OA17085A - Antibodies to matrix metalloproteinase 9. - Google Patents
Antibodies to matrix metalloproteinase 9. Download PDFInfo
- Publication number
- OA17085A OA17085A OA1201400386 OA17085A OA 17085 A OA17085 A OA 17085A OA 1201400386 OA1201400386 OA 1201400386 OA 17085 A OA17085 A OA 17085A
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- OA
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- seq
- amino acid
- mmp9
- antibody
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Abstract
The present disclosure provides compositions and methods of use involving binding proteins, e.g., antibodies and antigenbinding fragments thereof, that bind to the matrix metalloproteinase-9 (MMP9) protein (MMP9 is also known as gelatinase-B), such as where the binding proteins comprise an immunoglobulin (Ig) heavy chain (or functional fragment thereof) and an Ig light chain (or functional fragment thereof).
Description
ANTIBODIES TO MATRIX METALLOPROTEINASE 9
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB [0001] The entire content of the following electronic submission ofthe sequence listing via the USPTO EFS-WEB server, as authorized and set forth in MPEP §1730 H.B,2(a)(C), is incorporated herein by reference in its entirety for ail purposes. The sequence listing is identified on the electronically filed text file as follows:
File Name | Date of Création | Size (bytes) |
246l02008540Seqlîst | February 29,2012 | 65,102 bytes |
FIELD [0002] This disciosure is in the field ofextracellular enzymes, extracelîular matrix enzymes, proteases and immunology.
BACKGROUND [0003] Matrix metalloproteinases (MMPs) belong to a family of extracellular enzymes invoîved in forming and remodeîing the extracellular matrix. These enzymes contain a conserved catalytic domain in which a zinc atom is coordinated by three histidine residues. Over 20 members of this family are known, organized into a number of groups including collagénases, gelatinases, stromelysins, matrilysins, enamelysins and membrane MMPs.
[0004] MMP2 and MMP9 belong to the gélatinase group of matrix metalloproteinases. Besides containing signal peptide, propeptide, catalytic, zinc-binding and heamopexin-like domains common to most MMPs, the gelatinases also contain a pluraiity of fibronectin-like domains and an O-glycosylated domain.
[0005] MMPs are associated with a number of diseases. However, availabie inhibitors of MMPs hâve been unsuccessful, in part due to toxicity and lack of efficacy. Therefore, there is a need for spécifie and effective MMP inhibitors.
SUMMARY [0006] The présent disciosure provides compositions and methods of use involving binding proteins, e.g., antibodies and antigen-binding fragments thereof, that bind to matrix metalloproteinase-9 (MMP9) protein (also known as gelatinase-B). The binding proteins typically are antibodies or fragments (e.g., antigen-binding fragments) thereof and typically contain an immunoglobulin (1g) heavy chain (or functional fragment thereof) and an Ig light chain (or functional fragment thereof). The heavy chain is typically an IgG, typically a human IgG, such as an IgGl or IgG4, or other IgG such as an IgG2, or modified version thereof. The light chain typically is a kappa chain.
[0007] Among the MMP9 binding proteins, e.g., antibodies, are those that bind specifically to MMP9 and not to other matrix metalloproteinases. Such MMP9 binding proteins find use in applications in which it is necessary or désirable to obtain spécifie modulation (e.g., inhibition) of MMP9, e.g., without directly affecting the activity of other matrix metalloproteinases. Thus, in certain embodiments of the présent disclosure an antiMMP9 antibody or fragment thereof is a spécifie inhîbitor of the activity of MMP9. In some aspects, the MMP9 binding proteins disclosed herein will be useful for inhibition of MMP9 while allowing normal function of other, related matrix metalloproteinases.
[0008] The antibodies and fragments can be described with reference to their amino acid sequences or portions thereof, and/or various functions such as binding specificity to MMP9 or particular epitopes thereof or the ability to compete for binding to epitopes on MMP9 with particular antibodies, and/or activity, such as the ability to inhibit MMP9, e.g., noncompetitively.
[0009] The antibodies and fragments include those having a heavy chain variable (VH) région having a heavy chain complementary determining région (CDR) with an amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15; those having a light chain variable (VL) région having a light chain complementary determining région (CDR) with an amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. Exemplary antibodies and fragments include those having a heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 14, and a heavy chain CDR3 with the amino acid sequence of SEQ ID NO: 15, and those having a heavy chain CDR3 of SEQ ID NO: 15. Exemplary antibodies and fragments further include those with a light chain CDR1 with the amino acid sequence of SEQ ID NO: 16, a light chain CDR2 with the amino acid sequence of SEQ ID NO: 17, and a light chain CDR3 with the amino acid sequence of SEQ ID NO: 18, and those having a light chain CDR3 with the amino acid sequence of SEQ ID NO: 18, as well as those having heavy chain CDRs of SEQ ID NOs: 13,14, and 15, and ligjit chain CDRs of SEQ ID NOs: 16,17, and 18.
[0010] Exemplary antibodies and fragments further include those having a heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 34, a heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 35, and a heavy chain CDR3 with the amino acid sequence of SEQ ID NO: 36, those with a heavy chain CDR3 with the amino acid sequence of SEQ ID NO: 36, those with a light chain CDR1 with the amino acid sequence of SEQ ID NO: 37, a light chain CDR2 with the amino acid sequence of SEQ ID NO: 38, and a light chain CDR3 with the amino acid sequence of SEQ ID NO: 39, those with a light chain CDR3 with the amino acid sequence of SEQ ID NO: 39, as well as those having heavy chain CDRs of SEQ ID NOs: 34, 35, and 36, and light chain CDRs of SEQ ID NOs: 37,38, and 39.
[00Π] Exemplary antibodies and fragments further include those having a light chain CDR1 with the amino acid sequence of SEQ ID NO: 42, a light chain CDR2 with the amino acid sequence of SEQ ID NO: 43, and a light chain CDR3 with the amino acid sequence of SEQ ID NO: 44, and those with a light chain CDR3 with the amino acid sequence of SEQ ID NO: 44.
J0012] The antibodies and fragments further include those having a VH région with an amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, and those having a VL région with an amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, as well as antibodies and fragments having a VH région with an amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 and a VL région with an amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In a particular example, the antibodies or fragments hâve a VH région of SEQ ID NO: 7 and a VL région of SEQ ID NO: 12, or at least at or about 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more sequence identity with such sequences. They further include those having a VH région with an amino acid sequence set forth in SEQ ID NO: 32 or 47, and those with a VL région with an amino acid sequence set forth in SEQ ID NO: 33 or in SEQ ID NO: 41 or in SEQ ID NO: 48, and combinations thereof, and sequence having at least at or about 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more sequence identity with such sequences.
[0013] The antibodies and fragments further include those having a VH région with an amino acid sequence set forth in SEQ ID NO: I, and/or having a VL région with an amino acid sequence set forth in SEQ ID NO: 2, and/or a VH or VL région having at least at or about 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more sequence identity with such sequences.
[0014] In some cases, the heavy chain is encoded by a polynucleotide having a nucléotide sequence selected from the group consisting of SEQ ID NOs: 19-22 and the light chain is encoded by a polynucleotide having a nucléotide sequence selected from the group consisting ofSEQ ID NOs: 23-26.
[0015J In some embodiments, the antibodies or fragments thereof inhibit the enzymatic activity of MMP 9, such as by non-competitive inhibition.
[0016| The antibodies and fragments further include those that specifically bînd to a given epitope ofMMP9. In some cases, the epitope îs an epitope specifically bound by any 5 ofthe above-described antibodies. In one example, the epitope contains an amino acid residue (i.e., one or more amino acid residuefs)) outside of cysteine-switch active pocket of SEQ ID NO: 27. In certain examples, the epitope includes an amino acid residue (i.e., one or more amino acid residue(s)) within a given région of MMP9, for example, where the région is residues 104-202 of SEQ ID NO: 27. In some examples, the epitope includes an amino 10 acid residue (i.e., one or more amino acid residue(s)) within a given région of MMP9, for example, where the région i s residues 104-119, residues 159-166, or residues 191-202 of SEQ ID NO: 27. In one example, the epitope includes an amino acid residue (i.e., one or more amino acid residue) within a région of MMP9 that is residues 104-119 of SEQ ID NO: 27, an amino acid residue within a région of MMP9 that is residues 159-166 of SEQ ID NO: 15 27, and an amino acid residue within a région of MMP9 that is residues 191-202 of SEQ ID
NO: 27. In some cases, the epitope includes El 11, DI 13, R162, or 1198 of SEQ ID NO: 27. In some cases, it includes R162 of SEQ ID NO: 27. In some cases, it includes El 11, DI 13, RI 62, and 1198 of SEQ ID NO: 27.
[0017] In some cases, the antibody or fragment is human or is humanized.
[0018J In some examples, the antibodies and fragments specifically bindto human
MMP9 with a dissociation constant (1Q) equal to or lower than 100 nM, optionally lower than 10 nM, optionally lower than I nM, optionally lower than 0.5 nM, optionally lower than 0.1 nM, optionally lower than 0.01 nM, or optionally lower than 0.005 nM, in certain examples, between 0.1 and 0.2 nM, or between 0.1 and 10 pM, e.g., between 0.4 and 9 pm, such as between 0.4 and 8.8 pm, in the form of monoclonal antibody, scFv, Fab, or other form of antibody measured at a température of about 4°C, 25°C, 37°C or 42°C.
[0019] Also among the provided antibodies and fragments are those having at least at or about 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more sequence identity with any of the above-described antibodies or containing various portions with at least at or about 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more sequence identity with the respective portions of the antibodies described above, such as having a VH région with such identity with SEQ ID NO: 7 and a VL région with such identity with SEQ ID NO: 12. Also provided are antibodies that compete for binding to MMP9 with any of the above-described antibodies, such as those that compete for binding to MMP9 with an antibody having a VH région with the amino acid sequence set forth in SEQ ID NO: 7 and a VL région with the amino acid sequence set forth inSEQlD NO: 12.
[0020] Also provided are isolated nucleic acids encoding the antibodies and fragments, such as nucleic acids including a coding sequence for any of the above-described antibodies and fragments. Among the provided nucleic acids are those containing a nucléotide sequence encoding a heavy chain polypeptide comprising CDRs with the amino acid sequences set forth in SEQ ID NOs: 13-15, and/or a light chain polypeptide comprising CDRs with the amino acid sequences set forth in SEQ ID NOs: 16-18. In one example, the nucléotide sequence encodes the heavy chain polypeptide, which has an amino acid sequence selected from the group consisting of SEQ ID NOS: 1,3, and 5-8. ln another example, the nucléotide sequence encodes the light chain polypeptide, which has an amino acid sequence selected from the group consisting of SEQ ID NOS: 2,4, and 9-12. In one example, the nucléotide sequence includes a sequence selected from the group consisting of SEQ ID NOs: 19-26, such as SEQ ID NO: 21, SEQ ID NO: 26, or SEQ ID NOs: 21 and 26. Also provided are vectors containing such nucleic acids and cells including the same, such as host cells.
[0021 [ Also provided are pharmaceutical compositions including the antibodies, fragments, nucleic acids, vectors, and cells. In some examples, the pharmaceutical compositions further include a carrier or excipient, such as a pharmaceutically acceptable or biologically acceptable carrier or excipient, ln some cases, the pharmaceutical compositions are used in the provided therapeutic methods and uses.
[0022] Also provided are methods and uses ofthe antibodies, fragments, nucleic acids, vectors, cells, and compositions, for example in therapeutics, such as inhibiting MMP9 in a subject, and diagnostics, such as for detecting MMP9 in the subject.
