protein binds to human MMP-9 specifically, and not to MMP- 9 from another species (e.g., the protein does not bind to MMP-9 from another species with greater than background levels of binding).
In some embodiments, the protein binds MMP-9 specifically, and not to another matrix metalloproteinase (e.g., the protein does not bind to any other matrix metalloproteinase with greater than background levels of binding).
Such binding proteins can be conjugated to a drug (e.g., to form a MMP-9 binding protein-drug conjugate) and used therapeutically. This disclosure relates, in part, to MMP-9 binding protein-drug conjugates, the preparation of these conjugates, and uses thereof.
The protein can include one or more of the following characteristics: (a) a human CDR or human framework region; (b) the HC immunoglobulin variable domain sequence comprises one or more (e.g., 1, 2, or 3) CDRs that are at least 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a HC variable domain described herein; (c) the LC immunoglobulin variable domain sequence comprises one or more (e.g., 1, 2, or 3) CDRs that are at least 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a LC variable domain described herein; (d) the LC immunoglobulin variable domain sequence is at least 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a LC variable domain described herein; (e) the HC immunoglobulin variable domain sequence is at least 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a HC variable domain described herein; (f) the protein binds an epitope bound by a protein described herein, or an epitope that overlaps with such epitope; and (g) a primate CDR or primate framework region.
The protein can bind to MMP-9, e.g., human MMP-9, with a binding affinity of at least 105, 106, 107,108, 109, 1010 and 1011 M"1. In one embodiment, the protein binds to MMP-9 with a Koff slower than 1 X 10~3, 5 X 10 "4 s"1, or 1 X 10~4 s"1. In one embodiment, the protein binds to
MMP-9 with a K0n faster than 1 X 102, 1 X 103, or 5 X 103 M 1S 1. In one embodiment, the protein inhibits human MMP-9 activity, e.g., with a Ki of less than 10~5, 10~6, 10~7, 10~8, 10~9, and 10~10 M. The protein can have, for example, an IC50 of less than 100 nM, 10 nM or 1 nM. In some embodiments, the protein has an IC50 of about 1.8 nM. The affinity of the protein for MMP-9 can be characterized by a KD of less than 100 nm, less than 10 nM, or about 3 nM (e.g., 3.1 nM), about 5 nM (e.g., 5 nM), about 6 nm (e.g., 5.9 nM), about 7 nM (e.g., 7.1 nM), or about 10 nM (e.g., 9.6 nM).
In some embodiments, the protein has a KD <200 nM.
In some embodiments, the protein has a tl/2 of at least about 10 minutes (e.g., 11 minutes), at least about 20 minutes (e.g., 18 minutes), at least about 25 minutes(e.g., 25 minutes), at least about 35 minutes (e.g., 33 minutes), or at least about 60 minutes (e.g., 57 minutes).
In one embodiment, the protein binds the catalytic domain of human MMP-9, e.g., the protein contacts residues in or near the active site of MMP-9.
In some embodiments, the protein does not contact residues in or near the active site of MMP-9 but instead binds elsewhere on MMP-9 and causes a steric change in MMP-9 that affects (e.g., inhibits) its activity.
In one embodiment, the protein also binds to MMP-16 and/or MMP-24, e.g., with a binding affinity of at least 105, 106, 107 ,108, 109, 1010 and 1011 M"1. For example, the protein binds to both MMP-9 and to MMP-16 or MMP-24 with a binding affinity of at least 105, 106, 107, 108, 109, 1010 and 1011 M"1.
In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having the light and heavy chains of antibodies selected from the group consisting of DX-2802, 539A- M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06. In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having its heavy selected from the group consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081- D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288- C08, and M0281-F06.
In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having its light chain selected from the group consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs selected from the corresponding CDRs of the group of heavy chains consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs selected from the corresponding CDRs of the group of light chains consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs selected from the corresponding CDRs of the group of light chains consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06and one or more (e.g., 1, 2, or 3) heavy chain CDRs selected from the corresponding CDRs of the group of heavy chains consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06 (respectively). In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having light and heavy antibody variable regions of an antibody selected from the group consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075- D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a heavy chain antibody variable region of an antibody selected from the group consisting of: DX- 2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a light chain antibody variable region of an antibody selected from the group consisting of: DX- 2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
In a more preferred embodiment, the protein is an antibody (e.g., a human antibody) having the light and heavy chains of antibodies from M0166-F10. In another preferred embodiment, the protein is an antibody (e.g., a human antibody) having its heavy chain from M0166-F10. In yet another preferred embodiment, the protein is an antibody (e.g., a human antibody) having its light chain from M0166-F10.
In an even more preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain of M0166-F10. In another even more preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chain of M0166-F10. In an even more preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain of M0166-F10 and one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains of M0166- FlO.
In a more preferred embodiment, the protein is an antibody (e.g., a human antibody) having light and heavy antibody variable regions of M0166-F10. In another preferred embodiment, the protein is an antibody (e.g., a human antibody) having the heavy chain antibody variable region of M0166-F10. In yet another preferred embodiment, the protein is an antibody (e.g., a human antibody) having the light chain antibody variable region of M0166-F10.
In a more preferred embodiment, the protein is an antibody (e.g., a human antibody) having the light and heavy chains of antibodies from 539A-M0240-B03. In another preferred embodiment, the protein is an antibody (e.g., a human antibody) having its heavy chain from 539A-M0240-B03. In yet another preferred embodiment, the protein is an antibody (e.g., a human antibody) having its light chain from 539A-M0240-B03. In an even more preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain of 539A-M0240-B03. In another even more preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains of 539A-M0240-B03. In an even more preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain of 539A- M0240-B03 and one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains of 539A-M0240-B03. In a more preferred embodiment, the protein is an antibody (e.g., a human antibody) having light and heavy antibody variable regions of 539A-M0240-B03. In another preferred embodiment, the protein is an antibody (e.g., a human antibody) having the heavy chain antibody variable region of 539A-M0240-B03. In yet another preferred embodiment, the protein is an antibody (e.g., a human antibody) having the light chain antibody variable region of 539A- M0240-B03.
In a more preferred embodiment, the protein is an antibody (e.g., a human antibody) having the light and heavy chains of antibodies from X0034-C02 (DX-2802). In another preferred embodiment, the protein is an antibody (e.g., a human antibody) having its heavy chain from X0034-C02. In yet another preferred embodiment, the protein is an antibody (e.g., a human antibody) having its light chain from X0034-C02.
In an even more preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain of X0034-C02. In another even more preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains of X0034-C02. In an even more preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain of X0034-C02 and one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains of X0034- C02. In a more preferred embodiment, the protein is an antibody (e.g., a human antibody) having light and heavy antibody variable regions of X0034-C02. In another preferred embodiment, the protein is an antibody (e.g., a human antibody) having the heavy chain antibody variable region of X0034-C02. In yet another preferred embodiment, the protein is an antibody (e.g., a human antibody) having the light chain antibody variable region of X0034-C02.
In some embodiments, the protein may contain one or more (e.g., 1, 2, or 3) heavy chain and/or light chain CDR regions from another MMP-9 binding protein described herein. In some embodiments, the protein may contain the heavy chain and/or light chain variable domain from another MMP-9 binding protein described herein. In some embodiments, the protein may contain the heavy chain and/or light chain from another MMP-9 binding protein described herein.
In one embodiment, the HC and LC variable domain sequences are components of the same polypeptide chain. In another, the HC and LC variable domain sequences are components of different polypeptide chains. For example, the protein is an IgG, e.g., IgGl, IgG2, IgG3, or IgG4. The protein can be a soluble Fab (sFab). In other implementations the protein includes a Fab2', scFv, minibody, scFv::Fc fusion, Fab: :HS A fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen combining site of one of the binding proteins herein. The VH and VL regions of these Fabs can be provided as IgG, Fab, Fab2, Fab2', scFv, PEGylated Fab, PEGylated scFv, PEGylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA + VH::CH1, HSA: :LC + VH::CH1, or other appropriate construction.
In one embodiment, the protein is a human or humanized antibody or is non- immunogenic in a human. For example, the protein includes one or more human antibody framework regions, e.g., all human framework regions. In one embodiment, the protein includes a human Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or 99% identical to a human Fc domain.
In one embodiment, the protein is a primate or primatized antibody or is non- immunogenic in a human. For example, the protein includes one or more primate antibody framework regions, e.g., all primate framework regions. In one embodiment, the protein includes a primate Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or 99% identical to a primate Fc domain. "Primate" includes humans {Homo sapiens), chimpanzees {Pan troglodytes and Pan paniscus (bonobos)), gorillas {Gorilla gorilla), gibons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and tarsiers.
In certain embodiments, the protein includes no sequences from mice or rabbits (e.g., is not a murine or rabbit antibody). In one embodiment, the protein is capable of binding to tumor cells expressing MMP-9, e.g., to Colo205 (a human colorectal carcinoma cell line), or MCF-7 (a human breast adenocarcinoma cell line) cells.
In one embodiment, protein is physically associated with a nanoparticle, and can be used to guide a nanoparticle to a cell expressing MMP-9 on the cell surface. In one embodiment, the protein causes effector cells (CDC or ADCC) to kill a cell which expresses MMP-9.
In some aspects, the MMP-9 binding protein is administered (alone or in combination with a second agent (e.g., a second agent described herein)) to a subject to treat a symptom of systemic sclerosis, e.g., Raynaud's syndrome, swelling (eventually skin tightening and contractures of the fingers), polyarthralgia, dysphagia, dyspnea, lung fibrosis, acute alveolitis, pulmonary hypertension, heartburn, cardiac arrhythmias or renal crisis, or other symptom described herein. The more evident symptom is usually the hardening of the skin and associated scarring. Blood vessels may also be more visible. Many SSc patients (over 80%) have vascular symptoms and Raynaud's phenomenon. During an attack, there is discoloration of the hands and feet in response to cold. Raynaud's normally affects the fingers and toes. SSc and Raynaud's can cause painful ulcers on the fingers or toes which are known as digital ulcers. Calcinosis is also common in SSc, and is often seen near the elbows, knees or other joints. Diffuse scleroderma can cause musculoskeletal, pulmonary, gastrointestinal, renal and other complications. Patients with larger amounts of cutaneous involvement are more likely to have involvement of the internal tissues and organs. In some aspects, the MMP-9 binding protein is administered (alone or in combination with a second agent (e.g., a second agent described herein)) to a subject to treat Sjogren's syndrome associated with SSc.
In some aspects, the MMP-9 binding protein is administered (alone or in combination with a second agent (e.g., a second agent described herein)) to a subject at risk of developing systemic sclerosis, e.g., with a familial predisposition for the disease (e.g., a polymorphism in COL1A2 or TGF-βl), or with another risk factor, e.g., a prior cytomegalovirus (CMV) infection, or exposure to organic solvents or other chemical agents (e.g., vinyl chloride, bleomycin, pentazocine, epoxy and/or aromatic hydrocarbons, contaminated rapeseed oil or L-tryptophan).
In another aspect, the disclosure features an MMP-9 binding protein that is a competitive inhibitor of MMP-9. In some embodiments, the binding protein competes with an MMP-9 substrate (e.g., collagen), e.g., binds to the same epitope as the substrate, e.g., and prevents substrate binding.
In some aspects, the method includes inhibiting an interaction between MMP-9 and an
MMP-9 substrate (e.g., collagen). The method includes contacting an MMP-9 binding protein (e.g., an MMP-9 binding protein described herein) with MMP-9 (e.g., in vitro or in vivo), wherein the binding protein binds to MMP-9 and thereby prevents the binding of an MMP-9 substrate to MMP-9. In some embodiments, the binding protein binds to the same epitope on MMP-9 as the substrate, e.g., the binding protein is a competitive inhibitor. In some embodiments, the binding protein does not bind the same epitope as the substrate but causes a steric change in MMP-9 that decreases or inhibits the ability of the substrate to bind.
In one aspect, the method features a MMP-9 binding protein-drug conjugate that includes a MMP-9 binding protein (e.g., an MMP-9 binding protein described herein) and a drug.
In one embodiment, the binding protein comprises at least one immunoglobulin variable region, and/or the protein binds to and/or inhibits MMP-9, e.g., inhibits MMP-9 catalytic activity.
In one embodiment, the drug is a cytotoxic or cytostatic agent. The cytotoxic agent can be, e.g., selected from the group consisting of an auristatin, a DNA minor groove binding agent, a DNA minor groove alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a podophyllotoxin, a baccatin derivative, a cryptophysin, a combretastatin, a maytansinoid, and a vinca alkaloid. In one embodiment, the cytotoxic agent is an auristatin and, e.g., the auristatin is selected from AFP, MMAF, MMAE, AEB, AEVB and auristatin E. In one embodiment, the auristatin is AFP or MMAF. In another embodiment, the cytotoxic agent is a maytansinoid and, e.g., the maytansinoid is selected from a maytansinol, maytansine, DMl, DM2, DM3 and DM4. In one embodiment, the maytansinoid is DMl. In another embodiment, the cytotoxic agent is selected from paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatstatin, calicheamicin, and netropsin. In one embodiment, the cytotoxin is an auri statin, a maytansinoid, or calicheamicin.
In one embodiment, the cytotoxic agent is an antitubulin agent and, e.g., the antitubulin agent is selected from AFP, MMAP, MMAE, AEB, AEVB, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicines, colcimid, estramustine, cemadotin, discodermolide, maytansinol, maytansine, DMl, DM2, DM3, DM4 and eleutherobin.
In one embodiment, the MMP-9 binding protein (e.g., antibody) is conjugated to the cytotoxic agent via a linker. In one embodiment, the linker is cleavable under intracellular conditions, e.g., the cleavable linker is a peptide linker cleavable by an intracellular protease. In one embodiment, the linker is a peptide linker, e.g., a dipeptide linker, e.g., a val-cit linker or a phe-lys linker. In one embodiment, the cleavable linker is hydrolyzable at a pH of less than 5.5, e.g., the hydrolyzable linker is a hydrazone linker. In another embodiment, the cleavable linker is a disulfide linker.
A binding protein described herein can be provided as a pharmaceutical composition, e.g., including a pharmaceutically acceptable carrier. The composition can be at least 10, 20, 30, 50, 75, 85, 90, 95, 98, 99, or 99.9% free of other protein species. In some embodiments, the binding protein can be produced under GMP (good manufacturing practices). In some embodiments, the binding protein is provided in pharmaceutically acceptable carriers, e.g., suitable buffers or excipients. The dose of a binding protein (e.g., a pharmaceutical composition containing a binding protein described herein) is sufficient to block about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% of the activity of MMP-9 in the patient, e.g., at the site of disease. Depending on the disease, this may require a dose, e.g., of between about 0.01 mg/Kg to about 100 mg/Kg, e.g., between about 0.1 and about 10 mg/Kg. For example, the dose can be a dose of about 0.1, about 1, about 3, about 6, or about 10 mg/Kg. For example, for an IgG having a molecular mass of 150,000 g/mole (2 binding sites), these doses correspond to approximately 18 nM, 180 nM, 540 nM, 1.08 microM, and 1.8 microM, respectively, of binding sites for a 5 L blood volume. Medicine being partly an art, the optimal dose will be established by clinical trials, but will most likely lie in this range.
In another aspect, the disclosure features a method of detecting an MMP-9 in a sample, e.g., a sample from a patient (e.g., tissue biopsy or blood sample). The method includes: contacting the sample with an MMP-9 binding protein (e.g., an MMP-9 binding protein described herein); and detecting an interaction between the protein and the MMP-9, if present. In some embodiments, the protein includes a detectable label.
An MMP-9 binding protein can be used to detect MMP-9 in a subject. The method includes: administering an MMP-9 binding protein (e.g., an MMP-9 binding protein described herein) to a subject; and detecting the protein in the subject, e.g., detecting an interaction between the protein and the MMP-9, if present. In some embodiments, the protein further includes a detectable label.
In another aspect, the disclosure features a method of modulating MMP-9 activity. The method includes: contacting an MMP-9 with an MMP-9 binding protein (e.g., an MMP-9 binding protein described herein) (e.g., in a human subject), thereby modulating MMP-9 activity. In some embodiments, the binding protein inhibits MMP-9 activity (e.g., inhibits MMP-9 catalytic activity).
In another aspect, the disclosure features a method of treating systemic sclerosis. The method includes: administering, to a subject, an MMP-9 binding protein (e.g., an MMP-9 binding protein described herein) in an amount sufficient to treat systemic sclerosis in the subject.
MMP-9 binding proteins can be useful for modulating systemic sclerosis in a subject. The protein can be administered, to the subject, in an amount effective to modulate systemic sclerosis. In another aspect, the disclosure features a method of treating Sjogren's syndrome associated with SSc. The method includes: administering, to a subject, an MMP-9 binding protein (e.g., an MMP-9 binding protein described herein) in an amount sufficient to treat Sjogren's syndrome associated with SSc in the subject.
Other exemplary therapeutic methods that include administering an MMP-9 binding protein are described below. An MMP-9 binding protein described herein can be administered in combination with one or more other MMP inhibitors, e.g., small molecule inhibitors, e.g., broad specificity inhibitors. In one embodiment, the small molecule inhibitors are one or more of neovastat, marimastat, BAY 12-9566, or prinomastat. In another embodiment, the one or more MMP inhibitors include another MMP-9 binding protein (e.g., an MMP-9 binding protein described herein), or an MMP-12 binding protein, e.g., an MMP-12 binding protein described herein.
In one aspect, the disclosure features the use of an MMP-9 binding protein described herein for the manufacture of a medicament for the treatment of a disorder described herein, e.g., systemic sclerosis.
In another aspect, the disclosure features a method of treating or preventing systemic sclerosis in a subject, the method comprising: administering an isolated protein (e.g., antibody, e.g., human antibody) that binds MMP-
12 to the subject, wherein the antibody binds the same epitope or competes for binding with an antibody selected from the group consisting of DX-2712, a mutant or variant of DX-2712, 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, MOl 18-Fl 1, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11.
In some embodiments, the antibody competes with or binds the same epitope as DX- 2712.
In some embodiments, the antibody competes with or binds the same epitope as 539B- X0041-D02.
In some aspects, the disclosure features a method of treating or preventing systemic sclerosis in a subject, the method comprising: administering an isolated protein (e.g., antibody, e.g., human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence to the subject, wherein: the heavy chain immunoglobulin variable domain sequence comprises one, two, or three (e.g., three) CDR regions from the heavy chain variable domain of DX-2712, a mutant or variant of DX-2712, 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01,
M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134- D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134- F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-
HlO, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, MOl 30- AOl, M0130-C12, M0130-F06, M0130-H04, M0131- A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, MOl 18-Fl 1, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063- A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-
F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087- F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-
DOl, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11, and/or the light chain immunoglobulin variable domain sequence comprises one, two, or three (e.g., three) CDR regions from the light chain variable domain of DX-2712, a mutant or variant of DX-2712, 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09,
M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134- D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134- F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03,
M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135- HlO, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131- A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, MOl 18-Fl 1, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-
A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067- F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087- F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07,
M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016- DOl, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11 (respectively), and the protein binds to and inhibits MMP- 12.
In some embodiments, the one, two, or three (e.g., three) CDR regions from the heavy chain variable domain are from DX-2712 and/or the one, two, or three (e.g., three) CDR regions from the light chain variable domain are from DX-2712.
In some embodiments, the one, two, or three (e.g., three) CDR regions from the heavy chain variable domain are from 539B-X0041-D02and/or the one, two, or three (e.g., three) CDR regions from the light chain variable domain are from 539B-X0041-D02.
In some embodiments, the the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of DX-2712, a mutant or variant of DX-2712, 539B- X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134- BOl, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134- C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134- E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, M0118-F11, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11, and/or the the light chain immunoglobulin variable domain sequence comprises the light chain variable domain of DX-2712, a mutant or variant of DX-2712, 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, MOl 18-Fl 1, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11 (respectively).
In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of DX-2712, and/or the the light chain immunoglobulin variable domain sequence comprises the light chain variable domain of DX- 2712.
In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of 539B-X0041-D02, and/or the the light chain immunoglobulin variable domain sequence comprises the light chain variable domain of 539B- X0041-D02. In some embodiments, the protein comprises the heavy chain of DX-2712, a mutant or variant of DX-2712, 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, M0118-F11, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11, and/or the light chain of DX-2712, a mutant or variant of DX-2712, 539B-X0041-D02, M0134-A02, M134- A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, MOl 18-Fl 1, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-Ell (respectively).
In some embodiments, the protein comprises the heavy chain of DX-2712, and/or the light chain of DX-2712. In some embodiments, the protein comprises the heavy chain of 539B-X0041-D02, and/or the light chain of 539B-X0041-D02.
This disclosure also relates to a method of treating or preventing systemic sclerosis in a subject, the method comprising: administering an isolated protein that binds MMP- 12 (e.g., an MMP- 12 binding protein described herein) to the subject.
In some embodiments, the MMP- 12 binding protein is used to treat the diffuse form of systemic sclerosis.
In some embodiments, the MMP- 12 binding protein is used to treat the limited form of systemic sclerosis.
In some embodiments, the MMP- 12 binding protein is used in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is another MMP-12 binding protein, e.g., another MMP-12 binding protein described herein. In some embodiments, the second therapeutic agent is another inhibitor described herein, e.g., a small molecule inhibitor of MMP-12. In some embodiments, the second therapeutic agent is an MMP-9 binding protein, e.g., an MMP-9 binding protein described herein.
In some embodiments, the second therapeutic agent is e.g., cyclosporin, an NSAID (e.g., naproxen), a calcium channel blocker (e.g., nifedipine), an angiotensin receptor blocker (e.g., losartan), epoprostenol (prostacyclin), a prostacyclin analogue (e.g., iloprost, e.g., aerolized iloprost), a dual endothelin-receptor antagonist (e.g., bosentan), prostaglandin El (e.g., alprostadil), a proton pump inhibitor, an antibiotic (e.g., tetracycline), an immunosuppressant (e.g., cyclophosphamide, methotrexate or azathioprine), an ACE inhibitor, a sympathetic blocker, or surgery (e.g., lung transplantation).
These proteins include antibodies (including antibody fragments) (e.g., primate antibodies and Fabs, especially human antibodies and Fabs) that bind to MMP-12 (e.g., human MMP-12). In some embodiments, these proteins include antibodies (including antibody fragments) (e.g., primate antibodies and Fabs, especially human antibodies and Fabs) that inhibit MMP-12 (e.g., human MMP-12) (e.g., inhibit the catalytic activity of MMP-12). The MMP-12 binding proteins can be used in the treatment of systemic sclerosis (scleroderma), in which excess or inappropriate activity of MMP-12 features. In many cases, the proteins have tolerable low or no toxicity.
In some embodiments, the method uses a protein (e.g., an antibody, peptide or Kunitz domain protein) that binds MMP-12, in particular, a protein (e.g., an antibody (e.g., human antibody), peptide or Kunitz domain protein) that binds and inhibits MMP-12.
In one embodiment, the method uses an antibody (e.g., a human antibody) that binds to human MMP-12. In one embodiment, the human antibody is an inhibitor of the catalytic activity of MMP-12. The antibody can be, e.g., an IgGl, IgG2, IgG3, IgG4, Fab, Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen combining site of one of the antibodies herein listed. In one embodiment, the antibody is used to guide a nano-particle or toxin to a cell expressing MMP-12 on the cell surface. In one embodiment, the antibody causes effector functions (CDC or ADCC) to kill the cell which expresses MMP-12.
In some embodiments, the VH and VL regions of the binding proteins (e.g., Fabs) can be provided as IgG, Fab, Fab2, Fab2', scFv, PEGylated Fab, PEGylated scFv, PEGylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA + VH::CH1, HSA: :LC + VH::CH1, or other appropriate construct.
In another embodiment, the binding protein comprises a Kunitz domain protein or modified version (e.g., HSA fusion) or peptide-based MMP-12 binding protein that can inhibit MMP-12 activity.
In one embodiment, the method uses a protein (e.g., an isolated protein) that binds to MMP-12 (e.g., human MMP-12) and includes at least one immunoglobulin variable region. For example, the protein includes a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence. In one embodiment, the protein binds to and inhibits MMP-12 (e.g., inhibits MMP-12 catalytic activity), e.g., human MMP-12.
In some embodiments, the protein binds to human MMP-12 specifically, and not to MMP-12 from another species (e.g., the protein does not bind to MMP-12 from another species with greater than background levels of binding).
In some embodiments, the protein binds MMP-12 specifically, and not to another matrix metalloproteinase (e.g., the protein does not bind to any other matrix metalloproteinase with greater than background levels of binding). Such binding proteins can be conjugated to a drug (e.g., to form a MMP-12 binding protein-drug conjugate) and used therapeutically. This disclosure relates, in part, to MMP-12 binding protein-drug conjugates, the preparation of these conjugates, and uses thereof. The conjugates can be used, e.g., in the treatment of disorders, e.g., for the treatment of systemic sclerosis. The protein can include one or more of the following characteristics: (a) a human CDR or human framework region; (b) the HC immunoglobulin variable domain sequence comprises one or more (e.g., 1, 2, or 3) CDRs that are at least 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a HC variable domain described herein; (c) the LC immunoglobulin variable domain sequence comprises one or more (e.g., 1, 2, or 3) CDRs that are at least 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a LC variable domain described herein; (d) the LC immunoglobulin variable domain sequence is at least 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a LC variable domain described herein; (e) the HC immunoglobulin variable domain sequence is at least 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a HC variable domain described herein; (f) the protein binds an epitope bound by a protein described herein, or an epitope that overlaps with such epitope; and (g) a primate CDR or primate framework region.
The protein can bind to MMP-12, e.g., human MMP-12, with a binding affinity of at least 105, 106, 107,108, 109, 1010 and 1011 M"1. In one embodiment, the protein binds to MMP-12 with a Koff slower than 1 X 10"3, 5 X 10 ~4 s"1, or 1 X 10 ~4 s"1. In one embodiment, the protein binds to MMP-12 with a K0n faster than 1 X 102, 1 X 103, or 5 X 103 M 1S 1. In one embodiment, the protein inhibits human MMP-12 activity, e.g., with a Ki of less than 10"5, 10"6, 10"7, 10"8, 10"9, and 10"10 M. The protein can have, for example, an IC50 of less than 100 nM, 10 nM or 1 nM. In some embodiments, the protein has an IC50 of about 1.8 nM. The affinity of the protein for MMP-12 can be characterized by a KD of less than 100 nm, less than 10 nM, or about 3 nM (e.g., 3.1 nM), about 5 nM (e.g., 5 nM), about 6 nm (e.g., 5.9 nM), about 7 nM (e.g., 7.1 nM), or about 10 nM (e.g., 9.6 nM).
In some embodiments, the protein has a KD <200 nM.
In some embodiments, the protein has a tl/2 of at least about 10 minutes (e.g., 11 minutes), at least about 20 minutes (e.g., 18 minutes), at least about 25 minutes(e.g., 25 minutes), at least about 35 minutes (e.g., 33 minutes), or at least about 60 minutes (e.g., 57 minutes). In one embodiment, the protein binds the catalytic domain of human MMP-12, e.g., the protein contacts residues in or near the active site of MMP-12.
In some embodiments, the protein does not contact residues in or near the active site of MMP-12 but instead binds elsewhere on MMP-12 and causes a steric change in MMP-12 that affects (e.g., inhibits) its activity. In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having the light and heavy chains of antibodies selected from the group consisting of DX-2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134- BI l, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134- C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134- FOl, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135- A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135- F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135- HlO, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, MOIlO- G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, MODI- DOS, M0132-A04, M0133-B08, M0133-E05, M0121-E07, MOl 18-Fl 1, M0125-G07, M0124- E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063- BOl, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067- A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069- Al l, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071- D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089- COl, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041- B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, MOOIl- HI l, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014- GI l, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11.
In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having its heavy chain selected from the group consisting of DX-2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, M0118-F11, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11.
In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having its light chain selected from the group consisting of DX-2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, M0118-F11, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11.
In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs selected from the corresponding CDRs of the group of heavy chains consisting of DX-2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, MOl 18-Fl 1, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11.
In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs selected from the corresponding CDRs of the group of light chains consisting of DX-2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, MOl 18-Fl 1, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11. In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs selected from the corresponding CDRs of the group of light chains consisting of DX-2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, MOl 18-Fl 1, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11, and having one or more (e.g., 1, 2, or 3) heavy chain CDRs selected from the corresponding CDRs of the group of heavy chains consisting of DX-2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B- X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134- BOl, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134- C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134- E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, MOl 18-Fl 1, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11 (respectively).