[0023] For example, provided are diagnostic and prognostic methods involving détection of MMP9, and agents (such as any of the above-described anti-MMP9 antibodies and other MMP9 binding proteins) for use in such methods. In some cases, the diagnostic method detects MMP9 expression in a test sample from a subject. Such methods can be carried out, for example, by contacting the test sample with an antibody or fragment as described herein (such as any of the above-described antibodies or fragments) and detecting binding of the antibody or fragment to protein in the sample, thereby detecting the presence of MMP9. In some cases, a sample is first obtained or provided. In some examples, the methods include comparing the amount or level of MMP9 detected to a control level or amount, such as by comparing the amount of binding detected in the test sample with an amount of binding of the antibody or fragment to a control sample. In some cases, the methods involve simply comparing a test level and a control level of MMP9. In some cases, a higher test level (as compared to the control level) is indicative of the disease or condition.
[0024] In some cases, the MMP9 detected by the method indicates the presence ofa disease or condition in the subject, such as an MMP9-associated disease or condition. In some cases, the methods further include treating the subject or adjusting (i.e., altering or discontinuing) treatment of the subject based on the results of the method, e.g., based on the levels ofMMP9 detected in the sample. Among the biological samples are tissue, cells isolated from such tissues, and the like. In some cases, the methods are performed on liquid samples, such as blood, plasma, sérum, whole blood, saliva, urine, or semen. Tissue samples include, for example, formalin-fîxed or frozen tissue sections.
[0025] Also provided are methods of inhibiting MMP9 activity in a subject and/or treating a disease or condition in the subject, for example, using an agent that noncompetitively inhibits MMP9, and agents (such as any of the above-described anti-MMP9 antibodies and other MMP9 binding proteins) for use in such methods. The methods generally are carried out by administering to the subject an MMP9 binding protein, such as an MMP9-binding antibody or fragment thereof as provided herein, e.g., in an effective amount. The antibody or fragment generally specifically binds to and non-competitively inhibits MMP9, for example, such that MMP9 activity is inhibited in the subject. In some cases, the antibody or fragment is one that binds MMP9 outside of the cysteine-switch active pocket, such as in one of the epitopes described above. In some cases, the antibody or fragment does not substantially bind to an MMP protein other than MMP9 and/or does not substantially bind to MMP2.
[0026] The subject generally is one with a disease or condition, typically one associated with increased or decreased MMP9 expression and/or activity. In certain cases, the subject with a disease or condition associated with increased MMP9 expression and/or activity. In other cases, the subject with a disease or condition associated with decreased MMP9 expression and/or activity.
[0027] Also provided are MMP9 polypeptides, induding mutant MMP9 polypeptides, such as those containing residues 111-198 of SEQ ID NO: 27, and those having an amino acid sequence containing residues 111-198 of SEQ ID NO: 27 with an amino acid substitution at residue 111, 113,162, or 198 of SEQ ID NO 27, or with an amino acid substitution at all such residues.
[0028] Also provided are uses of any of the above-described antibodies, nucleic acids, vectors, cells, and compositions, in the therapeutic and diagnostic methods described above.
BRIEF DESCRIPTION OF THE DRAWINGS [0029] Figure 1 shows the amino acid sequence of the heavy chain variable région of a mouse monoclonal anti-MMP9 antibody (AB0041), along with the amino acid sequences of humanized variants of heavy chain (VH1-VH4), aligned to show différences în framework amino acid sequence resulting from humanization. CDRs are shown in italics, and amino acids that are different in the humanized variants, compared to the parent mouse monoclonal, are underlined.
[0030( Figure 2 shows the amino acid sequence of the light chain variable région of a mouse monoclonal anti-MMP9 antibody (AB0041), along with the amino acid sequences of humanized variants of this light chain (VHI-VH4), aligned to show différences in framework amino acid sequence resulting from humanization. CDRs are shown in italics, and amino acids that are different in the humanized variants, compared to the parent mouse monoclonal, are underlined.
[0031] Figure 3 shows a schematic diagram of the MMP9 protein.
[0032] Figure 4 shows a comparison between the amino acid sequences of the heavy and light chains of antibodies designated AB0041, M4, and Ml 2.
DETAILED DESCRIPTION [0033] Practice of the présent disclosure employs, unless otherwise indicated, standard methods and conventional techniques in the fields of cell biology, toxîcology, molecular biology, biochemistry, cell culture, immunology, oncology, recombinant DNA and related fields as are within the skîH of the art. Such techniques are described in the Hterature and thereby available to those of skill in the art. See, for example, Alberts, B. et al., “Molecular Biology of the Cell,” 5lh édition, Garland Science, New York, NY, 2008; Voet, D. et al. “Fundamentals of Biochemistry; Life at the Molecular Level,” 31111 édition, John Wiley & Sons, Hoboken, NJ, 2008; Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” 31 édition, Cold Spring Harbor Laboratory Press, 2001; Ausubel, F. étal., “Current Protocole in Molecular Biology,” John Wiley & Sons, New York, 1987 and periodic updates; Freshney, R.I., “Culture of Animal Cells: A Manual of Basic Technique,” 4,h édition, John Wiley & Sons, Somerset, NJ, 2000; and the sériés “Methods in Enzymology,” Academie
Press, San Diego, CA. See also, for example, “Current Protocols in Immunology,” (R. Coico, sériés editor), Wiley, last updated August 2010.
[0034] Certain MMPs play rôles in tumor growth, metastasis, inflammation, autoimmunity, and vascular disease. See, for example, Hu et al. (2007) Nature Reviews: Drug Discovery 6:480-498. Thus, it is désirable to inhibit the activity of one or more particular MMPs in certain therapeutic settings. While sharing significant homology at a sequence level, the expression and functional rôles of the two gelatinases MMP9 and MMP2 vary significantly. MMP9 expression is induced by a number of disease associated cytokines and growth factors. Also, the MMP9 knockout mouse is protected in a variety of disease models, whereas MMP2 is more constitutively expressed and the MMP2 knockout animais tend toward little protection. Some studies hâve shown that MMP2 knockout mouse exhibited worse disease in challenge models. For some diseases or disorders, the activity of more than one MMPs is inhibited. In clinical studies, the inhibitors to more than one MMPs hâve caused adverse effects, such as toxicity or lack of efficacy, that are not desired. It has been shown that the activity of certain MMPs, e.g., MMP2, is often required for normal tissue homeostasis and/or is protective against disease. Certain available MMP inhibitors hâve caused side effects.
[0035] Among the provided embodiments are agents, inciuding therapeutic reagents, such as antibodies and antigen-binding fragments thereof, that specifically inhibit the catalytic activity of a single MMP or a select plurality of MMPs, such as MMP9 and that do not react with or inhibit certain other MMPs or any other MMPs. Also among the provided embodiments are methods and uses ofthe same for treatment of various diseases.
MMP9 Binding Proteins [0036] The présent disclosure provides binding proteins, e.g., antibodies and fragments (e.g., antigen-binding fragments) thereof, that bind to the matrix metalloproteinase-9 (MMP9) protein (MMP9 is also known as gelatinase-B), e.g., human MMP9, such as the human MMP9 having an amino acid sequence set forth in SEQ ID NO: 27 or SEQ ID NO: 28. The binding proteins ofthe présent disclosure generally comprise an immunoglobulin (Ig) heavy chain (or functional fragment thereof) and an Ig light chain (or functional fragment thereof).
[0037] The disclosure further provides MMP9 binding proteins that bind specifically to MMP9 and not to other matrix metalloproteinases such as MMPI, MMP2, MMP3, MMP7, MMP9, MMP10, MMP12, and MMP13. Such spécifie MMP9 binding proteins are thus generally not significantly or detectably crossreactive with non-MMP9 matrix metalloproteinases. MMP9 binding proteins that specifïcally bind MMP9 find use in applications in which it is necessary or désirable to obtain spécifie modulation (e.g., inhibition) of MMP9, e.g., without directly afïecting the activity of other matrix metalloproteinases.
[0038] In certain embodiments of the présent disclosure, an anti-MMP9 antibody is an inhibitor of the activity of MMP9, and can be a spécifie inhibitor of MMP9. In one embodiment, the MMP9 binding proteins disclosed herein is useful for inhibition of MMP9 while not afïecting other matrix metalloproteinases. An inhibitor of MMP” or “inhibitor of 10 MMP9 activity can be an antibody or an antigen binding fragment thereof that directly or îndirectly inhibits activity of MMP9, including but not limited to enzymatic processing, inhibiting action of MMP9 on it substrate (e.g., by inhibiting substrate binding, substrate cleavage, and the like), and the like.
|0039] The présent disclosure also provides MMP9 binding proteins that specifically bind 15 to non-mouse MMP9, such as human MMP9, Cynomolgus monkey MMP9, and rat MMP9.
[0040] The présent disclosure also provides MMP9 binding proteins (e.g., antî-MMP9 antibodies and functional fragments thereof) that act as non-competitive inhibitors. A “noncompetitive inhibitor” refers to an inhibitor binds at site away from substrate binding site of an enzyme, and thus can bind the enzyme and effect inhibitory activity regardless of whether 20 or not the enzyme is bound to its substrate. The non-competitive or allosteric inhibition is generally independent of substrate association or concentration. Such non-competitive inhibitors can, for example, provide for a level of inhibition that can be substantially independent of substrate concentration.
[0041] MMP9 binding proteins (e.g., antibodies and functional fragments thereof) ofthe 25 présent disclosure include those that bind MMP9, particularly human MMP9, and having a heavy chain polypeptide (or functional fragment thereof) that has at least about 80%, 85%, 90%, 95% or more amino acid sequence identity to a heavy chain polypeptide disclosed herein.
[0042] MMP9 binding proteins (e.g., antibodies and functional fragments thereof) of the 30 présent disclosure include those that bind MMP9, particularly human MMP9, and having a light polypeptide (or functional fragment thereof) that has at least about 80%, 85%, 90%, 95% or more amino acid sequence identity to a heavy chain polypeptide disclosed herein.
[0043] MMP9 binding proteins (e.g., antibodies and functional fragments thereof) of the présent disclosure include those that bind MMP9, particularly human MMP9, and hâve a heavy chain polypeptide (or functional fragment thereof) having the complementarity determining régions (“CDRs”) of heavy chain polypeptide and the CDRs of a light chain polypeptide (or fùnctional fragment thereof) as disclosed herein.
[0044] Homology” or “identity” or “similarity” as used herein in the context ofnucleic acids and polypeptides refers to the relationship between two polypeptides or two nucleic acid molécules based on an alignment of the amino acid sequences or nucleic acid sequences, respectively. Homology and identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an équivalent position in the compared sequences is occupied by the same base or amino acid, then the molécules are J 0 identical at that position; when the équivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molécules can be referred to as homologous (similar) at that position. Expression as a percentage of homology/similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. In comparing two sequences, the 15 absence of residues (amino acids or nucleic acîds) or presence of extra residues also decreases the identity and homology/similarity.
[0045] As used herein, “identity” means the percentage of identical nucléotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., takîng into account gaps and insertions.
Sequences are generally aligned for maximum correspondence over a designated région, e.g., a région at least about 20,25,30,35,40,45,50,55,60,65 or more amino acids or nucléotides in length, and can be up to the full-length of the reference amino acid or nucléotide. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculâtes the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. [0046] Examples ofalgorithme that are suitable for determining percent sequence identity 30 are the BLAST and BLAST 2.0 algorithme, which are described in Altschul et al. (1990) J.
Mol. Biol. 215:403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). Further exemplary algorithms include ClustalW (Higgins D., et al. (1994) Nucleic Acids Res 22:4673-4680), available at www.ebi.ac.uk/Tools/clustalw/index.html.