In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having light and heavy antibody variable regions of an antibody selected from the group consisting of DX-2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134- A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, MOl 18-Fl 1, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11.
In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a heavy chain antibody variable region of an antibody selected from the group consisting of: DX- 2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134- A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, MOl 18-Fl 1, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11. In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a light chain antibody variable region of an antibody selected from the group consisting of: DX- 2712, a mutant or variant of DX-2712 (e.g., as described herein), 539B-X0041-D02, M0134- A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, MOl 18-Fl 1, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11.
In a more preferred embodiment, the protein is an antibody (e.g., a human antibody) having the light and heavy chains of antibody M0008-H09, M0131-A06 or M0121-E07. In another preferred embodiment, the protein is an antibody (e.g., a human antibody) having its heavy chain from M0008-H09, M0131-A06 or M0121-E07. In yet another preferred embodiment, the protein is an antibody (e.g., a human antibody) having its light chain from M0008-H09, M0131-A06 or M0121-E07.
In an even more preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain of M0008-H09, M0131-A06 or M0121-E07. In another even more preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains of M0008-H09, M0131- A06 or M0121-E07. In an even more preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs and one or more (e.g., 1, 2, or3 ) light chain CDRs from the corresponding CDRs of the heavy chain and light chain of M0008-H09, M0131-A06 or M0121-E07 (respectively). In a more preferred embodiment, the protein is an antibody (e.g., a human antibody) having the light and heavy chain variable regions of antibody M0008-H09, M0131-A06 or M0121-E07. In another preferred embodiment, the protein is an antibody (e.g., a human antibody) having its heavy chain variable region from M0008-H09, M0131-A06 or M0121-E07. In yet another preferred embodiment, the protein is an antibody (e.g., a human antibody) having its light chain variable region from M0008-H09, M0131-A06 or M0121-E07.
In a more preferred embodiment, the protein is an antibody (e.g., human antibody) having the light and heavy chains of antibody DX-2712 (also referred to as M0131-A06-GA-S). In another preferred embodiment, the protein is a human antibody having its heavy chain from antibody DX-2712. In yet another preferred embodiment, the protein is a human antibody having its light chain from antibody DX-2712.
In an even more preferred embodiment, the protein is a human antibody having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain from antibody DX-2712. In another even more preferred embodiment, the protein is a human antibody having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains from antibody DX-2712. In another even more preferred embodiment, the protein is a human antibody having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains from antibody DX-2712 and having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain from antibody DX- 2712.
In a more preferred embodiment, the protein is an antibody (e.g., human antibody) having the light and heavy chain variable regions of antibody DX-2712 (also referred to as M0131-A06- GA-S). In another preferred embodiment, the protein is a human antibody having its heavy chain variable region from antibody DX-2712. In yet another preferred embodiment, the protein is a human antibody having its light chain variable region from antibody DX-2712.
In another embodiment, the protein is a human antibody having the light and heavy chains of a mutant or variant of DX-2712, e.g., a mutant or variant described herein. In another preferred embodiment, the protein is a human antibody having its heavy chain from a mutant or variant of DX-2712, e.g., a mutant or variant described herein. In yet another preferred embodiment, the protein is a human antibody having its light chain from a variant of DX-2712, e.g., a mutant or variant described herein.
In an even more preferred embodiment, the protein is a human antibody having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain from a mutant or variant of DX-2712, e.g., a mutant or variant described herein. In another even more preferred embodiment, the protein is a human antibody having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains from a mutant or variant of DX- 2712, e.g., a mutant or variant described herein. In another even more preferred embodiment, the protein is a human antibody having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains from a mutant or variant of DX-2712, e.g., a mutant or variant described herein and having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain from a mutant or variant of DX-2712, e.g., a mutant or variant described herein.
In a more preferred embodiment, the protein is an antibody (e.g., human antibody) having the light and heavy chain variable regions of a mutant or variant of DX-2712, e.g., a mutant or variant described herein. In another preferred embodiment, the protein is a human antibody having its heavy chain variable region from a mutant or variant of DX-2712, e.g., a mutant or variant described herein. In yet another preferred embodiment, the protein is a human antibody having its light chain variable region from a mutant or variant of DX-2712, e.g., a mutant or variant described herein.
In a more preferred embodiment, the protein is a human antibody having the light and heavy chains of antibody 539B-X0041-D02. In another preferred embodiment, the protein is a human antibody having its heavy chain from antibody 539B-X0041-D02. In yet another preferred embodiment, the protein is a human antibody having its light chain from antibody 539B-X0041-D02.
In an even more preferred embodiment, the protein is a human antibody having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain from antibody 539B-X0041-D02. In another even more preferred embodiment, the protein is a human antibody having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains from antibody 539B-X0041-D02. In another even more preferred embodiment, the protein is a human antibody having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains from antibody 539B-X0041-D02 and having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain from antibody 539B-X0041-D02. In a more preferred embodiment, the protein is an antibody (e.g., human antibody) having the light and heavy chain variable regions of antibody 539B-X0041-D02. In another preferred embodiment, the protein is a human antibody having its heavy chain variable region from antibody 539B-X0041-D02. In yet another preferred embodiment, the protein is a human antibody having its light chain variable region from antibody 539B-X0041-D02.
In another embodiment, the protein is a human antibody having the light and heavy chains of a variant of 539B-X0041-D02, e.g., a variant described herein. In another preferred embodiment, the protein is a human antibody having its heavy chain from a variant of 539B- X0041-D02, e.g., a variant described herein. In yet another preferred embodiment, the protein is a human antibody having its light chain from a variant of 539B-X0041-D02, e.g., a variant described herein.
In an even more preferred embodiment, the protein is a human antibody having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain from a variant of 539B-X0041-D02, e.g., a variant described herein. In another even more preferred embodiment, the protein is a human antibody having one or more (e.g., 1, 2, or 3) light chain
CDRs from the corresponding CDRs of the light chains from a variant of 539B-X0041-D02, e.g., a variant described herein. In another even more preferred embodiment, the protein is a human antibody having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chains from a variant of 539B-X0041-D02, e.g., a variant described herein and having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain from a variant of 539B-X0041-D02, e.g., a variant described herein.
In a more preferred embodiment, the protein is an antibody (e.g., human antibody) having the light and heavy chain variable regions of a variant of 539B-X0041-D02, e.g., a variant described herein. In another preferred embodiment, the protein is a human antibody having its heavy chain variable region from a variant of 539B-X0041-D02, e.g., a variant described herein. In yet another preferred embodiment, the protein is a human antibody having its light chain variable region from a variant of 539B-X0041-D02, e.g., a variant described herein.
In one embodiment, the HC and LC variable domain sequences are components of the same polypeptide chain. In another, the HC and LC variable domain sequences are components of different polypeptide chains. For example, the protein is an IgG, e.g., IgGl, IgG2, IgG3, or IgG4. The protein can be a soluble Fab (sFab). In other implementations the protein includes a Fab2', scFv, minibody, scFv::Fc fusion, Fab: :HS A fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen combining site of one of the binding proteins herein. The VH and VL regions of these Fabs can be provided as IgG, Fab, Fab2, Fab2', scFv, PEGylated Fab, PEGylated scFv, PEGylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA + VH::CH1, HSA: :LC + VH::CH1, or other appropriate construction.
In one embodiment, the protein is a human or humanized antibody or is non- immunogenic in a human. For example, the protein includes one or more human antibody framework regions, e.g., all human framework regions. In one embodiment, the protein includes a human Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or 99% identical to a human Fc domain.
In one embodiment, the protein is a primate or primatized antibody or is non- immunogenic in a human. For example, the protein includes one or more primate antibody framework regions, e.g., all primate framework regions. In one embodiment, the protein includes a primate Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or 99% identical to a primate Fc domain. "Primate" includes humans {Homo sapiens), chimpanzees {Pan troglodytes and Pan paniscus (bonobos)), gorillas {Gorilla gorilla), gibons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and tarsiers.
In certain embodiments, the protein includes no sequences from mice or rabbits (e.g., is not a murine or rabbit antibody).
In one embodiment, protein is physically associated with a nanoparticle, and can be used to guide a nanoparticle to a cell expressing MMP- 12 on the cell surface. In one embodiment, the protein causes effector cells (CDC or ADCC) to kill a cell which expresses MMP- 12. In some aspects, the MMP-12 binding protein is administered (alone or in combination with a second agent described herein) to a subject to treat a symptom of systemic sclerosis, e.g., Raynaud's syndrome, swelling (eventually skin tightening and contractures of the fingers), polyarthralgia, dysphagia, dyspnea, lung fibrosis, acute alveolitis, pulmonary hypertension, heartburn, cardiac arrhythmias or renal crisis, or other symptom described herein. The more evident symptom is usually the hardening of the skin and associated scarring. Blood vessels may also be more visible. Many SSc patients (over 80%) have vascular symptoms and Raynaud's phenomenon. During an attack, there is discoloration of the hands and feet in response to cold. Raynaud's normally affects the fingers and toes. SSc and Raynaud's can cause painful ulcers on the fingers or toes which are known as digital ulcers. Calcinosis is also common in SSc, and is often seen near the elbows, knees or other joints. Diffuse scleroderma can cause musculoskeletal, pulmonary, gastrointestinal, renal and other complications. Patients with larger amounts of cutaneous involvement are more likely to have involvement of the internal tissues and organs. In some aspects, the MMP-12 binding protein is administered (alone or in combination with a second agent (e.g., a second agent described herein)) to a subject to treat Sjogren's syndrome associated with SSc.
In some aspects, the MMP-12 binding protein is administered (alone or in combination with a second agent (e.g., a second agent described herein)) to a subject at risk of developing systemic sclerosis, e.g., with a familial predisposition for the disease (e.g., a polymorphism in COLl A2 or TGF-βl), or with another risk factor, e.g., a prior cytomegalovirus (CMV) infection, or exposure to organic solvents or other chemical agents (e.g., vinyl chloride, bleomycin, pentazocine, epoxy and/or aromatic hydrocarbons, contaminated rapeseed oil or L-tryptophan).
In another aspect, the method uses an MMP-12 binding protein that is a competitive inhibitor of MMP-12. In some embodiments, the binding protein competes with an MMP-12 substrate (e.g., lung extracellular matrix, elastin, gelatin, fibronectin, apo[a], apoB-100, collagen, osteonectin, TFPI, alpha 1-protease inhibitor, uPAR and CD14), e.g., binds to the same epitope as the substrate, e.g., and prevents substrate binding.
In some aspects, the method includes inhibiting an interaction between MMP-12 and an
MMP-12 substrate (e.g., lung extracellular matrix, elastin, gelatin, fibronectin, apo[a], apoB-100, collagen, osteonectin, TFPI, alpha 1-protease inhibitor, uPAR and CD14). The method includes contacting an MMP- 12 binding protein described herein with MMP- 12 (e.g., in vitro or in vivo), wherein the binding protein binds to MMP- 12 and thereby prevents the binding of an MMP- 12 substrate to MMP- 12. In some embodiments, the binding protein binds to the same epitope on MMP- 12 as the substrate, e.g., the binding protein is a competitive inhibitor. In some embodiments, the binding protein does not bind the same epitope as the substrate but causes a steric change in MMP- 12 that decreases or inhibits the ability of the substrate to bind.
In one aspect, the disclosure features an MMP-12 binding protein-drug conjugate that includes a MMP-12 binding protein (e.g., an MMP-12 binding protein described herein) and a drug.
In one embodiment, the binding protein comprises at least one immunoglobulin variable region, and/or the protein binds to and/or inhibits MMP-12, e.g., inhibits MMP-12 catalytic activity. In one embodiment, the drug is a cytotoxic or cytostatic agent. The cytotoxic agent can be, e.g., selected from the group consisting of an auristatin, a DNA minor groove binding agent, a DNA minor groove alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a podophyllotoxin, a baccatin derivative, a cryptophysin, a combretastatin, a maytansinoid, and a vinca alkaloid. In one embodiment, the cytotoxic agent is an auristatin and, e.g., the auristatin is selected from AFP, MMAF, MMAE, AEB, AEVB and auristatin E. In one embodiment, the auristatin is AFP or MMAF. In another embodiment, the cytotoxic agent is a maytansinoid and, e.g., the maytansinoid is selected from a maytansinol, maytansine, DMl, DM2, DM3 and DM4. In one embodiment, the maytansinoid is DMl. In another embodiment, the cytotoxic agent is selected from paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin- 10, echinomycin, combretatstatin, calicheamicin, and netropsin. In one embodiment, the cytotoxin is an auristatin, a maytansinoid, or calicheamicin.
In one embodiment, the cytotoxic agent is an antitubulin agent and, e.g., the antitubulin agent is selected from AFP, MMAP, MMAE, AEB, AEVB, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicines, colcimid, estramustine, cemadotin, discodermolide, maytansinol, maytansine, DMl, DM2, DM3, DM4 and eleutherobin.
In one embodiment, the MMP-12 binding protein (e.g., antibody) is conjugated to the cytotoxic agent via a linker. In one embodiment, the linker is cleavable under intracellular conditions, e.g., the cleavable linker is a peptide linker cleavable by an intracellular protease. In one embodiment, the linker is a peptide linker, e.g., a dipeptide linker, e.g., a val-cit linker or a phe-lys linker. In one embodiment, the cleavable linker is hydrolyzable at a pH of less than 5.5, e.g., the hydrolyzable linker is a hydrazone linker. In another embodiment, the cleavable linker is a disulfide linker.
A binding protein described herein can be provided as a pharmaceutical composition, e.g., including a pharmaceutically acceptable carrier. The composition can be at least 10, 20, 30, 50, 75, 85, 90, 95, 98, 99, or 99.9% free of other protein species. In some embodiments, the binding protein can be produced under GMP (good manufacturing practices). In some embodiments, the binding protein is provided in pharmaceutically acceptable carriers, e.g., suitable buffers or excipients.
The dose of a binding protein (e.g., a pharmaceutical composition containing a binding protein described herein) is sufficient to block about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% of the activity of MMP-12 in the patient, e.g., at the site of disease. Depending on the disease, this may require a dose, e.g., of between about 0.01 mg/Kg to about 100 mg/Kg, e.g., between about 0.1 and about 10 mg/Kg. For example, the dose can be a dose of about 0.1, about 1, about 3, about 6, or about 10 mg/Kg. For example, for an IgG having a molecular mass of 150,000 g/mole (2 binding sites), these doses correspond to approximately 18 nM, 180 nM, 540 nM, 1.08 microM, and 1.8 microM, respectively, of binding sites for a 5 L blood volume. Medicine being partly an art, the optimal dose will be established by clinical trials, but will most likely lie in this range.
An MMP-12 binding protein can be used to detect MMP-12 in a subject. The method includes: administering an MMP-12 binding protein (e.g., an MMP-12 binding protein described herein) to a subject; and detecting the protein in the subject, e.g., detecting an interaction between the protein and the MMP- 12, if present. In some embodiments, the protein further includes a detectable label.
In another aspect, the disclosure features a method of modulating MMP- 12 activity. The method includes: contacting an MMP-12 with an MMP-12 binding protein (e.g., an MMP-12 binding protein described herein) (e.g., in a human subject), thereby modulating MMP-12 activity. In some embodiments, the binding protein inhibits MMP-12 activity (e.g., inhibits MMP-12 catalytic activity).
In another aspect, the disclosure features a method of treating systemic sclerosis. The method includes: administering, to a subject, an MMP-12 binding protein (e.g., an MMP-12 binding protein described herein) in an amount sufficient to treat systemic sclerosis in the subject.
In another aspect, the disclosure features a method of treating Sjogren's syndrome associated with SSc. The method includes: administering, to a subject, an MMP-9 binding protein (e.g., an MMP-12 binding protein described herein) in an amount sufficient to treat Sjogren's syndrome associated with SSc in the subject.
Other exemplary therapeutic methods that include administering an MMP-12 binding protein are described below. An MMP-12 binding protein described herein can be administered in combination with one or more other MMP inhibitors, e.g., small molecule inhibitors, e.g., broad specificity inhibitors. In one embodiment, the small molecule inhibitors are one or more of the small molecule inhibitors described herein. In another embodiment, the one or more MMP inhibitors include another MMP-12 binding protein, e.g., another MMP-12 binding protein described herein, or an MMP-9 binding protein, e.g., an MMP-9 binding protein described herein.
In one aspect, the disclosure features the use of an MMP-12 binding protein described herein for the manufacture of a medicament for the treatment of a disorder described herein, e.g., systemic sclerosis. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
DESCRIPTION OF DRAWINGS FIGURE 1 is a graph depicting inhibition assays of MMP- 12 using non-inhibitor binding proteins.
FIGURES 2A, 2B and 2C: FIGURE 2A is a line graph showing human MMP-12 activity (Fluo/min) in the presence of increasing concentrations (nM) of an MMP-12 binding protein (539B-M08H09). FIGURE 2B is a line graph showing the binding rate of elastin (dF/min) in the presence of increasing concentrations (nM) of an MMP-12 binding protein (539B-M08H09). FIGURE 2C is a line graph showing murine MMP-12 activity (Fluo/sec) in the presence of increasing concentrations (nM) of an MMP-12 binding protein (539B-M08H09).
FIGURES 3 A and 3B: FIGURE 3 A is a line graph showing measured IC50 (nM) of an MMP-12 binding protein (M08H09) versus substrate concentration (μM) of human MMP-12. FIGURE 3B is a line graph showing measured IC50 (nM) of an MMP-12 binding protein (M08H09) versus substrate concentration (μM) of murine MMP-12.
FIGURES 4 A and 4B: FIGURES 4 A and 4B are line graphs showing murine MMP-12 activity (Fluo/sec) in the presence of increasing concentrations (nM) of an MMP-12 binding protein (539B-M11H11). FIGURE 4A: Fab; FIGURE 4B: IgG FIGURE 5 is a bar graph showing ELISA competition assays with four different MMP-
12 binding proteins, M08H09, M0013-G12, M0016-A11 and R0062-E11.
FIGURE 6 is a bar graph showing the effect of various doses of MMP-12 binding proteins M08-H09 and Mil -HIl on peribronchial inflammation score in OVA-challenged mouse model of airway inflammation. FIGURES 7A and 7B are bar graphs showing eosinophil percentages (FIGURE 7A) and counts (FIGURE 7B) in OVA-challenged mouse model of airway inflammation administered various doses of MMP-12 binding proteins M08-H09 and Mil -HIl.
FIGURE 8 is a bar graph showing the effect of an MMP-12 binding protein (M08-H09) on inflammatory cell infiltration into a carrageenan-stimulated mouse air pouch. FIGURE 9 is a line graph showing human MMP- 12 activity (Fluo/sec) in the presence of increasing concentrations (nM) of an MMP-12 binding protein (539B-M131 A06) and an MMP- 12 binding protein (539B-M131A06-GA-S) which is a version of 539B-M131A06 that has been modified to remove a glycosylation site. FIGURES 1OA abd 1OB summarize the identification of amino acid changes in affinity matured variant HV-CDRs (cycles 1 and 2) that contribute to improvement in affinity and inhibition properties.
FIGURES 11 A and HB: FIGURE 11 A is a line graph showing human MMP-9 activity (Fluo/sec) in the presence of increasing concentrations (nM) of an MMP-9 binding protein (539A-M0166-F10). FIGUREIlB is a table showing that an MMP-9 binding protein (539A- M0166-F10) is specific for human MMP-9.
FIGURE 12 is a line graph showing measured IC50 (nM) versus substrate concentration (μM).
FIGURE 13 is a series of line graphs showing IC50 measurements at various concentrations of substrate.
DETAILED DESCRIPTION
Subjects with systemic sclerosis (SSc) have higher serum concentrations of MMP-9 and TIMP-I, and a higher ratio of MMP-9 to TIMP-I than healthy controls. The serum concentrations of MMP-9 correlated well with the degree of skin involvement, as determined by the Rodnan score (thickness). Circulating TGF-β strongly correlated with MMP-9 concentrations. Dermal fibroblasts from patients with SSc produced more MMP-9 than those from healthy controls upon stimulation with IL-Ib, TNF-α, or TGF-β.
The overproduced MMP-9 may induce microvascular damage and leakage of substances that further augment endothelial cell damage or fibroblast activation in SSc.
Failure of endothelial cells to develop new vessels in response to hypoxia (caused by excessive collagen deposition) is a distinctive feature of systemic sclerosis (SSc). Matrix metalloproteinase-12 (MMP-12) blocks angiogenesis by cleavage of the endothelial urokinase- type plasminogen activator receptor (uPAR). The over-expression of MMP-12 by both SSc fibroblasts and SSc endothelial cells indicates that MMP-12 over-production may have a critical pathogenic role in SSc-associated vascular alterations. SSc MVEC-conditioned medium impaired uPA-dependent proliferation and invasion as well as capillary morphogenesis in normal microvascular endothelial cells (MVECs) in vitro. An anti-MMP-12 antibody can restore SSc MVEC function.
Excess tissue matrix accumulates in systemic sclerosis (SSc), accounting for both visceral and dermal fibrosis. Either decreased serum levels of matrix metalloproteinases (MMPs) or increased levels of tissue inhibitors of matrix metalloproteinases (TIMPs) may account for this matrix accumulation. TIMP-I levels are significantly raised in the serum of diffuse cutaneous SSc and limited cutaneous SSc patients compared with the normal controls. Serum MMP-I levels do not differ significantly between either of the SSc groups or between SSc groups and normal controls.
Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are 72- and 92-kD, respectively, type IV collagenases that are members of a group of secreted zinc metalloproteases which, in mammals, degrade the collagens of the extracellular matrix. Other members of this group include interstitial collagenase (MMP-I) and stromelysin (MMP-3).
MMP-2, the 72-kD type IV collagenase (also known as CLG4A), is secreted from normal skin fibroblasts, whereas MMP-9, the 92-kD collagenase (also known as CLG4B), is produced by normal alveolar macrophages and granulocytes. The present disclosure provides proteins that bind to MMP-9 and, in some instances, inhibit MMP-9 activity. Matrix metalloproteinase-12 (MMP- 12) is a type IV collagenase that is a member of a group of secreted zinc metalloproteases which, in mammals, degrade various proteins of the extracellular matrix. Other members of this group include interstitial collagenase (MMP-I) and stromelysin (MMP-3). MMP- 12 (a.k.a. macrophage elastase, macrophage metalloelastase, or matrix metalloproteinase 12) is thought to be involved in many disease states. Many small molecule inhibitors of MMP- 12 have been tested for safety and efficacy in cancer and other diseases. So far, all have lacked either sufficient potency or sufficient specificity or both. The present disclosure provides proteins that bind MMP-12, and in some instances, inhibit MMP-12 activity. In many instances, the disclosed MMP-12 binding proteins bind and inhibit human and mouse MMP12 enzyme activity. In other instances, MMP-12 binding proteins are disclosed that bind MMP-12 but do not inhibit MMP-12 activity. Such MMP-12 binding proteins are useful, e.g., to determine the presence of MMP-12. The term "binding protein" refers to a protein that can interact with a target molecule. This term is used interchangeably with "ligand." An "MMP-9 binding protein" refers to a protein that can interact with MMP-9, and includes, in particular, proteins that preferentially interact with and/or inhibit MMP-9. For example, the MMP-9 binding protein is an antibody. An "MMP-12 binding protein" refers to a protein that can interact with MMP-12, and includes, in particular, proteins that preferentially interact with and/or inhibit MMP-12. For example, the MMP-12 binding protein is an antibody.
The term "antibody" refers to a protein that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (de Wildt et al., Eur J Immunol. 1996; 26(3):629-39.)) as well as complete antibodies. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). Antibodies may be from any source, but primate (human and non-human primate) and primatized are preferred The VH and VL regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" ("FR"). The extent of the framework region and CDRs has been precisely defined (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. MoI. Biol. 196:901-917, see also www.hgmp.mrc.ac.uk). Kabat definitions are used herein. Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.
As used herein, an "immunoglobulin variable domain sequence" refers to an amino acid sequence which can form the structure of an immunoglobulin variable domain such that one or more (e.g., 1, 2, or 3) CDR regions are positioned in a conformation suitable for an antigen binding site. 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 omit 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 include other alterations. In one embodiment, a polypeptide that includes immunoglobulin variable domain sequence can associate with another immunoglobulin variable domain sequence to form an antigen binding site, e.g., a structure that preferentially interacts with an MMP-9 protein, e.g., the MMP-9 catalytic domain.
The VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. In IgGs, the heavy chain constant region includes three immunoglobulin domains, CHl, CH2 and CH3. The light chain constant region includes a CL domain. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system. The light chains of the immunoglobulin may be of types kappa or lambda. In one embodiment, the antibody is glycosylated. An antibody can be functional for antibody-dependent cytotoxicity and/or complement-mediated cytotoxicity.
One or more regions of an antibody can be human or effectively human. For example, one or more of the variable regions can be human or effectively human. For example, one or more of the CDRs can be human, e.g., HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3. Each of the light chain CDRs can be human. HC CDR3 can be human. One or more of the framework regions can be human, e.g., FRl, FR2, FR3, and FR4 of the HC or LC. For example, the Fc region can be human. In one embodiment, all the framework regions are human, e.g., derived from a human somatic cell, e.g., a hematopoietic cell that produces immunoglobulins or a non-hematopoietic cell. In one embodiment, the human sequences are germline sequences, e.g., encoded by a germline nucleic acid. In one embodiment, the framework (FR) residues of a selected Fab can be convertered to the amino-acid type of the corresponding residue in the most similar primate germline gene, especially the human germline gene. One or more of the constant regions can be human or effectively human. For example, at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of an immunoglobulin variable domain, the constant region, the constant domains (CHl, CH2, CH3, CLl), or the entire antibody can be human or effectively human. All or part of an antibody can be encoded by an immunoglobulin gene or a segment thereof. Exemplary human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the many immunoglobulin variable region genes. Full-length immunoglobulin "light chains" (about 25 KDa or about 214 amino acids) are encoded by a variable region gene at the NH2- terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH- terminus. Full-length immunoglobulin "heavy chains" (about 50 KDa or about 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids). The length of human HC varies considerably because HC CDR3 varies from about 3 amino-acid residues to over 35 amino-acid residues.
The term "antigen-binding fragment" of a full length antibody refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments encompassed within the term "antigen-binding fragment" of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab')2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (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 functionality. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv). See e.g., US patents 5,260,203, 4,946,778, and 4,881,175; Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. ScL USA 85:5879-5883. Antibody fragments can be obtained using any appropriate technique including conventional techniques known to those with skill in the art. The term "monospecific antibody" refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes a "monoclonal antibody" or "monoclonal antibody composition," which as used herein refer to a preparation of antibodies or fragments thereof of single molecular composition, irrespective of how the antibody was generated.
An "effectively human" immunoglobulin variable region is an immunoglobulin variable region that includes a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. An "effectively human" antibody is an antibody that includes a sufficient number of human amino acid positions such that the antibody does not elicit an immunogenic response in a normal human.
A "humanized" immunoglobulin variable region is an immunoglobulin variable region that is modified to include a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. Descriptions of "humanized" immunoglobulins include, for example, US 6,407,213 and US 5,693,762.
As used herein, "binding affinity" refers to the apparent association constant or K3. The K3 is the reciprocal of the dissociation constant (Kd). A binding protein may, for example, have a binding affinity of at least 105, 106, 107 ,108, 109, 1010 and 1011 M"1 for a particular target molecule, e.g., MMP-9, MMP-16, or MMP-24. Higher affinity binding of a binding protein to a first target relative to a second target can be indicated by a higher K3 (or a smaller numerical value Kd) for binding the first target than the K3 (or numerical value Kd) for binding the second target. In such cases, the binding protein has specificity for the first target (e.g., a protein in a first conformation or mimic thereof) relative to the second target (e.g., the same protein in a second conformation or mimic thereof; or a second protein). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, or 105 fold.
Binding affinity can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in TRIS-buffer (5OmM TRIS, 15OmM NaCl, 5mM CaCl2 at pH7.5). These techniques can be used to measure the concentration of bound and free binding protein as a function of binding protein (or target) concentration. The concentration of bound binding protein ([Bound]) is related to the concentration of free binding protein ([Free]) and the concentration of binding sites for the binding protein on the target where (N) is the number of binding sites per target molecule by the following equation:
[Bound] = N • [Free]/((1/Ka) + [Free]).
It is not always necessary to make an exact determination of K3, though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to K3, and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.
An "isolated composition" refers to a composition that is removed from at least 90% of at least one component of a natural sample from which the isolated composition can be obtained. Compositions produced artificially or naturally can be "compositions of at least" a certain degree of purity if the species or population of species of interests is at least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure on a weight-weight basis.
An "epitope" refers to the site on a target compound that is bound by a binding protein (e.g., an antibody such as a Fab or full length antibody). In the case where the target compound is a protein, the site can be entirely composed of amino acid components, entirely composed of chemical modifications of amino acids of the protein (e.g., glycosyl moieties), or composed of combinations thereof. Overlapping epitopes include at least one common amino acid residue, glycosyl group, phosphate group, sulfate group, or other molecular feature. An (first) antibody "binds to the same epitope" as another (second) antibody if the antibody binds to the same site on a target compound that the second antibody binds, or binds to a site that overlaps (e.g., 50%, 60%, 70%, 80%, 90%, or 100% overlap, e.g., in terms of amino acid sequence or other molecular feature (e.g., glycosyl group, phosphate group, or sulfate group) with the site that the second antibody binds. An (first) antibody "competes for binding" with another (second) antibody if the binding of the first antibody to its epitope decreases (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) the amount of the second antibody that binds to its epitope. The competition can be direct (e.g., the first antibody binds to an epitope that is the same as, or overlaps with, the epitope bound by the second antibody), or indirect (e.g., the binding of the first antibody to its epitope causes a steric change in the target compound that decreases the ability of the second antibody to bind to its epitope).
Calculations of "homology" or "sequence identity" between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.
In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. For example, the reference sequence may be the length of the immunoglobulin variable domain sequence. As used herein, the term "substantially identical" (or "substantially homologous") is used herein to refer to a first amino acid or nucleic acid sequence that contains a sufficient number of identical or equivalent (e.g., with a similar side chain, e.g., conserved amino acid substitutions) amino acid residues or nucleotides to a second amino acid or nucleic acid sequence such that the first and second amino acid or nucleic acid sequences have (or encode proteins having) similar activities, e.g., a binding activity, a binding preference, or a biological activity. In the case of antibodies, the second antibody has the same specificity and has at least 50%, at least 25%, or at least 10% of the affinity relative to the same antigen.
Sequences similar or homologous (e.g., at least about 85% sequence identity) to the sequences disclosed herein are also part of this application. In some embodiments, the sequence identity can be about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. In addition, substantial identity exists when the nucleic acid segments hybridize under selective hybridization conditions (e.g., highly stringent hybridization conditions), to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. 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. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: (1) low stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1% SDS at least at 500C (the temperature of the washes can be increased to 55°C for low stringency conditions); (2) medium stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 600C; (3) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and (4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified. The disclosure includes nucleic acids that hybridize with low, medium, high, or very high stringency to a nucleic acid described herein or to a complement thereof, e.g., nucleic acids encoding a binding protein described herein. The nucleic acids can be the same length or within 30, 20, or 10% of the length of the reference nucleic acid. The nucleic acid can correspond to a region encoding an immunoglobulin variable domain sequence described herein. An MMP-9 binding protein may have mutations (e.g., at least one, two, or four, and/or less than 15, 10, 5, or 3) relative to a binding protein described herein (e.g., conservative or nonessential amino acid substitutions), which do not have a substantial effect on protein function. Whether or not a particular substitution will be tolerated, i.e., will not adversely affect biological properties, such as binding activity can be predicted, e.g., by evaluating whether the mutation is conservative or by the method of Bowie, et al. (1990) Science 247:1306-1310.
A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). It is possible for many framework and CDR amino acid residues to include one or more conservative substitutions.
Motif sequences for biopolymers can include positions which can be varied amino acids. For example, the symbol "X" in such a context generally refers to any amino acid (e.g., any of the twenty natural amino acids or any of the nineteen non-cysteine amino acids). Other allowed amino acids can also be indicated for example, using parentheses and slashes. For example, "(A/W/F/N/Q)" means that alanine, tryptophan, phenylalanine, asparagine, and glutamine are allowed at that particular position. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of the binding agent, e.g., the antibody, without abolishing or more preferably, without substantially altering a biological activity, whereas changing an "essential" amino acid residue results in a substantial loss of activity.
The term "cognate ligand" refers to a naturally occurring ligand of an MMP-9, including naturally occurring variants thereof (e.g., splice variants, naturally occurring mutants, and isoforms).
Statistical significance can be determined by any art known method. Exemplary statistical tests include: the Students T-test, Mann Whitney U non-parametric test, and Wilcoxon non-parametric statistical test. Some statistically significant relationships have a P value of less than 0.05 or 0.02. Particular binding proteins may show a difference, e.g., in specificity or binding, that are statistically significant (e.g., P value < 0.05 or 0.02). The terms "induce", "inhibit", "potentiate", "elevate", "increase", "decrease" or the like, e.g., which denote distinguishable qualitative or quantitative differences between two states, and may refer to a difference, e.g., a statistically significant difference, between the two states.
The singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.
The term "agonist", as used herein, is meant to refer to an agent that mimics or up- regulates (e.g., potentiates or supplements) the bioactivity of a protein. An agonist can be a wild- type protein or derivative thereof having at least one bioactivity of the wild-type protein. An agonist can also be a compound that upregulates expression of a gene or which increases at least one bioactivity of a protein. An agonist can also be a compound which increases the interaction of a polypeptide with another molecule, e.g., a target peptide or nucleic acid.
"Antagonist" as used herein is meant to refer to an agent that downregulates (e.g., suppresses or inhibits) at least one bioactivity of a protein. An antagonist can be a compound which inhibits or decreases the interaction between a protein and another molecule, e.g., a target peptide or enzyme substrate. An antagonist can also be a compound that downregulates expression of a gene or which reduces the amount of expressed protein present.
A "patient", "subject" or "host" to be treated by the subject method may mean either a human or non-human animal.
The term "preventing" or to "prevent" a disease in a subject refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease is prevented, that is, administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) so that it protects the host against developing the unwanted condition. "Preventing" a disease may also be referred to as "prophylaxis" or "prophylactic treatment." Sequences similar or homologous (e.g., at least about 85% sequence identity) to the sequences disclosed herein are also part of this application. In some embodiments, the sequence identity can be about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. In addition, substantial identity exists when the nucleic acid segments hybridize under selective hybridization conditions (e.g., highly stringent hybridization conditions), to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. The term "treat" or "treatment" refers to the application or administration of an agent, alone or in combination with one or more other agents (e.g., a second agent) to a subject, e.g., a patient, e.g., a patient who has a disorder (e.g., a disorder as described herein), a symptom of a disorder or a predisposition for a disorder, e.g., to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder. Treating a cell refers to a reduction in an activity of a cell. A reduction does not necessarily require a total elimination of activity, but a reduction, e.g., a statistically significant reduction, in the activity or the number of cells.
MMP-9 Binding Proteins The disclosure provides proteins that bind to MMP-9 (e.g., human MMP-9) and include at least one immunoglobin variable region. For example, the MMP-9 binding protein includes a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence. A number of exemplary MMP-9 binding proteins are described herein. The MMP-9 binding protein may be an isolated protein (e.g., at least 70, 80, 85, 90, 91,
92, 93, 94, 95, 96, 97, 98, or 99% free of other proteins).
The MMP-9 binding protein may additionally inhibit MMP-9, e.g., human MMP-9. The binding protein can inhibit the catalytic activity of MMP-9 (e.g., human MMP-9). In one embodiment, the protein binds the catalytic domain of human MMP-9, e.g., the protein contacts residues in or near the active site of MMP-9. In some embodiments, the protein does not contact residues in or near the active site of MMP-9 but instead binds elsewhere on MMP-9 and causes a steric change in MMP-9 that affects (e.g., inhibits) its activity.
Exemplary MMP-9 binding proteins include DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06, or proteins that comprise the HC and/or LC CDRs of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, and M0166-F10, or proteins that comprise the HC and/or LC variable regions of DX-2802, 539A- M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06MMP-9 binding proteins may be antibodies. MMP-9 binding antibodies may have their HC and LC variable domain sequences included in a single polypeptide (e.g., scFv), or on different polypeptides (e.g., IgG or Fab). In some aspects, MMP-9/MMP-2 dual binding prioteins (e.g., antibodies, e.g., human antibodies) can be used in the methods described herein.
Matrix Metalloproteinase 9 (MMP-9) MMP-9 Sequences. MMP-9 is encoded by a gene designated as MMP9 with full name
Matrix metalloproteinase-9 precursor. Synonyms for MMP-9 include matrix metalloproteinase 9, gelatinase B (GELB), 92kDa gelatinase (CLG4B), 92kDa type IV collagenase (EC 3.4.24.35). The DNA sequence is known for Homo sapiens and Mus musculus. An exemplary cDNA sequence encoding human MMP9 and the amino acid sequence are shown below. Exemplary cDNA sequences encoding murine MMP9 and amino acid sequences are also shown below. An exemplary MMP-9 protein can include the human or mouse MMP-9 amino acid sequence, a sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to one of these sequences, or a fragment thereof, e.g., a fragment without the signal sequence or prodomain.
Table A shows the similar genes in other organisms and the percentage of similarity with human MMP-9. No similarity-to-human data found for MMP9 for: chimpanzee {Pan troglodytes), pig (Sus scrofa), cow (Bos taurus), fruit fly (Drosophila melanogaster), worm (Caenorhabditis elegans), baker's yeast (Saccharomyces cerevisiae), tropical clawed frog (Silurana tropicalis), African malaria mosquito {Anopheles gambiae), green algae (Chlamydomonas reinhardtii), soybean (Glycine max), barley (Hordeum vulgare), tomato (Lycopersicon esculentum), rice blast fungus (Magnaporthe grisea), sugarcane (Saccharum officinarum), loblolly pine (Pinus taeda), corn (Zea mays), wheat (Triticum aestivum), Alicante grape (Vitis vinifera), bread mold (Neurospora crassa), fission yeast (Schizosaccharomyces pombe), sea squirt (Ciona intestinalis), amoeba (Dictyostelium discoideum), A. gosspyii yeast (Ashbya gossypii), K. lactis yeast (Kluyveromyces lactis), medicago trunc (Medicago truncatula), malaria parasite {Plasmodium falciparum), schistosome parasite {Schistosoma mansoni), sorghum {Sorghum bicolor), toxoplasmosis {Toxoplasma gondii). cDNA and amino acid sequences of human MMP9
ACCESSION AK123156
VERSION AK123156.1 GI:34528630
translation^1 MARKGARRP RQGPGSHKWLQPGSRRE KERIPQPPPPARPPRD AA PRRVLVPAVRRVPESGHFAGRPWAPQCHPKGLRRP SAESHSVAQAGVQCHDLGSLQPP PPSSGDSPASASRVAGITSTVPGTLSALDDCCLITELPYKPPAVLY"
1 acactttgcg ttccgcggcc ccggcccctt ggtttcctag tcctggctcc attcccctct 61 caggcctagg gctgggaccc ctccccgccc ccggtcttgg ccctgccccc ttcaacagac
121 ggtccgcccc ggcccctccc cctcgtcccg cccggccctg gcaggccccg ccccctgcgg
181 cctctacctt tgacgtcttc ccccgggagg tggcgggggt ctgcgaccga atgccggcgg
241 gactctgggt cagggcttct ggcgggccct gcggggggca gcgaggtgac cgtgaacctg
301 cggctcatgg cgcggaaagg agccaggcgg ccgcggcaag gtccgggatc gcacaagtgg 361 ctgcaaccag gctctaggag ggagaaagag cggatccccc aaccccctcc gcccgcccgc
421 cccccgcgag acgcggcgcc gcgcagggtc ctagtgcccg ctgtgcgaag ggttcctgaa
481 tctggccact tcgctgggag gccctgggct ccccagtgcc acccgaaggg cctgaggagg
541 ccatctgcag aatctcactc tgtcgcccag gccggagtgc agtgtcatga tcttggctca
601 ctgcaacctc cgcctcccag ttcaggagat tctcctgcct cagcctcccg ggtggctggg 661 attacaagca cagtgcctgg cacattatcg gcacttgatg actgttgtct aataactgag
721 cttccataca aaccacctgc cgtcctgtac tgaaggagaa agagcttcca gccggggagg
781 caggaaatct gggtcctggt cttggttgca tccctgactt cctaaatgac ctggagaagg
841 cctctgcctc tgctgggatc ttgtctgtgc tggggcattt gtttccattt ccaagggctt
901 tttcttcctc gctcagaatt tgaccactca ctaagaggag cttagtgtgg tgtctcacga 961 agggatcctc ctcagccctc acctcggtac tggaagacgt cgtgcgtgtc caaaggcacc
1021 ccggggaaca tccggtccac ctcgctggcg ctccggggat ccaccatctg cgccttcacg
1081 tcgaacctgc gggcaggcgc ggaggagaca ggtgctgagc cggctagcgg acggaccgac
1141 ggcgcccggg ctccccctgc cggcggccgc ggcggcgctc acctccagag gcgccgcccg
1201 ctgaacagca gcatcttccc cctgccactc cggagggccc cggtcacctg ggccacgtcg 1261 gcgcccaggc ccagcttgtc cagacgcctc gggcccagca ccgacgcgcc tgtgtacacc
1321 cacacctggc gccctgcagg ggaggagggt cacgtcggtt tgggggcgca gagggagcac
1381 gtactcctag aacgcgagga gggagattcc ggcgaggcct ttcctagccc gcgtgcccgc
1441 agtccctgca acccaggggc agaggcgctg ggtagagcga cgcgagggcg tggagaggag
1501 ggggcagaaa ctcagccgcc cctacgtttg ctaaactgcg tccgccaggg ggcgtatttt 1561 tctaaaacgc acaagacgtt tcgtgggtta tcgatggtct cttgagcctc cttgactgat
1621 ggggattgac cgggcggggg agggaaagta ggtaactaac cagagaagaa gaaaagcttc
1681 ttggagagcg gctcctcaaa gaccgagtcc agcttgcggg gcagcgcggg ccacttgtcg
1741 gcgataagga aggggccctg cggccggctc cccctgccct cagagaatcg ccagtacttc
1801 ctgagaaagc gaggagggaa aggacgggct ctaagccttg gacacagggc cagtgggcgg 1861 gaagggacgg gcagcccctc cgcaaagccc cctcccgcat ccacacaacc ccgcctcctc
1921 acccatcctt gaacaaatac agctggttcc caatc cDNA and amno acid sequences of mouse MMP9
ACCESSION NM_013599
VERSION NM_013599.2 GI : 31560795 translation= "MSPWQPLLLALLAFGCS SAAPYQRQP TFVVFPKDLKTSNLTDTQ LAEAYLYRYGYTRAAQMMGEKQSLRPALLMLQKQLSLPQTGELDSQTLKAIRTPRCGV PDVGRFQTFKGLKWDHHNITYWIQNYSEDLPRDMIDDAFARAFAVWGEVAPLTFTRVY GPEADIVIQFGVAEHGDGYPFDGKDGLLAHAFPPGAGVQGDAHFDDDELWSLGKGVVI PTYYGNSNGAPCHFPFTFEGRSYSACTTDGRNDGTPWCSTTADYDKDGKFGFCPSERL YTEHGNGEGKPCVFPFIFEGRSYSACTTKGRSDGYRWCATTANYDQDKLYGFCPTRVD ATWGGNSAGELCVFPFVFLGKQYSSCTSDGRRDGRLWCATTSNFDTDKKWGFCPDQG YSLFLVAAHEFGHALGLDHSSVPEALMYPLYSYLEGFPLNKDDIDGIQYLYGRGSKPD PRPPATTTTEPQPTAPPTMCPTIPPTAYPTVGPTVGPTGAPSPGPTSSPSPGPTGAPS PGPTAPPTAGSSEASTESLSPADNPCNVDVFDAIAEIQGALHFFKDGWYWKFLNHRGS PLQGPFLTARTWPALPATLDSAFEDPQTKRVFFFSGRQMWVYTGKTVLGPRSLDKLGL GPEVTHVSGLLPRRLGKALLFSKGRVWRFDLKSQKVDPQSVIRVDKEFSGVPWNSHDI FQYQDKAYFCHGKFFWRVSFQNEVNKVDHEVNQVDDVGYVTYDLLQCP "
1 ctcaccatga gtccctggca gcccctgctc ctggctctcc tggctttcgg ctgcagctct 61 gctgcccctt accagcgcca gccgactttt gtggtcttcc ccaaagacct gaaaacctcc 121 aacctcacgg acacccagct ggcagaggca tacttgtacc gctatggtta cacccgggcc 181 gcccagatga tgggagagaa gcagtctcta cggccggctt tgctgatgct tcagaagcag 241 ctctccctgc cccagactgg tgagctggac agccagacac taaaggccat tcgaacacca 301 cgctgtggtg tcccagacgt gggtcgattc caaaccttca aaggcctcaa gtgggaccat 361 cataacatca catactggat ccaaaactac tctgaagact tgccgcgaga catgatcgat 421 gacgccttcg cgcgcgcctt cgcggtgtgg ggcgaggtgg cacccctcac cttcacccgc 481 gtgtacggac ccgaagcgga cattgtcatc cagtttggtg tcgcggagca cggagacggg 541 tatcccttcg acggcaagga cggccttctg gcacacgcct ttccccctgg cgccggcgtt 601 cagggagatg cccatttcga cgacgacgag ttgtggtcgc tgggcaaagg cgtcgtgatc 661 cccacttact atggaaactc aaatggtgcc ccatgtcact ttcccttcac cttcgaggga 721 cgctcctatt cggcctgcac cacagacggc cgcaacgacg gcacgccttg gtgtagcaca 781 acagctgact acgataagga cggcaaattt ggtttctgcc ctagtgagag actctacacg 841 gagcacggca acggagaagg caaaccctgt gtgttcccgt tcatctttga gggccgctcc 901 tactctgcct gcaccactaa aggccgctcg gatggttacc gctggtgcgc caccacagcc 961 aactatgacc aggataaact gtatggcttc tgccctaccc gagtggacgc gaccgtagtt 1021 gggggcaact cggcaggaga gctgtgcgtc ttccccttcg tcttcctggg caagcagtac 1081 tcttcctgta ccagcgacgg ccgcagggat gggcgcctct ggtgtgcgac cacatcgaac 1141 ttcgacactg acaagaagtg gggtttctgt ccagaccaag ggtacagcct gttcctggtg 1201 gcagcgcacg agttcggcca tgcactgggc ttagatcatt ccagcgtgcc ggaagcgctc 1261 atgtacccgc tgtatagcta cctcgagggc ttccctctga ataaagacga catagacggc 1321 atccagtatc tgtatggtcg tggctctaag cctgacccaa ggcctccagc caccaccaca 1381 actgaaccac agccgacagc acctcccact atgtgtccca ctatacctcc cacggcctat 1441 cccacagtgg gccccacggt tggccctaca ggcgccccct cacctggccc cacaagcagc 1501 ccgtcacctg gccctacagg cgccccctca cctggcccta cagcgccccc tactgcgggc 1561 tcttctgagg cctctacaga gtctttgagt ccggcagaca atccttgcaa tgtggatgtt 1621 tttgatgcta ttgctgagat ccagggcgct ctgcatttct tcaaggacgg ttggtactgg 1681 aagttcctga atcatagagg aagcccatta cagggcccct tccttactgc ccgcacgtgg 1741 ccagccctgc ctgcaacgct ggactccgcc tttgaggatc cgcagaccaa gagggttttc 1801 ttcttctctg gacgtcaaat gtgggtgtac acaggcaaga ccgtgctggg ccccaggagt 1861 ctggataagt tgggtctagg cccagaggta acccacgtca gcgggcttct cccgcgtcgt 1921 ctcgggaagg ctctgctgtt cagcaagggg cgtgtctgga gattcgactt gaagtctcag 1981 aaggtggatc cccagagcgt cattcgcgtg gataaggagt tctctggtgt gccctggaac 2041 tcacacgaca tcttccagta ccaagacaaa gcctatttct gccatggcaa attcttctgg 2101 cgtgtgagtt tccaaaatga ggtgaacaag gtggaccatg aggtgaacca ggtggacgac 2161 gtgggctacg tgacctacga cctcctgcag tgcccttgaa ctagggctcc ttctttgctt 2221 caaccgtgca gtgcaagtct ctagagacca ccaccaccac caccacacac aaaccccatc 2281 cgagggaaag gtgctagctg gccaggtaca gactggtgat ctcttctaga gactgggaag 2341 gagtggaggc aggcagggct ctctctgccc accgtccttt cttgttggac tgtttctaat 2401 aaacacggat ccccaacctt ttccagctac tttagtcaat cagcttatct gtagttgcag 2461 atgcatccga gcaagaagac aactttgtag ggtggattct gaccttttat ttttgtgtgg 2521 cgtctgagaa ttgaatcagc tggcttttgt gacaggcact tcaccggcta aaccacctct 2581 cccgactcca gcccttttat ttattatgta tgaggttatg ttcacatgca tgtatttaac 2641 ccacagaatg cttactgtgt gtcgggcgcg gctccaaccg ctgcataaat attaaggtat 2701 tcagttgccc ctactggaag gtattatgta actatttctc tcttacattg gagaacacca 2761 ccgagctatc cactcatcaa acatttattg agagcatccc tagggagcca ggctctctac 2821 tgggcgttag ggacagaaat gttggttctt ccttcaagga ttgctcagag attctccgtg 2881 tcctgtaaat ctgctgaaac cagaccccag actcctctct ctcccgagag tccaactcac 2941 tcactgtggt tgctggcagc tgcagcatgc gtatacagca tgtgtgctag agaggtagag 3001 ggggtctgtg cgttatggtt caggtcagac tgtgtcctcc aggtgagatg acccctcagc 3061 tggaactgat ccaggaagga taaccaagtg tcttcctggc agtctttttt aaataaatga 3121 ataaatgaat atttacttaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3181 aaaaa / /
ACCESSION NP_038627 VERSION NP_038627 . 1 GI : 7305277
1 mspwqpllla llafgcssaa pyqrqptfvv fpkdlktsnl tdtqlaeayl yrygytraaq
61 mmgekqslrp allmlqkqls lpqtgeldsq tlkairtprc gvpdvgrfqt fkglkwdhhn
121 itywiqnyse dlprdmidda farafavwge vapltftrvy gpeadiviqf gvaehgdgyp 181 fdgkdgllah afppgagvqg dahfdddelw slgkgvvipt yygnsngapc hfpftfegrs
241 ysacttdgrn dgtpwcstta dydkdgkfgf cpserlyteh gngegkpcvf pfifegrsys
301 acttkgrsdg yrwcattany dqdklygfcp trvdatvvgg nsagelcvfp fvflgkqyss
361 ctsdgrrdgr lwcattsnfd tdkkwgfcpd qgyslflvaa hefghalgld hssvpealmy
421 plysylegfp lnkddidgiq ylygrgskpd prppatttte pqptapptmc ptipptaypt 481 vgptvgptga pspgptssps pgptgapspg ptapptagss easteslspa dnpcnvdvfd
541 aiaeiqgalh ffkdgwywkf lnhrgsplqg pfltartwpa lpatldsafe dpqtkrvfff
601 sgrqmwvytg ktvlgprsld klglgpevth vsgllprrlg kallfskgrv wrfdlksqkv
661 dpqsvirvdk efsgvpwnsh difqyqdkay fchgkffwrv sfqnevnkvd hevnqvddvg
721 yvtydllqcp / /
Figure imgf000058_0001
Figure imgf000059_0001
Domains of MMP-9. MMP-9 belongs to the peptidase MlOA family. MMP-9 consists of five domains; the amino-terminal and zinc-binding domains shared by all members of the secreted metalloprotease gene family, the collagen-binding fibronectin-like domain also present in the 72-kDa type IV collagenase, a carboxyl-terminal hemopexin-like domain shared by all known enzymes of this family with the exception of PUMP-I, and a unique 54-amino-acid- long proline-rich domain homologous to the alpha 2 chain of type V collagen (Wilhelm et al. (1989) /. Biol. Chem. 264, 17213-17221) (Table B).
Figure imgf000059_0002
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Factors that regulate MMP-9. The catalytic activity of MMP-9 is inhibited by histatin-3 1/24 (histatin-5). MMP-9 is activated by urokinase-type plasminogen activator; plasminogen; IL-lbeta, 4-aminophenylmercuric acetate and phorbol ester. MMP-9 exists as monomer, disulfide-linked homodimer, and as a heterodimer with a 25 kDa protein. Macrophages and transformed cell lines produce only the monomeric MMP-9, the hetrodimeric form is produced by normal alveolar macrophages and granulocytes. The processing of the precursor yields different active forms of 64, 67 and 82 kDa. Sequentially processing by MMP-3 yields the 82 kDa matrix metalloproteinase-9. In arthritis patients, this enzyme can contribute to the pathogenesis of joint destruction and can be a useful marker of disease status.
Endogenous inhibitors of MMP-9. MMP-9 has a number of endogenous inhibitors. Like other MMPs, MMP-9 is inhibited by TIMPs (Murphy, G., and Willenbrock, F. (1995) Methods Enzymol. 248, 496-510). A characteristic of MMP-9 (and MMP-2) is the ability of their zymogens to form tight non-covalent and stable complexes with TIMPs. It has been shown that pro-MMP-2 binds TIMP-2 (Goldberg et al. (1989) Proc. Natl. Acad. ScL U. S. A. 86, 8207- 8211), whereas pro-MMP-9 binds TIMP-I (Wilhelm et al. (1989) /. Biol. Chem. 264, 17213- 17221). TIMPs typically are slow, tight binding inhibitors. A MMP-9 binding protein (e.g., antibody) selected from a library of phage-displayed proteins can be selected have more rapid kinetics. For example, recombinant TIMP-I can be administered to inhibit MMP-9, e.g., in combination with a MMP-9 binding protein decscribed herein.
Small molecule inhibitors of MMP-9. Skiles et al. (2004, Curr Med Chem, 11:2911-77) reported that first generation small-molecule MMP inhibitors had poor bioavailability and the second generation had caused musculoskeletal pain and inflammation. Most small-molecule MMP inhibitors interact with the catalytic zinc but have fairly low affinity. Thus, a higher concentration is needed to have effect. The interaction with the catalytic zinc leads to inhibition of other MMPs and toxic side effects. A MMP-9 binding protein described herein can be used in combination with a small molecule inhibitor. For example, because the inhibitors are used in combination, the dose of the small molecule used can be decreased and therefore result in fewer side effects. Examples of small molecule MMP-9 inhibitors include small synthetic anthranilic acid-based inhibitors (see, e.g., Calbiochem Inhibitor-I, catalogue #444278 and Levin et al., 2001, Bioorg. Med. Chem. Lett. 11:2975-2978).
Small interfering RNA inhibitors of MMP-9. MMP-9 can be inhibited by small interfering RNA (siRNA). Examples of siRNA that can be used include:
MMP-9 siRNA
5'- GACUUGCCGCGAGACAUGAtt -3' 3'- ttCUGAACGGCGCUCUGUACU -5'
Control RNA (mismatch)
5'- GACUUCGCGGGACACAUGAtt -3'
3'- ttCUGAAGCGCCCUGUGUACU -5'
See also Kawasaki et al., Feb. 10, 2008, Nat. Med. advance on-line publication doi:10.1038/nml723. The siRNA can be administered to inhibit MMP-9, e.g., in combination with a MMP-9 binding protein decscribed herein.
MMP-12 Binding Proteins
The disclosure provides proteins that bind to MMP-12 (e.g., human MMP-12 and/or murine MMP-12) and include at least one immunoglobin variable region. For example, the MMP-12 binding protein includes a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence. A number of exemplary MMP- 12 binding proteins are described herein.
The MMP- 12 binding protein may be an isolated protein (e.g., at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% free of other proteins). The MMP-12 binding protein may additionally inhibit MMP-12, e.g., human and/or murine MMP-12. The binding protein can inhibit the catalytic activity of MMP-12 (e.g., human MMP-12). In one embodiment, the protein binds the catalytic domain of human MMP-12, e.g., the protein contacts residues in or near the active site of MMP-12. In some embodiments, the protein does not contact residues in or near the active site of MMP-12 but instead binds elsewhere on MMP-12 and causes a steric change in MMP-12 that affects (e.g., inhibits) its activity.
Exemplary MMP-12 binding proteins include M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135- A06M0135-A07M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, MOl 18-Fl 1, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027- EIl. Preferably, the MMP-12 binding protein is M0030-A10, M0032-H09, M0038-A03, M0038-H04, M0039-B02, M0040-B05, M0041-A05, R011-B11, M0007-A10 (also referred to as M7A10) or M0008-E08 (also referred to as M8E8). Others include DX-2712 (also referred to as M0131-A06-GA-S), a mutant or variant of DX-2712 (e.g., as described herein), and 539B- X0041-D02. Others include proteins that comprise the HC and/or LC CDRs of these antibodies, or proteins that comprise the HC and/or LC variable regions of any of these antibodies.
MMP-12 binding proteins may be antibodies. MMP-12 binding antibodies may have their HC and LC variable domain sequences included in a single polypeptide (e.g., scFv), or on different polypeptides (e.g., IgG or Fab).
Matrix Metalloproteinase 12 (MMP-12)
MMP-12 Sequences. MMP-12 is encoded by a gene designated as MMP 12 with full name Matrix metalloproteinase- 12 precursor. Synonyms for MMP-12 include matrix metalloproteinase 12, macrophage elastase, macrophage metalloelastase. The DNA sequence is known for Homo sapiens and Mus musculus. An exemplary cDNA sequence encoding human MMP 12 and the amino acid sequence are shown below. Exemplary cDNA sequences encoding murine MMP 12 and amino acid sequences are also shown below. An exemplary MMP-12 protein can include the human or mouse MMP-12 amino acid sequence, a sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to one of these sequences, or a fragment thereof, e.g., a fragment without the signal sequence or prodomain.
cDNA and amino acid sequences of human MMP12
>embl I BC112301 I BC112301 Homo sapiens matrix metallopeptidase 12 (macrophage elastase), mRNA (cDNA clone MGC:138506 IMAGE : 8327769) , complete cds atgaagtttcttctaatactgct cctgcaggccactgcttctggagctcttcccctgaacagctctacaagcctggaaaaaaa taatgtgctatttggtgaaagatacttagaaaaattttatggccttgagataaacaaact tccagtgacaaaaatgaaatatagtggaaacttaatgaaggaaaaaatccaagaaatgca gcacttcttgggtctgaaagtgaccgggcaactggacacatctaccctggagatgatgca cgcacctcgatgtggagtccccgatgtccatcatttcagggaaatgccaggggggcccgt atggaggaaacattatatcacctacagaatcaataattacacacctgacatgaaccgtga ggatgttgactacgcaatccggaaagctttccaagtatggagtaatgttacccccttgaa attcagcaagattaacacaggcatggctgacattttggtggtttttgcccgtggagctca tggagacttccatgcttttgatggcaaaggtggaatcctagcccatgcttttggacctgg atctggcattggaggggatgcacatttcgatgaggacgaattctggactacacattcagg aggcacaaacttgttcctcactgctgttcacgagattggccattccttaggtcttggcca ttctagtgatccaaaggccgtaatgttccccacctacaaatatgttgacatcaacacatt tcgcctctctgctgatgacatacgtggcattcagtccctgtatggagacccaaaagagaa ccaacgcttgccaaatcctgacaattcagaaccagctctctgtgaccccaatttgagttt tgatgctgtcactaccgtgggaaataagatctttttcttcaaagacaggttcttctggct gaaggtttctgagagaccaaagaccagtgttaatttaatttcttccttatggccaacctt gccatctggcattgaagctgcttatgaaattgaagccagaaatcaagtttttctttttaa agatgacaaatactggttaattagcaatttaagaccagagccaaattatcccaagagcat acattcttttggttttcctaactttgtgaaaaaaattgatgcagctgtttttaacccacg tttttataggacctacttctttgtagataaccagtattggaggtatgatgaaaggagaca gatgatggaccctggttatcccaaactgattaccaagaacttccaaggaatcgggcctaa aattgatgcagtcttctactctaaaaacaaatactactatttcttccaaggatctaacca atttgaatatgacttcctactccaacgtatcaccaaaacactgaaaagcaatagctggtt tggttgttagaaatggtgtaattaatggtttttgttagttcacttcagcttaataagtat ttattgcatatttgctatgtcctcagtgtaccactacttagagatatgtatcataaaaat aaaatctgtaaaccataggtaatgattatataaaatacataatatttttcaattttgaaa actctaattgtccattcttgcttgactctactattaagtttgaaaatagttaccttcaaa ggccaagagaattctatttgaagcatgctctgtaagttgcttcctaacat
>Amino acid sequence of human MMP12 (AAI12302.1)
MKFLLILLLQATASGALPLNSSTSLEKNNVLFGERYLEKFYGLEINKLPVTKMKYSGNLM KEKIQEMQHFLGLKVTGQLDTS TLEMMHAPRCGVPDVHHFREMPGGPVWRKHYITYRINN
YTPDMNREDVDYAIRKAFQVWSNVTPLKFSKINTGMADILWFARGAHGDFHAFDGKGGI
LAHAFGPGSGIGGDAHFDEDEFWTTHSGGTNLFLTAVHEIGHSLGLGHSSDPKAVMFPTY
KYVD INTFRLSADD IRGIQSLYGDPKENQRLPNPDNSEPALCDPNLSFDAVT TVGNKIFF
FKDRFFWLKVSERPKTSVNLISSLWPTLPSGIEAAYEIEARNQVFLFKDDKYWLISNLRP EPNYPKSIHSFGFPNFVKKIDAAVFNPRFYRTYFFVDNQYWRYDERRQMMDPGYPKLITK
NFQGIGPKIDAVFYSKNKYYYFFQGSNQFEYDFLLQRITKTLKSNSWFGC
Polymorphisms in the MMP- 12 gene are described, for example, in (2002) J Am Coll Cardiol. 3:40(l):43-8, (2002) Hum MoI Genet. l:ll(5):569-76., QXX)I) Stroke 32(9):2198-202., and (2000) Circ Res. 86(9):998-1003. cDNA and amno acid sequences of mouse MMP12
> Mouse MMP-12 cDNA sequence (NM_008605)
1 ACTCTGCTGAAAGGAGTCTGCACAATGAAATTTCTCATGATGATTGTGTTCTTACAGGTA 61 TCTGCCTGTGGGGCTGCTCCCATGAATGACAGTGAATTTGCTGAATGGTACTTGTCAAGA 121 TTTTATGATTATGGAAAGGACAGAATTCCAATGACAAAAACAAAAACCAATAGAAACTTC 181 CTAAAAGAAAAACTCCAGGAAATGCAGCAGTTCTTTGGGCTAGAAGCAACTGGGCAACTG
241 GACAACTCAACTCTGGCAATAATGCACATCCCTCGATGTGGAGTGCCCGATGTACAGCAT
301 CTTAGAGCAGTGCCCCAGAGGTCAAGATGGATGAAGCGGTACCTCACTTACAGGATCTAT
361 AATTACACTCCGGACATGAAGCGTGAGGATGTAGACTACATATTTCAGAAAGCTTTCCAA 421 GTCTGGAGTGATGTGACTCCTCTAAGATTCAGAAAGCTTCATAAAGATGAGGCTGACATT
481 ATGATACTTTTTGCATTTGGAGCTCACGGAGACTTCAACTATTTTGATGGCAAAGGTGGT 541 ACACTAGCCCATGCTTTTTATCCTGGACCTGGTATTCAAGGAGATGCACATTTTGATGAG 601 GCAGAAACGTGGACTAAAAGTTTTCAAGGCACAAACCTCTTCCTTGTTGCTGTTCATGAA
661 CTTGGCCATTCCTTGGGGCTGCAGCATTCCAATAATCCAAAGTCAATAATGTACCCCACC 721 TACAGATACCTTAACCCCAGCACATTTCGCCTCTCTGCTGATGACATACGTAACATTCAG
781 TCCCTCTATGGAGCCCCAGTGAAACCCCCATCCTTGACAAAACCTAGCAGTCCACCATCA 841 ACTTTCTGTCACCAAAGCTTGAGTTTTGATGCTGTCACAACAGTGGGAGAGAAAATCTTT 901 TTCTTTAAAGACTGGTTCTTCTGGTGGAAGCTTCCTGGGAGTCCAGCCACCAACATTACT 961 TCTATTTCTTCCATATGGCCAAGCATCCCATCTGGTATTCAAGCTGCTTACGAAATTGAA 1021 AGCAGAAATCAACTTTTCCTTTTTAAAGATGAGAAGTACTGGTTAATAAACAACTTAGTA 1081 CCAGAGCCACACTATCCCAGGAGCATATATTCCCTGGGCTTCTCTGCATCTGTGAAGAAG 1141 GTTGATGCAGCTGTCTTTGACCCACTTCGCCAAAAGGTTTATTTCTTTGTGGATAAACAC 1201 TACTGGAGGTATGATGTGAGGCAGGAGCTCATGGACCCTGCTTACCCCAAGCTGATTTCC 1261 ACACAC TTCC CAGGAAT C AAGC C T AAAAT T GAT GCAGTCCTC TAT T T C AAAAGACAC TAC 1321 TACATCTTCCAAGGAGCCTATCAATTGGAATATGACCCCCTGTTCCGTCGTGTCACCAAA 1381 ACATTGAAAAGTACAAGCTGGTTTGGTTGT
>Amino acid sequence of mouse MMP12 (based on accession number NM_008605)
MKFLMMIVFLQVSACGAAPMNDSEFAEWYLSRFYDYGKDRIPMTKTKTN RNFLKEKLQEMQQFFGLEATGQLDNSTLAIMHIPRCGVPDVQHLRAVPQ RSRWMKRYLTYRIYNYTPDMKREDVDYIFQKAFQVWSDVTPLRFRKLHK DEADIMILFAFGAHGDFNYFDGKGGTLAHAFYPGPGIQGDAHFDEAETW TKSFQGTNLFLVAVHELGHSLGLQHSNNPKSIMYPTYRYLNPSTFRLSA DDIRNIQSLYGAPVKPPSLTKPSSPPSTFCHQSLSFDAVTTVGEKIFFF KDWFFWWKLPGSPATNITSISSIWPSIPSGIQAAYEIESRNQLFLFKDE KYWLINNLVPEPHYPRSIYSLGFSASVKKVDAAVFDPLRQKVYFFVDKH YWRYDVRQELMDPAYPKLI STHFPGIKPKIDAVLYFKRHYYIFQGAYQL EYDP LFRRVTKTLKST SWFGC
Factors that regulate MMP- 12. Expression of MMP- 12 is regulated by many factors. Reports of upregulation include: Oncogene. 2004 Jan 22;23(3):845-51. (recurrence in stage I lung cancer, 2/10 cases), Ann Neurol. 2003 Jun;53(6):731-42. (collagenase-induced rat model of intracerebral hemorrhage), Cancer Res. 2005 May 15;65(10):4261-72. (protein kinase C/p53 resistant cells), Br J Dermatol. 2005 Apr;152(4):720-6. (Samples from nine patients with squamous cell carcinoma), Cardiovasc Res. 2005 May l;66(2):410-9. (Aging), J Immunol. 2005 Apr 15;174(8):4953-9. (Surfactant protein D -/- mice), J Biol Chem. 2005 Jun 3 ;280(22):21653-60. (Corneal wound repair ), Surgery. 2005 Apr;137(4):457-62. (periaortic application Of CaCl2 in mice), J Virol. 2005 Apr;79(8):4764-73. (murine viral encephalitis), Biochem Biophys Res Commun. 2005 Apr 29;330(l): 194-203. (cigarette smoke condensate in mice), Inflamm Res. 2005 Jan;54(l):31-6. (bronchoalveolar lavage and lung tissue from COPD patients), Am J Respir Cell MoI Biol. 2005 Apr;32(4):311-8. (Induction of human IL-lbeta in transgenic mice), Toxicol Pathol. 2004 May-Jun;32(3):351-6. (Cigarette smoke in mice), Am J Physiol Lung Cell MoI Physiol. 2004 Apr;286(4):L801-7. (lysosomal acid lipase gene knockout mice), J Neurosci. 2004 Feb 11;24(6): 1521-9. (Al adenosine receptor null mice), J Neuroimmunol. 1998 JuI 1;87(1- 2):62-72. (experimentally-induced delayed type hypersensitivity model of MS), Scand J Immunol. 2005 Jan;61(l): 10-7. (IL-4), J Pediatr Gastroenterol Nutr. 2005 Jan;40(l):60-6. (Enterocolitis), J Cell Physiol. 2005 Jul;204(l): 139-45. (Statins), J Immunol. 2004 Oct 15;173(8):5209-18. (experimental autoimmune encephalomyelitis), J Cardiovasc Pharmacol.
2004 Jul;44(l):57-65. (hypercholesterolemic hamsters with endothelial injury in the carotid artery), Cancer Metastasis Rev. 2004 Jan- Jun;23(l-2): 101-17. (colorectal cancer), Free Radic Biol Med. 2004 Mar 15;36(6):782-801. (oxidative stress), Chest. 2004 Feb;125(2):466-72. (Wood smoke, cigarette smoke, CPOD).
Down regulation or no upregulation is reported in Inflammation. 2003 Apr;27(2):107-13. (Mice immunized with type II collagen), Cancer Res. 2003 Jan l;63(l):256-62. (Epstein-Barr virus proteins; nasopharyngeal carcinoma), Curr Eye Res. 1998 Feb;17(2):132-40. (human interphotoreceptor matrix and vitreous from postmortem human eyes), and Scand J Immunol.
2005 Jan;61(l):10-7. (dexamethasone). Endogenous inhibitors of MMP- 12. MMP-12 has a number of endogenous inhibitors. Like other MMPs, MMP-12 is inhibited by TIMPs (Murphy, G., and Willenbrock, F. (1995) Methods Enzymol 248, 496-510).
Small molecule inhibitors of MMP-12. Small molecule inhibitors of MMP-12 have been synthesized and tested. Most of these have either insufficient potency or insufficient specificity, or both. The reports include: Proc Natl Acad Sci USA. 2005 Apr 12;102(15):5334-9. (acetohydroxamic acid and N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid); Arthritis Rheum. 2004 Oct;50(10):3275-85. (a general hydroxamate inhibitor of MMP activity); Arch Biochem Biophys. 2003 Jan 15;409(2):335-40. (peptide Iin24); J MoI Biol. 2001 Sep 28;312(4):743-51. (hydroxamic acid inhibitor, CGS27023A); J MoI Biol. 2001 Sep 28;312(4):731-42. (batimastat (BB-94)); Anticancer Res. 2001 Jan-Feb;21(l A): 145-55. (AE- 941, an orally bioavailable extract made of cartilage); J Comb Chem. 2000 Nov-Dec;2(6):624- 38. (XXX-Gpsi(PO2H-CH2)L-XXX library on beads); Biochim Biophys Acta. 2000 Mar 16;1478(l):51-60. (green tea polyphenols); J Leukoc Biol. 1984 May;35(5):449-57. (peptide chloromethyl ketone); Am Rev Respir Dis. 1982 Feb;125(2):203-7. (a battery of elastase inhibitors); Mem Inst Oswaldo Cruz. 2005 Mar;100: 167-172. (marimastat); J MoI Biol. 2004 Aug 20;341(4): 1063-76. (CP-271485, PF-00356231, and PD-0359601); Inflamm Res. 2003 Mar;52(3):95-100.; Bioorg Med Chem Lett. 2004 Oct 4;14(19):4935-9. (inhibitors with novel oxazoline zinc binding groups); and J Cardiovasc Pharmacol. 2004 Jul;44(l):57-65. (ONO-
4817). Other small molecule inhibitors of MMP-12 are described, e.g., in US Patent Application No: 20050014817 (Fluorothiophene derivatives), US Patent Application No.: 20050014816 (Thiophene amino acid derivatives), US Patent 6,770,640 (l-Carboxylmethyl-2-oxo-azepan derivatives), PCT Publication No.: WO200040577 (l-Carboxymethyl-2-Oxo-Azepan Derivatives), PCT Publication No.: WO 200532541 (Substituted Heterocyclic Mercaptosulfide Inhibitors), PCT Publication No.: WO 200183431, US Patent Application 20030158155, European Patent No.: 1288199, PCt Publication No.: WO 200040600 and US Patent 6,352,976, US Patent 6,350,907, US Patent 6,924,276, US Patent 6,916,807, US Patent 6,686,355, US Patent 6,548,477 and US Patent 5,506,242. The small molecule can be administered to inhibit MMP-12, e.g., in combination with a MMP-12 binding protein described herein. Small interfering RNA inhibitors of MMP-12. MMP- 12 can be inhibited by small interfering RNA (siRNA). Examples of siRNA that can be used are described in US Patent Publication No.: 20040087533 and PCT Publication No.: WO 200409098. The siRNA can be administered to inhibit MMP-12, e.g., in combination with a MMP-12 binding protein described herein.
Drug Conjugates
The MMP-9 binding proteins described herein can be conjugated to a drug (e.g., a cytotoxic, cytostatic, or immunomodulatory agent). The conjugates can be used therapeutically or prophylactically, e.g., the binding protein can target the drug, e.g., in vivo, e.g., to a site of disease (e.g., a tumor or site of inflammation), e.g., such that the drug affects the site of disease (e.g., causes a cytostatic or cytotoxic effect on targeted cells).
In some embodiments, the binding protein itself has therapeutic or prophylactic efficacy (e.g., the protein can modulate (e.g., antagonize) MMP-9 or -12, or cause a cytostatic or cytotoxic effect on a cell that expresses MMP-9 or -12 (e.g., an endothelial cell or tumor cell)). The binding protein-drug conjugate can be used such that the binding protein and drug both contribute (e.g., additively or synergistically) to an effect on MMP-9 or -12 (e.g., a therapeutic effect, e.g., in vivo, e.g., to a site of disease (e.g., a tumor or site of undesired angiogenesis or vascularization). The drug and/or binding protein can be, for example, cytotoxic, cytostatic or otherwise prevent or reduce the ability of a targeted cell to divide and/or survive (e.g., when the drug is taken up or internalized by the targeted cell and/or upon binding of the binding protein to MMP-9 or -12). For example, if the targeted cell is a cancer cell, the drug and/or binding protein can prevent or reduce the ability of the cell to divide and/or metastasize. Useful classes of drugs that can be used in the binding protein-drug conjugates described herein include cytotoxic or immunomodulatory agents such as, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, pre-forming compounds, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like.