[0047] Residue positions which are not identical can differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatichydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanîne, tyrosine, and tryptophan; a group of amino acids having basic side chains is ! 0 lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and méthionine.
[0048] Sequence identlty between two nucleic acids can also be described in terms of hybridization of two molécules to each other under stringent conditions. The hybridization conditions are selected following standard methods in the art (see, for example, Sambrook, et 15 al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor,
N.Y.). An example of stringent hybridization conditions is hybridization at 50°C or higher and 0.1 χ SSC (15 mM sodium ch!oride/1.5 mM sodium citrate). Another example of stringent hybridization conditions is ovemight incubation at 42 °C in a solution: 50 % formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate 20 (pH7.6), 5 x Denhardt’s solution, 10% dextran sulfate, and 20 mg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65 °C. Stringent hybridization conditions are hybridization conditions that are at least as stringent as the above représentative conditions, where conditions are considered to be at least as stringent if they are at least about 80% as stringent, typically at least 90% as stringent as the above spécifie 25 stringent conditions.
[0049] Accordingly, the présent disclosure provides, for example, antibodies or antigen binding fragments thereof, comprising a heavy chain variable région polypeptide having at least 80%, 85%, 90%, 95%, or greater amino acid sequence identity to an amino acid sequence of a heavy chain variable région described herein (e.g., SEQ ID NOS:1 or 5-8), and 30 a variable light chain polypeptide having at least 80%, 85%, 90%, 95%, or greater amino acid sequence identity to an amino acid sequence of a light chain polypeptide as set forth herein (e.g., SEQ ID NOS:2 or 9-12).
[0050] Examples ofanti-MMP9 antibodies ofthe présent disclosure are described in more detail below.
Antibodies [0051] The MMP9 binding proteins include antibodies and functional fragments thereof, such as those that specifically bind to MMP9. As used herein, the term antibody means an isolated or recombinant polypeptide binding agent that comprises peptide sequences (e.g., variable région sequences) that specifically bind an antigenic epitope. The term is used in its broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments including but not limited to Fv, scFv, Fab, Fab’ F(ab')2 and Fabî, so long as they exhibit the desired biological activity. The term “human antibody” refers to antibodies containing sequences of human origin, except for possible non-human CDR régions, and does not imply that the full structure of an immunoglobulin molécule be présent, only that the antibody has minimal immunogenic effect in a human (i.e.t does not induce the production of antibodies to itself).
[0052] An antibody fragment comprises a portion of a full-length antibody, for example, the antigen binding or variable région of a full-length antibody. Such antibody fragments may also be referred to herein as “functional fragments: or “antigen-binding fragments”. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al. (1995) Protein Eng. 8(10): 1057-1062); singlechain antibody molécules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site, and a residual Fc fragment, a désignation reflecting the abilïty to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.
[0053] Fv is a minimum antibody fragment containing a complété antigen-recognition and -binding site. This région consists of a dimer ofone heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three complementarity-determining régions (CDRs) of each variable domain interact to define an antigen-binding site on the surface of the Vh-Vl dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or an isolated Vh or Vl région comprising only three of the six CDRs spécifie for an antigen) has the ability to recognize and bind antigen, although generally at a lower affinity than does the entire Fv fragment.
[0054[ The “Ftb” fragment also contains, in addition to heavy and light chain variable régions, the constant domain of the light chain and the first constant domain (CHi) of the heavy chain. Fab fragments were originally observed following papain digestion of an antibody. Fab’ fragments differ from Fab fragments in that F(ab') fragments contain severai additional residues at the carboxy terminus of the heavy chain CHi domain, including one or more cysteines from the antibody hinge région. Ffab'h fragments contain two Fab fragments joined, near the hinge région, by disulfîde bonds, and were originally observed following pepsin digestion of an antibody. Fab'-SH is the désignation herein for Fab' fragments in which the cysteine residue(s) of the constant domains bear a free thiol group. Other chemical couplings of antibody fragments are also known.
[0055] The light chains of antibodies (immunogîobulins) from any vertebrate species can be assigned to one of two clearfy distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Dependîng on the amino acid sequence of the constant domain of their heavy chains, immunogîobulins can be assigned to five major classes: IgA, IgD, IgE, IgG, and IgM, and severai of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and lgA2.
[0056] Single-chain Fv or sFv or scFv antibody fragments comprise the Vh and Vl domains of antibody, wherein these domains are présent in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the Vh and Vl domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, in The Pharmacology ofMonoclonal Antibodies, vol. 113 (Rosenburg and Moore eds.) Springer-Verlag, New York, pp. 269-315 (1994).
]0057] The term diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (Vh) connected to a lightchain variable domain (Vl) in the same polypeptide chain (Vh-Vl). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen-binding sites. Diabodies are additionally described, for example, in EP 404,097; WO 93/11161 and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.
]0058] An isolated antibody is one that has been identified and separated and/or recovered from a component of its naturel environment. Components of its naturel environment may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutés. In some embodiments, an isolated antibody is purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, for example, more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internai amino acid sequence, e.g., by use of a spinning cup sequenator, or (3) to homogeneity by gel electrophoresîs (e.g., SDS-PAGE) under reducing or nonreducing conditions, with détection by Coomassie blue or silver stain. The term isolated antibody includes an antibody in situ within recombinant cells, since at least one component of the antibody*s natural environment will not be présent. In certain embodiments, isolated antibody is prepared by at least one purification step.
[0059] As used herein, “immunoreactive” refers to antibodies or fragments thereof that are spécifie to a sequence of amino acid residues (“binding site” or “epitope”), yet if are 10 cross-reactive to other peptides/proteins, are not toxic at the levels at which they are formulated for administration to human use. Epitope refers to that portion of an antigen capable of forming a binding interaction with an antibody or antigen binding fragment thereof. An epitope can be a linear peptide sequence (i.e., “continuous”) or can be composed of noncontiguous amino acid sequences (i.e., “conformational” or “discontinuous”). The term 15 “preferentially binds” means that the binding agent binds to the binding site with greater affinity than it binds unrelated amino acid sequences.
[0060] Anti-MMP9 antibodies can be described in terms of the CDRs of the heavy and light chains. As used herein, the term CDR or complementarity determining région is intended to mean the non-contiguous antigen combining sites found within the variable 20 région of both heavy and light chain polypeptides. These particular régions hâve been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, Sequences of proteins of immunological interest (1991); by Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the définitions include overiapping or subsets of amino acid 25 residues when compared against each other. Nevertheless, application ofeither définition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited référencés are set forth below in Table 1 as a comparison.
Table lî CDR Définitions
Kabat' Cbothia1 MacCallum1
vhcdri | 31-35 | 26-32 | 30-35 |
VhCDR2 | 50-65 | 53-55 | 47-58 |
Vu CDR3 | 95-102 | 96-101 | 93-101 |
VlCDR1 | 24-34 | 26-32 | 30-36 |
VlCDR2 | 50-56 | 50-52 | 46-55 |
VlCDR3 | 89-97 | 91-96 | 89-96 |
Residue numbering follows the nomenclature of Kabat et al., supra ’Residue numbering follows the nomenclature of Chothia et al., supra ’Residue numbering follows the nomenclature of MacCallum et al., supra [0061] As used herein, the term framework when used in reference to an antibody variable région is intended to mean ail amino acid residues outside the CDR régions within the variable région of an antibody. A variable région framework is generally a disconlinuous amino acid sequence between about 100-120 amino acids in length but is intended to reference only those amino acids outside of the CDRs. As used herein, the term framework région is inlended to mean each domain of the framework that is separated by the CDRs.
[0062] In some embodiments, an antibody is a humanized antibody or a human antibody. Humanized antibodies include human immununoglobulins (récipient antibody) in which residues from a complementary-determining région (CDR) of the récipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. Thus, humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins which contain minimal sequence derived from non-human immunoglobulin. The non-human sequences are located primarily in the variable régions, particularly in the complementarity-determining régions (CDRs). In some embodiments, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues that are found neither in the récipient antibody nor in the imported CDR or framework sequences. In certain embodiments, a humanized antibody comprises substantially ail of at least one, and typically two, variable domains, in which ail or substantially ail of the CDRs correspond to those ofa non-human immunoglobulin and ail or substantially ail ofthe framework régions are those of a human immunoglobulin consensus sequence. For the purposes of the présent disciosure, humanized antibodies can also include immunoglobulin fragments, such as Fv, Fab, Fab’, F(ab*)2 or other antigen-binding subsequences of antibodies.
[0063] The humanized antibody can also comprise at least a portion of an immunoglobulin constant région (Fc), typically that of a human immunoglobulin. See, for example, Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323329; and Presta (1992) Curr. Op. Struct. Biol. 2:593-596.
[0064] Methods for humanizing non-human antibodies are known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as import or “donor” residues, which are typically obtained from an import or “donor” variable domain. For example, humanization can be performed essentially according to the method of Winter and co-workers, by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. See, for example, Jones et al., supra', Riechmann et al., supra and Verhoeyen et al. (1988) Science 239:1534-1536. Accordingly, such humanized antibodies include chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantiaily less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In certain embodiments, humanized antibodies are human antibodies in which some CDR residues and optionally some framework région residues are substituted by residues from analogous sites in rodent antibodies (e.g., murine monoclonal antibodies).
[0065] Human antibodies can also be produced, for example, by using phage display libraries. Hoogenboom et al. (1991) J. Mol. Biol, 227:381; Marks étal. (1991) J. Mol. Biol. 222:581. Other methods for preparing human monoclonal antibodies are described by Cole et al. (1985) “Monoclonal Antibodies and Cancer Therapy,” Alan R. Liss, p. 77 and Boemer étal. (1991) J. Immunol. 147:86-95.
[0066] Human antibodies can be made by introducing human immunoglobulin loci into transgenic animais (e.g., mice) in which the endogenous immunoglobulin genes hâve been partially or completely inactivated. Upon immunological challenge, human antibody production is observed, which closely resembles that seen in humans in ail respects, including gene rearrangement, assembly, and antibody répertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the foliowing scientific publications: Marks et al. (1992) Bio/Technology 10:779-783(1992); Lonberg cZ a/. (1994) Afa/t/re368: 856-859; Morrison (\994) Nature 368:812-813; Fishwald et al. (1996) Nature Biotechnology 14:845-851; Neuberger (1996) Nature Biotechnology 14:826; and Lonberg et al. (1995) Intem. Rev. Immunol. 13:65-93.
[0067] Antibodies can be affinity matured using known sélection and/or mutagenesis methods as described above. ln some embodiments, affinity matured antibodies hâve an affinity which is fîve times or more, ten times or more, twenty times or more, or thirty times or more than that of the starting antibody (generally murine, rabbît, chicken, humanized or human) from which the matured antibody is prepared.
[0068] An antibody can also be a bispecific antibody. Bispecific antibodies are monoclonal, and may be human or humanized antibodies that hâve binding specificities for at least two different antigens. In the présent case, the two different binding specificities can be directed to two different MMPs, or to two different epitopes on a single MMP (e.g., MMP9).
[0069] An antibody as disclosed herein can also be an immunoconjugate. Such immunoconjugates comprise an antibody (e.g., to MMP9) conjugated to a second molécule, such as a reporter An immunoconjugate can also comprise an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
[0070] An antibody that specifically binds to” or is “spécifie for” a particular polypeptide or an epitope refers to the sélective binding of the antibody to the target antigen or epitope; these terms, and methods for determining spécifie binding, are well understood in the art. An antibody exhibits “spécifie binding” for a particular target antigen or epitope if it binds with greater affinity, avidity, more readily, and/or with greater duration to that target antigen or epitope than it does with other substances. In some embodiments, the antibody that specifically binds to the polypeptide or epitope is one that that binds to that particular polypeptide or epitope without substantially binding to any other polypeptide or polypeptide epitope.