Individual cytotoxic or immunomodulatory agents include, for example, an androgen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (actinomycin), daunorubicin, decarbazine, docetaxel, doxorubicin, an estrogen, 5- fluorodeoxyuridine, 5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, plicamycin, procarbazine, rapamycin (Sirolimus), streptozotocin, tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine, vincristine, vinorelbine, VP- 16 and VM-26.
In some typical embodiments, the drug comprises a cytotoxic agent. Suitable cytotoxic agents include, for example, dolastatins (e.g., auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (e.g., enediynes and lexitropsins), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide, eleutherobin, and mitoxantrone. In some embodiments, the drug is a cytotoxic agent such as AFP, MMAF, MMAE, AEB,
AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatstatin, chalicheamicin, maytansine, DM-I, or netropsin.
In some embodiments, the drug is a cytotoxic agent that comprises a conventional chemotherapeutic such as, for example, doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C or etoposide. In some embodiments, the drug can be a combined therapy, such as CHOP (Cyclophosphamide, Doxorubicin, Prednisolone and Vincristine), CHOP-R (Cyclophosphamide, Doxorubicin Vincristine, Prednisolone, and rituximab) or ABVD (Doxorubicin, Bleomycin, Vinblastine and Dacarbazine). Agents such as CC-1065 analogues (e.g., DCl), calicheamicin, maytansine, analogues of dolastatin 10, rhizoxin, and palytoxin can also be used. In specific embodiments, the drug can be a cytotoxic or cytostatic agent that comprises auristatin E (also known in the art as dolastatin-10) or a derivative thereof. Typically, the auristatin E derivative is, e.g., an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other auristatin derivatives include AFP, MMAF, and MMAE. The synthesis and structure of auristatin E and its derivatives are described in US 20030083263 and US 20050009751, and U.S. Pat. Nos. 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414. In some preferred embodiments, MMAF or AFP is used.
In specific embodiments, the drug is a cytotoxic agent that comprises a DNA minor groove binding agent. See, e.g., U.S. Pat. No. 6,130,237. For example, in some embodiments, the minor groove binding agent is a CBI compound. In other embodiments, the minor groove binding agent is an enediyne (e.g., calicheamicin). Examples of anti-tubulin agents that can be used in the MMP-9binding protein-drug conjugates include, but are not limited to, taxanes (e.g., TAXOL® (paclitaxel), TAXOTERE® (docetaxel)), T67 (Tularik), vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), and dolastatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB). Other antitubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine, cryptophysins, cemadotin, maytansinoids, combretastatins, discodermolide, eleutherobin, rhizoxin/maytansine, auristatin dolastatin 10 MMAE, and peloruside A.
In some embodiments, the drug is a cytotoxic agent such as an anti-tubulin agent. In some embodiments, the anti-tubulin agent is an auristatin, a vinca alkaloid, a podophyllotoxin, a taxane, a baccatin derivative, a cryptophysin, a maytansinoid, a combretastatin, or a dolastatin. In some embodiments, the antitubulin agent is AFP, MMAP, MMAE, AEB, AEVB, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP- 16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicines, colcimid, estramustine, cemadotin, discodermolide, maytansine, DMl, DM2, DM3, DM4, or eleutherobin. In some embodiments, the cytotoxic agent comprises a maytansinoid, another group of anti-tubulin agents. For example, in specific embodiments, the maytansinoid is maytansine or DM-I (ImmunoGen, Inc.; see also Chari et al. Cancer Res. 52:127-131 (1992)). In some embodiments, sterically hindered thiol and disulfide-containing maytansinoids in which the alpha-carbon atom bearing the sulfur atom bears one or two alkyl substituents are used in the binding protein-drug conjugate, e.g., US 2007-0292422; US 2007-0264266. In some embodiments, the drug comprises an agent that acts to disrupt DNA. The drug may be selected from enediynes (e.g., calicheamicin and esperamicin) and non-enediyne small molecule agents (e.g., bleomycin, methidiumpropyl-EDTA-Fe(II)). Other useful drugs include daunorubicin, doxorubicin, distamycin A, cisplatin, mitomycin C, ecteinascidins, duocarmycin/CC-1065, and bleomycin/pepleomycin. In other embodiments, the drug can comprise an alkylating agent such as Asaley NSC
167780, AZQ NSC 182986, BCNU NSC 409962, Busulfan NSC 750, carboxyphthalatoplatinum NSC 271674, CBDCA NSC 241240, CCNU NSC 79037, CHIP NSC 256927, chlorambucil NSC 3088, chlorozotocin NSC 178248, cis-platinum NSC 119875, clomesone NSC 338947, cyanomorpholinodoxorubicin NSC 357704, cyclodisone NSC 348948, dianhydrogalactitol NSC 132313, fluorodopan NSC 73754, hepsulfam NSC 329680, hycanthone NSC 142982, melphalan NSC 8806, methyl CCNU NSC 95441, mitomycin C NSC 26980, mitozolamide NSC 353451, nitrogen mustard NSC 762, PCNU NSC 95466, piperazine NSC 344007, piperazinedione NSC 135758, pipobroman NSC 25154, porfiromycin NSC 56410, spirohydantoin mustard NSC 172112, teroxirone NSC 296934, tetraplatin NSC 363812, thio-tepa NSC 6396, triethylenemelamine NSC 9706, uracil nitrogen mustard NSC 34462, or Yoshi-864 NSC 102627.
In some embodiments, the drug can comprise an antimitotic agent such as allocolchicine NSC 406042, Halichondrin B NSC 609395, colchicine NSC 757, colchicine derivative NSC 33410, dolastatin 10 NSC 376128 (NG-auristatin derived), maytansine NSC 153858, rhizoxin NSC 332598, taxol NSC 125973, taxol derivative NSC 608832, thiocolchicine NSC 361792, trityl cysteine NSC 83265, vinblastine sulfate NSC 49842, or vincristine sulfate NSC 67574.
In other embodiments, the drug can comprise an topoisomerase I inhibitor such as camptothecin NSC 94600, camptothecin, Na salt NSC 100880, aminocamptothecin NSC 603071, camptothecin derivative NSC 95382, camptothecin derivative NSC 107124, camptothecin derivative NSC 643833, camptothecin derivative NSC 629971, camptothecin derivative NSC 295500, camptothecin derivative NSC 249910, camptothecin derivative NSC 606985, camptothecin derivative NSC 374028, camptothecin derivative NSC 176323, camptothecin derivative NSC 295501, camptothecin derivative NSC 606172, camptothecin derivative NSC 606173, camptothecin derivative NSC 610458, camptothecin derivative NSC 618939, camptothecin derivative NSC 610457, camptothecin derivative NSC 610459, camptothecin derivative NSC 606499, camptothecin derivative NSC 610456, camptothecin derivative NSC 364830, camptothecin derivative NSC 606497, or morpholinodoxorubicin NSC 354646.
In other embodiments, the drug can comprise an topoisomerase II inhibitor such as doxorubicin NSC 123127, amonafide NSC 308847, m-AMSA NSC 249992, anthrapyrazole derivative NSC 355644, pyrazoloacridine NSC 366140, bisantrene HCL NSC 337766, daunorubicin NSC 82151, deoxydoxorubicin NSC 267469, mitoxantrone NSC 301739, menogaril NSC 269148, N,N-dibenzyl daunomycin NSC 268242, oxanthrazole NSC 349174, rubidazone NSC 164011, VM-26 NSC 122819, or VP-16 NSC 141540.
In other embodiments, the drug can comprise an RNA or DNA antimetabolite such as L- alanosine NSC 153353, 5-azacytidine NSC 102816, 5-fluorouracil NSC 19893, acivicin NSC 163501, aminopterin derivative NSC 132483, aminopterin derivative NSC 184692, aminopterin derivative NSC 134033, an antifol NSC 633713, an antifol NSC 623017, Baker's soluble antifol NSC 139105, dichlorallyl lawsone NSC 126771, brequinar NSC 368390, ftorafur (pro-drug) NSC 148958, 5,6-dihydro-5-azacytidine NSC 264880, methotrexate NSC 740, methotrexate derivative NSC 174121, N-(phosphonoacetyl)-L-aspartate (PALA) NSC 224131, pyrazofurin NSC 143095, trimetrexate NSC 352122, 3-HP NSC 95678, 2'-deoxy-5-fluorouridine NSC
27640, 5-HP NSC 107392, alpha-TGDR NSC 71851, aphidicolin glycinate NSC 303812, ara-C NSC 63878, 5-aza-2'-deoxycytidine NSC 127716, beta-TGDR NSC 71261, cyclocytidine NSC 145668, guanazole NSC 1895, hydroxyurea NSC 32065, inosine glycodialdehyde NSC 118994, macbecin 11 NSC 330500, pyrazoloimidazole NSC 51143, thioguanine NSC 752, or thiopurine NSC 755. See also US 2007-0292441.
The abbreviation "AFP" refers to dimethylvaline-valine-dolaisoleuine-dolaproine- phenylalanine-p-phenylened-iamine (e.g., see Formula XVI in US 2006-0233794).
The abbreviation "MAE" refers to monomethyl auristatin E (see Formula XI in US 2006- 0233794). The abbreviation "AEB" refers to an ester produced by reacting auristatin E with paraacetyl benzoic acid (e.g., see Formula XX in US 2006-0233794) The abbreviation "AEVB" refers to an ester produced by reacting auristatin E with benzoylvaleric acid (e.g., see Formula XXI in US 2006-0233794).
The abbreviation "MMAF" refers to dovaline-valine-dolaisoleunine-dolaproine- phenylalanine (e.g., see Formula IVIV in US 2006-0233794). The abbreviations "fk" and "phe-lys" refer to the linker phenylalanine-lysine.
The abbreviations "vc" and "val-cit" refer to the linker valine-citrulline.
In some embodiments, the drug is a cytotoxic agent selected from the group consisting of an auristatin, a DNA minor groove binding agent, a DNA minor groove alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid.
In some embodiments, the drug is a cytotoxic agent such as AFP or MMAF.
In some embodiments, the drug is an immunosuppressive agent such as gancyclovir, etanercept, cyclosporine, tacrolimus, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, Cortisol, aldosterone, dexamethasone, a cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, or a leukotriene receptor antagonist.
See generally US 2007-0292441; US 2007-0292422; US 2007-0264266; and US 2006- 0233794.
Linkers The binding proteins described herein can be associated with a drug to form a binding protein-drug conjugate by being linked to the drug directly. In some embodiments, the binding protein is directly conjugated to the drug. Alternatively, the binding proteins described herein can be associated with a drug to form a binding protein-drug conjugate by use of a linker region between the drug and the binding protein. In some embodiments, the binding protein is conjugated to the drug via a linker. The linker can be cleavable under intracellular conditions, e.g., such that cleavage of the linker releases the drug from the binding protein in the intracellular environment. In some embodiments, the cleavable linker is a peptide linker cleavable by an intracellular protease. In some embodiments, the peptide linker is a dipeptide linker. In some embodiments, the dipeptide linker is a val-cit (vc) linker or a phe-lys (fk) linker.
In some embodiments, the cleavable linker is hydrolyzable at a pH of less than 5.5. In some embodiments, the hydrolyzable linker is a hydrazone linker. In some embodiments, the cleavable linker is a disulfide linker.
For example, in some embodiments, the linker is cleavable by a cleaving agent that is present in the intracellular environment (e.g., within a lysosome or endosome or caveolea). The linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleaving agents can include cathepsins B and D and plasmin, which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside target cells (see, e.g., Dubowchik and Walker Pharm. Therapeutics 83:67-123 (1999)). In some embodiments, peptidyl linkers are cleavable by enzymes that are present in targeted cells (e.g., cancer cells). For example, a peptidyl linker that is cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancerous tissue, can be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Gly linker). Other such linkers are described, e.g., in U.S. Pat. No. 6,214,345. In some embodiments, the peptidyl linker cleavable by an intracellular protease is a Val-Cit (vc) linker or a Phe-Lys linker (fk) (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the val-cit linker). One advantage of using intracellular proteolytic release of the drug is that the drug can be attenuated when conjugated and the serum stabilities of the conjugates are typically high. In some preferred embodiments, a vc linker is used in the binding protein-drug conjugates described herein. For example, a binding protein- vcAFP or a binding protein- vcMMAF conjugate (e.g., a MMP-9 or -12 binding protein-vcAFP or a MMP-9 or -12 binding protein- vcMMAF conjugate) is prepared.
In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. For example, the pH-senstive linker is hydrolyzable under acidic conditions. For example, an acid-labile linker that is hydrolyzable in the lysosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal., ketal., or the like) can be used. See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker Pharm. Therapeutics 83:67-123 (1999); Neville et al. Biol. Chem. 264:14653-14661 (1989). Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929)).
In yet other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N- succinimidyl-3-(2-pyridyldithio propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)- , SPDB and SMPT (See, e.g., Thorpe et al. Cancer Res. 47:5924-5931 (1987); Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987). See also U.S. Pat. No. 4,880,935.
In yet other embodiments, the linker is a malonate linker (Johnson et al. Anticancer Res. 15:1387-93 (1995)), a maleimidobenzoyl linker (Lau et al. Bioorg-Med-Chem. 3(10):1299-1304 (1995), or a 3'-N-amide analog (Lau et al. Bioorg-Med-Chem. 3(10):1305-12 (1995)).
In some embodiments, the linker is not substantially sensitive to the extracellular environment. As used herein, "not substantially sensitive to the extracellular environment," in the context of a linker, means that no more than about 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linkers, in a sample of a binding protein- drug conjugate, are cleaved when the binding protein-drug conjugate is present in an extracellular environment (e.g., in plasma). Whether a linker is not substantially sensitive to the extracellular environment can be determined, for example, by incubating independently with plasma both (a) the binding protein-drug conjugate (the "conjugate sample") and (b) an equal molar amount of unconjugated binding protein or drug (the "control sample") for a predetermined time period (e.g., 2, 4, 8, 16, or 24 hours) and then comparing the amount of unconjugated binding protein or drug present in the conjugate sample with that present in control sample, as measured, for example, by high performance liquid chromatography.
In other, non-mutually exclusive embodiments, the linker promotes cellular internalization. In certain embodiments, the linker promotes cellular internalization when conjugated to the drug (i.e., in the milieu of the linker-drug moiety of the binding protein-drug conjugate described herein). In yet other embodiments, the linker promotes cellular internalization when conjugated to both the drug and the binding protein. A variety of linkers that can be used with the present compositions and methods are described in WO 2004010957.
In some embodiments, the binding protein-drug conjugates described herein are used therapeutically in the treatment of a disorder (e.g., cancer or inflammation). In certain embodiments, it is desirable to only target a binding protein-drug conjugate to a cell that expresses the target to which the binding protein binds (e.g., to only target a MMP-9 expressing cell to which a MMP-9 binding protein binds, and not target a nearby "bystander" cell), e.g., to minimize toxicity. In other embodiments, it is desirable to target a binding protein-drug conjugate to a cell expressing the target to which the binding protein binds and also to bystander cells (e.g., to elicit a "bystander effect"). In some embodiments, a binding protein-drug conjugate (e.g., a MMP-9 binding protein-drug conjugate can be engineered to exert a precise killing of only antigen-presenting cells without damaging proximal antigen-negative tissues, e.g., by preparing thioether-linked conjugates. Alternatively, it can be engineered to produce a bystander effect, e.g., by preparing disulfide-linked conjugates. For example, many solid tumors express targets (e.g., antigens) in a heterogeneous fashion and are populated with both target-positive and target-negative cells. The bystander cytotoxicity associated with disulfide linker-containing conjugates provides a rationale for treatment of sites of a disorder (e.g., tumors) with binding protein-drug conjugates even if the sites exhibit heterogeneous target expression. The bystander effect adds a degree of nonselective killing activity. Potentially, this could be a drawback if normal cells in tissues surrounding the site of disorder (e.g., tumor) are affected. However, as a potential advantage, the bystander cytotoxicity may damage tissues intricately involved in supporting the disorder, such as endothelial cells and pericytes of tumor neovasculature, or tumor stromal cells, resulting, for example, in enhanced antitumor activity of the binding protein-drug conjugate against tumors expressing the antigen either homogeneously or heterogeneously. See also Kovtum et al. Cancer Res. 66:3214 (2006).
Techniques for conjugating therapeutic agents to proteins (such as binding proteins, e.g., MMP-9 binding proteins) are known. See, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al eds., Alan R. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (Robinson et al. eds., Marcel Deiker, Inc., 2nd ed. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al. Immunol. Rev. 62: 119-58 (1982). See also, e.g., US 2006-0233794 and PCT publication WO 89/12624.
Display Libraries
A display library is a collection of entities; each entity includes an accessible polypeptide component and a recoverable component that encodes or identifies the polypeptide component. The polypeptide component is varied so that different amino acid sequences are represented.
The polypeptide component can be of any length, e.g. from three amino acids to over 300 amino acids. A display library entity can include more than one polypeptide component, for example, the two polypeptide chains of an sFab. In one exemplary implementation, a display library can be used to identify proteins that bind to MMP-9 or -12. In a selection, the polypeptide component of each member of the library is probed with MMP-9 or -12 (e.g., the catalytic domain of MMP-9 or -12 or other fragment) and if the polypeptide component binds to the MMP-9, the display library member is identified, typically by retention on a support.
Retained display library members are recovered from the support and analyzed. The analysis can include amplification and a subsequent selection under similar or dissimilar conditions. For example, positive and negative selections can be alternated. The analysis can also include determining the amino acid sequence of the polypeptide component and purification of the polypeptide component for detailed characterization.
A variety of formats can be used for display libraries. Examples include the following. Phage Display: The protein component is typically covalently linked to a bacteriophage coat protein. The linkage results from translation of a nucleic acid encoding the protein component fused to the coat protein. The linkage can include a flexible peptide linker, a protease site, or an amino acid incorporated as a result of suppression of a stop codon. Phage display is described, for example, in U.S. 5,223,409; Smith (1985) Science 228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809; de Haard et al. (1999) /. Biol. Chem 274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-8 and Hoet et al. (2005) Nat BiotechnoL 23(3)344-8. Bacteriophage displaying the protein component can be grown and harvested using standard phage preparatory methods, e.g. PEG precipitation from growth media. After selection of individual display phages, the nucleic acid encoding the selected protein components can be isolated from cells infected with the selected phages or from the phage themselves, after amplification. Individual colonies or plaques can be picked, the nucleic acid isolated and sequenced.
Other Display Formats. Other display formats include cell based display (see, e.g., WO 03/029456), protein-nucleic acid fusions (see, e.g., US 6,207,446), ribosome display (See, e.g., Mattheakis et al. (1994) P roc. Natl. Acad. Sci. USA 91 : 9022 and Hanes et al. (2000) Nat
BiotechnoL 18:1287-92; Hanes et al. (2000) Methods Enzymol. 328:404-30; and Schaffitzel et al. (1999) / Immunol Methods. 231(1-2): 119-35), and E. coli periplasmic display (J Immunol Methods. 2005 Nov 22;PMID: 16337958).
Scaffolds. Scaffolds useful for display include: antibodies (e.g., Fab fragments, single chain Fv molecules (scFV), single domain antibodies, camelid antibodies, and camelized antibodies); T-cell receptors; MHC proteins; extracellular domains (e.g., fibronectin Type III repeats, EGF repeats); protease inhibitors (e.g., Kunitz domains, ecotin, BPTI, and so forth); TPR repeats; trifoil structures; zinc finger domains; DNA-binding proteins; particularly monomeric DNA binding proteins; RNA binding proteins; enzymes, e.g., proteases (particularly inactivated proteases), RNase; chaperones, e.g., thioredoxin and heat shock proteins; intracellular signaling domains (such as SH2 and SH3 domains); linear and constrained peptides; and linear peptide substrates. Display libraries can include synthetic and/or natural diversity. See, e.g., US 2004-0005709.
Display technology can also be used to obtain binding proteins (e.g., antibodies) that bind particular epitopes of a target. This can be done, for example, by using competing non-target molecules that lack the particular epitope or are mutated within the epitope, e.g., with alanine. Such non-target molecules can be used in a negative selection procedure as described below, as competing molecules when binding a display library to the target, or as a pre-elution agent, e.g., to capture in a wash solution dissociating display library members that are not specific to the target. Iterative Selection. In one preferred embodiment, display library technology is used in an iterative mode. A first display library is used to identify one or more binding proteins for a target. These identified binding proteins are then varied using a mutagenesis method to form a second display library. Higher affinity binding proteins are then selected from the second library, e.g., by using higher stringency or more competitive binding and washing conditions.
In some implementations, the mutagenesis is targeted to regions at the binding interface. If, for example, the identified binding proteins are antibodies, then mutagenesis can be directed to the CDR regions of the heavy or light chains as described herein. Further, mutagenesis can be directed to framework regions near or adjacent to the CDRs. In the case of antibodies, mutagenesis can also be limited to one or a few of the CDRs, e.g., to make precise step-wise improvements. Exemplary mutagenesis techniques include: error-prone PCR, recombination, DNA shuffling, site-directed mutagenesis and cassette mutagenesis.
In one example of iterative selection, the methods described herein are used to first identify a protein from a display library that binds MMP-9 with at least a minimal binding specificity for a target or a minimal activity, e.g., an equilibrium dissociation constant for binding of less than 1 nM, 10 nM, or 100 nM. The nucleic acid sequence encoding the initial identified proteins are used as a template nucleic acid for the introduction of variations, e.g., to identify a second protein that has enhanced properties (e.g., binding affinity, kinetics, or stability) relative to the initial protein. Off-Rate Selection. Since a slow dissociation rate can be predictive of high affinity, particularly with respect to interactions between polypeptides and their targets, the methods described herein can be used to isolate binding proteins with a desired (e.g., reduced) kinetic dissociation rate for a binding interaction to a target.
To select for slow dissociating binding proteins from a display library, the library is contacted to an immobilized target. The immobilized target is then washed with a first solution that removes non-specifically or weakly bound biomolecules. Then the bound binding proteins are eluted with a second solution that includes a saturating amount of free target or a target specific high-affinity competing monoclonal antibody, i.e., replicates of the target that are not attached to the particle. The free target binds to biomolecules that dissociate from the target. Rebinding is effectively prevented by the saturating amount of free target relative to the much lower concentration of immobilized target. The second solution can have solution conditions that are substantially physiological or that are stringent. Typically, the solution conditions of the second solution are identical to the solution conditions of the first solution. Fractions of the second solution are collected in temporal order to distinguish early from late fractions. Later fractions include biomolecules that dissociate at a slower rate from the target than biomolecules in the early fractions.
Further, it is also possible to recover display library members that remain bound to the target even after extended incubation. These can either be dissociated using chaotropic conditions or can be amplified while attached to the target. For example, phage bound to the target can be contacted to bacterial cells. Selecting or Screening for Specificity. The display library screening methods described herein can include a selection or screening process that discards display library members that bind to a non-target molecule. Examples of non-target molecules include streptavidin on magnetic beads, blocking agents such as bovine serum albumin, non-fat bovine milk, any capturing or target immobilizing monoclonal antibody, or non-transfected cells which do not express the human MMP-9 or -12 target.
In one implementation, a so-called "negative selection" step is used to discriminate between the target and related non-target molecule and a related, but distinct non-target molecules. The display library or a pool thereof is contacted to the non-target molecule. Members of the sample that do not bind the non-target are collected and used in subsequent selections for binding to the target molecule or even for subsequent negative selections. The negative selection step can be prior to or after selecting library members that bind to the target molecule.
In another implementation, a screening step is used. After display library members are isolated for binding to the target molecule, each isolated library member is tested for its ability to bind to a non-target molecule (e.g., a non-target listed above). For example, a high-throughput ELISA screen can be used to obtain this data. The ELISA screen can also be used to obtain quantitative data for binding of each library member to the target as well as for cross species reactivity to related targets or subunits of the target (e.g., mouse MMP-9) and also under different condition such as pH6 or pH 7.5. The non-target and target binding data are compared (e.g., using a computer and software) to identify library members that specifically bind to the target. Other Exemplary Expression Libraries
Other types of collections of proteins (e.g., expression libraries) can be used to identify proteins with a particular property (e.g., ability to bind MMP-9 or -12 and/or ability to modulate MMP-9 or -12), including, e.g., protein arrays of antibodies (see, e.g., De Wildt et al. (2000) Nat. Biotechnol. 18:989-994), lambda gtll libraries, two-hybrid libraries and so forth.
Exemplary Libraries
It is possible to immunize a non-human primate and recover primate antibody genes that can be displayed on phage (see below). From such a library, one can select antibodies that bind the antigen used in immunization. See, for example, Vaccine. (2003) 22(2):257-67 or Immunogenetics. (2005) 57(10):730-8. Thus one could obtain primate antibodies that bind and inhibit MMP-9 by immunizing a chimpanzee or macaque and using a variety of means to select or screen for primate antibodies that bind and inhibit MMP-9 or -12. One can also make chimeras of primatized Fabs with human constant regions, see Curr Opin MoI Ther. (2004) 6(6):675-83. "PRIMATIZED antibodies, genetically engineered from cynomolgus macaque monkey and human components, are structurally indistinguishable from human antibodies. They may, therefore, be less likely to cause adverse reactions in humans, making them potentially suited for long-term, chronic treatment " Curr Opin Investig Drugs. (2001) 2(5):635-8.
One exemplary type of library presents a diverse pool of polypeptides, each of which includes an immunoglobulin domain, e.g., an immunoglobulin variable domain. Of interest are display libraries where the members of the library include primate or "primatized" (e.g., such as human, non-human primate or "humanized") immunoglobin domains (e.g., immunoglobin variable domains) or chimeric primatized Fabs with human constant regions. Human or humanized immunoglobin domain libraries may be used to identify human or "humanized" antibodies that, for example, recognize human antigens. Because the constant and framework regions of the antibody are human, these antibodies may avoid themselves being recognized and targeted as antigens when administered to humans. The constant regions may also be optimized to recruit effector functions of the human immune system. The in vitro display selection process surmounts the inability of a normal human immune system to generate antibodies against self- antigens. A typical antibody display library displays a polypeptide that includes a VH domain and a VL domain. An "immunoglobulin domain" refers to a domain from the variable or constant domain of immunoglobulin molecules. Immunoglobulin domains typically contain two β-sheets formed of about seven β-strands, and a conserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay, 1988, Ann. Rev. Immunol. 6:381-405). The display library can display the antibody as a Fab fragment (e.g., using two polypeptide chains) or a single chain Fv (e.g., using a single polypeptide chain). Other formats can also be used.
As in the case of the Fab and other formats, the displayed antibody can include one or more constant regions as part of a light and/or heavy chain. In one embodiment, each chain includes one constant region, e.g., as in the case of a Fab. In other embodiments, additional constant regions are displayed.
Antibody libraries can be constructed by a number of processes (see, e.g., de Haard et al., 1999, /. Biol. Chem. 274:18218-30; Hoogenboom et al., 1998, Immunotechnology 4:1-20; Hoogenboom et al., 2000, Immunol. Today 21:371-378, and Hoet et al. (2005) Nat Biotechnol. 23(3)344-8. Further, elements of each process can be combined with those of other processes. The processes can be used such that variation is introduced into a single immunoglobulin domain (e.g., VH or VL) or into multiple immunoglobulin domains (e.g., VH and VL). The variation can be introduced into an immunoglobulin variable domain, e.g., in the region of one or more of CDRl, CDR2, CDR3, FRl, FR2, FR3, and FR4, referring to such regions of either and both of heavy and light chain variable domains. The variation(s) may be introduced into all three CDRs of a given variable domain, or into CDRl and CDR2, e.g., of a heavy chain variable domain. Any combination is feasible. In one process, antibody libraries are constructed by inserting diverse oligonucleotides that encode CDRs into the corresponding regions of the nucleic acid. The oligonucleotides can be synthesized using monomeric nucleotides or trinucleotides. For example, Knappik et al., 2000, J. MoI. Biol. 296:57-86 describe a method for constructing CDR encoding oligonucleotides using trinucleotide synthesis and a template with engineered restriction sites for accepting the oligonucleotides.
In another process, an animal, e.g., a rodent, is immunized with MMP-9 or -12. The animal is optionally boosted with the antigen to further stimulate the response. Then spleen cells are isolated from the animal, and nucleic acid encoding VH and/or VL domains is amplified and cloned for expression in the display library. In yet another process, antibody libraries are constructed from nucleic acid amplified from naϊve germline immunoglobulin genes. The amplified nucleic acid includes nucleic acid encoding the VH and/or VL domain. Sources of immunoglobulin-encoding nucleic acids are described below. Amplification can include PCR, e.g., with primers that anneal to the conserved constant region, or another amplification method.
Nucleic acid encoding immunoglobulin domains can be obtained from the immune cells of, e.g., a primate (e.g., a human), mouse, rabbit, camel, or rodent. In one example, the cells are selected for a particular property. B cells at various stages of maturity can be selected. In another example, the B cells are naϊve. In one embodiment, fluorescent-activated cell sorting (FACS) is used to sort B cells that express surface-bound IgM, IgD, or IgG molecules. Further, B cells expressing different isotypes of IgG can be isolated. In another preferred embodiment, the B or T cell is cultured in vitro. The cells can be stimulated in vitro, e.g., by culturing with feeder cells or by adding mitogens or other modulatory reagents, such as antibodies to CD40, CD40 ligand or CD20, phorbol myristate acetate, bacterial lipopolysaccharide, concanavalin A, phytohemagglutinin, or pokeweed mitogen.
In another embodiment, the cells are isolated from a subject that has a disease of condition described herein, e.g., a cancer (e.g., metastatic cancer, e.g., metastatic breast cancer), an inflammatory disease (e.g., synovitis, atherosclerosis), rheumatoid arthritis, osteoarthritis, an ocular condition (e.g., macular degeneration), diabetes, Alzheimer's Disease, cerebral ischemia, endometriosis, fibrin-invasive activity, angiogenesis, or capillary tube formation In another embodiment, the cells are isolated from a transgenic non-human animal that includes a human immunoglobulin locus.
In one preferred embodiment, the cells have activated a program of somatic hypermutation. Cells can be stimulated to undergo somatic mutagenesis of immunoglobulin genes, for example, by treatment with antiimmunoglobulin, anti-CD40, and anti-CD38 antibodies (see, e.g., Bergthorsdottir et al., 2001, /. Immunol. 166:2228). In another embodiment, the cells are naϊve.
The nucleic acid encoding an immunoglobulin variable domain can be isolated from a natural repertoire by the following exemplary method. First, RNA is isolated from the immune cell. Full length (i.e., capped) mRNAs are separated (e.g. by degrading uncapped RNAs with calf intestinal phosphatase). The cap is then removed with tobacco acid pyrophosphatase and reverse transcription is used to produce the cDNAs.
The reverse transcription of the first (antisense) strand can be done in any manner with any suitable primer. See, e.g., de Haard et al., 1999, /. Biol. Chem. 274:18218-30. The primer binding region can be constant among different immunoglobulins, e.g., in order to reverse transcribe different isotypes of immunoglobulin. The primer binding region can also be specific to a particular isotype of immunoglobulin. Typically, the primer is specific for a region that is 3' to a sequence encoding at least one CDR. In another embodiment, poly-dT primers may be used (and may be preferred for the heavy-chain genes). A synthetic sequence can be ligated to the 3' end of the reverse transcribed strand. The synthetic sequence can be used as a primer binding site for binding of the forward primer during PCR amplification after reverse transcription. The use of the synthetic sequence can obviate the need to use a pool of different forward primers to fully capture the available diversity.
The variable domain-encoding gene is then amplified, e.g., using one or more rounds. If multiple rounds are used, nested primers can be used for increased fidelity. The amplified nucleic acid is then cloned into a display library vector.
Secondary Screening Methods
After selecting candidate library members that bind to a target, each candidate library member can be further analyzed, e.g., to further characterize its binding properties for the target, e.g., MMP-9 or -12, or for binding to other protein, e.g., another metalloproteinase. Each candidate library member can be subjected to one or more secondary screening assays. The assay can be for a binding property, a catalytic property, an inhibitory property, a physiological property (e.g., cytotoxicity, renal clearance, immunogenicity), a structural property (e.g., stability, conformation, oligomerization state) or another functional property. The same assay can be used repeatedly, but with varying conditions, e.g., to determine pH, ionic, or thermal sensitivities.
As appropriate, the assays can use a display library member directly, a recombinant polypeptide produced from the nucleic acid encoding the selected polypeptide, or a synthetic peptide synthesized based on the sequence of the selected polypeptide. In the case of selected Fabs, the Fabs can be evaluated or can be modified and produced as intact IgG proteins. Exemplary assays for binding properties include the following.
ELISA. Binding proteins can be evaluated using an ELISA assay. For example, each protein is contacted to a microtitre plate whose bottom surface has been coated with the target, e.g., a limiting amount of the target. The plate is washed with buffer to remove non- specifically bound polypeptides. Then the amount of the binding protein bound to the target on the plate is determined by probing the plate with an antibody that can recognize the binding protein, e.g., a tag or constant portion of the binding protein. The antibody is linked to a detection system (e.g. , an enzyme such as alkaline phosphatase or horse radish peroxidase (HRP) which produces a colorimetric product when appropriate substrates are provided).
Homogeneous Binding Assays. The ability of a binding protein described herein to bind a target can be analyzed using a homogenous assay, i.e., after all components of the assay are added, additional fluid manipulations are not required. For example, fluorescence resonance energy transfer (FRET) can be used as a homogenous assay (see, for example, Lakowicz et al., U.S. Patent No. 5,631,169; Stavrianopoulos, et al., U.S. Patent No. 4,868,103). A fluorophore label on the first molecule (e.g., the molecule identified in the fraction) is selected such that its emitted fluorescent energy can be absorbed by a fluorescent label on a second molecule (e.g., the target) if the second molecule is in proximity to the first molecule. The fluorescent label on the second molecule fluoresces when it absorbs to the transferred energy. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal. A binding event that is configured for monitoring by FRET can be conveniently measured through standard fluorometric detection means, e.g., using a fluorimeter. By titrating the amount of the first or second binding molecule, a binding curve can be generated to estimate the equilibrium binding constant.
Another example of a homogenous assay is ALPHASCREEN™ (Packard Bioscience, Meriden CT). ALPHASCREEN ™ uses two labeled beads. One bead generates singlet oxygen when excited by a laser. The other bead generates a light signal when singlet oxygen diffuses from the first bead and collides with it. The signal is only generated when the two beads are in proximity. One bead can be attached to the display library member, the other to the target. Signals are measured to determine the extent of binding.
Surface Plasmon Resonance (SPR). The interaction of binding protein and a target can be analyzed using SPR. SPR or Biomolecular Interaction Analysis (BIA) detects biospecific interactions in real time, without labeling any of the interactants. Changes in the mass at the binding surface (indicative of a binding event) of the BIA chip result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)). The changes in the refractivity generate a detectable signal, which are measured as an indication of real-time reactions between biological molecules. Methods for using SPR are described, for example, in U.S. Patent No. 5,641,640; Raether, 1988, Surface Plasmons Springer Verlag; Sjolander and Urbaniczky, 1991, Anal. Chem. 63:2338-2345; Szabo et al., 1995, Curr. Opin. Struct. Biol. 5:699-705 and on-line resources provide by BIAcore International AB (Uppsala, Sweden). BIAcore Flexchip can be used to compare and rank interactions in real time, in terms of kinetics, affinity or specificity without the use of labels. Information from SPR can be used to provide an accurate and quantitative measure of the equilibrium dissociation constant (Kd), and kinetic parameters, including K0n and KOff, for the binding of a binding protein to a target. Such data can be used to compare different biomolecules. For example, selected proteins from an expression library can be compared to identify proteins that have high affinity for the target or that have a slow KOff. This information can also be used to develop structure- activity relationships (SAR). For example, the kinetic and equilibrium binding parameters of matured versions of a parent protein can be compared to the parameters of the parent protein. Variant amino acids at given positions can be identified that correlate with particular binding parameters, e.g., high affinity and slow KOff. This information can be combined with structural modeling (e.g., using homology modeling, energy minimization, or structure determination by x-ray crystallography or NMR). As a result, an understanding of the physical interaction between the protein and its target can be formulated and used to guide other design processes.
Cellular Assays. Binding proteins can be screened for ability to bind to cells which transiently or stably express and display the target of interest on the cell surface. For example, MMP-9 binding proteins can be fluorescently labeled and binding to MMP-9 in the presence of absence of antagonistic antibody can be detected by a change in fluorescence intensity using flow cytometry e.g., a FACS machine.
Other Exemplary Methods for Obtaining MMP-9 or MMP-12 binding antibodies
In addition to the use of display libraries, other methods can be used to obtain a MMP-9 or -12 binding antibody. For example, MMP-9 or -12 protein or a region thereof can be used as an antigen in a non-human animal, e.g., a rodent.
In one embodiment, the non-human animal includes at least a part of a human immunoglobulin gene. For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci. Using the hybridoma technology, antigen-specific monoclonal antibodies (Mabs) derived from the genes with the desired specificity may be produced and selected. See, e.g., XENOMOUSE™, Green et al., 1994, Nat. Gen. 7:13-21; U.S. 2003-0070185, WO 96/34096, published Oct. 31, 1996, and PCT Application No. PCT/US96/05928, filed Apr. 29, 1996.
In another embodiment, a monoclonal antibody is obtained from the non-human animal, and then modified, e.g., humanized or deimmunized. Winter describes a CDR-grafting method that may be used to prepare the humanized antibodies (UK Patent Application GB 2188638A, filed on March 26, 1987; US Patent No. 5,225,539. All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen.
Humanized antibodies can be generated by replacing sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. US Patent Nos. 5,585,089, US 5,693,761 and US 5,693,762. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Numerous sources of such nucleic acid are available. For example, nucleic acids may be obtained from a hybridoma producing an antibody against a predetermined target, as described above. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.
Reducing Immunogenicity of MMP-9 or -12 Binding Proteins
Immunoglobin MMP-9 or -12 binding proteins (e.g., IgG or Fab MMP-9 or -12 binding proteins) may be modified to reduce immunogenicity. Reduced immunogenicity is desirable in MMP-9 or -12 binding proteins intended for use as therapeutics, as it reduces the chance that the subject will develop an immune response against the therapeutic molecule. Techniques useful for reducing immunogenicity of MMP-9 binding proteins include deletion/modification of potential human T cell epitopes and 'germlining' of sequences outside of the CDRs (e.g., framework and Fc).
An MMP-9 or -12 -binding antibody may be modified by specific deletion of human T cell epitopes or "deimmunization" by the methods disclosed in WO 98/52976 and WO 00/34317. Briefly, the heavy and light chain variable regions of an antibody are analyzed for peptides that bind to MHC Class II; these peptides represent potential T-cell epitopes (as defined in WO 98/52976 and WO 00/34317). For detection of potential T-cell epitopes, a computer modeling approach termed "peptide threading" can be applied, and in addition a database of human MHC class II binding peptides can be searched for motifs present in the VH and VL sequences, as described in WO 98/52976 and WO 00/34317. These motifs bind to any of the 18 major MHC class II DR allotypes, and thus constitute potential T cell epitopes. Potential T-cell epitopes detected can be eliminated by substituting small numbers of amino acid residues in the variable regions, or preferably, by single amino acid substitutions. As far as possible conservative substitutions are made, often but not exclusively, an amino acid common at this position in human germline antibody sequences may be used. Human germline sequences are disclosed in Tomlinson, LA. et al., 1992, /. MoL Biol. 227:776-798; Cook, G. P. et al., 1995, Immunol. Today Vol. 16 (5): 237-242; Chothia, D. et al., 1992, /. MoI. Bio. 227:799-817. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, LA. et al. MRC Centre for Protein Engineering, Cambridge, UK). After the deimmunizing changes are identified, nucleic acids encoding VH and VL can be constructed by mutagenesis or other synthetic methods (e.g., de novo synthesis, cassette replacement, and so forth). Mutagenized variable sequence can, optionally, be fused to a human constant region, e.g., human IgGl or K constant regions.
In some cases a potential T cell epitope will include residues which are known or predicted to be important for antibody function. For example, potential T cell epitopes are usually biased towards the CDRs. In addition, potential T cell epitopes can occur in framework residues important for antibody structure and binding. Changes to eliminate these potential epitopes will in some cases require more scrutiny, e.g., by making and testing chains with and without the change. Where possible, potential T cell epitopes that overlap the CDRs were eliminated by substitutions outside the CDRs. In some cases, an alteration within a CDR is the only option, and thus variants with and without this substitution should be tested. In other cases, the substitution required to remove a potential T cell epitope is at a residue position within the framework that might be critical for antibody binding. In these cases, variants with and without this substitution should be tested. Thus, in some cases several variant deimmunized heavy and light chain variable regions were designed and various heavy/light chain combinations tested in order to identify the optimal deimmunized antibody. The choice of the final deimmunized antibody can then be made by considering the binding affinity of the different variants in conjunction with the extent of deimmunization, i.e., the number of potential T cell epitopes remaining in the variable region. Deimmunization can be used to modify any antibody, e.g., an antibody that includes a non-human sequence, e.g., a synthetic antibody, a murine antibody other non-human monoclonal antibody, or an antibody isolated from a display library.
MMP-9 or -12 binding antibodies are "germlined" by reverting one or more non-germline amino acids in framework regions to corresponding germline amino acids of the antibody, so long as binding properties are substantially retained. Similar methods can also be used in the constant region, e.g., in constant immunoglobulin domains. Antibodies that bind to MMP-9, or -12 e.g., an antibody described herein, may be modified in order to make the variable regions of the antibody more similar to one or more germline sequences. For example, an antibody can include one, two, three, or more amino acid substitutions, e.g., in a framework, CDR, or constant region, to make it more similar to a reference germline sequence. One exemplary germlining method can include identifying one or more germline sequences that are similar (e.g., most similar in a particular database) to the sequence of the isolated antibody. Mutations (at the amino acid level) are then made in the isolated antibody, either incrementally or in combination with other mutations. For example, a nucleic acid library that includes sequences encoding some or all possible germline mutations is made. The mutated antibodies are then evaluated, e.g., to identify an antibody that has one or more additional germline residues relative to the isolated antibody and that is still useful (e.g., has a functional activity). In one embodiment, as many germline residues are introduced into an isolated antibody as possible.
In one embodiment, mutagenesis is used to substitute or insert one or more germline residues into a framework and/or constant region. For example, a germline framework and/or constant region residue can be from a germline sequence that is similar (e.g., most similar) to the non-variable region being modified. After mutagenesis, activity (e.g., binding or other functional activity) of the antibody can be evaluated to determine if the germline residue or residues are tolerated (i.e., do not abrogate activity). Similar mutagenesis can be performed in the framework regions.
Selecting a germline sequence can be performed in different ways. For example, a germline sequence can be selected if it meets a predetermined criteria for selectivity or similarity, e.g., at least a certain percentage identity, e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity. The selection can be performed using at least 2, 3, 5, or 10 germline sequences. In the case of CDRl and CDR2, identifying a similar germline sequence can include selecting one such sequence. In the case of CDR3, identifying a similar germline sequence can include selecting one such sequence, but may including using two germline sequences that separately contribute to the amino-terminal portion and the carboxy-terminal portion. In other implementations more than one or two germline sequences are used, e.g., to form a consensus sequence.
In one embodiment, with respect to a particular reference variable domain sequence, e.g., a sequence described herein, a related variable domain sequence has at least 30, 40, 50, 60, 70, 80, 90, 95 or 100% of the CDR amino acid positions that are not identical to residues in the reference CDR sequences, residues that are identical to residues at corresponding positions in a human germline sequence (i.e., an amino acid sequence encoded by a human germline nucleic acid). In one embodiment, with respect to a particular reference variable domain sequence, e.g., a sequence described herein, a related variable domain sequence has at least 30, 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of the FR regions identical to FR sequence from a human germline sequence, e.g., a germline sequence related to the reference variable domain sequence.
Accordingly, it is possible to isolate an antibody which has similar activity to a given antibody of interest, but is more similar to one or more germline sequences, particularly one or more human germline sequences. For example, an antibody can be at least 90, 91, 92, 93, 94, 95,
96, 97, 98, 99, or 99.5% identical to a germline sequence in a region outside the CDRs (e.g., framework regions). Further, an antibody can include at least 1, 2, 3, 4, or 5 germline residues in a CDR region, the germline residue being from a germline sequence of similar (e.g., most similar) to the variable region being modified. Germline sequences of primary interest are human germline sequences. The activity of the antibody (e.g., the binding activity as measured by KA) can be within a factor or 100, 10, 5, 2, 0.5, 0.1, and 0.001 of the original antibody. Germline sequences of human immunoglobin genes have been determined and are available from a number of sources, including the international ImMunoGeneTics information system® (IMGT), available via the world wide web at imgt.cines.fr, and the V BASE directory
(compiled by Tomlinson, LA. et al. MRC Centre for Protein Engineering, Cambridge, UK, available via the world wide web at vbase.mrc-cpe.cam.ac.uk).
Exemplary germline reference sequences for Vkappa include: 012/02, 018/08, A20,
A30, L14, Ll, L15, L4/18a, L5/L19, L8, L23, L9 ,L24, LI l, L12, Ol l/Ol, A17, Al, A18, A2, A19/A3, A23, A27, Al l, L2/L16, L6, L20, L25, B3, B2, A26/A10, and A14. See, e.g.,
Tomlinson et al., 1995, EMBO J. 14(18):4628-3.
A germline reference sequence for the HC variable domain can be based on a sequence that has particular canonical structures, e.g., 1-3 structures in the Hl and H2 hypervariable loops.
The canonical structures of hypervariable loops of an immunoglobulin variable domain can be inferred from its sequence, as described in Chothia et al., 1992, /. MoI. Biol. 227:799-817;
Tomlinson et al., 1992, /. MoL Biol. 227:776-798); and Tomlinson et al., 1995, EMBO J.
14(18):4628-38. Exemplary sequences with a 1-3 structure include: DP-I, DP-8, DP-12, DP-2,
DP-25, DP-15, DP-7, DP-4, DP-31, DP-32, DP-33, DP-35, DP-40, 7-2, hv3005, hv3005f3, DP-
46, DP-47, DP-58, DP-49, DP-50, DP-51, DP-53, and DP-54. Protein Production
Standard recombinant nucleic acid methods can be used to express a protein that binds to MMP-9 or -12. Generally, a nucleic acid sequence encoding the protein is cloned into a nucleic acid expression vector. Of course, if the protein includes multiple polypeptide chains, each chain can be cloned into an expression vector, e.g., the same or different vectors, that are expressed in the same or different cells.
Antibody Production. Some antibodies, e.g., Fabs, can be produced in bacterial cells, e.g., E. coli cells. For example, if the Fab is encoded by sequences in a phage display vector that includes a suppressible stop codon between the display entity and a bacteriophage protein (or fragment thereof), the vector nucleic acid can be transferred into a bacterial cell that cannot suppress a stop codon. In this case, the Fab is not fused to the gene III protein and is secreted into the periplasm and/or media.
Antibodies can also be produced in eukaryotic cells. In one embodiment, the antibodies (e.g., scFv's) are expressed in a yeast cell such as Pichia (see, e.g., Powers et al., 2001, /. Immunol. Methods. 251:123-35), Hanseula, or Saccharomyces.
In one preferred embodiment, antibodies are produced in mammalian cells. Preferred mammalian host cells for expressing the clone antibodies or antigen-binding fragments thereof include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, 1980, Proc. Natl. Acad. ScL USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, 1982, MoI. Biol. 159:601 621), lymphocytic cell lines, e.g., NSO myeloma cells and SP2 cells, COS cells, HEK293T cells (J. Immunol. Methods (2004) 289(l-2):65-80.), and a cell from a transgenic animal, e.g., a transgenic mammal. For example, the cell is a mammary epithelial cell.
In addition to the nucleic acid sequence encoding the diversified immunoglobulin domain, the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhff host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
In an exemplary system for recombinant expression of an antibody, or antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhff CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/ AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element) to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.
For antibodies that include an Fc domain, the antibody production system may produce antibodies in which the Fc region is glycosylated. For example, the Fc domain of IgG molecules is glycosylated at asparagine 297 in the CH2 domain. This asparagine is the site for modification with biantennary-type oligosaccharides. It has been demonstrated that this glycosylation is required for effector functions mediated by Fcg receptors and complement CIq (Burton and Woof, 1992, Adv. Immunol. 51:1-84; Jefferis et al., 1998, Immunol. Rev. 163:59-76). In one embodiment, the Fc domain is produced in a mammalian expression system that appropriately glycosylates the residue corresponding to asparagine 297. The Fc domain can also include other eukaryotic post-translational modifications.
Antibodies can also be produced by a transgenic animal. For example, U.S. Patent No. 5,849,992 describes a method of expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter and nucleic acids encoding the antibody of interest and a signal sequence for secretion. The milk produced by females of such transgenic mammals includes, secreted-therein, the antibody of interest. The antibody can be purified from the milk, or for some applications, used directly.
Characterization of MMP-9 or -12 Binding Proteins
MMP-9. Binding of MMP-9 binding proteins to cells expressing MMP-9 can be characterized in a number assays known in the art, including FACS (Fluorescence Activated Cell Sorting), immunofluorescence, and immunocytochemistry. MMP-9 binding protein is contacted with cells and/or tissues which express or contain MMP-9 and binding is detected in accordance with the method being used. For example, a fluorescent detection system (e.g., fluorescent- labeled secondary antibody) employed for FACS and immunofluorescence analysis, or a enzymatic system is used for immunocytochemistry are generally used in these assayscan be performed on non-perm. MMP-9 binding proteins can be characterized as to cellular binding by FACS (Fluorescence Activated Cell Sorting) using cells expressing MMP-9. Individual cells held in a thin stream of fluid are passed through one or more laser beams cause light to scatter and fluorescent dyes to emit light at various frequencies. Photomultiplier tubes (PMT) convert light to electrical signals and cell data is collected. Forward and side scatter are used for preliminary identification of cells. Forward and side scatter are used to exclude debris and dead cells. Fluorescent labeling allows investigation of cell structure and function. Cell autofluorescence is generated by labeling cell structures with fluorescent dyes. FACS collects fluorescence signals in one to several channels corresponding to different laser excitation and fluorescence emission wavelength. Immunofluorescence, the most widely used application, involves the staining of cells with antibodies conjugated to fluorescent dyes such as fluorescein and phycoerythrin (PE). This method can be used to label MMP-9 on the cell surface of MDA- MB-231 cells using biotinylated MMP-9 binding proteins. Biotin is used in this two-step detection systems in concert with conjugated steptavidin. Biotin is typically conjugated to proteins via primary amines (i.e., lysines). Usually, between 1.5 and 3 biotin molecules are conjugated to each antibody. A second fluorescently conjugated antibody (streptavidin/PE) is added which is specific for biotin.
MMP-9 binding proteins can be characterized in cultured cells expressing the MMP-9 antigen. The method generally used is immunocytochemistry. Immunocytochemistry involves the use of antibodies that recognize parts of the receptor that are exposed to the outside environment when expressed at the cell surface (the 'primary antibody'). If the experiment is carried out in intact cells, such an antibody will only bind to surface expressed receptors. Biotinylated or non-biotinylated MMP-9 binding proteins can be used. The secondary antibody is then either a streptavidin/HRP antibody (for biotinylated MMP-9 binding protein) or an anti- human IgG/HRP (for non-biotinylated MMP-9 binding protein). The staining can then be detected using an inverted microscope. The assay can be performed in the absence of MMP-9 binding protein and in presence of lOμg/mL of MMP-9 binding protein.
MMP-9 binding proteins can be characterized in assays that measure their modulatory activity toward MMP-9 or fragments thereof in vitro or in vivo. For example, MMP-9 can be combined with a substrate such as Mca-Pro-Leu-Ala-Cys(Mob)-Trp-Ala-Arg-Dap(Dnp)-NH? under assay conditions permitting cleavage by MMP-9. The assay is performed in the absence of the MMP-9 binding protein, and in the presence of increasing concentrations of the MMP-9 binding protein. The concentration of binding protein at which 50% of the MMP-9 activity (e.g., binding to the substrate) is inhibited is the IC50 value (Inhibitory Concentration 50%) or EC50 (Effective Concentration 50%) value for that binding protein. Within a series or group of binding proteins, those having lower IC50 or EC50 values are considered more potent inhibitors of MMP-9 than those binding proteins having higher IC50 or EC50 values. Exemplary binding proteins have an IC50 value of less than 800 nM, 400 nM, 100 nM, 25 nM, 5 nM, or 1 nM, e.g., as measured in an in vitro assay for inhibition of MMP-9 activity when the MMP-9 is at 2 pM. MMP-9 binding proteins may also be characterized with reference to the activity of
MMP-9 on substrates (e.g., collagen, gelatin). For example, cleavage of gelatin by MMP-9 can be detected in zymography. The method is based on a SDS gel impregnated with a substrate, which is degraded by the proteases resolved during the incubation period. Coomassie blue staining of the gels reveals proteolytic fragments as white bands on a dark blue background. Within a certain range, the band intensity can be related linearly to the amount of the protease loaded. Cells expressing MMP-9 are used in this assay. The assay is performed in the absence of the MMP-9 binding protein, and in the presence of increasing concentrations of the MMP-9 binding protein. The concentration of binding protein at which 50% of the MMP-9 activity (e.g., binding to the substrate) is inhibited is the IC50 value (Inhibitory Concentration 50%) or EC50 (Effective Concentration 50%) value for that binding protein. Within a series or group of binding proteins, those having lower IC50 or EC50 values are considered more potent inhibitors of MMP-9 than those binding proteins having higher IC50 or EC50 values. Exemplary binding proteins have an IC50 value of less than 800 nM, 400 nM, 100 nM, 25 nM, 5 nM, or 1 nM, e.g., as measured in an in vitro assay for inhibition of MMP-9 activity.
The binding proteins can also be evaluated for selectivity toward MMP-9. For example, a MMP-9 binding protein can be assayed for its potency toward MMP-9 and a panel of MMPs and other enzymes, e.g., human and/or mouse enzymes, e.g., MMP-I, -2, -3, -7, -8, -12, -13, -14, -16, -17, -24, and TACE, and an IC50 value or EC50 value can be determined for each MMP. In one embodiment, a compound that demonstrates a low IC50 value or EC50 value for the MMP-9, and a higher IC50 value or EC50 value, e.g., at least 2-, 5-, or 10- fold higher, for another MMP within the test panel (e.g., MMP-I, -10) is considered to be selective toward MMP-9.
MMP-9 binding proteins can be evaluated for their ability to inhibit MMP-9 in a cell based assay, e.g., in situ zymography, e.g., in Colo205 cells or MCF-7 cells.
A pharmacokinetics study in rat, mice, or monkey can be performed with MMP-9 binding proteins for determining MMP-9 half-life in the serum. Likewise, the effect of the binding protein can be assessed in vivo, e.g., in an animal model for a disease, for use as a therapeutic, for example, to treat a disease or condition described herein, e.g., systemic sclerosis, a cancer (e.g., metastatic cancer, e.g., metastatic breast cancer), an inflammatory disease (e.g., chronic obstructive pulmonary disease (COPD), asthma, rhinitis (e.g., allergic rhinitis), inflammatory bowel disease, synovitis, rheumatoid arthritis), heart failure, septic shock, neuropathic pain, inflammatory pain, osteoarthritis, or an ocular condition (e.g., macular degeneration).
MMP-12. Binding of MMP- 12 binding proteins to cells expressing MMP- 12 can be characterized in a number assays known in the art, including FACS (Fluorescence Activated Cell Sorting), immunofluorescence, and immunocytochemistry. MMP-12 binding protein is contacted with cells and/or tissues which express or contain MMP-12, and binding is detected in accordance with the method being used. For example, a fluorescent detection system (e.g., fluorescent-labeled secondary antibody) employed for FACS and immunofluorescence analysis, or a enzymatic system is used for immunocytochemistry are generally used in these assays can be performed on non-perm. MMP-12 binding proteins can be characterized as to cellular binding by FACS (Fluorescence Activated Cell Sorting) using cells expressing MMP-12.
Individual cells held in a thin stream of fluid are passed through one or more laser beams cause light to scatter and fluorescent dyes to emit light at various frequencies. Photomultiplier tubes (PMT) convert light to electrical signals and cell data is collected. Forward and side scatter are used for preliminary identification of cells. Forward and side scatter are used to exclude debris and dead cells. Fluorescent labeling allows investigation of cell structure and function. Cell autofluorescence is generated by labeling cell structures with fluorescent dyes. FACS collects fluorescence signals in one to several channels corresponding to different laser excitation and fluorescence emission wavelength. Immunofluorescence, the most widely used application, involves the staining of cells with antibodies conjugated to fluorescent dyes such as fluorescein and phycoerythrin (PE). This method can be used to label MMP- 12 on the cell surface of MDA- MB-231 cells using biotinylated MMP- 12 binding proteins. Biotin is used in this two-step detection systems in concert with conjugated steptavidin. Biotin is typically conjugated to proteins via primary amines (i.e., lysines). Usually, between 1.5 and 3 biotin molecules are conjugated to each antibody. A second fluorescently conjugated antibody (streptavidin/PE) is added which is specific for biotin. MMP- 12 binding proteins can be characterized in cultured cells expressing the MMP- 12 antigen. The method generally used is immunocytochemistry. Immunocytochemistry involves the use of antibodies that recognize parts of the receptor that are exposed to the outside environment when expressed at the cell surface (the 'primary antibody'). If the experiment is carried out in intact cells, such an antibody will only bind to surface expressed receptors. Biotinylated or non-biotinylated MMP- 12 binding proteins can be used. The secondary antibody is then either a streptavidin/HRP antibody (for biotinylated MMP- 12 binding protein) or an anti- human IgG/HRP (for non-biotinylated MMP- 12 binding protein). The staining can then be detected using an inverted microscope. The assay can be performed in the absence of MMP-12 binding protein and in presence of lOμg/mL of MMP-12 binding protein. MMP-12 binding proteins can be characterized in assays that measure their modulatory activity toward MMP-12 or fragments thereof in vitro or in vivo. For example, MMP-12 can be combined with a substrate such as Mca-Pro-Leu-Ala-Cys(Mob)-Trp-Ala-Arg-Dap(Dnp)-NH? under assay conditions permitting cleavage by MMP-12. The assay is performed in the absence of the MMP-12 binding protein, and in the presence of increasing concentrations of the MMP-12 binding protein. The concentration of binding protein at which 50% of the MMP-12 activity (e.g., binding to the substrate) is inhibited is the IC50 value (Inhibitory Concentration 50%) or EC50 (Effective Concentration 50%) value for that binding protein. Within a series or group of binding proteins, those having lower IC50 or EC50 values are considered more potent inhibitors of MMP- 12 than those binding proteins having higher IC50 or EC50 values. Exemplary binding proteins have an IC50 value of less than 800 nM, 400 nM, 100 nM, 25 nM, 5 nM, or 1 nM, e.g., as measured in an in vitro assay for inhibition of MMP- 12 activity when the MMP- 12 is at 2 pM.
MMP- 12 binding proteins may also be characterized with reference to the activity of MMP-12 on substrates (e.g., lung extracellular matrix, elastin, gelatin, etc.). For example, cleavage of gelatin by MMP-12 can be detected in zymography. The method is based on a SDS gel impregnated with a substrate, which is degraded by the proteases resolved during the incubation period. Coomassie blue staining of the gels reveals proteolytic fragments as white bands on a dark blue background. Within a certain range, the band intensity can be related linearly to the amount of the protease loaded. Cells expressing MMP-12 are used in this assay. The assay is performed in the absence of the MMP-12 binding protein, and in the presence of increasing concentrations of the MMP-12 binding protein. The concentration of binding protein at which 50% of the MMP-12 activity (e.g., binding to the substrate) is inhibited is the IC50 value (Inhibitory Concentration 50%) or EC50 (Effective Concentration 50%) value for that binding protein. Within a series or group of binding proteins, those having lower IC50 or EC50 values are considered more potent inhibitors of MMP-12 than those binding proteins having higher IC50 or EC50 values. Exemplary binding proteins have an IC50 value of less than 800 nM, 400 nM, 100 nM, 25 nM, 5 nM, or 1 nM, e.g., as measured in an in vitro assay for inhibition of MMP-12 activity.
The binding proteins can also be evaluated for selectivity toward MMP-12. For example, a MMP-12 binding protein can be assayed for its potency toward MMP-12 and a panel of MMPs and other enzymes, e.g., human and/or mouse enzymes, e.g., MMP-I, -2, -3, -7, -8, -9, -13, -14, - 16, -17, -24, and TACE, and an IC50 value or EC50 value can be determined for each MMP. In one embodiment, a compound that demonstrates a low IC50 value or EC50 value for the MMP-12, and a higher IC50 value or EC50 value, e.g., at least 2-, 5-, or 10- fold higher, for another MMP within the test panel (e.g., MMP-I, -10) is considered to be selective toward MMP-12.
MMP-12 binding proteins can be evaluated for their ability to inhibit MMP-12 in a cell based assay, e.g., in situ zymography, e.g., in Colo205 cells or MCF-7 cells. A pharmacokinetics study in rat, mice, or monkey can be performed with MMP- 12 binding proteins for determining MMP- 12 half-life in the serum. Likewise, the effect of the binding protein can be assessed in vivo, e.g., in an animal model for a disease, for use as a therapeutic, for example, to treat a disease or condition described herein, e.g., systemic sclerosis, a cancer (e.g., metastatic cancer, e.g., metastatic colorectal, lung, or hepatocellular cancer), an inflammatory disease (e.g., chronic obstructive pulmonary disease (COPD), asthma, rhinitis (e.g., allergic rhinitis), atherosclerosis, multiple sclerosis, rheumatoid arthritis), cardiovascular disease, aneurysym, wound healing, aging and nerve damage associated with excess or inappropriate activity of MMP- 12.
Pharmaceutical Compositions
In another aspect, the disclosure provides compositions, e.g., pharmaceutically acceptable compositions or pharmaceutical compositions, which include an MMP-9 or -12-binding protein, e.g., an antibody molecule, other polypeptide or peptide identified as binding to MMP-9 or -12 described herein. The MMP-9 or -12 binding protein can be formulated together with a pharmaceutically acceptable carrier. Pharmaceutical compositions include therapeutic compositions and diagnostic compositions, e.g., compositions that include labeled MMP-9 or -12 binding proteins for in vivo imaging.
A pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion), although carriers suitable for inhalation and intranasal administration are also contemplated. Depending on the route of administration, the MMP-9 or -12 binding protein may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