[0071] In some embodiments, the provided antibodies specifically bind to human MMP9 with a dissociation constant (Kj) equal to or lower than 100 nM, optionally lower than 10 nM, optionally lower than 1 nM, optionally lower than 0.5 nM, optionally lower than 0.1 nM, optionally lower than 0.01 nM, or optionally lower than 0.005 nM, in certain examples, between 0.1 and 0.2 nM, or between 0.1 and 10 pM, e.g., between 0.4 and 9 pm, such as between 0.4 and 8.8 pm, in the form of monoclonal antibody, scFv, Fab, or other form of antibody measured at a température of about 4°C, 25°C, 37°C or 42”C.
|0072] In certain embodiments, an antibody of the présent disclosure binds to one or more processing sites (e.g., sites of proteolytic cleavage) in MMP9, thereby effectively blocking processing ofthe proenzyme or preproenzyme to the catalytically active enzyme, and thus reducing the proteolytic activity ofthe MMP9.
[0073] In certain embodiments, an antibody according to the présent disclosure binds to MMP9 with an affinity at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times greater than its binding affinity for another MMP. Binding afïïnity can be measured by any method known in the art and can be expressed as, for example, on-rate, off-rate, dissociation constant (Kd), equilibrium constant (K»,) or any term in the art.
[0074] In certain embodiments, an antibody according to the présent disclosure is one that inhibits the enzymatic (i.e., catalytic) activity of MMP9, such as a non-compétitive inhibitor of the catalytic activity of MMP9. In certain embodiments, an antibody according to the présent disclosure binds within the catalytic domain of MMP9. In additional embodiments, an antibody according to the présent disclosure binds outside the catalytic domain of MMP9.
[0075] Also provided are antibodies or antigen binding fragments thereofthat compete with any one or more of the antï-MMP9 antibodies or antigen binding fragments thereof described herein for binding to MMP9. Thus, the présent disclosure contemplâtes anti-MMP9 antibodies, and functional fragments thereof, that compete for binding with, for example, an antibody having a heavy chain polypeptide of any of SEQ ID NOS: I or 5-8, a light chain polypeptide of SEQ ID NOS: 2 or 9-12, or combinations thereof. In one embodiment, the antî-MMP9 antibody, or functional fragment thereof, competes for binding to human MMP9 with the antibody described herein as AB004I.
Epitope Binding [0076] Also provided are antibodies and fragments thereofthat bind to the same epitope, e.g., MMP9 epitope as any one or more of the antibodies described herein. Also provided are antibodies and fragments that specifically bind to an epitope of MMP9, where the epitope includes an amino acid residue within a particular région of MMP9 or multiple régions of MMP9. Such régions can include, for example, structural loops and/or other structural domains of MMP9, such as those shown to be important for binding to exemplary antibodies described herein. Typically, the régions are defined according to amino acid residue positions on the full-length MMP9 sequence, e.g., SEQ ID NO: 27. In some examples, the epitope is outside of cysteîne-switch active pocket of SEQ ID NO: 27. In some example, the epitope contains an amino acid residue (i.e., one or more amino acid residue(s)) within a région that is residues 104-202 of SEQ ID NO: 27. In one example, the epitope contains an amino acid residue (i.e., one or more amino acid residue(s)) within a région that is residues 104-119, residues 159-166, or residues 191-202 of SEQ ID NO: 27. In some aspects, the epitope includes an amino acid residue (i.e., one or more amino acid residue(s)) within a région of MMP9 that is residues 104-119 of SEQ ID NO: 27, an an amino acid residue within a région of MMP9 that is residues 159-166 of SEQ ID NO: 27, and an amino acid residue within a région of MMP9 that is residues 191-202 of SEQ ID NO: 27. In some cases, the epitope includes El 11, DI 13, RI62, or 1198 of SEQ ID NO: 27. In some cases, it includes
RI62 of SEQ ID NO: 27. In some cases, it includes El II, DI 13, R162, and 1198 of SEQ ID NO: 27.
MMP9 sequence [0077] The amino acid sequence ofhuman MMP9 protein is as foliows:
MSLWQPLVLV LLVLGCCFAA PRQRQSTLVL FPGDLRTNLT DRQLAEEYLY 50
RYGYTRVAEM RGESKSLGPA LLLLQKQLSL PETGELDSAT LKAMRTPRCG 100
VPDLGRFQTF EGDLKWHHHN ITYWIQNYSE DLPRAVIDDA FARAFALWSA 150
VTPLTFTRVY SRDADIVIQF GVAEHGDGYP FDGKDGLLAH AFPPGPGIQG 200
DAHFDDDELW SLGKGWVPT RFGNADGAAC HFPFIFEGRS YSACTTDGRS 250
DGLPWCSTTA NYDTDDRFGF CPSERLYTRD GNADGKPCQF PFIFQGQSYS 300
ACTTDGRSDG YRWCATTANY DRDKLFGFCP TRADSTVMGG NSAGELCVFP 350
FTFLGKEYST CTSEGRGDGR LWCATTSNFD SDKKWGFCPD QGYSLFLVAA 400
HEFGHALGLD HSSVPEALMY PMYRFTEGPP LHKDDVNGIR HLYGPRPEPE 450
PRPPTTTTPQ PTAPPTVCPT GPPTVHPSER PTAGPTGPPS AGPTGPPTAG 500
PSTATTVPLS PVDDACNVNI FDAIAEIGNQ LYLFKDGKYW RFSEGRGSRP 550
QGPFLIADKW PALPRKLDSV FEEPLSKKLF FFSGRQVWVY TGASVLGPRR 600
LDKLGLGADV AQVTGALRSG RGKMLLFSGR RLWRFDVKAQ MVDPRSASEV 650
DRMFPGVPLD THDVFQYREK AYFCQDRFYW RVSSRSELNQ VDQVGYVTYD 700 ILQCPED (SEQ ID NO:27) [00781 Protein domains are shown schematically in Figure 3 and are indicated below:
Amino Acid # 1-19 | Feature Signal Peptide | |
30 | 38-98 | Peptidoglycan Binding Domain |
R98/C99 | Cysteine-switch active pocket | |
112-445 | Zn dépendent metalloproteinase domain | |
223-271 | Fibronectin type 1! domain (gelatin binding domain) | |
281-329 | Fibronectin type II domain (gelatin binding domain) | |
35 | 340-388 | Fibronectin type II domain (gelatin binding domain) |
400-411 | Zn binding région | |
521-565 | Hemopexin-like domain | |
567-608 | Hemopexin-like domain | |
613-659 | Hemopexin-like domain | |
40 | 661-704 | Hemopexin-like domain |
[0079] The amino acid sequence ofmature fùll-length human MMP9 (which is the amino acid sequence of the propolypeptide of SEQ ID NO:27 without the signal peptide) is:
APRQRQSTLVL FPGDLRTNLT DRQLAEEYLY RYGYTRVAEM RGESKSLGPA
LLLLQKQLSL ITYWIQNYSE GVAEHGDGYP RFGNADGAAC CPSERLYTRD DRDKLFGFCP LWCATTSNFD PMYRFTEGPP GPPTVHPSER FDAIAEIGNQ FEEPLSKKLF RGKMLLFSGR AYFCQDRFYW
PETGELDSAT DLPRAVIDDA FDGKDGLLAH HFPFIFEGRS GNADGKPCQF TRADSTVMGG SDKKWGFCPD LHKDDVNGIR PTAGPTGPPS LYLFKDGKYW FFSGRQVWVY RLWRFDVKAQ RVSSRSELNQ
LKAMRTPRCG FARAFALWSA AFPPGPGIQG YSACTTDGRS PFIFQGQSYS NSAGELCVFP QGYSLFLVAA HLYGPRPEPE AGPTGPPTAG RFSEGRGSRP TGASVLGPRR MVDPRSASEV VDQVGYVTYD
VPDLGRFQTF VTPLTFTRVY dahfdddelw DGLPWCSTTA ACTTDGRSDG FTFLGKEYST HEFGHALGLD PRPPTTTTPQ PSTATTVPLS QGPFLIADKW LDKLGLGADV DRMFPGVPLD ILQCPED (!
EGDLKWHHHN SRDADIVÏQF SLGKGVWPT NYDTDDRFGF YRWCATTANY CTSEGRGDGR HSSVPEALMY PTAPPTVCPT PVDDACNVNI PALPRKLDSV AQVTGALRSG THDVFQYREK IEQ ID NO:28) [0080] The amino acid sequence of the signal peptide is MSLWQPLVLV LLVLGCCFA (SEQ ID NO:29).
[0081] Also provided are MMP9 polypeptides, including mutant MMP9 polypeptides. Such peptides are useful, for example, in generating and selecting antibodies and fragments as provided herein. Exemplary polypeptides include those having an amino acid sequence containing residues 104-202 of SEQ ID NO: 27, and those having an amino acid sequence of SEQ ID NO: 27 with an amino acid substitution at residue 111,113,162, or 198 of SEQ ID NO 27 or with an amino acid substitution at ail such residues. Other exemplary polypeptides include those having an amino acid sequence containing residues 111-198 of SEQ ID NO: 27, and those having an amino acid sequence containing residues 111-198 of SEQ ID NO: 27 with an amino acid substitution at residue 111, 113,162, or 198 of SEQ ID NO 27 or with an amino acid substitution at ail such residues. Such polypeptides fmd use, for exemple, in selecting antibodies that bind to epitopes containing such residues and/or for which such residues of MMP9 are important for binding, such as those described herein.
[0082] The présent disclosure contemplâtes MMP9 binding proteins that bind any portion of MMP9, e.g., human MMP9, with MMP9 binding proteins that preferentially bind MMP9 relative to other MMPs being of particular interest.
[0083] Antî-MMP9 antibodies, and fùnctiona! fragments thereof, can be generated accordingly to methods well known in the art. Exemplary anti-MMP9 antibodies are provided below.
Mouse monoclonal antI-MMP9 antibodies [0084] A mouse monoclonal antibody to human MMP9 was obtained as described in Example 1. This antibody contains a mouse IgG2b heavy chain and a mouse kappa light 5 chain, and is denoted AB0041.
[0085] The amino acid sequence of the AB0041 heavy chain is as follows:
MAVLVLFLCLVAFPSCVLSOVOLKESGPGLVAPSOSLSITCTVSGFSLL
SYGVHWVRQPPGKGLEWLGVIWTGGTTNYNSALMSRLS1SKDDSKSQ
VFLKMNSLQTDDTAIYYCARYYYGMDYWGQGTSVTVSS/fÂ'7T/’/’SPT
PLAPGCGDTTGSSVTLGCL VKGYFPESVTVTIVNSGSLSSSVHTFPALLQSGL
YTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGP1ST1NPCPPCKE
CHKCPAPNLEGGPSVF1FPPNIKDVLM1SLTPKVTCWVDVSEDDPDVRIS
WFVNNVEVHTAQTQTHREDYNSTIRWSALPIQUQDWMSGKEFKCKVNN
KDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLWGFNPGDIS
VEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLD1KTSKWEKTDSFSCNVRH
EGLKNYYLKKT1SRSPGK (SEQIDNO:I) [0086] The signal sequence is underlined, and the sequence of the IgG2b constant région is presented italics.