A pharmaceutically acceptable salt is a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al., 1977, /. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.
The compositions may be in a variety of forms. These include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The form can depend on the intended mode of administration and therapeutic application. Many compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for administration of humans with antibodies. An exemplary mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In one embodiment, the MMP- 9 binding protein is administered by intravenous infusion or injection. In another preferred embodiment, the MMP-9 binding protein is administered by intramuscular or subcutaneous injection.
The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the binding protein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
An MMP-9 or -12 binding protein can be administered by a variety of methods, although for many applications, the preferred route/mode of administration is intravenous injection or infusion. For example, for therapeutic applications, the MMP-9 or -12 binding protein can be administered by intravenous infusion at a rate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m2 or 7 to 25 mg/m2. The route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are available. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., 1978, Marcel Dekker, Inc., New York.
Pharmaceutical compositions can be administered with medical devices. For example, in one embodiment, a pharmaceutical composition disclosed herein can be administered with a device, e.g., a needleless hypodermic injection device, a pump, or implant.
In certain embodiments, an MMP-9 or -12 binding protein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds disclosed herein cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties that are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade, 1989, /. Clin. Pharmacol. 29:685).
Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody disclosed herein is 0.1-20 mg/kg, more preferably 1-10 mg/kg. An anti- MMP-9 or -12 antibody can be administered, e.g., by intravenous infusion, e.g., at a rate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m or about 5 to 30 mg/m . For binding proteins smaller in molecular weight than an antibody, appropriate amounts can be proportionally less. Dosage values may vary with the type and severity of the condition to be alleviated. For a particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
The pharmaceutical compositions disclosed herein may include a "therapeutically effective amount" or a "prophylactically effective amount" of an MMP-9 or -12 binding protein disclosed herein. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the protein to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition is outweighed by the therapeutically beneficial effects.
A "therapeutically effective dosage" preferably modulates a measurable parameter, e.g., levels of circulating IgG antibodies or enzymatic activity, by a statistically significant degree or at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to modulate a measurable parameter, e.g., a disease-associated parameter, can be evaluated in an animal model system predictive of efficacy in human disorders and conditions, e.g., systemic sclerosis. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to modulate a parameter in vitro.
A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, because a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
Stabilization and Retention
In one embodiment, an MMP-9 or -12 binding protein is physically associated with a moiety that improves its stabilization and/or retention in circulation, e.g., in blood, serum, lymph, or other tissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold. For example, an MMP-9 or -12 binding protein can be associated with a polymer, e.g., a substantially non-antigenic polymer, such as polyalkylene oxides or polyethylene oxides. Suitable polymers will vary substantially by weight. Polymers having molecular number average weights ranging from about 200 to about 35,000 (or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used. For example, an MMP-9 or -12 binding protein can be conjugated to a water soluble polymer, e.g., hydrophilic polyvinyl polymers, e.g. polyvinylalcohol and polyvinylpyrrolidone. A non-limiting list of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained.
An MMP-9 or -12 binding protein can also be associated with a carrier protein, e.g., a serum albumin, such as a human serum albumin. For example, a translational fusion can be used to associate the carrier protein with the MMP-9 or -12 binding protein.
Kits An MMP-9 or -12 binding protein described herein can be provided in a kit, e.g., as a component of a kit. For example, the kit includes (a) an MMP-9 or -12 binding protein, e.g., a composition (e.g., a pharmaceutical composition) that includes an MMP-9 or -12 binding protein, and, optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of an MMP-9 or -12 binding protein for the methods described herein. The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to using the binding protein to treat, prevent, or diagnosis of disorders and conditions, e.g., systemic sclerosis.
In one embodiment, the informational material can include instructions to administer an MMP-9 or -12 binding protein in a suitable manner to perform the methods described herein, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). In another embodiment, the informational material can include instructions to administer an MMP-9 or -12 binding protein to a suitable subject, e.g., a human, e.g., a human having, or at risk for, a disorder or condition described herein, e.g., systemic sclerosis. For example, the material can include instructions to administer an MMP-9 or -12 binding protein to a patient with a disorder or condition described herein, e.g., systemic sclerosis. The informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in print but may also be in other formats, such as computer readable material.
An MMP-9 or -12 binding protein can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that an MMP-9 or -12 binding protein be substantially pure and/or sterile. When an MMP-9 or -12 binding protein is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred. When an MMP-9 or -12 binding protein is provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.
The kit can include one or more containers for the composition containing an MMP-9 or - 12 binding protein. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained association with the container. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of an MMP-9 or -12 binding protein. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of an MMP-9 or -12 binding protein. The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.
The kit optionally includes a device suitable for administration of the composition, e.g., a syringe, inhalant, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device. In one embodiment, the device is an implantable device that dispenses metered doses of the binding protein. The disclosure also features a method of providing a kit, e.g., by combining components described herein.
Treatments
Proteins that bind to MMP-9 or -12 and identified by the method described herein and/or detailed herein have therapeutic and prophylactic utilities, particularly in human subjects. These binding proteins are administered to a subject to treat, prevent, and/or diagnose systemic sclerosis. Treating includes administering an amount effective to alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder. The treatment may also delay onset, e.g., prevent onset, or prevent deterioration of a disease or condition.
As used herein, an amount of an target-binding agent effective to prevent a disorder, or a prophylactically effective amount of the binding agent refers to an amount of a target binding agent, e.g., an MMP-9 or -12 binding protein, e.g., an anti-MMP-9 or -12 antibody described herein, which is effective, upon single- or multiple-dose administration to the subject, for preventing or delaying the occurrence of the onset or recurrence of a disorder, e.g., a disorder described herein, e.g., systemic sclerosis. A binding agent described herein can be used to reduce angiogenesis in a subject, e.g., to treat systemic sclerosis. The method includes administering the binding protein (e.g., an MMP-9 or -12 binding protein, e.g., an anti-MMP-9 or -12 binding protein described herein) to the subject, e.g., in an amount effective to modulate systemic sclerosis, a symptom of the disorder, or progression of the disorder. The agent (e.g., an MMP-9 or -12 binding protein, e.g., an anti- MMP-9 or -12 antibody) may be administered multiple times (e.g., at least two, three, five, or ten times) before a therapeutically effective amount is attained.
Methods of administering MMP-9 or -12 binding proteins and other agents are also described in "Pharmaceutical Compositions." Suitable dosages of the molecules used can depend on the age and weight of the subject and the particular drug used. The binding proteins can be used as competitive agents to inhibit, reduce an undesirable interaction, e.g., between a natural or pathological agent and the MMP-9 or -12. The dose of the MMP-9 or -12 binding protein can be the amount sufficient to block 90%, 95%, 99%, or 99.9% of the activity of MMP- 9 or -12 in the patient, especially at the site of disease. Depending on the disease, this may require 0.1, 1.0, 3.0, 6.0, or 10.0 mg/Kg. For an IgG having a molecular mass of 150,000 g/mole (two binding sites), these doses correspond to approximately 18 nM, 180 nM, 540 nM, 1.08 μM, and 1.8 μM of binding sites for a 5 L blood volume.
In one embodiment, the MMP-9 or -12 binding proteins are used to inhibit an activity of a cell, e.g., a fibroblast or endothelial cell in vivo. The binding proteins can be used by themselves or conjugated to an agent, e.g., a cytotoxic drug, cytotoxin enzyme, or radioisotope. This method includes: administering the binding protein alone or attached to an agent (e.g., a cytotoxic drug), to a subject requiring such treatment. For example, MMP-9 or -12 binding proteins that do not substantially inhibit MMP-9 or -12 may be used to deliver nanoparticles containing agents, such as toxins, to MMP-9 or -12 associated cells or tissues, e.g., fibroblasts or endothelial cells.
The binding proteins may be used to deliver an agent (e.g., any of a variety of cytotoxic and therapeutic drugs) to cells and tissues where MMP-9 or -12 is present. Exemplary agents include a compound emitting radiation, molecules of plants, fungal, or bacterial origin, biological proteins, and mixtures thereof. The cytotoxic drugs can be intracellularly acting cytotoxic drugs, such as toxins short range radiation emitters, e.g., short range, high energy α-emitters.
To target MMP-9 or -12 expressing cells, a prodrug system can be used. For example, a first binding protein is conjugated with a prodrug which is activated only when in close proximity with a prodrug activator. The prodrug activator is conjugated with a second binding protein, preferably one which binds to a non competing site on the target molecule. Whether two binding proteins bind to competing or non competing binding sites can be determined by conventional competitive binding assays. Exemplary drug prodrug pairs are described in Blakely et al., (1996) Cancer Research, 56:3287 3292.
The MMP-9 or -12 binding proteins can be used directly in vivo to eliminate antigen- expressing cells via natural complement-dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC). The binding proteins described herein can include complement binding effector domain, such as the Fc portions from IgGl, -2, or -3 or corresponding portions of IgM which bind complement. In one embodiment, a population of target cells is ex vivo treated with a binding agent described herein and appropriate effector cells. The treatment can be supplemented by the addition of complement or serum containing complement. Further, phagocytosis of target cells coated with a binding protein described herein can be improved by binding of complement proteins. In another embodiment target, cells coated with the binding protein which includes a complement binding effector domain are lysed by complement.
Methods of administering MMP-9 or -12 binding proteins are described in "Pharmaceutical Compositions." Suitable dosages of the molecules used will depend on the age and weight of the subject and the particular drug used. The binding proteins can be used as competitive agents to inhibit or reduce an undesirable interaction, e.g., between a natural or pathological agent and the MMP-9 or -12.
The MMP-9 or -12 binding protein can be used to deliver macro and micromolecules, e.g., a gene into the cell for gene therapy purposes into the endothelium or epithelium and target only those tissues expressing the MMP-9 or -12. The binding proteins may be used to deliver a variety of cytotoxic drugs including therapeutic drugs, a compound emitting radiation, molecules of plants, fungal, or bacterial origin, biological proteins, and mixtures thereof. The cytotoxic drugs can be intracellularly acting cytotoxic drugs, such as short range radiation emitters, including, for example, short range, high energy α emitters, as described herein. In the case of polypeptide toxins, recombinant nucleic acid techniques can be used to construct a nucleic acid that encodes the binding protein (e.g., antibody or antigen-binding fragment thereof) and the cytotoxin (or a polypeptide component thereof) as translational fusions. The recombinant nucleic acid is then expressed, e.g., in cells and the encoded fusion polypeptide isolated. Alternatively, the MMP-9 or -12 binding protein can be coupled to high energy radiation emitters, for example, a radioisotope, such as I, a γ-emitter, which, when localized at a site, results in a killing of several cell diameters. See, e.g., S.E. Order, "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy", Monoclonal Antibodies for Cancer Detection and Therapy, R.W. Baldwin et al. (eds.), pp 303 316 (Academic Press 1985). Other suitable radioisotopes include a emitters, such as 212Bi, 213Bi, and 211At, and b emitters, such as 186Re and 90Y. Moreover, 177 Lu may also be used as both an imaging and cytotoxic agent.
Radioimmunotherapy (RIT) using antibodies labeled with 1311 ,90Y, and 177Lu is under intense clinical investigation. There are significant differences in the physical characteristics of these three nuclides and as a result, the choice of radionuclide is very critical in order to deliver maximum radiation dose to a tissue of interest. The higher beta energy particles of 90Y may be good for bulky tumors. The relatively low energy beta particles of I are ideal, but in vivo dehalogenation of radioiodinated molecules is a major disadvantage for internalizing antibody. In contrast, Lu has low energy beta particle with only 0.2-0.3 mm range and delivers much lower radiation dose to bone marrow compared to Y. In addition, due to longer physical half- life (compared to Y), the residence times are higher. As a result, higher activities (more mCi amounts) of Lu labeled agents can be administered with comparatively less radiation dose to marrow. There have been several clinical studies investigating the use of Lu labeled antibodies in the treatment of various cancers. (Mulligan T et al., 1995, Clin. Cane. Res. 1: 1447- 1454; Meredith RF, et al., 1996, /. Nucl. Med. 37: 1491-1496; Alvarez RD, et al., 1997, Gynecol. Oncol. 65: 94-101).
Exemplary Diseases and Conditions
The MMP-9 or -12 binding proteins described herein are useful to treat diseases or conditions in which MMP-9 or -12 is implicated (respectively), e.g., a disease or condition described herein, or to treat one or more symptoms associated therewith. In some embodiments, the MMP-9 or -12 binding protein (e.g., MMP-9 or -12 binding IgG or Fab) inhibits MMP-9 or - 12 activity (respectively), e.g., catalytic activity.
Examples of such diseases and conditions include systemic sclerosis. A therapeutically effective amount of a MMP-9 or -12 binding protein is administered to a subject having or suspected of having a disorder in which MMP-9 or -12 (respectively) is implicated, thereby treating (e.g., ameliorating or improving a symptom or feature of a disorder, slowing, stabilizing or halting disease progression) the disorder.
The MMP-9 or -12 binding protein is administered in a therapeutically effective amount. A therapeutically effective amount of an MMP-9 binding protein or an MMP- 12 binding protein is the amount which is effective, upon single or multiple dose administration to a subject, in treating a subject, e.g., curing, alleviating, relieving or improving at least one symptom of a disorder in a subject to a degree beyond that expected in the absence of such treatment. A therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition is outweighed by the therapeutically beneficial effects.
A therapeutically effective amount can be administered, typically an amount of the compound which is effective, upon single or multiple dose administration to a subject, in treating a subject, e.g., curing, alleviating, relieving or improving at least one symptom of a disorder in a subject to a degree beyond that expected in the absence of such treatment. A therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition is outweighed by the therapeutically beneficial effects. A therapeutically effective dosage preferably modulates a measurable parameter, favorably, relative to untreated subjects. The ability of a compound to inhibit a measurable parameter can be evaluated in an animal model system predictive of efficacy in a human disorder.
Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
Systemic Sclerosis Systemic sclerosis (SSc) is the generalized type of scleroderma, which is a chronic disease characterized by excessive deposits of collagen in the skin or other organs such as joints, esophagus, lower gastrointestinal (GI) tract, lung, heart and kidney. SSc can be fatal as a result of heart, kidney, lung, or intestinal damage.
There are three major forms of scleroderma: diffuse, limited and morphea/linear. Diffuse and limited sclerodermas are both systemic diseases. There is also a subset of the systemic form known as "systemic scleroderma sine scleroderma", in which the usual skin involvement is not present.
People with systemic sclerosis often have all or some of the symptoms that some doctors call CREST, which stands for the following: Calcinosis: Calcinosis is the formation of calcium deposits in the connective tissues, which can be detected by x ray. They are typically found on the fingers, hands, face, and trunk and on the skin above elbows and knees. When the deposits break through the skin, painful ulcers can result.
Raynaud's phenomenon: Raynaud's phenomenon is a condition in which the small blood vessels of the hands and/or feet contract in response to cold or anxiety. As the vessels contract, the hands or feet turn white and cold, then blue. As blood flow returns, they become red. Fingertip tissues may suffer damage, leading to ulcers, scars, or gangrene.
Esophageal dysfunction: impaired function of the esophagus that occurs when smooth muscles in the esophagus lose normal movement. In the upper esophagus, the result can be swallowing difficulties; in the lower esophagus, the problem can cause chronic heartburn or inflammation.
Sclerodactyly: thick and tight skin on the fingers, resulting from deposits of excess collagen within skin layers. The condition makes it harder to bend or straighten the fingers. The skin may also appear shiny and darkened, with hair loss. Telangiectasias: small red spots on the hands and face that are caused by the swelling of tiny blood vessels. While not painful, these red spots can create cosmetic problems. An an MMP-9 and/or -12 binding protein (e.g., an anti-MMP-9 or -12 binding protein described herein) can be used to treat or prevent one or more of these symptoms.
Limited scleroderma typically comes on gradually and affects the skin only in certain areas: the fingers, hands, face, lower arms, and legs. Many people with limited disease have Raynaud's phenomenon for years before skin thickening starts. Others start out with skin problems over much of the body, which improves over time, leaving only the face and hands with tight, thickened skin. Telangiectasias and calcinosis often follow. Because of the predominance of CREST in people with limited disease, some doctors refer to limited disease as the CREST syndrome. Diffuse scleroderma typically comes on suddenly. Skin thickening occurs quickly and over much of the body, affecting the hands, face, upper arms, upper legs, chest, and stomach in a symmetrical fashion (for example, if one arm or one side of the trunk is affected, the other is also affected). Some people may have more area of their skin affected than others. Internally, it can damage key organs such as the heart, lungs, and kidneys. Scleroderma affects the skin, and in more serious cases it can affect the blood vessels and internal organs. Common symptoms include, e.g., Raynaud's syndrome, swelling (eventually skin tightening and contractures of the fingers), polyarthralgia, dysphagia, dyspnea, lung fibrosis, acute alveolitis, pulmonary hypertension, heartburn, cardiac arrhythmias and renal crisis. The more evident symptom is usually the hardening of the skin and associated scarring. Blood vessels may also be more visible. Many SSc patients (over 80%) have vascular symptoms and Raynaud's phenomenon. During an attack, there is discoloration of the hands and feet in response to cold. Raynaud's normally affects the fingers and toes. SSc and Raynaud's can cause painful ulcers on the fingers or toes which are known as digital ulcers. Calcinosis is also common in SSc, and is often seen near the elbows, knees or other joints. Diffuse scleroderma can cause musculoskeletal, pulmonary, gastrointestinal, renal and other complications. Patients with larger amounts of cutaneous involvement are more likely to have involvement of the internal tissues and organs.
Immunologic mechanisms and familial predisposition contribute to etiology. Polymorphisms in COLl A2 and TGF-βl may influence severity and development of the disease. There is evidence implicating cytomegalovirus (CMV) as the original epitope of the immune reaction, and organic solvents and other chemical agents (e.g., vinyl chloride, bleomycin, pentazocine, epoxy and/or aromatic hydrocarbons, contaminated rapeseed oil or L-tryptophan) have been linked with scleroderma.
Treatments for some of the symptoms of scleroderma include drugs that soften the skin and reduce inflammation. Topical treatment for the skin changes of scleroderma do not alter the disease course, but may improve pain and ulceration. The skin tightness may be treated systemically with methotrexate and cyclosporin. A range of NSAIDs (nonsteroidal anti- inflammatory drugs) such as naproxen can be used to ease painful symptoms. Episodes of Raynaud's phenomenon can respond to calcium channel blockers, e.g., nifedipine; angiotensin receptor blockers, e.g., losartan; or dual endothelin-receptor antagonists, e.g., bosentan. Severe digital ulceration (ischemia) may respond to epoprostenol (prostacyclin or its analogue iloprost), prostaglandin El (alprostadil) or sympathetic blockers. Reflux esophagitis can be treated with proton pump inhibitors. Broad-spectrum antibiotics, e.g., tetracycline can suppress overgrowth of intestinal flora and alleviate malabsorption symptoms. Scleroderma renal crisis, the occurrence of acute renal failure and malignant hypertension (very high blood pressure with evidence of organ damage) in people with scleroderma, is effectively treated with drugs from the class of the ACE (Angiotensin Converting Enzyme) inhibitors. Active alveolitis is often treated with pulses of immunosuppressants,e.g., cyclophosphamide, methotrexate and azathioprine often together with a small dose of steroids. Surgery, e.g., lung transplantation can also be performed. Pulmonary hypertension may be treated, e.g., with epoprostenol (prostacyclin), bosentan and possibly aerolized iloprost.
The disclosure provides methods of treating or preventing SSc (e.g., ameliorating symptoms or the worsening of SSc) by administering a therapeutically effective amount of a MMP-9 and/or MMP-12 binding protein (e.g., an MMP-9 or MMP-12 binding proteindescribed herein; an inhibitory MMP-9 or MMP-12 binding protein, e.g., an anti-MMP-9 or anti-MMP-12 IgG or Fab) to a subject having or suspected of having SSc. Also provided are methods of treating SSc by administering a therapeutically effective amount of a MMP-9 and/or MMP-12 binding protein (e.g., an MMP-9 or MMP-12 binding proteindescribed herein) with another SSc treatment: e.g., cyclosporin, NSAIDs (e.g., naproxen), calcium channel blockers (e.g., nifedipine), angiotensin receptor blockers (e.g., losartan), epoprostenol (prostacyclin), prostacyclin analogues (e.g., iloprost), dual endothelin-receptor antagonists (e.g., bosentan), prostaglandin El, proton pump inhibitors, antibiotics (e.g., tetracycline), immunosuppressants (e.g., cyclophosphamide, methotrexate or azathioprine), ACE inhibitors, and surgery (e.g., lung transplantation).
Guidance regarding the efficacy and dosage an MMP-9 or MMP- 12 binding protein which will deliver a therapeutically effective amount of the protein can be obtained from animal models of SSc, see e.g., those described in Wu et al. Curr Rheumatol Rep. 2008;10(3): 173-82 and references cited therein.
Sjogren's syndrome
Sjogren's syndrome can be associated with SSc. Sjogren's syndrome is an autoimmune disorder in which immune cells attack and destroy the exocrine glands that produce tears and saliva.
Nine out of ten Sjogren's patients are women and the average age of onset is late 40s, although Sjogren's occurs in all age groups in both women and men. It is estimated to strike as many as 4 million people in the United States alone making it the second most common autoimmune rheumatic disease. Sjogren's syndrome can exist as a disorder in its own right
(Primary Sjogren's syndrome) or it may develop years after the onset of an associated rheumatic disorders such as rheumatoid arthritis, systemic lupus erythematosus, scleroderma, primary biliary cirrhosis etc. (Secondary Sjogren's syndrome).
The hallmark symptoms of the disorder are dry mouth and dry eyes (part of what are known as sicca symptoms). In addition, Sjogren's syndrome may cause skin, nose, and vaginal dryness, and may affect other organs of the body, including the kidneys, blood vessels, lungs, liver, pancreas, and brain.
An an MMP-9 and/or -12 binding protein (e.g., an anti-MMP-9 or -12 binding protein described herein) (alone or in combination with a second agent, e.g., a second agent dercribed herein or a second treatment for Sjorgen's syndrome) can be used to treat or prevent Sjogren's syndrome associated with systemic sclerosis.
Combination Therapies
The MMP-9 binding proteins described herein, e.g., anti-MMP-9 Fabs or IgGs, can be administered in combination with one or more of the other therapies for treating a disease or condition associated with MMP-9 activity, e.g., a disease or condition described herein, e.g., systemic sclerosis. For example, an MMP-9 binding protein can be used therapeutically or prophylactically with surgery, another MMP-9 inhibitor, e.g., a small molecule inhibitor, another anti-MMP-9 Fab or IgG (e.g., another Fab or IgG described herein), peptide inhibitor, or small molecule inhibitor. Examples of MMP-9 inhibitors that can be used in combination therapy with an MMP-9 binding protein are described herein.
One or more small-molecule MMP inhibitors can be used in combination with one or more MMP-9 binding proteins described herein. For example, the combination can result in a lower dose of the small-molecule inhibitor being needed, such that side effects are reduced.
The MMP-12 binding proteins described herein, e.g., anti-MMP-12 Fabs or IgGs, can be administered in combination with one or more of the other therapies for treating a disease or condition associated with MMP-12 activity, e.g., a disease or condition described herein, e.g., systemic sclerosis. For example, an MMP-12 binding protein can be used therapeutically or prophylactically with surgery, another MMP-12 inhibitor, e.g., a small molecule inhibitor, another anti-MMP-12 Fab or IgG (e.g., another Fab or IgG described herein), peptide inhibitor, or small molecule inhibitor. Examples of MMP-12 inhibitors that can be used in combination therapy with an MMP-12 binding protein are described herein.
One or more small-molecule MMP inhibitors can be used in combination with one or more MMP-12 binding proteins described herein. For example, the combination can result in a lower dose of the small-molecule inhibitor being needed, such that side effects are reduced. The MMP-9 or -12 binding proteins described herein can be administered in combination with one or more current therapies for treating systemic sclerosis. For example, proteins that inhibit IGF-II or that inhibit a downstream event of IGF-II/IGF-IIE activity can also be used in combination with another treatment for SSc-associated pulmonary fibrosis, such as surgery or administration of a second agent. For example, the second agent can include certain anti- inflammatory drugs (e.g., steroids), cytotoxic drugs, immunosuppressive agents, collagen synthesis inhibitors, endothelin receptor antagonist or surgery. For example, high doses of oral corticosteroids (e.g., prednisone, 40 to 80 mg daily) are the usual treatment. Cytotoxic drugs such as cyclophosphamide and immunosuppressants such as azathioprine (cyclophosphamide is also an immunosuppressant) have also been used. Collagen synthesis inhibitors such as Pirfenidone and endothelin receptor antagonists such as Bosentan may also be effective. Lung transplantation for highly selected patients with end-stage pulmonary fibrosis has been reported. In particular, cyclophosphamide or azathioprine can be used to treat SSc-associated pulmonary fibrosis. As further examples, pulses of cyclophosphamide, often together with a small dose of steroids; epoprostenol, bosentan or iloprost (e.g, aerolized iloprost) can be used.
The term "combination" refers to the use of the two or more agents or therapies to treat the same patient, wherein the use or action of the agents or therapies overlap in time. The agents or therapies can be administered at the same time (e.g., as a single formulation that is administered to a patient or as two separate formulations administered concurrently) or sequentially in any order. Sequential administrations are administrations that are given at different times. The time between administration of the one agent and another agent can be minutes, hours, days, or weeks. The use of an MMP-9 or -12 binding protein described herein can also be used to reduce the dosage of another therapy, e.g., to reduce the side-effects associated with another agent that is being administered, e.g., to reduce the side-effects of the second therapy. Accordingly, a combination can include administering a second agent at a dosage at least 10, 20, 30, or 50% lower than would be used in the absence of the MMP-9 or -12 binding protein.
The second agent or therapy can also be another agent for systemic sclerosis, e.g., cyclosporin, an NSAID (e.g., naproxen), a calcium channel blocker (e.g., nifedipine), an angiotensin receptor blocker (e.g., losartan), epoprostenol (prostacyclin), a prostacyclin analogue (e.g., iloprost, e.g., aerolized iloprost), a dual endothelin-receptor antagonist (e.g., bosentan), prostaglandin El (e.g., alprostadil), a proton pump inhibitor, an antibiotic (e.g., tetrocycline), an immunosuppressant (e.g., cyclophosphamide, methotrexate or azathioprine), an ACE inhibitor, a sympathetic blocker, or surgery (e.g., lung transplantation).
EXAMPLES Example 1: Selection and Screening of Anti-MMP-12 Fabs and IgGs
See also WO 2009/111507.
Selection strategies employed to identify anti-MMP-12 antibodies are:
(1) Selection with capture of a biotinylated form of MMP- 12 catalytic domain on a streptavidin coated surface; (2) Selection with capture of a biotinylated form of proMMP-12 (APMA-activated ) on a streptavidin coated surface; (3) Phage, suitably depleted (e.g., previous contact with streptavidin) were allowed to interact with the target, unbound phage washed away and the output sampled and/or amplified for the next round of selection. This was repeated until the output phage in ELISA analysis indicate a high percentage of binders. The phage clones were converted into sFabs. 88/518 unique sFab were identified by ELISA and sequencing (campaign A) and 168 by sequencing (campaign B). Their ability to inhibit human MMP-12, murine MMP-12 and other MMPs (1, 2, 3, 7, 8, 9, 10, 13, 14, 16, 17 and 24) was determined by usual means. The sFabs were converted to IgGIs.
Table I
Figure imgf000118_0001
Campaign A:
Table II. hMMP-12 inhibition Screen: IC50 Values
Figure imgf000118_0002
There was no crossreactivity to mouse MMP 12.
Table III: Ki values of four of the Campaign A MMP- 12 binding proteins that act as inhibitors:
Figure imgf000119_0001
The Campaign A MMP- 12 binding proteins were crossreacted against other human proteases. The results are provided in Table IV. Table IV
Figure imgf000119_0002
Crossreactivity of some of the campaign A MMP- 12 binding proteins was assessed against other human proteases. The results are provided below. Table V
Figure imgf000120_0001
Campaign B:
Three selection strategies were used. When the phage outputs in ELISA analysis indicate a high percentage of binders, the phage outputs were converted to sFabs. A high through-put sequencing campaign was done instead of high through-put ELISA and unique sFabs were recovered:
1. Three rounds on mouse MMP12: HT sequencing : 153 unique clones
2. Alternation (human-mouse MMP12 ): HT sequencing : 120 unique clones 3. Alternation (5 human peptides-mouse MMP 12): not pursued
13 inhibitors of mouse MMP- 12 were identified including 1 that is cross reactive with human MMP- 12. The following peptides were designed: Table VI
Figure imgf000121_0001
BIOTIN - CG XXXXXXXXXXXXXXXXXX - COOH
Several MMP- 12 non-inhibitor antibodies were identified for Campaign B. These antibodies are described in FIGURE 1.
In addition, several MMP- 12 antibody inhibitors of huMMP12 were identified (100 nM): Table VII
Figure imgf000121_0002
Figure imgf000122_0001
As shown in FIGURE 2, 539B-M0008-H09 is cross reactive with human MMP-12 and murine MMP-12. 539B-M0008-H09 showed a linear relationship between IC50 and concentration (μM) for both human MMP-12 and murione MMP-12, see FIGURE 3. The Ki of 539B-M0008-H09 for human MMP-12 is 2.8 + 0.8 nM and the Km is 16 ^6 μM. The Ki of 539B-M0008-H09 for murine MMP-12 is 2.2 ± 0.6 nM and the Km is 42 ^17 μM.
In addition, 539B-M0011-Hl 1 was found to be an inhibitor of murine MMP-12 but not human MMP-12. This is shown in FIGURE 4.
The cross reactivity of several MMP-12 binding proteins from Campaign B was assessed for cross reactivity with other MMPs. Table VIII
Figure imgf000122_0002
Figure imgf000123_0001
As shown in FIGURE 5, using ELIA competition binding assays, it was shown that M08H09 cross reacts with human and murine MMP-12, M0013-G12 and M0016-A11 inhibit human MMP-12 and R0062-E11 inhibits murine MMP-12.
Example 2: Evaluation of M08-H09 on Inflammation
The purpose of the following experiments were to determine the effect of 539B-M008- H09 (M08-H09) on inflammation and specifically on inflammatory cell infiltration into the carrageenan-stimulated mouse air pouch and on the OVA-challenged mouse model.
For the OVA-challenged mouse model, the mice were sensitized by IP administration of OVA/ Alum on day 0 and day 7. Six hours prior to OVA challenge, M08-H09 was administered IP to the mice. The mice were challenged by pulmonary administration of OVA. On day 26, metacholine induction and airway hyperresponsiveness was measured. Six different groups were tested. Group 1 was a control group that received PBS. Grous 2, 3, 4 and 5 were administered M08-H09 at doses of 1 mg/kg, 5 mg/kg, 10 mg/kg and 25 mg/kg. Group 6 was administered 25 mg/kg of Mll-Hll.