[0087] The amino acid sequence of theAB0041 light chain is as follows:
MESOIOVFVFVFLWLSGVDGDIVMTOSHKFMSTSVGDRVSITCKASOD
VRNTVAWYQQKTGQSPKLLIYSSSYRNTGVPDRFTGSGSGTDFTFTISS
VQAEDLAVYFCQQHYITPYTFGGGTKLEIK/MDÆiPmZFPPSSKeira
GGASWCFLNNFYPKDINVKWK1DGSERQNGVLNSWTDQDSKDSTYSMSS
TLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:2) [0088] The signal sequence is underlined, and the sequence of the kappa constant région is presented in italics.
[0089] The following amino acid sequence comprises the framework régions and complementarity-determining régions (CDRs) of the variable région of the IgG2b heavy chain of AB004I (with CDRs underlined):
QVQLKESGPGLVAPSQSLSITCTVSGFSLLSYGVHWVROPPGKGLEWL
GV1WTGGTTNYNSALMSRLSISKDDSKSOVFLKMNSLOTDDTAIYYCA
RYYYGMDYWGOGTSVTVSS (SEQ ID NO:3) [0090] The following amino acid sequence comprises the framework régions and complementarity-determining régions (CDRs) of the variable région of the kappa light chain of AB0041 (with CDRs underlined):
DIVMTOSHKFMSTSVGDRVSrrCKASODVRNTVAWYOQKTGQSPKLL IYSSSYRNTGVPDRFTGSGSGTDFTFTISSVOAEDLAVYFCOOHYITPYT FGGGTKLEIK (SEQ ID NO:4)
Other exemplary mouse anti-human MMP9 antibodies (e.g., M4 and M12) are described in Example IB. An exemplary anti-mouse MMP9 antibody (AB0046) is described in Example I C. Other exemplary mouse anti-human MMP9 antibodies include antibodies comprise the variable régions having the sequence of SEQ ID NO: 3, and the constant régions having 95% similarity as the sequences of the IgG2b constant régions. In addition, the exemplary mouse anti-human MMP9 antibodies include antibodies comprise the variable régions having the sequence of SEQ ID NO: 4, and the constant régions having 95% similarity as the sequences of the IgG2b constant régions. Other exemplary mouse anti-human MMP9 antibodies include antibodies comprise the variable régions having the sequences of SEQ ID NOs: 3 and 4, and the constant régions having 95% similarity as the sequences of the IgG2b constant régions. Such anti-mouse antibodies are suitable for testing and assessing the MMP9inhibition methods.
Heavy-chain variants [0091] The amino acid sequences of the variable régions of the AB004I heavy and light chains were separately modified, by altering framework région sequences in the heavy and iight chain variable régions. The effect of these sequence alterations was to deplete the antibody of human T-cell epitopes, thereby reducing or abolishing its immunogenicity in humans.
[0092] Four heavy-chain variants were constructed, in a human IgG4 heavy chain background containing a S24 i P amino acid change that stabilizes thehinge domain (Angal et al. (1993) Molec. Immunol. 30:105-108), and are denoted VH1, VH2, VH3 and VH4. The amino acid sequences of their framework régions and CDRs are as follows:
VHl
QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWL
GVIWTGGTTNYNSALMSRLTISKDDSKSTVYLKMNSLKTEDTAIYYCA
RYYYGMDYWGQGTSVTVSS (SEQ ID N0:5)
VH2
QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWL GVIWTGGTTNYNSALMSRLTISKDDSKNTVYLKMNSLKTEDTAIYYC ARYYYGMDYWGQGTLVTVSS (SEQ ID N0:6)
VII3
QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWL
GVIWTGGTTNYNSALMSRFTISKDDSKNTVYLKMNSLKTEDTAIYYC
ARYYYGMDYWGQGTLVTVSS (SEQIDNO:7)
VH4
QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWL
GVIWTGGTTNYNSALMSRFTISKDDSKNTLYLKMNSLKTEDTAIYYCA
RYYYGMDYWGQGTLVTVSS (SEQ ID NO:8) [0093] Figure 1 shows an alignment of the amino acid sequences of the variable régions of the humanized heavy chains and indicates the différences in amino acid sequences in the 20 framework régions among the four variants.
Llght-chain variants [0094] Four light-chain variants were constructed, in a human kappa chain background, and are denoted Vkl, Vk2, Vk3 and Vk4. The amino acid sequences of their framework 25 régions and CDRs are as follows:
Vkl
DIVMTQSPSFLSASVGDRVTITCKASQDVRNTVAWYQQKTGKAPKLLi
YSSSYRNTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYFCQQHYITPYT
FGGGTKVEIK (SEQ ID NO:9)
Vk2
DIVMTQSPSSLSASVGDRVTITCKASQDVRNTVAWYQQKPGKAPKLLI YSSSYRNTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYFCQQHYITPYT FGGGTKVEIK (SEQIDNO:10)
Vk3
DIQMTQSPSSLSASVGDRVT1TCKASQDVRNTVAWYQQKPGKAPKLLI YSSSYRNTGVPDRFSGSGSGTDFTLT1SSLQAEDVAVYFCQQHYITPYT FGGGTKVEIK (SEQ ID NO: 11 )
Vk4
DIQMTQSPSSLSASVGDRVTITCKASQDVRNTVAWYQQKPGKAPKLL1 YSSSYRNTGVPDRFSGSGSGTDFTLT1SSLQAEDVAVYYCQQHYITPYT FGGGTKVEIK (SEQIDNO: 12)
J0095J Figure 2 shows an alignment of the amino acid sequences of the variable régions of the humanized light chains and indicates the différences in amino acid sequences in the framework régions among the four variants.
[0096] The humanized heavy and light chains are combined in ail possible pair-wise combinations to generate a number of functional humanized anti-MMP9 antibodies. For example, provided are antibodies with a heavy chain variable (VH) région having the amino acid sequence set forth in any of SEQ ID NOs: 3,5,6,7, and 8; antibodies having a light chain variable (VL) région having the amino acid sequence set forth in any of SEQ ID NOs:
4.9.10.11, and 12; and antibodies with a heavy chain variable (VH) région having the amino acid sequence set forth in any of SEQ ID NOs: 3,5,6,7, and 8 and a light chain variable (VL) région having the amino acid sequence set forth in any of SEQ ID NOs: 4,9,
10.11, and 12, as well as antibodies that compete for binding to MMP9 with such antibodies and antibodies having at least at or about 75 %, 80 %, 85 %, 90%, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more sequence identity with such antibodies. In one example, the antibody has a VH région with an amino acid sequence having at least at or about 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more sequence identity with SEQ ID NO: 7 and a VL région with an amino acid sequence having at least at or about 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more sequence identity with SEQ ID NO: 12, or a VH région of SEQ ID NO; 7 and a VL région of SEQ ID NO: 12.
(0097[ Additional heavy chain variable région amino acid sequences having 75% or more, 80% or more, 90% or more, 95% or more, or 99% or more homology to the heavy chain variable région sequences disclosed herein are also provided. Furthermore, additional light chain variable région amino acid sequences having 75% or more, 80% or more, 90% or more, 95% or more, or 99% or more homology to the light chain variable région sequences disclosed herein are also provided.
[0098] Additional heavy chain variable région amino acid sequences having 75% or more, 80% or more, 90% or more, 95% or more, or 99% or more sequence identity to the heavy chain variable région sequences disclosed herein are also provided. Furthermore, additional light chain variable région amino acid sequences having 75% or more, 80% or more, 90% or more, 95% or more, or 99% or more sequence identity to the light chain variable région sequences disclosed herein are also provided.
Complementarlty-determlning régions (CDRs) [0099] In some embodiments, the CDRs of the heavy chain ofexempîary provided antiMMP9 antibodies as disclosed herein hâve the following amino acid sequences:
CDR1: GFSLLSYGVH (SEQ ID NO: 13)
CDR2: VIWTGGTTNYNSALMS (SEQ ID NO: 14) CDR3:YYYGMDY (SEQ ID NO: 15) [0100] Thus, among the provided anti-MMP9 antibodies are antibodies having a heavy chain CDR1 région with an amino acid sequence as set forth in SEQ ID NO: 13, antibodies having a heavy chain CDR2 région with an amino acid sequence set forth in SEQ ID NO: 14, and antibodies having a heavy chain CDR3 région with an amino acid sequence as set forth in SEQ ID NO: 15, and antibodies that compete for binding with or bind to the same epitope on
MMP9 as such antibodies. In some cases, the antibodies contaîn VH CDRs having the sequences set forth in SEQ ID NO: 13,14, and 15.
[0101] In some embodiments, the CDRs ofthe light chain ofexempîary anti-MMP9 antibodies as disclosed herein hâve the following amino acid sequences:
CDR1: KASQDVRNTVA (SEQ ID NO: 16)
CDR2: SSSYRNT (SEQ ID NO: 17)
CDR3: QQHYITPYT (SEQ ID NO: 18) [0102] Thus, among the provided anti-MMP9 antibodies are antibodies having a light chain CDR1 région with an amino acid sequence as set forth in SEQ ID NO: 16, antibodies having a light chain CDR2 région with an amino acid sequence set forth in SEQ ID NO: 17, and antibodies having a light chain CDR3 région with an amino acid sequence as set forth in
SEQ ID NO: 18, and antibodies that compete for binding with or bind to the same epitope on MMP9 as such antibodies. In some cases, the antibodies contain VL CDRs having the sequences set forth in SEQ ID NO: 16, 17, and 18.
Nucleic acids encoding antI-MMP9 antibodies [0103] The présent disclosure provides nucleic acids encoding anti-MMP9 antibodies and functional fragments thereof. Accordingly, the présent disclosure provides an isolated polynucleotide (nucleic acid) encoding an antibody or antigen-binding fragment as described herein, vectors containing such polynucleotides, and host cells and expression Systems for transcribing and translating such polynucleotides into polypeptides.
[01041 The présent disclosure also contemplâtes constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide as above.
[0105| The présent disclosure also provides a recombinant host cell which comprises one or more constructs as above, as well as methods of production of the antibody or antigen20 binding fragments thereof described herein which method comprises expression of nucleic acid encoding a heavy chain polypeptide and a light chain polypeptide (in the same or different host cells, and from the same or different constructs) in a recombination host cell. Expression can be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression, an antibody or antigen25 binding fragment can be isolated and/or purified using any suitable technique, then used as appropriate.
[01061 Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus Systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. A common bacterial host is E. coli.
(01071 Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including operably linked promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or other sequences as appropriate. Vectors can be plasmids, viral e.g. ‘phage, or phagemid, as appropriate. For further details see, for exemple, Molecular Cloning: a Laboratory Manual: 2nd édition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocole for manipulation of nucleic acid, for example in préparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference in their entirety.
[0108] The nucleic acid encoding a polypeptide of interest is integrated into the genome of the host cell or can be maintained as a stable or transient episomal element.
[0109] Any of a wide variety of expression control sequences - sequences that control the expression of a DNA sequence operatîvely linked to it - can be used in these vectors to express the DNA sequences. For example, a nucleic acid encoding a polypeptide of interest can be operably linked to a promoter, and provided in an expression construct for use in methods of production of recombinant MMP9 proteins or portions thereof.
[0110] Those of skilt in the art are aware that nucleic acids encoding the antibody chains disclosed herein can be synthesized using standard knowledge and procedures in molecular biology.
[0111] Examples ofnucléotide sequences encoding the heavy and light chain amino acid sequences disclosed herein, are as follows:
VIII: CAGGTGCAGC TGCAGGAATC CGGCCCTGGC CTGGTCAAGC CCTCCGAGAC ACTGTCCCTG ACCTGCACCG TGTCCGGCTT CTCCCTGCTG TCCTACGGCG TGCACTGGGT CCGACAGCCT CCAGGGAAGG GCCTGGAATG GCTGGGCGTG ATCTGGACCG GCGGCACCAC CAACTACAAC TCCGCCCTGA TGTCCCGGCT GACCATCTCC AAGGACGACT CCAAGTCCAC CGTGTACCTG AAGATGAACT CCCTGAAAAC CGAGGACACC GCCATCTACT ACTGCGCCCG GTACTACTAC GGCATGGACT ACTGGGGCCA GGGCACCTCC GTGACCGTGT CCT CA (SEQ ID NO: 19)
VH2: CAGGTGCAGC TGCAGGAATC CGGCCCTGGC CTGGTCAAGC CCTCCGAGAC ACTGTCCCTG ACCTGCACCG TGTCCGGCTT CTCCCTGCTG TCCTACGGCG TGCACTGGGT CCGACAGCCT
CCAGGCAAAG | GCCTGGAATG GCTGGGCGTG ATCTGGACCG |
GCGGCACCAC | CAACTACAAC TCCGCCCTGA TGTCCCGGCT |
GACCATCTCC | AAGGACGACT CCAAGAACAC CGTGTACCTG |
AAGATGAACT | CCCTGAAAAC CGAGGACACC GCCATCTACT |
ACTGCGCCCG | GTACTACTAC GGCATGGACT ACTGGGGCCA |
GGGCACCCTG | GTCACCGTGT CCTCA (SEQIDNO:20) |
VH3: CAGGTGCAGC TGCAGGAATC CGGCCCTGGC CTGGTCAAGC
CCTCCGAGAC | ACTGTCCCTG ACCTGCACCG TGTCCGGCTT |
CTCCCTGCTG | TCCTACGGCG TGCACTGGGT CCGACAGCCT |
CCAGGCAAAG | GCCTGGAATG GCTGGGCGTG ATCTGGACCG |
GCGGCACCAC | CAACTACAAC TCCGCCCTGA TGTCCCGGTT |
CACCATCTCC | AAGGACGACT CCAAGAACAC CGTGTACCTG |
AAGATGAACT | CCCTGAAAAC CGAGGACACC GCCATCTACT |
ACTGCGCCCG | GTACTACTAC GGCATGGACT ACTGGGGCCA |
GGGCACCCTG | GTCACCGTGT CCTCA (SEQIDNO:21) |
VH4: CAGGTGCAGC TGCAGGAATC CGGCCCTGGC CTGGTCAAGC
CCTCCGAGAC | ACTGTCCCTG ACCTGCACCG TGTCCGGCTT |
CTCCCTGCTG | TCCTACGGCG TGCACTGGGT CCGACAGCCT |
CCAGGCAAAG | GCCTGGAATG GCTGGGCGTG ATCTGGACCG |
GCGGCACCAC | CAACTACAAC TCCGCCCTGA TGTCCCGGTT |
CACCATCTCC | AAGGACGACT CCAAGAACAC CCTGTACCTG |
AAGATGAACT | CCCTGAAAAC CGAGGACACC GCCATCTACT |
ACTGCGCCCG | GTACTACTAC GGCATGGACT ACTGGGGCCA |
GGGCACCCTG | GTCACCGTGT CCTCA (SEQ ID NO:22) |
Vkl: GACATCGTGA TGACCCAGTC CCCCAGCTTC CTGTCCGCCT
CCGTGGGCGA | CAGAGTGACC ATCACATGCA AGGCCTCTCA |
GGACGTGCGG | AACACCGTGG CCTGGTATCA GCAGAAAACC |
GGCAAGGCCC | CCAAGCTGCT GATCTACTCC TCCTCCTACC |
GGAACACCGG | CGTGCCCGAC CGGTTTACCG GCTCTGGCTC |
CGGCACCGAC | TTTACCCTGA CCATCAGCTC CCTGCAGGCC |
GAGGACGTGG | CCGTGTACTT CTGCCAGCAG CACTACATCA |
CCCCCTACAC | CTTCGGCGGA GGCACCAAGG TGGAAATAAA A (SEQ |
ID NO:23)
Vk2: GACATCGTGA TGACCCAGTC CCCCTCCAGC CTGTCCGCCT CTGTGGGCGA CAGAGTGACC ATCACATGCA AGGCCTCTCA GGACGTGCGG AACACCGTGG CCTGGTATCA GCAGAAGCCC GGCAAGGCCC CCAAGCTGCT GATCTACTCC TCCTCCTACC GGAACACCGG CGTGCCCGAC CGGTTTACCG GCTCTGGCTC CGGCACCGAC TTTACCCTGA CCATCAGCTC CCTGCAGGCC GAGGACGTGG CCGTGTACTT CTGCCAGCAG CACTACATCA CCCCCTACAC CTTCGGCGGA GGCACCAAGG TGGAAATAAA A (SEQ ID NO:24)
Vk3: GACATCCAGA TGACCCAGTC CCCCTCCAGC CTGTCCGCCT CTGTGGGCGA CAGAGTGACC ATCACATGCA AGGCCTCCCA GGACGTGCGG AACACCGTGG CCTGGTATCA GCAGAAGCCC GGCAAGGCCC CCAAGCTGCT GATCTACTCC TCCTCCTACC GGAACACCGG CGTGCCCGAC CGGTTCTCTG GCTCTGGAAG CGGCACCGAC TTTACCCTGA CCATCAGCTC CCTGCAGGCC GAGGACGTGG CCGTGTACTT CTGCCAGCAG CACTACATCA CCCCCTACAC CTTCGGCGGA GGCACCAAGG TGGAAATAAA A (SEQID NO:25)
Vk4: GACATCCAGA TGACCCAGTC CCCCTCCAGC CTGTCCGCCT CTGTGGGCGA CAGAGTGACC ATCACATGCA AGGCCTCTCA GGACGTGCGG AACACCGTGG CCTGGTATCA GCAGAAGCCC GGCAAGGCCC CCAAGCTGCT GATCTACTCC TCCTCCTACC GGAACACCGG CGTGCCCGAC CGGTTCTCTG GCTCTGGAAG CGGCACCGAC TTTACCCTGA CCATCAGCTC CCTGCAGGCC GAGGACGTGG CCGTGTACTA CTGCCAGCAG CACTACATCA CCCCCTACAC CTTCGGCGGA GGCACCAAGG TGGAAATAAA A(SEQID NO:26) [0112] Because the structure of antibodies, including the juxtaposition of CDRs and framework régions in the variable région, the structure of framework régions and the structure of heavy- and light-chain constant régions, is well-known in the art; it is well within the skill of the art to obtain related nucleic acids that encode anti-MMP-9 antibodies. Accordingly, polynucleotides comprising nucleic acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% and at least 99% homology to any of the nucléotide sequences disclosed herein are also provided. Accordingly, polynucleotides comprising nucleic acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% and at least 99% identity to any of the nucléotide sequences disclosed herein are also provided. In one example, the polynucleotide contains at least at or about 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more sequence identity with SEQ ID NO: 21 or includes or is SEQ ID NO: 21 and/or contains at least at or about 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more sequence identity with SEQ ID NO: 26 or includes or is SEQ ID NO: 26.
Pharmaceutical Compositions |0113] MMP9 binding proteins, as well as nucleic acid (e.g., DNA or RNA) encoding MMP9 binding proteins, can be provided as a pharmaceutical composition, e.g., combined with a pharmaceutically acceptable carrier or excipient. Such pharmaceutical compositions are useful for, for example, administration to a subject in vivo or ex vivo, and for diagnosing and/or treating a subject with the MMP9 binding proteins, such as in any of the therapeutic or diagnostic methods provided herein.
10114] Pharmaceutically acceptable carriers are physiologically acceptable to the administered patient and retain the therapeutic properties of the antibodies or peptides with which it is administered. Pharmaceutically-acceptable carriers and their formulations are and generally described in, for example, Remington’ pharmaceutical Sciences (18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA 1990). One exemplary pharmaceutical carrier is physiological saline. Each carrier is “pharmaceutically acceptable” in the sense of being compatible with the other ingrédients of the formulation and not substantially injurious to the patient.
[0115[ Pharmaceutical compositions can be formulated to be compatible with a particular route of administration, systemic or local. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.
[0116] Pharmaceutical compositions can include pharmaceutically acceptable additives. Examples of additives include, but are not limited to, a sugar such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose and mixtures thereof. Pharmaceutically acceptable additives can be combined with pharmaceutically acceptable carriers and/or excipients such as dextrose. Additives also include surfactants such as polysorbate 20 or polysorbate 80.
[0117] The formulation and delivery methods will generally be adapted according to the site and the disease to be treated. Exemplary formulations include, but are not limited to, those suitable for parentéral administration, e.g., intravenous, intra-arterial, intramuscular, or subcutaneous administration, or oral administration.
[0118] Pharmaceutical compositions for parentéral delivery include, for example, water, saline, phosphate buffered saline, Hank’s solution, Ringer’s solution, dextrose/saline, and glucose solutions. The formulations can contain auxiliary substances to approximate physiologicat conditions, such as buffering agents, tonicity adjusting agents, wetting agents, détergents and the like. Additives can also include additional active ingrédients such as bactericidal agents, or stabilizers. For example, the solution can contain sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate or triethanolamine oleate. Additional parentéral formulations and methods are described in Bai (1997) J. Neuroimmunol. 80:65 75; Warren (1997) J. Neurol. Sci. 152:31 38; and Tonegawa (1997) J. Exp. Med. 186:507 515. The parentéral préparation can be enclosed in amputes, disposable syringes or multiple dose viats made of gtass or plastic.
[0119] Pharmaceutical compositions for intradermal or subcutaneous administration can include a stérile diluent, such as water, saline solution, fixed oils, polyethylene glycols, glycérine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyt alcohot or methyi parabens; antioxidants such as ascorbic acid, glutathione or sodium bisulfite; chelating agents such as ethylenedîaminetetraacetic acid; buffers such as acétates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
[0120] Pharmaceutical compositions for injection inctude aqueous solutions (where water soluble) or dispersions and stérile powders for the extemporaneous préparation of stérile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiotogical satine, bacteriostatic water, Cremophor ELTM (BASF, Parsîppany, N.J.) or phosphate buffered satine (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, éthanol, polyol (for example, glycerot, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Fluidity 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 ofdispersion and by the use ofsurfactants. Antibacterial and antifungat agents inctude, for example, parabens, chlorobutanol, phénol, ascorbic acid and thimerosal. Isotonie agents, for exampte, sugars, polyalcohots such as manitol, sorbitol, and sodium chloride may be inctuded in the composition. The resulting solutions can be packaged for use as is, or lyophilized; the lyophilized préparation can later be combined with a stérile solution prior to administration.
[0121] Pharmaceutically acceptable carriers can contain a compound that stabilizes, increases, or delays absorption or clearance. Such compounds include, for example, carbohydrates, such as glucose, sucrose, or dextrans; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of peptides; or excipients or other stabilizers and/or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Détergents can also be used to stabïlize or to increase or decrease the absorption ofthe pharmaceutical composition, including liposomal carriers. To protect from digestion the compound can be complexed with a composition to render it résistant to acidic and enzymatic hydrolysis, or the compound can be complexed in an appropriately résistant carrier such as a liposome. Means of protecting compounds from digestion are known in the art (see, e.g., Fix (1996) Pharm Res. 13:1760 1764; Samanen (1996) J. Pharm. Pharmacol. 48:119 135; and U.S. Pat. No. 5,391,377, describing lipid compositions for oral delivery of therapeutic agents).
[0122] Compositions of the présent invention can be combined with other therapeutic moieties or imaging/dîagnostic moieties as provided herein. Therapeutic moieties and/or imaging moieties can be provided as a separate composition, or as a conjugated moiety présent on an MMP9 binding protein.