The OVA challenged mice were assessed for BAL inflammation based upon differential cell counts, lung histology by quantifying global inflammation, measurement of airway responsiveness to metacholine challenge, IL-4, IL-5 and IL- 13 ELISA measurement of BAL or protein lung extracts, serum specific IgE measurement, lung histology using congo red staining to determine eosinophil counts around bronchi and measurement of MMP-12 activity in the lungs using a fluorogenic substrate. As shown in FIGURE 6, M08-H09 at all doses resulted in a decrease in the peribronchial inflammation score. A significant decrease was seen at 10 mg/kg and 25 mg/kg doses of M08- H09.
As shown in FIGURE 7, the differential cell counts showed that M08-H09 results in eosinophil percentages that are decreased.
For the carrageenan-stimulated mouse air pouch model, subcutaneous injection of air into the hind flank or back of mice produces an air pouch in a week, the interior surface of which contains both fibroblast-like and macrophage-like cells. Inflammatory stimulation of the pouch results in leukocyte recruitment to the pouch and release of mediators (cytokines) into the exudate. Preventing inflammatory cell infiltration into the air pouch may translate into preventing inflammation of the synovium in rheumatoid arthritis.
The effect of M08-H09 on inflammatory cell infiltration was compared to inflammatory cell infiltration indomethacin and a control (PBS). In addition, inflammatory cell recruitment was compared to an unstimulated mouse. As shown in FIGURE 8, M08-H09 significantly decreased total white blood cell infiltration and specifically neutrophil and lymphocyte infiltration.
Example 3: Affinity Matured Variants of M08-H09 Table IX summarizes cycle 1 of the affinity maturation of M08-H09.
Table IX:
Figure imgf000125_0001
In cycle II of the affinity maturation, 109 Fabs were analyzed and 26 Fabs were selected as improved inhibitors. The IC50 on human and murine MMP- 12 was measured and an affinity ranking on human and murine MMP- 12 was performed. For this analysis, 4 Fabs were selected and reformatted as IgGs. These four IgGs are described below in Table X:
Figure imgf000127_0001
Figure imgf000128_0001
539B-M131A06 was cross reactive with human and murine MMP-12. It has a IC50 or Ki of <0.3 nM and a KD of 180 pM for human MMP-12 and 73-150 pM for murine MMP-12.
Example 4: Modification of 539B-M131-A06 (M131A06) to remove a glycosylation site in CDRl of the variable light chain.
As shown in FIGURE 9, CDR 1 of the variable light chain of M131 A06 was modified to remove a glycosylation site. Removal of the glycosylation site in CDRl did not effect binding of M131A06 to MMP-12. the MMP-12 antibody with the glycosylation site removed is referred to as 539B-M131A06-GA-S.
Example 5
FIGURES 1OA and 1OB summarize the identification of amino acid changes in affinity matured variant HV-CDRs (cycles 1 and 2) that contribute to improvement in affinity and inhibition properties.
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
EVQLLESGGGLVQPGGSLRLSC
EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYNMSWVRQAPGKGLEWVSGISPSGGPTGYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKDIRGAYSSGLFDYWGRGTLVTVSS
EVQLLESGGGLVQPGGSLRLSCAASGFTFSLYHMHWVRQAPGKGLE WVSG IGP SGGWT I YADSVKGRFT I SRDNSKNTLYLQMNSLRAEDTAVYYCAKD IRGAYS SGLFDYWGRGTLVTVSS EVQLLESGGGLVQPGGSLRLSCAASGFTF SE YNMHWVRQAPGKGLE WVSGISPSGGMTHYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKDIRGAYSSGLFDYWGRGTLVTVSS
Figure imgf000137_0001
EVQLLESGGGLVQPGGSLRLSCAASGFTF SP YWMHWVRQAPGKGLE WVSGIGPSGGP TF YADS IKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKDIRGAYSSGLFDYWGRGTLVTVSS
Figure imgf000137_0002
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYWMQWVRQAPGKGLE WVSG IVP SGGLTMYADSVKGRFT I SRDNSKNTLYLQMNSLRAEDTAVYYCAKD IRGAYS SGLFDYWGRGTLVTVSS
EVQLLESGGGLVQPGGSLRLSC2
EVQLLESGGGLVQPGGSLRLSC2
Cycle 2 Inhibitors-Light Chain Variable Region Sequences
Initial Name LV-FR1 LV-CDR1 LV-FR2 LV-CDR2 LV-FR3 LV-CDR3 LV-FR4
539B-M0105-
C05 QD IQMTQSPSSLSASVGD RVTITC RANQSIYTYLN WYQQKPGKAPELLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQAGI FGQGTKLEIK
539B-M0105-
E1 1 QDIQMTQSPSSLSASVGDRVTITC RANQSIYTYLN WYQQKPGKAPELLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQAGI FGQGTKLEIK
539B-M0105-
F08 QDIQMTQSPSSLSASVGDRVTITC RANQSIYTYLN WYQQKPGKAPELLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQAGI FGQGTKLEIK
539B-M0107-
A12 QDIQMTQSPSSLSASVGDRVTITC RANQSIYTYLN WYQQKPGKAPELLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQAGI FGQGTKLEIK
539B-M0108-
A02 QDIQMTQSPSSLSASVGDRVTITC RANQSIYTYLN WYQQKPGKAPELLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQAGI FGQGTKLEIK
539B-M0109-
G1 1 QDIQMTQSPSSLSASVGDRVTITC RANQSIYTYLN WYQQKPGKAPELLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQAGI FGQGTKLEIK
539B-M01 10-
G05 QDIQMTQSPSSLSASVGDRVTITC RANQSIYTYLN WYQQKPGKAPELLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQAGI FGQGTKLEIK
539B-M0129-
B1 1 QDIQMTQSPSSLSASVGDRVTITC RANQSIYTYLN WYQQKPGKAPELLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQAGI FGQGTKLEIK
H
E E E E E E
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
EVQ
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
Figure imgf000149_0001
Figure imgf000150_0001
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Figure imgf000154_0001
Figure imgf000155_0001
Figure imgf000156_0001
Example 6: Additional MMP- 12 Binding Proteins Twenty-one additional mutants were prepared: Eighteen mutants of DX-2712 were made by changing residues in the CDRl and 2 of the HC of DX-2712. A residue in the light chain of each of DX-2712, M0121-E07, and M0008-H09 (the parental antibody) was glycosylated.
18 mutants of DX-2712:
Figure imgf000157_0001
LC S25N glycosylated variants of DX-2712, M008-H09, M0121-E07:
Figure imgf000158_0001
Figure imgf000159_0001
Figure imgf000160_0001
Figure imgf000161_0001
Figure imgf000162_0001
Figure imgf000163_0001
Figure imgf000163_0002
Figure imgf000164_0001
Figure imgf000165_0001
Figure imgf000166_0001
Figure imgf000167_0001
Figure imgf000168_0001
Figure imgf000169_0001
Figure imgf000170_0001
Example 7: Selection and Screening of Anti-MMP-9 Fabs and IgGs
See also WO 2009/111450.
Two strategies were employed to identify anti-MMP-9 antibodies: (1) Capture of a non-biotinylated form of MMP-9 (PMA-activated) by a biotinylated binding but not inhibiting Fab with the subsequent immobilization of the biotinylated entity on a streptavidin coated surface; and
(2) MMP-9 (PMA activated) in solution. Phage, suitably depleted (e.g., previous contact with streptavidin) were allowed to interact with the target, unbound phage washed away and the output sampled and/or amplified for the next round of selection. This was repeated until the output phage in ELISA analysis indicate a high percentage of binders. 128/2076 unique sFabs were identified by ELISA and sequencing.
After sequencing analysis, the phage display were converted into sFabs and then into IgGIs. Their ability to inhibit MMP-9 and other MMPs (1, 3, 7, 8, 9, 10, 12, 13, 14) was determined by usual means. Compounds were initially screened at 1 μM against MMP-9 and those compounds that inhibited MMP-9 >80% were subjected to additional screens against purified recombinant human MMPs. For these additional screens, an IC50 value was determined.
Example 8: Exemplary Clone Identified
539A-M0166-F10 is a selective inhibitor of human MMP-9 (IC50=I.8 +0.3nM). 539A- M0166-F10 does not inhibit activity of mouse MMP-9. 539A-M0166-F10 potently inhibits activity of hMMP-9 on tumor sections.
Example 9: CDR Amino Acid Sequences of MMP-9 Binding Anti-MMP-9 Binding Fabs
Unique Fab on phage sequences 55 Fabs and 25 Fabs from the two screens were determined. The results are summarized in Table 3.
Figure imgf000172_0001
Figure imgf000173_0001
Figure imgf000174_0001
Figure imgf000175_0001
Figure imgf000176_0001
Figure imgf000177_0001
Example 10: CDR Amino Acid Sequences of MMP-9 Binding Anti-MMP-9 Binding Fabs
Unique Fab on phage sequences SC-OU SR-OOl 539A-M00166, SC-Ol 5 SR-OOl 539A- M0167, SC-016 SR-OOl 539A-M0168)
1. SC-014 SR-OOl 539A-M00166 (phage was depleted on biotinylated Fab M0076- D03 immobilized streptavidin beads were selected against MMP-9 captured by Fab D03 immobilized on streptavidin beads)
42 intact clones (both LV and HV); all clones unique to this selection arm, except for 1 clone that was found also in SC-015 SR-001 (plate MO 167)
2. SC-015 SR-001 539A-M0167 (a similar procedure as above was followed but the Fab used during depletion and selection was M0078-G07)
24 intact clones (both LV and HV); all clones unique to this selection arm, except for 1 clone described above and second clone found in previous selection attempts (more on that later)
3. SC-016 SR-001 539A-M0168 (phage depleted on D03 streptavidin beads were incubated with MMP-9 in solution and subsequently the MMP-9 with or without phage captured onto D03 streptavidin beads)
18 intact clones (both LV and HV); all clones unique to this selection arm Results are summarized in Table 4.
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
Figure imgf000182_0001
Figure imgf000183_0001
Figure imgf000184_0001
Figure imgf000185_0001
Figure imgf000186_0001
Figure imgf000187_0001
Example 11 : CDR Sequences of MMP-9 Binding Fabs
Unique Fab on phage sequences SC-017 SR-OOl 539A-M00166, SC-018 SR-001 539A- MOl 67, SC-019 SR-001 539A-M0168: Results are summarized in Table 5.
Figure imgf000189_0001
Figure imgf000190_0001
Example 12: Affinity Ranking of MMP-9 Binding Fabs
Affinity ranking of 24 Fabs from the ROO 17 plate was performed by Flexchip. Results are summarized in Table 6.
Figure imgf000191_0001
Figure imgf000192_0002
Example 13: Competition Experiments
Results of the compertition experiments are summarized in Table 7.
Figure imgf000192_0001
R0025-B12_M0131-F06 used for competition.
Example 14:
As shown in FIGURE HA, antibody 539A-M0166-F10 has an IC50 of 4.3 ± 1.9 nM on human MMP-9 activity. The IC50 is ~ 33nM for the 539A-M0166-F10 Fab.
FIGURE HB shows that 539A-M0166-F10 is specific for human MMP-9 (hMMP-9) as compared to the other human (h) and murine (m) MMPs tested. The residual enzyme activity was measured in the presence of 1 μM antibody (Fab or hlgG-l, as indicated in FIG. 1 IB). The human MMP-I, -2, -3, -7, -8, -9, -10, -12, -13, and -14 were obtained from BIOMOL (Human MMP-9: SE-244, BIOMOL; Human MMP-14: SE-259, BIOMOL; Human MMP-I, -2, -8, -13: MMP MultiPack-1 from BIOMOL; Human MMP-3, -7, -10, -12: MMP MultiPack-2 from BIOMOL). The mouse MMP-2 and -9 were from R&D (Mouse MMP-9: 909-MM, R&D Mouse MMP-2: 924-MP, R&D). The substrate was Mca-Pro-Lys-Pro-Leu-Ala-Leu-Dap(Dnp)- Ala-Arg-NH2 (M-2225, Bachem) for human MMP-3, and Mca-Lys-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala- Arg-NH2 (M-2350, Bachem) for all the other enzymes. The substrate concentration in the assay was 10 μM.
Example 15:
539A-M0166-F10 also decreases MMP-9 activity in MCF-7 and Colo205 tumors, as measured by in situ zymography (data not shown).
Example 16: The DNA and amino acid sequences of variable regions of 539A-M0166-F10 sFAB are as follows:
539A-M0166-F10 (phage/SFAB) VL leader +VL
Figure imgf000193_0001
539A-M0166-F10 (phage/SFAB) VH leader +VH
Figure imgf000194_0001
Example 17:
Experiments were performed to characterize the interaction of the M0166-F10 hlgGl with human MMP-9. The Ki was measured and the inhibition mechanism was determined. The results show that inhibition of human MMP-9 by M0166-F10 appears to follow a competitive model, with a Ki value equal to 0.3 + 0.5nM.
The experiments were performed as follows: Materials : - Substrate : Mca -KPLGL-Dap(Dnp)-AR-NH2 (M-2350) from BACHEM (521575). A 1OmM stock solution was prepared in DMSO.
- Human MMP-9 catalytic domain (BIOMOL, SE-244), stock solution at 0.24 mg/ml.
- M0166-F10 MgGl : 2551-095. Dialysed against TCN. Stock solution at 0.226 mg/ml.
- Experiments were performed in TCNB: 50 mM Tris/HCl, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij 35, pH 7.5.
- 96-well black plates from Perkin Elmer (6005270).
Spectramax M2e to measure fluorescence emission of the substrate upon hydrolysis (temperature control set at 300C; λeXC=328 nm and λem=393 nm). Procedure :
90 μl of the enzyme (final concentration = 0.6 nM) was preincubated with 90 μl of various concentrations (0-100 nM final) of M0166-F10 for 1.5 h at 300C. 20 μl the substrate was then added to a final concentration ranging from 3 to 15μM, and initial rates were recorded. Each data point was measured in triplicate, and initial rates were averaged. Averaged initial rates were plotted against the MOl 66-Fl 0 concentration for each substrate concentration, and ICso's were calculated using the following equation :
Figure imgf000195_0001
The IC50 values were then plotted against the substrate concentration.
Results :
The plot of the measured IC50 (nM) vs. the substrate concentration (μM) is shown in FIGURE 12. The IC50 increases linearly with the substrate concentration, which indicates that M0166-F10 behaves as a competitive inhibitor of the human MMP-9. For a competitive inhibition model, the following equation applies:
Figure imgf000195_0002
and therefore the value of the K1 can be calculated from the intercept. Here, K1 = 0.3 + 0.5 nM.
The IC50 measurements at various concentrations of substrate (3 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, and 15 μM) are shown in FIGURE 13.
Example 18: Exemplary Clone Identified
539A-M0240-B03 is a selective inhibitor of MMP-9. 539A-M0240-B03 can decrease or inhibit the activity of human and mouse MMP-9.
The sequences of the complememtarity determining regions (CDRs) of 539A-M0240-B03 light chain (LC) and heavy chain (HC) are as follows:
LC CDRl: TGTSSDVGGYNYVS LC CDR2: DVSKRPS LC CDR3: CS YAGS YTLV
HC CDRL TYQMV
HC CDR2: VIYPS GGPTVYADS VKG
HC CDR3: GEDYYDSSGPGAFDI
Example 19: Additional MMP-9 binding proteins
A protein containing the HC CDR sequences of 539A-M0240-B03 (M0240-B03) and the light chain sequence shown below can be used in the methods described herein. A protein containing the LC CDRs shown below and the HC CDRs of 539A-M0240-B03, or a protein containing the LC variable region (light V gene) shown below and the 539A-M0240-B03 HC CDRs can also be used in the methods described herein. The protein can include a constant region sequence, such as the constant region (LC- lambda 1) shown below. Light V gene = VL2_2e; J gene = JL3
FRl-L CDRl-L FR2 -L CDR2 -L
QSALTQPRSVSGSP GQSVT I SC TGTSSDVGGYNYVS WYQQHP GKAPKLMIY DVSKRPS GVPD
FR3-L CDR3-L FR4 -L RF SGSKSGNTASLT I SGLQAEDEADYYC CSYAGSYTLV F GGGTKLTVL
LC-lambdal
GQPKAAP SVTI CQVTHEGSTVEKTVAPTECS
CDR regions are in bold.
The amino acid and nucleic acid sequences for another exemplary protein that can be used in the methods described herein are provided below. A protein containing the LC and HC CDRs shown below, or a protein containing the light chain and heavy chain variable regions (LV and HV, respectively) shown below can also be used in the methods described herein. 539A-M0240-B03: Parental isolate (sFab; IgG-pBhl(f)).
539A-X0034-C02 (GS clone) (X0034-C02): DX-2802: Germlined, sequence optimized. The entire antibody fragment, containing the signal sequence, variable region and constant region of both the light and heavy chains were sequenced. The sequence data is available in 539A-R0108-A01 (539A-X0034-C02).
Figure imgf000198_0001
Figure imgf000199_0001
The amino acid and nucleic acid sequences for another exemplary protein that can be used in the methods described herein are provided below. A protein containing the LC and HC CDRs shown below, or a protein containing the light chain and heavy chain variable regions (LV and HV, respectively) shown below can also be used in the methods described herein. A protein containing the light chain and heavy chain (designated as LV+ LC and HV + HC, respectively, below) sequences can also be used.
Figure imgf000201_0001
Figure imgf000202_0001
Figure imgf000203_0001
Example 20: Additional MMP-9 binding proteins
Table 80 gives the LV and HV CDR sequences of M0279-A03, M0279-B02, M0288-C08, and M0281-F06.
Table 80
Figure imgf000204_0001
VL and VH for Fabs affinity matured for binding to hMMP9 and mMMP9
M0279-A03-LC SEQ ID NO : 37
QDIQMTQSPATLSVSPGERVTLSC KASHSISRNLA WYQQKPGQAPRLLIF GASTRAT GIPARFSGSGSGTEFTLTISSLEAEDFAVYYC QQRRNWPVT FGPGTKLDFK
M0279-A03-HC SEQ ID NO : 38
EVQLLESGGGLVQPGGSLRLSCAASGFTFS QYPMW WVRQAPGKGLEWVS YIVPSGGRTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DRAYGDYVGWNGFDY WGQGTLVTVSS
M0279-B02-LC SEQ ID NO : 39
QDIQMTQSPATLSVSPGERATLSC RASQSVSSNLA WYQQKPGQAPRLLIY GASTRAT GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQRRNWPVT FGQGTRLEII
M0279-B02-HC SEQ ID NO : 40
EVQLLESGGGLVQPGGSLRLSCAASGFTFS QYPMW WVRQAPGKGLEWVS YIVPSGGRTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DRAYGDYVGWNGFDY WGQGTLVTVSS
M0288-C08-LC SEQ ID NO : 41 QD IQMTQSP GTLSVSPGERVTLSC RASQSVSSYLA WYQQKPGQAPRLLIY GASTRVT GIPARF SGSGSGTEFTLT I SSLQSEDFAVYYC QQRSSWP IT FGQGTRLE IK
M02 88-C 08 -HC SEQ ID NO : 42
EVQLLESGGGLVQPGGSLRLSCAASGFTFS QYPMW WVRQAPGKGLEWVS YIVPSGGRTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DRAYGDYVGWNGFDY WGQGTLVTVSS
LC for Fab M281-F06 (SEQ ID NO: 44) QDIQMTQSPA ALSLSPGERA TLSCRASQSV SSDLAWYQQK PGQAPRLLIY GASTRATGIP 60 ARFSGSRSGT AFTLTISSLE PEDFAVYYCQ QRSNWPVTFG QGTKLEIK 108
HC for Fab M281-F06 (SEQ ID NO: 45)
EVQLLESGGG LVQPGGSLRL SCAASGFTFS QYPMWWVRQA PGKGLEWVSY IVPSGGRTYY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDR AYGDYVGWNG FDYWGQGTLV 120
TVSS 124
Figure imgf000205_0001
Example 21: Additional MMP-9 binding proteins
See also WO 2009/111508.
M237-D02 (also referred to herein as 539A-M0237-D02) was used as the parent antibody for affinity maturation. Two libraries were built and Fabs that bind all of the targets (hMMP9, hMMP2, mMMP9, and mMMP2) were selected. One library allows the selected LC of M237-D02 to be replaced with any LC of the FAB -310 library (Hoet et al., Nat Biotechnol. 2005 23:344-348). The other library allowed HC CDRl-2 to be replaced by any HC CDRl-2 of the FAB-310 library. Table 50 gives the LV and HV CDR sequences of the affinity matured variants of M237-D02. Following Table 5 is a listing of the full sequence of LV and HV for these antibodies. Sequences for VL, VH and part of the constant region for M0237-D02, M0275-D03, M0273-G07, M0273-C10, M0273-A11, M0276-F11, M0275-E12, M0274-G08, M0272-H08, M0273-B10, M0301- D12, M0307-F04, M0299-A12, M0301-A09, M256-D11, M299-C08, M281-F06, and M306-D04 are listed afterwards.
Table 60 shows the selected FABs from the HC-CDRl-2 library at positions 25- 66. Non-standard position 58a was allowed so that sequences having an insert could be displayed. In Table 60, "-" means that the sequence is identical to M237-D02; "#" means there is a deletion.
Table 70 shows the VLs of M0237-D02 and M0275-D02 compared to germline. In Table 70, "-" means that the sequence is identical to germline L6::JK5.
Antibodies having the sequences described herein can be expressed by joining DNA encoding a signal sequence to DNA encoding the sequences given as is known to persons with skill in the art.
The VH CDRl and CDR2 sequences of M0237-D02, M0275-D03, M0273-G07, M0273-C10, M0273-A11, M0276-F11, M0275-E12, M0274-G08, M0272-H08, M0273- BlO, M0301-D12, M0307-F04, M0299-A12, M0301-A09, and M256-D11 as shown in Table 6 were analyzed and the following sequence characteristics were observed.
VH CDRl contains residues 31-35 and only residues 31, 33, and 35 were allowed to vary. All but one of the isolates have W35, suggesting the importance for binding all four targets. The P33 of the parental M0237-D02 is preferentially replaced with D, evidently preferred for binding all four targets. At position 31, there is no strong preference although four isolates have H. Antibodies that are more likely to have suitable solubility properties could be picked.
VH CDR2 contains residues 50, 51, 52, 52a, and 53-65. Positions 50, 52, 52a, 56, and 58 were varied. At position 50, the parental Y has mostly changed to V (only VGYRSW were allowed). At position 52, the parental V has mostly changed to Y (only VGYRSW were allowed). At 52a, only P and S were allowed and both seem to be accepted. M0275-D03 lacks an amino acid at position 55. At position 56, all amino acids except C were allowed. The parental R has been rejected but there is no clear preference; there are 4 Ys, 3 Ps, 3 As, 2 Ts, 1 F, and 1 G. At position 58, all amino acids except C were allowed. Two of the isolates retain the parental Y, 5 have F, 2 have G, 2 have S, and one has A.
There are only two VLs in this collection of isolates (SEQ ID NO: 1-30). One is the VL with SEQ ID NO:3, which was originally isolated from M0237-D02. The other is shown in Table 80 as M0275-D03-LC. The departures from germline in the framework regions was changed to give the sequence M0237-D02 (SEQ ID NO: 1) shown in Table 800.
Figure imgf000207_0001
Figure imgf000208_0001
Figure imgf000209_0001
Figure imgf000210_0001
Figure imgf000211_0001
Figure imgf000212_0001
Figure imgf000213_0001
Figure imgf000214_0001
Figure imgf000215_0001
Figure imgf000216_0001
Figure imgf000217_0001
Figure imgf000218_0001
Figure imgf000219_0001
Figure imgf000220_0001
Figure imgf000221_0001
Table 80 : Abs that bind MMP-9 but do not inhibit
M0076-D03-LC SEQ ID NO :
QDIQMTQSPSSLSASVGDRVTITC RASQGIRNDLD WYQQKPGTAPKRLIY SASNLQS GVPSRFSGSGSGTEFTLTISNLQPEDLATYFC LQHNSFPLT FGQGTKVEIK
M0076-D03-HC SEQ ID NO :
EVQLLESGGGLVQPGGSLRLSCAASGFTFS LYRMN WVRQAPGKGLEWVS YI GSSGGATAYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GAWYLDS WGQGTLVTVSS
M0078-G07-LC SEQ ID NO :
QDIQMTQSPGTLSVSPGERATLSC RASQSVSSDLA WYQHKPGQAPRLLIY GVSTKAT GVPARFSGSGSGTEFTLTISSLQSEDLAVYYC QQYHNWPPLT FGGGTKVE IK
M0078-G07-HC SEQ ID NO :
EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYTME WVRQAPGKGLEWVS WISPSGGYTFYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTALYYCAR GYSYGSIDL WGRGTLVTVSS
Figure imgf000222_0001
Figure imgf000224_0001
Figure imgf000225_0001
Figure imgf000226_0001
Figure imgf000227_0001
Figure imgf000228_0001
Figure imgf000229_0001
Figure imgf000230_0001
Figure imgf000231_0001
Figure imgf000232_0001
Figure imgf000233_0001
Figure imgf000234_0001
Figure imgf000235_0001
Figure imgf000236_0001
Figure imgf000237_0001
Figure imgf000238_0001
Figure imgf000239_0001
Figure imgf000240_0001
Figure imgf000241_0001
Figure imgf000242_0001
Figure imgf000243_0001
Figure imgf000244_0001
Figure imgf000245_0001
Figure imgf000246_0001
Figure imgf000247_0001
Figure imgf000248_0001
Figure imgf000249_0001
Figure imgf000250_0001
Figure imgf000251_0001
Figure imgf000252_0001
Figure imgf000253_0001
Figure imgf000254_0001
Figure imgf000255_0001
Figure imgf000256_0001
Figure imgf000257_0001
Figure imgf000258_0001
Figure imgf000259_0001
Figure imgf000260_0001
Figure imgf000261_0001
Figure imgf000262_0001
Figure imgf000263_0001
Figure imgf000264_0001
Figure imgf000265_0001
Figure imgf000266_0001
Figure imgf000267_0001
Figure imgf000268_0001
Figure imgf000269_0001
Figure imgf000270_0001
Figure imgf000271_0001
Figure imgf000272_0001
Figure imgf000273_0001
Figure imgf000274_0001
Figure imgf000275_0001
Figure imgf000276_0001
Figure imgf000277_0001
Figure imgf000278_0001
Figure imgf000279_0001
Figure imgf000280_0001
Figure imgf000281_0001
Figure imgf000282_0001
Figure imgf000283_0001
Figure imgf000284_0001
Figure imgf000285_0001
Figure imgf000286_0001
Figure imgf000287_0001
Figure imgf000288_0001
Figure imgf000289_0001
Figure imgf000290_0001
Figure imgf000291_0001
Figure imgf000292_0001
Figure imgf000293_0001
Figure imgf000294_0001
Figure imgf000295_0001
Figure imgf000296_0001
Figure imgf000297_0001
Figure imgf000298_0001
Figure imgf000299_0001
Figure imgf000300_0001
Figure imgf000301_0001
Figure imgf000302_0001
Figure imgf000303_0001
Figure imgf000304_0001
Figure imgf000305_0001
Figure imgf000306_0001
Figure imgf000307_0001
Figure imgf000308_0001
Figure imgf000309_0001
Figure imgf000310_0001
Figure imgf000311_0001
Figure imgf000312_0001
Figure imgf000313_0001
Figure imgf000314_0001
Figure imgf000315_0001
Figure imgf000316_0001
Figure imgf000317_0001
Figure imgf000318_0001
Figure imgf000319_0001
Figure imgf000320_0001
VL. VH and part of the constant region for M0237-D02. M0275-D03. M0273-G07. M0273-C10, M0273-A11, M0276-F11, M0275-E12, M0274-G08, M0272-H08, M0273- BlO1 M0301-D12, M0307-F04, M0299-A12, M0301-A09, M256-D11, M299-C08, M281-F06. and M306-D04 Fabs affinity matured for binding to hMMP9. hMMP2. mMMP9, and mMMP2
Figure imgf000320_0002
Figure imgf000321_0001
Figure imgf000322_0001
Figure imgf000323_0001
Figure imgf000324_0001
Figure imgf000325_0001
Figure imgf000326_0001
Figure imgf000327_0001
Figure imgf000328_0001
Figure imgf000329_0001
Figure imgf000330_0001
Figure imgf000331_0001
Figure imgf000332_0001
Figure imgf000333_0001
Figure imgf000334_0001
Example 22. Germlined, optimized MMP-9/2 specific antibodies
Antibodies X0106-A01, X0106-B02, X0106-C04, X0106-E4, and X0106-F05 were produced as IgGIs and tested for inhibition of human and mouse MMP-9 and MMP-2. X0106-A01, X0106-B02, X0106-C03, X0106-E4, and X0106-F05 are the germlined, optimized version of M0256-D11, M0276-F11, M0274-G08, M0275-D03, and M0307-F04, respectively.
Table 110 gives the LV and HV CDR sequences of the germlined, optimized MMP-9/MMP-2 specific antibodis (X0106-A01, X0106-B02, X0106-C04, X0106-E4, and X0106-F05). Following Table 10 is a listing of the sequences of VL and VH plus part of the constant regions for these antibodies.
Figure imgf000335_0001
VL and VH and part of constant region for Fabs germlined, optimized for binding to hMMP9, hMMP2, mMMP9, and mMMP2
X0106-A01-LC SEQ ID NO: 43
EIVLTQSPATLSLSPGERATLSC RASQSISSFLA WYQQKPGQAPRLLIY DASYRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRGNWPIT FGQGTRLEIK
X0106-A01-HC SEQ ID NO: 44
EVQLLESGGGLVQPGGSLRLSCAASGFTFS HYDMW WVRQAPGKGLEWVS VIYSSGGPTFYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DRAYGDYVGWNGFDY WGQGTLVTVSS
Figure imgf000336_0001
X0106-F05-HC SEQ ID NO: 52
EVQLLESGGGLVQPGGSLRLSCAASGFTFS LYDMW WVRQAPGKGLEWVS VIYSSGGYTGYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DRAYGDYVGWNGFDY WGQGTLVTVSS
Figure imgf000337_0001
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
EQUIVALENTS
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. A method of treating or preventing systemic sclerosis in a subject, the method comprising: administering an isolated antibody that binds MMP-9 to the subject, wherein the antibody binds the same epitope or competes for binding with an antibody selected from the group consisting of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288- C08, and M0281-F06.
2. The method of claim 1, wherein the antibody competes with or binds the same epitope as DX-2802.
3. A method of treating or preventing systemic sclerosis in a subject, the method comprising: administering an isolated protein comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence to the subject, wherein: the heavy chain immunoglobulin variable domain sequence comprises three CDR regions from the heavy chain variable domain of DX-2802, 539A-M0240-B03,
M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06, and/or the light chain immunoglobulin variable domain sequence comprises three CDR regions from the light chain variable domain of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10,
M0279-A03, M0279-B02, M0288-C08, and M0281-F06 (respectively), and the protein binds to and inhibits MMP-9.
4. The method of claim 3, wherein the three CDR regions from the heavy chain variable domain are from DX-2802 and/or the three CDR regions from the light chain variable domain are from DX-2802.
5. The method of claim 3, wherein the the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of DX-2802, 539 A-M0240- B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06, and/or the the light chain immunoglobulin variable domain sequence comprises the light chain variable domain of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06 (respectively).
6. The method of claim 3, wherein the the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of DX-2802, and/or the the light chain immunoglobulin variable domain sequence comprises the light chain variable domain of DX-2802.
7. The method of claim 3, wherein the protein comprises the heavy chain of DX- 2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075- D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06, and/or the light chain of DX-2802, 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03, M0072-H07, M0075-D12, M0166-F10, M0279-A03, M0279-B02, M0288-C08, and M0281-F06 (respectively).
8. The method of claim 3, wherein the protein comprises the heavy chain of DX- 2802, and/or the light chain of DX-2802.
9. A method of treating or preventing systemic sclerosis in a subject, the method comprising: administering an isolated antibody that binds MMP- 12 to the subject, wherein the antibody binds the same epitope or competes for binding with an antibody selected from the group consisting of DX-2712, a mutant or variant of DX-2712, 539B-X0041-D02,
M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134- C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134- G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135- G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129- BIl, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, M0118-F11, M0125-G07, M0124- E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04,
M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065- H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088- F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006- BlO, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-El l.
10. The method of claim 9, wherein the antibody competes with or binds the same epitope as DX-2712.
11. A method of treating or preventing systemic sclerosis in a subject, the method comprising: administering an isolated protein comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence to the subject, wherein: the heavy chain immunoglobulin variable domain sequence comprises three
CDR regions from the heavy chain variable domain of DX-2712, a mutant or variant of DX-2712, 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134- COl, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03,
M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03,
M0132-A04, M0133-B08, M0133-E05, M0121-E07, M0118-F11, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10,
M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12,
M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11, and/or the light chain immunoglobulin variable domain sequence comprises three CDR regions from the light chain variable domain of DX-2712, a mutant or variant of DX-2712, 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09,
M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134- COl, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01,
M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, MOl 18-Fl 1, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123-G07, M0063-A02,
M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01,
M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11 (respectively), and the protein binds to and inhibits MMP- 12.
12. The method of claim 11, wherein the three CDR regions from the heavy chain variable domain are from DX-2712 and/or the three CDR regions from the light chain variable domain are from DX-2712.
13. The method of claim 11, wherein the the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of DX-2712, a mutant or variant of DX-2712, 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134- C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135- A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135- GI l, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130- F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, M0118-F11, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122- C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067- B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071- H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041- B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08,
M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014- C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11, and/or the the light chain immunoglobulin variable domain sequence comprises the light chain variable domain of DX-2712, a mutant or variant of DX-2712, 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134- AlO, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134-C12, M0134- D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135-A05, M0135- A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03,
M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135-G11, M0135- H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130-F06, M0130- H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, M0118-F11, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122-C06, M0123- G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067-B09, M0067- ClO, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071-H03, M0071- H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05,
M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041-B05, M0041- GOl, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014-C09, M0014- GI l, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11 (respectively).
14. The method of claim 11, wherein the the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of DX-2712, and/or the the light chain immunoglobulin variable domain sequence comprises the light chain variable domain of DX-2712.
15. The method of claim 11, wherein the protein comprises the heavy chain of DX-2712, a mutant or variant of DX-2712, 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134-A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134- Cl 1, M0134-C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11, M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135- A03, M0135-A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135- G07, M0135-G11, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130- C12, M0130-F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, M0118-F11, M0125-G07, M0124-E07, M0119-D01, M0119- A02, M0122-C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067- B06, M0067-B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11,
M0069-C02, M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071- D09, M0071-H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039- FOl, M0041-B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013- H06, M0014-C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-E11, and/or the light chain of DX-2712, a mutant or variant of DX-2712, 539B-X0041-D02, M0134-A02, M134-A05, M134-A07, M134- A09, M134-A10, M134-A11, M0134-B01, M134-B04, M0134-B08, M0134-B11, M0134-C01, M0134-C02, M0134-C06, M0134-C09, M0134-C10, M0134-C11, M0134- C12, M0134-D02, M0134-DO3, M0134-E04, M0134-E07, M0134-E08, M134-E11,
M0134-F01, M0134-F05, M0134-G02, M0134-G04, M0134-G07, M0135-A03, M0135- A05, M0135-A06, M0135-A07, M0135-B02, M0135-B08, M0135-C01, M0135-C11, M0135-E03, M0135-F03, M0135-F11, M0135-G02, M0135-G03, M0135-G07, M0135- GI l, M0135-H03, M0135-H10, M0105-C05, M0105-E11, M0105-F08, M0107-A12, M0108-A02, M0109-G11, M0110-G05, M0129-B11, M0130-A01, M0130-C12, M0130- F06, M0130-H04, M0131-A06, M0131-D03, M0132-A04, M0133-B08, M0133-E05, M0121-E07, M0118-F11, M0125-G07, M0124-E07, M0119-D01, M0119-A02, M0122- C06, M0123-G07, M0063-A02, M0063-A04, M0063-B01, M0063-B11, M0063-C07, M0063-G01, M0065-E12, M0065-G03, M0065-H05, M0067-A02, M0067-B06, M0067- B09, M0067-C10, M0067-F02, M0067-F06, M0069-A04, M0069-A11, M0069-C02,
M0069-D10, M0069-G07, M0071-A01, M0071-B07, M0071-D05, M0071-D09, M0071- H03, M0071-H06, M0087-F09, M0088-F07, M0088-G10, M0088-H10, M0089-C01, M0089-F05, M0089-B07, M0089-H11, m0032-E01, M0034-C04, M0039-F01, M0041- B05, M0041-G01, M0042-B06, M0006-B10, M0007-H06, M0008-H09, M0009-H08, M0011-H11, M0015-F02, M0016-D01, M0013-D11, M0013-G12, M0013-H06, M0014- C09, M0014-G11, M0016-A11, M0016-H05, M0019-C05, M0020-B01, M0022-C07, M0025-D04 and M0027-Ell (respectively).
16. The method of claim 11 , wherein the protein comprises the heavy chain of DX-2712, and/or the light chain of DX-2712.
PCT/US2009/060716 2008-10-14 2009-10-14 Use of mmp-9 and mmp-12 binding proteins for the treatment and prevention of systemic sclerosis WO2010045388A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US10537308P 2008-10-14 2008-10-14
US61/105,373 2008-10-14