[0123J Formulations for in vivo administration are generally stérile. In one embodiment, the pharmaceutical compositions are formulated to be free of pyrogens such that they are acceptable for administration to human patients.
[0124] Various other pharmaceutical compositions and techniques for their préparation and use will be known to those of skill in the art in light of the présent disdosure. For a 25 detailed listing of suitable pharmacological compositions and associated administrative techniques one can refer to the detailed teachings herein, which can be further supplemented by texts such as Remington: The Science and Practice of Pharmacy 20th Ed. (Lippincott, Williams & Wilkins 2003).
[0125] Pharmaceutical compositions can be formulated based on the physical characteristics of the patient/subject needing treatment, the route of administration, and the like. Such can be packaged in a suitable pharmaceutical package with appropriate labels for the distribution to hospitals and dinics wherein the label is for the indication of treating a disorder as described herein in a subject. Médicaments can be packaged as a single or multiple units. Instructions for the dosage and administration of the pharmaceutical compositions of the présent invention can be included with the pharmaceutical packages and kits described below.
Methods ofUse [0126] The MMP9 binding proteins, including anti-MMP9 antibodies and fragments thereof, of the présent disclosure can be used, for example, in therapeutic and diagnostic methods, such as methods of détection of MMP9 in a sample, methods of treatment (e.g., as in methods of inhibition ofangiogenesis), and methods ofdiagnosis and prognosis. Thus, provided are diagnostic and therapeutic methods and uses ofthe anti-MMP9 antibodies. Examples of methods of use are described below.
Methods of Treatment [0127] Provided herein are methods oftreatment, including methods oftreating diseases and disorders associated with MMP9 expression and/or activity, as well as uses ofthe provided antibodies and compositions in such methods. The diseases and disorders inciude, but are not limited to cancer, e.g., tumors (e.g., primary or metastatic tumors), such as those that express or are disposed in a tissue which expresses MMP9, and inflammatory diseases, such as inflammatory bowe! diseases, rheumatoid arthritis and inflammatory myopathies.
[0128] As used herein, “treat” or treatment” means stasis or a postponement of development of one or more symptoms associated with a disease or disorder described herein, or ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, or ameliorating or preventing the underlying metabolic causes of symptoms. Thus, the terms dénoté that a bénéficiai resuit has been conferred on a mammalian subject with a disease or symptom, or with the potential to develop such disease or symptom. A response is achieved when the patient expériences partial or total alleviation, or réduction of signs or symptoms of illness, and can inciude, without limitation, prolongation of survival. The expected progression-free survival times can be measured in months to years, depending on prognostic factors including the number of relapses, stage of disease, and other factors.
[0129] Also provided are pharmaceutical compositions for use in connection with such methods, such as those containing any of the antibodies or fragments thereof described herein. Compositions can be suitable for administration locally or systemically by any suitable route.
[0130] In general, MMP9 binding proteins are administered in a therapeutically effective amount, e.g., in an amount to effect inhibition of tumor growth in a subject, to inhibit metastasis, to inhibit inflammation, to inhibit tissue destruction, to inhibit MMP9 activity, or to treat the particular disease or condition associated with MMP9.
[0131] As used herein, unless otherwise specified, the term “therapeutically effective amount” or “effective amount” refers to an amount of an agent or compound or composition that when administered (either alone or in combination with another therapeutic agent, as may be specified) to a subject is effective to prevent or ameliorate the disease condition or the progression of the disease, or resuit in amelioration of symptoms, e.g., treatment, healing, prévention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prévention or amelioration of such conditions. When applied to an individual active ingrédient administered alone, a therapeutically effective dose refers to that ingrédient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingrédients that resuit in the therapeutic effect, whether administered in combination, serially or simultaneously. In one example, when in vivo administration of an antî-MMP9 antibody is employed, normal dosage amounts can vary from about 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day, preferably about 1 pg/kg/day to 50 mg/kg/day, optionally about 100 pg/kg/day to 20 mg/kg/day, 500 pg/kg/day to 10 mg/kg/day, or 1 mg/kg/day to 10 mg/kg/day, depending upon the route of administration. In one embodiment, intravenous dosage range from about 1 mg/kg to about 30 mg/kg. In some embodiments, intravenous dosages range from at or about 1 mg/kg to at or about 14 mg/kg, such as from at or about 2 mg/kg to at or about 14 mg/kg, ql4d, once every 14 days. In other embodiments, subcutaneous dosages range from at or about 1 mg/kg to at or about 28 mg/kg, such as from at or about 2 mg/kg to at or about 28 mg/kg, ql4d, once every 14 days. In some embodiments, the effective amount ofdosage is administered once every 7 to 28 days. In one embodiment, the effective amount ofdosage is administered once every 7 days. In another embodiment, the effective amount ofdosage is administered once every 28 days.
[0132] The selected dosage regimen will dépend upon a variety offactors induding the activity of the MMP9 binding protein, the route of administration, the time of administration, the rate of excrétion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the âge, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
[0133] A clinician having ordinary skill in the art can readily détermine and prescribe the effective amount of the pharmaceuticai composition required. For example, the physicien or veterinarian can start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
[0134] In some cases, the methods of treatment include parentéral administration, e.g., intravenous, intra-arterial, intramuscular, or subcutaneous administration, or oral administration of the agent, e.g., anti-MMP9 antibody or composition containing the same.
[0135] As used herein, the term subject means a mammalian subject. Exempîary subjects include, but are not limited to humans, monkeys, dogs, cats, mice, rats, cows, horses, goats and sheep. In some embodiments, the subject has cancer, an inflammatory disease or condition, or an autoimmune disease or condition, and can be treated with the agent of the présent invention as described below.
[0136] If needed, for treatments, methods can further include additional thérapies, such as in the case of cancer, surgical removal of the cancer and/or administration of an anti-cancer agent or treatment in addition to an MMP9 binding protein. Administration of such an anticancer agent or treatment can be concurrent with administration of the compositions disclosed herein.
Methods of Détection of MMP9 [0137] The présent disclosure also contemplâtes methods ofdetecting MMP9 în a subject, e.g., to detect tumor or tumor-associated tissue expressing MMP9, or tissue or fluid or other biological sample associated with a disease as described herein, such as autoimmune or inflammatory disease. Thus, methods of diagnosing, monitoring, staging or detecting a tumor having MMP9 activity are provided.
[0138] Samples (e.g., test biological samples) from a subject (e.g., an individual suspected of having or known to hâve a tumor associated with MMP9 expression, or suspected of having or known to hâve another disease or condition), can be analyzed for MMP9 presence, absence, expression, and/or levels. For example, such samples can be collected and analyzed by detecting the presence or absence of binding of an MMP9 binding protein, such as an antibody or fragment as described herein, to substance (e.g., protein) in the sample. In some examples, the methods further include comparing the amount ofbinding detected to an amount of binding to a control sample, or comparing the detected level of MMP9 to a control level of MMP9. In some cases, the methods indicate the presence, absence, or severity of an MMP9-associated disease or condition, such as one described herein.
[0139] This analysis can be performed prior to the initiation oftreatment using an MMP9 binding protein as described herein, or can be done as part of monitoring of progress of cancer treatment. In some embodiments, provided are methods of treatment, carried out by performing the détection assays and initiating, altering, or discontinuing treatment of the subject, for example, based on the results of the diagnostic assay. Such diagnostic analysis can be performed using any sample, including but not limited to tissue, cells isolated from such tissues, and the like. In some cases, the methods are performed on liquid samples, such as blood, plasma, sérum, whole blood, saliva, urine, or semen. Tissue samples include, for example, formalin-fixed or frozen tissue sections.
[0140] Any suitable method for détection and analysis of MMP9 can be employed. Various diagnostic assay techniques known in the art can be adapted for such purpose, such as compétitive binding assays, direct or indirect sandwich assays and immunoprécipitation assays conducted in either heterogeneous or homogeneous phases.
[0141] MMP9 binding proteins for use in détection methods can be labeled with a détectable moiety. The détectable moiety directly or indirectly produces a détectable signal. For example, the détectable moiety can be any of those described herein such as, for example, a radioisotope, such as 3H, 14C, 32P, 35S, or 1251, a fluorescent or chemiluminescent compound, such as fluoresceîn isothiocyanate (FITC), Texas red, cyanin, photocyan, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, β-galactosidase or horseradish peroxidase.
[0142] Détection can be accomplished by contacting a sample under conditions suitable for MMP9 binding protein binding to MMP9, and assessing the presence (e.g., level) or absence of MMP9 binding protein-MMP9 complexes. A level of MMP9 in the sample in comparison with a level of a reference sample can indicate the presence of a tumor or tumorassocîated tissues having MMP9 activity. The reference sample can be a sample taken from the subject at an earlier time point or a sample from another individual.
[0143] Various aspects of the invention are further described and illustrated by way ofthe several examples which follow, none ofwhich are intended to limit the scope ofthe invention.
EXAMPLES
Example 1A: Préparation of antibodies to human MMP-9.
[0144] The full-length human MMP9 protein without a signal peptide (SEQ ID NO. 28) was used to îmmunize mice. Spleen cells from immunized mice were fused with myeloma cells to generate a hybridoma library. Monoclonal cultures were prepared and screened to identify cultures expressing an anti-MMP9 monoclonal antibody.
[0145] An antibody (AB0041 ) was purified from one ofthe cultures and characterized. This antibody contained an IgG2b heavy chain and a kappa light chain. Characterization included testing for the binding of AB0041 to other human MMPs and to MMP9 proteins from other species, including cynomolgus monkey, rat and mouse. As shown in Table 2, the AB0041 antibody had greater affinity to human and cynomolgus MMP9, that it had lower affinity to rat MMP9. In addition, the AB0041 antibody did not bind to murine MMP9 or to many human non-MMP matrix metalloproteinases.
Table 2: Cross reactivity of AB0041 and AB0045
MMP Tested | Dissociation constant (Kd) | |
AB0045 | AB0041 | |
Human MMPI | >100 nM | >100 nM |
Human MMP2 | >i00nM | >100 nM |
Mouse MMP2 | >100 nM | >100 nM |
Human MMP3 | >100 nM | >100 nM |
Human MMP7 | >100 nM | >100 nM |
Human MMP8 | >100 nM | >100 nM |
Human MMP9 | 0.168 ±0.117 nM | 0.133 ± 0.030 nM |
Cynomolgus monkey MMP9 | 0.082 ± 0.022 nM | 0.145 ± 0.16 nM |
Mouse MMP9 | >100 nM | >100 nM |
Rat MMP9 | 0.31 i± 0.017 nM | 0.332 ± 0,022 nM |
Human MMP 10 | >100 nM | >100 nM |
Human MMP 12 | >i00nM | >100 nM |
Human MMP 13 | >100 nM | >100 nM |
[0146] Additional characterization included assaying the binding of AB0041 to mutant mouse and human MMP9 proteins. Non-identical residues in the catalytic domain of mouse and human MMP9 proteins were identified, and forty-six non-identical amino acid residues were selected for mutagenesis. Most mutations were generated in mouse MMP9: the mouse amino acid residues were mutated to match those of human MMP9. Other mutations were generated in human MMP9: the human amino acid residues were mutated to match those of mouse MMP9. The mutated mouse or human MMP9 proteins were used in an EL1SA assay.