Publications (2)

Publication Number Publication Date
WO2010045388A2 true WO2010045388A2 (en) 2010-04-22
WO2010045388A3 WO2010045388A3 (en) 2010-09-30

Family

ID=42107220

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2009/060716 WO2010045388A2 (en) 2008-10-14 2009-10-14 Use of mmp-9 and mmp-12 binding proteins for the treatment and prevention of systemic sclerosis

Country Status (1)

Country Link
WO (1) WO2010045388A2 (en)

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011028883A2 (en) * 2009-09-03 2011-03-10 Dyax Corp. Metalloproteinase 9 and metalloproteinase 2 binding proteins
US8013125B2 (en) 2008-03-03 2011-09-06 Dyax Corp. Metalloproteinase 9 and metalloproteinase 2 binding proteins
US8455205B2 (en) 2008-03-03 2013-06-04 Dyax Corp. Metalloproteinase 9 binding proteins
US8501181B2 (en) 2007-12-17 2013-08-06 Dyax Corp. Compositions and methods for treating osteolytic disorders comprising MMP-14 binding proteins
WO2014006118A1 (en) 2012-07-04 2014-01-09 F. Hoffmann-La Roche Ag Anti-theophylline antibodies and methods of use
WO2015018805A1 (en) * 2013-08-05 2015-02-12 Immatics Biotechnologies Gmbh Novel immunotherapy against several tumors, such as lung cancer, including nsclc
WO2016005295A1 (en) * 2014-07-07 2016-01-14 Protagen Ag Marker sequences for diagnosing and stratifying systemic sclerosis patients
EP2985296A1 (en) * 2014-08-13 2016-02-17 Calypso Biotech SA Antibodies specific for MMP9
EP2985295A1 (en) * 2014-08-13 2016-02-17 Calypso Biotech SA Antibodies specific for MMP9
US9765153B2 (en) 2012-07-04 2017-09-19 Hoffmann-La Roche Inc. Anti-biotin antibodies and methods of use
WO2018152452A1 (en) * 2017-02-17 2018-08-23 Mapp Biopharmaceutical, Inc. Monoclonal antibodies and cocktails for treatment of ebola infections
US10160786B1 (en) 2013-08-05 2018-12-25 Immatics Biotechnologies Gmbh Immunotherapy against several tumors, such as lung cancer, including NSCLC
US10314909B2 (en) 2011-10-21 2019-06-11 Dyax Corp. Combination therapy comprising an MMP-14 binding protein
US10407511B2 (en) 2014-01-03 2019-09-10 Hoffmann-La Roche Inc. Covalently linked helicar-anti-helicar antibody conjugates and uses thereof
US10517945B2 (en) 2012-07-04 2019-12-31 Hoffman-La Roche Inc. Covalently linked antigen-antibody conjugates
US10519249B2 (en) 2014-01-03 2019-12-31 Hoffmann-La Roche Inc. Covalently linked polypeptide toxin-antibody conjugates
US10561737B2 (en) 2014-01-03 2020-02-18 Hoffmann-La Roche Inc. Bispecific anti-hapten/anti-blood brain barrier receptor antibodies, complexes thereof and their use as blood brain barrier shuttles
EP3434690A4 (en) * 2016-03-23 2020-04-01 Seoul National University R&DB Foundation Antibody that binds to envelope glycoprotein of severe fever with thrombocytopenia syndrome virus, and use for same
WO2022052968A1 (en) * 2020-09-09 2022-03-17 信达生物制药(苏州)有限公司 Monoclonal antibody for coronavirus spike protein, and use thereof
CN116063478A (en) * 2022-07-15 2023-05-05 北京顺元天生物制品有限公司 Preparation method of stem cell cytokine and pharmaceutical application of antibody combination thereof

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6455323B1 (en) * 1997-07-03 2002-09-24 Pharmacia & Upjohn Company Anti-bacterial methods and materials
US20020159971A1 (en) * 2001-02-23 2002-10-31 Michel Houde Methods and compositions for preventing and treating neutrophil-mediated diseases
US20020183500A1 (en) * 2000-11-20 2002-12-05 Roberto Macina Compositions and methods relating to lung specific genes and proteins
US20040214272A1 (en) * 1999-05-06 2004-10-28 La Rosa Thomas J Nucleic acid molecules and other molecules associated with plants
US20050136053A1 (en) * 2003-08-12 2005-06-23 Dyax Corporation Tie1-binding ligands
US20060036076A1 (en) * 2003-11-19 2006-02-16 Dyax Corp. Metalloproteinase-binding proteins
US20060088534A1 (en) * 1998-08-28 2006-04-27 Genentech, Inc. Human anti-factor ix/ixa antibodies
WO2009079585A2 (en) * 2007-12-17 2009-06-25 Dyax Corp. Compositions and methods for treating osteolytic disorders comprising mmp-14 binding proteins

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6455323B1 (en) * 1997-07-03 2002-09-24 Pharmacia & Upjohn Company Anti-bacterial methods and materials
US20060088534A1 (en) * 1998-08-28 2006-04-27 Genentech, Inc. Human anti-factor ix/ixa antibodies
US20040214272A1 (en) * 1999-05-06 2004-10-28 La Rosa Thomas J Nucleic acid molecules and other molecules associated with plants
US20020183500A1 (en) * 2000-11-20 2002-12-05 Roberto Macina Compositions and methods relating to lung specific genes and proteins
US20020159971A1 (en) * 2001-02-23 2002-10-31 Michel Houde Methods and compositions for preventing and treating neutrophil-mediated diseases
US20050136053A1 (en) * 2003-08-12 2005-06-23 Dyax Corporation Tie1-binding ligands
US20060036076A1 (en) * 2003-11-19 2006-02-16 Dyax Corp. Metalloproteinase-binding proteins
WO2009079585A2 (en) * 2007-12-17 2009-06-25 Dyax Corp. Compositions and methods for treating osteolytic disorders comprising mmp-14 binding proteins

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
'Anti-MMP-9, Catalytic domain.' MILLIPORE, [Online] 2008, Retrieved from the Internet: <URL:http://www.cpg- biotech.com/coa.nsf/a73664f9f981af8c852569b 9005b4eee/b065f7af50d3374e882576bd0064b0 c3/$FILE/AB19016_NMM1679627.pdf> [retrieved on 2010-06-02] *
KIM ET AL.: 'Elevated matrix metalloproteinase-9 in patients with systemic sclerosis.' ARTHRITIS RES THER. vol. 7, no. 1, 2005, pages R71 - R79 *
ROEB ET AL.: 'An MMP-9 mutant without gelatinolytic activity as a novel TIMP-1-antagonist.' THE FASEB JOUMAL. vol. 14, 2000, pages 1671 - 1673 *

Cited By (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8501181B2 (en) 2007-12-17 2013-08-06 Dyax Corp. Compositions and methods for treating osteolytic disorders comprising MMP-14 binding proteins
US8013125B2 (en) 2008-03-03 2011-09-06 Dyax Corp. Metalloproteinase 9 and metalloproteinase 2 binding proteins
US8455205B2 (en) 2008-03-03 2013-06-04 Dyax Corp. Metalloproteinase 9 binding proteins
WO2011028883A3 (en) * 2009-09-03 2011-05-05 Dyax Corp. Metalloproteinase 9 and metalloproteinase 2 binding proteins
WO2011028883A2 (en) * 2009-09-03 2011-03-10 Dyax Corp. Metalloproteinase 9 and metalloproteinase 2 binding proteins
US10314909B2 (en) 2011-10-21 2019-06-11 Dyax Corp. Combination therapy comprising an MMP-14 binding protein
RU2630664C2 (en) * 2012-07-04 2017-09-11 Ф. Хоффманн-Ля Рош Аг Theophylline antibodies and methods for their application
WO2014006118A1 (en) 2012-07-04 2014-01-09 F. Hoffmann-La Roche Ag Anti-theophylline antibodies and methods of use
US10517945B2 (en) 2012-07-04 2019-12-31 Hoffman-La Roche Inc. Covalently linked antigen-antibody conjugates
US9925272B2 (en) 2012-07-04 2018-03-27 Hoffmann-La Roche Inc. Anti-theophylline antibodies and methods of use
US9765153B2 (en) 2012-07-04 2017-09-19 Hoffmann-La Roche Inc. Anti-biotin antibodies and methods of use
US10479818B2 (en) 2013-08-05 2019-11-19 Immatics Biotechnologies Gmbh Immunotherapy against several tumors, such as lung cancer, including NSCLC
US10487116B2 (en) 2013-08-05 2019-11-26 Immatics Biotechnologies Gmbh Immunotherapy against several tumors, such as lung cancer, including NSCLC
US11859017B2 (en) 2013-08-05 2024-01-02 Immatics Biotechnologies Gmbh Immunotherapy against several tumors including lung cancer
US11161879B2 (en) 2013-08-05 2021-11-02 Immatics Biotechnologies Gmbh Immunotherapy against several tumors, such as lung cancer, including NSCLC
US11161878B2 (en) 2013-08-05 2021-11-02 Immatics Biotechnologies Gmbh Immunotherapy against several tumors including lung cancer
US9943579B2 (en) 2013-08-05 2018-04-17 Immatics Biotechnologies Gmbh Immunotherapy against several tumors, such as lung cancer, including NSCLC
US11939400B2 (en) 2013-08-05 2024-03-26 Immatics Biotechnologies Gmbh Immunotherapy against several tumors including lung cancer
US10071148B2 (en) 2013-08-05 2018-09-11 Immatics Biotechnologies Gmbh Immunotherapy against several tumors, such as lung cancer, including NSCLC
US10160786B1 (en) 2013-08-05 2018-12-25 Immatics Biotechnologies Gmbh Immunotherapy against several tumors, such as lung cancer, including NSCLC
IL262509A (en) * 2013-08-05 2018-12-31 Immatics Biotechnologies Gmbh Novel immunotherapy against several tumors, such as lung cancer, including nsclc
US11161877B2 (en) 2013-08-05 2021-11-02 Immatics Biotechnologies Gmbh Immunotherapy against several tumors including lung cancer
US10316062B1 (en) 2013-08-05 2019-06-11 Immatics Biotechnologies Gmbh Immunotherapy against several tumors, such as lung cancer, including NSCLC
US10316063B1 (en) 2013-08-05 2019-06-11 Immatics Biotechnologies Gmbh Immunotherapy against several tumors, such as lung cancer, including NSCLC
US10323065B1 (en) 2013-08-05 2019-06-18 Immatics Biotechnologies Gmbh Immunotherapy against several tumors, such as lung cancer, including NSCLC
US11161880B2 (en) 2013-08-05 2021-11-02 Immatics Biotechnologies Gmbh Immunotherapy against several tumors, such as lung cancer, including NSCLC
US11866517B2 (en) 2013-08-05 2024-01-09 Immatics Biotechnologies Gmbh Immunotherapy against several tumors including lung cancer
US11939401B2 (en) 2013-08-05 2024-03-26 Immatics Biotechnologies Gmbh Immunotherapy against several tumors including lung cancer
US11814446B2 (en) 2013-08-05 2023-11-14 Immatics Biotechnologies Gmbh Immunotherapy against several tumors including lung cancer
WO2015018805A1 (en) * 2013-08-05 2015-02-12 Immatics Biotechnologies Gmbh Novel immunotherapy against several tumors, such as lung cancer, including nsclc
US10793602B2 (en) 2013-08-05 2020-10-06 Immatics Biotechnologies Gmbh Immunotherapy against several tumors, such as lung cancer, including NSCLC
EA035362B1 (en) * 2013-08-05 2020-06-02 Имматикс Байотекнолоджиз Гмбх Novel immunotherapy against several tumors, such as lung cancer, including nsclc
US10407511B2 (en) 2014-01-03 2019-09-10 Hoffmann-La Roche Inc. Covalently linked helicar-anti-helicar antibody conjugates and uses thereof
US10561737B2 (en) 2014-01-03 2020-02-18 Hoffmann-La Roche Inc. Bispecific anti-hapten/anti-blood brain barrier receptor antibodies, complexes thereof and their use as blood brain barrier shuttles
US10519249B2 (en) 2014-01-03 2019-12-31 Hoffmann-La Roche Inc. Covalently linked polypeptide toxin-antibody conjugates
WO2016005295A1 (en) * 2014-07-07 2016-01-14 Protagen Ag Marker sequences for diagnosing and stratifying systemic sclerosis patients
US10571477B2 (en) 2014-07-07 2020-02-25 Protagen Gmbh Marker sequences for diagnosing and stratifying systemic sclerosis patients
AU2015303183B2 (en) * 2014-08-13 2021-01-28 Calypso Biotech Sa Antibodies specific for MMP9
RU2714043C2 (en) * 2014-08-13 2020-02-11 Эа Фарма Ко., Лтд. Antibodies specific to mmp9
CN106573984A (en) * 2014-08-13 2017-04-19 克里普索生物科技公司 Antibodies specific for MMP9
US10364296B2 (en) 2014-08-13 2019-07-30 Calypso Biotech Sa Antibodies specific for MMP9
EP2985296A1 (en) * 2014-08-13 2016-02-17 Calypso Biotech SA Antibodies specific for MMP9
EP2985295A1 (en) * 2014-08-13 2016-02-17 Calypso Biotech SA Antibodies specific for MMP9
WO2016023972A1 (en) * 2014-08-13 2016-02-18 Calypso Biotech Sa Antibodies specific for mmp9
WO2016023979A1 (en) * 2014-08-13 2016-02-18 Calypso Biotech Sa Antibodies specific for mmp9
EP3434690A4 (en) * 2016-03-23 2020-04-01 Seoul National University R&DB Foundation Antibody that binds to envelope glycoprotein of severe fever with thrombocytopenia syndrome virus, and use for same
US10947299B2 (en) 2016-03-23 2021-03-16 Seoul National University R&Db Foundation Antibody that binds to envelope glycoprotein of severe fever with thrombocytopenia syndrome virus and use for same
US11254732B2 (en) 2017-02-17 2022-02-22 Mapp Biopharmaceutical, Inc. Monoclonal antibodies and cocktails for treatment of Ebola infections
US11407816B2 (en) 2017-02-17 2022-08-09 Mapp Biopharmaceutical, Inc. Monoclonal antibodies and cocktails for treatment of Ebola infections
US11407817B2 (en) 2017-02-17 2022-08-09 Mapp Biopharmaceutical, Inc. Monoclonal antibodies and cocktails for treatment of Ebola infections
US11242379B2 (en) 2017-02-17 2022-02-08 Mapp Biopharmaceutical, Inc. Monoclonal antibodies and cocktails for treatment of Ebola infections
US11242378B2 (en) 2017-02-17 2022-02-08 Mapp Biopharmaceutical, Inc. Monoclonal antibodies and cocktails for treatment of Ebola infections
US10836810B2 (en) 2017-02-17 2020-11-17 Mapp Biopharmaceutical, Inc. Monoclonal antibodies and cocktails for treatment of ebola infections
WO2018152452A1 (en) * 2017-02-17 2018-08-23 Mapp Biopharmaceutical, Inc. Monoclonal antibodies and cocktails for treatment of ebola infections
WO2022052968A1 (en) * 2020-09-09 2022-03-17 信达生物制药(苏州)有限公司 Monoclonal antibody for coronavirus spike protein, and use thereof
CN116063478A (en) * 2022-07-15 2023-05-05 北京顺元天生物制品有限公司 Preparation method of stem cell cytokine and pharmaceutical application of antibody combination thereof
CN116063478B (en) * 2022-07-15 2023-10-24 珠海凤凰高科生物制药有限公司 Preparation method of stem cell cytokine and pharmaceutical application of antibody combination thereof

Also Published As

Publication number Publication date
WO2010045388A3 (en) 2010-09-30

Similar Documents

Publication Publication Date Title
WO2010045388A2 (en) Use of mmp-9 and mmp-12 binding proteins for the treatment and prevention of systemic sclerosis
US8013125B2 (en) Metalloproteinase 9 and metalloproteinase 2 binding proteins
US8008445B2 (en) Metalloproteinase 9 binding proteins
US8114968B2 (en) Metalloproteinase-12 specific monoclonal antibody
US20110135573A1 (en) Metalloproteinase 9 and metalloproteinase 2 binding proteins
TWI471334B (en) Antibody-drug conjugates
JP2011517662A5 (en)
JP2011517320A5 (en)
JP2021106599A (en) Humanized antibody to liv-1 and use of the same to treat cancer
US8501181B2 (en) Compositions and methods for treating osteolytic disorders comprising MMP-14 binding proteins
WO2021061867A1 (en) Anti-cd47 antibodies, activatable anti-cd47 antibodies, and methods of use thereof
US11827709B2 (en) Anti-AVB6 antibodies and antibody-drug conjugates
JP2023510349A (en) Site-directed antibody-drug conjugates with peptide-containing linkers
WO2009097397A2 (en) Metalloproteinase binding proteins
TW202221034A (en) Anti-cd228 antibodies and antibody-drug conjugates

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09821216

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09821216

Country of ref document: EP

Kind code of ref document: A2