[0147] In the ELIS A assay, the AB0041 antibody was used as the primary antibody and a goat anti-mouse IgG antibody conjugated to horseradish peroxidase was used to detect the binding. The wild-type human MMP9 was used a positive control and the wild-type mouse MMP9 was used as a négative control. The results of the ELISA assay showed an arginine residue at position 162 ofthe MMP9 amino acid sequence (RI 62) as important for the MMP9 binding ofthe AB0041 antibody. The results also showed the amino acid residues El 11, DI 13, and 1198 were important forthe MMP9 binding ofthe AB0041 antibody. Based on the crysta! structure of MMP9, El 11, DI 13, R162, and 1198 are grouped near each other around a Ca2+ ion binding pocket ofMMP9. In this study, the AB0041 antibody was shown to specificaliy bind to an epitope containing amino acid residues within régions of MMP9 containing amino acid residues 104-119,159-166, and 191-202.
[0148] In an enzymatic assay for MMP9, the AB0041 antibody was found to act as a noncompetitive inhibitor of MMP9.
Example IB: Préparation of additional antibodies to human MMP-9, [0149] Additional hybridomas were generated, which produced antibodies having variable régions that shared identity with AB0041. One such hybridoma, designated M4, expressed an antibody containing the heavy chain (!gG2b) sequence:
[0150] MAVLVLFLCLVAFPSCVLSOVQLKESGPGLVAPSOSLSITCTVSGFSLLSY GVHWVRQPPGKGLEWLGV1WTGGSTNYNSALMSRLSISKDDSKSQVFLKMNSLQTD DTAMYYCARYYYAMDYWGQGTSVTVSS/tXTTPPSJTPZJPGCGDTTGSSf'TZGCLPX' GYFPESVTrTWNSGSLSSSVHTFPAUQSGLYTMSSSVTYPSSTWPSQTVTCSVAHPASSTTV DKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVWDV SEDDPDVR1SWFVNNVEVHTAQTQTHREDYNSTIRWSALPIQHQDWMSGKEFKCKVNNK DLPSPIERTISKIKGL VRAPQVYILPPPAEQLSRKD VSLTCL VVGFNPGDISVEiVTSNGHTEE NYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRHEGLKNYYLKKT1SRSPGK (SEQIDNO:30) [0151] (signal peptide set forth in underlined text, variable région set forth in plain text, and constant région set forth in italics), and the light chain (kappa) sequence:
[0152] MESQIOVFVFVFLWLSGVDGDIVMTOSHKFMFTSVGDRVSITCKASODVR NTVAWYQQKTGQSPKLLIYSASYRNTGVPDRFTGSISGTDFTFTISSVQAEDLALYYC G^WYSYYYTFGGGYKLENKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKW KIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN
RNEC (signal peptide set forth in underlined text, variable région set forth in plain text, and constant région set forth in italics) (SEQ ID NO: 31).
[01531 The M4 antibody had a variable heavy chain with an amino acid sequence:
[0154] QVOLKESGPGLVAPSOSLSITCTVSGFSLLSYGVHWVRQPPGKGLEWr.Gy rWTGGSTNYNSALMSRLSlSKDDSKSOVFLKMNSLOTDDTAMYYCARYYYAMDYW GQGTSVTVSS (CDRs 1,2, and 3 (SEQ ID NOs: 34,35, and 36, respectively) underlined) (SEQ ID NO: 32) [0155] and a variable light chain with the amino acid sequence [0156] DIVMTOSHKFMFTSVGDRVSITCKASODVRNTVAWYOQKTGQSPKLLÏYS 10 ASYRNTGVPDRFTGSISGTDFTFTISSVOAEDLALYYCOQHYSTPYTFGGGTKLEVK (CDRs 1,2, and 3 (SEQ ID NOs: 37,38, and 39, respectively) underlined) (SEQ ID NO: 33).
[0157] Another such hybridoma, designated Μ12, expressed only a kappa chain, having the sequence:
[0158] OVFVYMLLWLSGVDGDIVMTQSOKFMSTSVGDRVSVTCK A SQNVGTNV
AWYQQKPGQSPKALIYSASYRFSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQ YNSYPYTFGGGrKLElKRADAAPTTSIFPPSSEQLTSGGASWCFLNNFYPKDINVKlVKID GSERQNGPLNSWTDQDSKDSTYSMSSTETLTKDEYERHNSYTCEATHKTSTSPIVKSFNRN EC (signal peptide set forth in underlined text, variable région set forth in plain text, and constant région set forth in italics) (SEQ ID NO: 40).
[0159] The M12 antibody had a variable light chain with the amino acid sequence (0160] DIVMTOSOKFMSTSVGDRVSVTCKASONVGTNVAWYQOKPGQSPKALIY SASYRFSGVPDRFTGSGSGTDFTLTISNVOSEDLAEYFCOOYNSYPYTFGGGTKLEIK (CDRs 1,2, and 3 (SEQ ID NOs: 42,43, and 44, respectively) underlined) (SEQ ID NO: 41).
(0161 ] A sequence comparison, showing différences between the M4 and M12 heavy and 25 light chains as compared with AB0041 antibody is shown in Figure 4.
[0162] An enzymatic assay was carried out. The results demonstrated that the antibodies produced by the M4 and Ml 2 hybridomas acted as non-competitive inhibitors of MMP9 (data not shown).
Example IC: Préparation of antibodies to mouse MMP-9.
[0163] Another mouse antibody, AB0046, was generated. Using a process similar to that described in Example 1 A, the MMP9-knockout mice (strain B6.FVB (Cg)- Afmp9/7V7J) was immunized using targeted domains of the pro/catalytîc domain fragment of murine MMP9. The AB0046 antibody had a kappa light chain with an amino acid sequence
MSSAOFLGLLLLCFQGTRCDIOMTOTTSSLSASLGDRVTISCSASQG1SNYLNWYQQK PDGTFKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYGWLPRTF GGG1KLE\KRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWK1DGSERQNGV LNSIVTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 45) (signal peptide set forth in underlined text, variable région set forth in plain text, and constant région set forth in italics) and an IgGI heavy chain with an amino acid sequence MGWSSIILFLVATATGVHSOVQLQOPGSVLVRPGASVKLSCTASGYTFTSYWMNWV KQRPGQGLEWIGEIYPISGRTNYNEKFKVKATLTVDTSSSTAYMDLNSLTSEDSAVY VCARSRAWWOYWGQGTrLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPE PVTVTWNSGSLSSGVHTFPA VLQSDL YTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIV PRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCWVDISKDDPEVQFSWFVDDV EVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEICnSKTKGRP KAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQIVNGQPAENYKNTQPIMDTDG SYFVYSKLNVQKSNIVEAGNTFTCSVUfEGLHNHHTEKSLSHSPGK(SEQ IDNO: 46) (signal peptide set forth in underlined text, variable région set forth in plain text, and constant région set forth in italics).
[0164] The following amino acid sequence comprises the framework régions and complementarity-determining régions (CDRs) of the variable région of the IgGI heavy chain of AB0046 (with CDRs underlined):
[0165[ OVOLOOPGSVLVRPGASVKLSCTASGYTFTSYWMNWVKQRPGOGLEWÏ GEIYPISGRTNYNEKFKVKATLTVDTSSSTAYMDLNSLTSEDSAVYYCARSRANWDD YWGQGTTLTVSS (SEQ ID No: 47).
[0166] The following amino acid sequence comprises the framework régions and complementarity-determining régions (CDRs) of the variable région of the kappa light chain of AB0046 (with CDRs underlined):
[0167] DIOMTOTrSSLSASLGDRVTISCSASQGISNYLNWYOOKPDGTFKLLIYYT SILHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCOOYGWLPRTFGGGTKLEIK (SEQ ID No: 48) [0168] Additional characterizations showed that the AB0046 antibody bound to mouse MMP9 non-competitively or its binding was not dépendant on the concentration of mouse MMP9. The AB0046 antibody did not bind to human MMP9 or MMP2, mouse MMP2,3,7, 8, or 12. Using epitope analysis as described in Example ! A, it was shown that the proline residue at position 162 of the mouse MMP9 amino acid sequence (P 162) (corresponding to RI62 of human MMP9) was important for the MMP9 binding of the AB0046 antibody. The results suggested that the AB0046 antibody specifically bound to an epitope containing a residue within a portion of mouse MMP9 corresponding to the portion containing amino acids 159-166 ofhuman MMP9. Thus, the AB0046 antibody was an inhibitory antibody spécifie to mouse MMP9 and had similar kinetics of binding and inhibition as those of AB0041. Because AB0046 îs spécifie to mouse MMP9 and binds to an epitope as AB0041/AB0045, AB0046 is suitable for assays which uses either AB0041 or AB0045.
[0169] Further characterization showed that the AB0046 antibody was a murine IgG 1 isotype, having a limited effector function in mouse.
[0170] Three other mouse anti-MMP9 antibodies were generated using similar methods, which were non-inhibîtory and for which PI 62 was important for binding.
Example 2: Humanlzatlon of antibodies to human MMP9 [0171] The amino acid sequences ofthe heavy chain and light chain ofthe mouse AB0041 antibody were altered at certain locations in the framework (i.e., non-CDR) portion of their variable régions to generate proteins that are less immunogenic in humans. These amino acid sequence changes were shown in Figures 1 and 2. The cross-reactivity of one humanized antibody, referred to as AB0045, is shown in Table 2A above.
[0172] The humanized variant anti-MMP9 antibody, AB0045 (humanized, modified lgG4 (S241P); see Example 2, above) contained the humanized AB0041 heavy chain variant VH3 (having the sequence set forth in SEQ ID NO: 7 [0173] (QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWLG VIWTGGTTNYNSALMSRFT1SKDDSKNTVYLKMNSLKTEDTAIYYCARYYYGMDY WGQGTLVTVSS) [0174] and the humanized AB0041 light chain variant VH4 (having the light chain sequence set forth in Vk4 (having the sequence set forth in SEQ ID NO: 12 [0175] (D1QMTQSPSSLSASVGDRVT1TCKASQDVRNTVAWYQQKPGKAPKLL1YS SSYRNTGVPDRFSGSGSGTDFTLT1SSLQAEDVAVYYCQQHY1TPYTFGGGTKVE1K)).
[0176] The heavy chain of the AB0045 antibody has the sequence set forth in SEQ ID NO: 49 (MGWSLILLFLVAVATRVHSQVQLOESGPGLVKPSETLSLTCTVSGFSLLSYGVHWV RQPPGKGLEWLGV1WTGGTTNYNSALMSRFT1SKDDSKNTVYLKMNSLKTEDTAIY VCARVYVGMDYWGQGTLVrVSSASTKGPSVFPLAPCSRSTSESTAALGCLFKDYFPEPy TVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV
DGVEVHNAKTKPREEQFNSTYRW'SVLTVUIQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAWNHYTQKSLSLSLGK (signal sequence underlined; sequence of the constant région presented italics)); the light chain of the AB0045 antibody has the sequence set forth in SEQ ID NO: 50 (MRVPAQLLGLLLLWLPGARCDIOMTQSPSSLSASVGDRVTITCKASODVRNTVAWY QQKPGKAPKLLIYSSSYRNTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYIT PYTEGGGYYLVElYiRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQlVKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (signal sequence underlined; sequence ofthe constant région presented italics)). The antibody contains 1312 amino acids in length, is composed of two heavy chains and two light chains, and has a theoretical pi of about 7.90, extinction coefficient of about 1.50 AU/cm at 280 nm for 1 g/L, a molecular weight of about 144 kDa, and density of about 1 g/mL in formulation buffer (50-100 mg/mL product concentration).
[0177[ Further characterization of this antibody is described in Example 3, below.
Example 3: Characterization of variant MMP9 antibody AB0045 and comparlson to AB0041 and AB0046 [0178] As described above, AB0045 and AB0041 antibodies are non-competitive inhibitors of MMP9. Thus, both antibodies inhibit MMP9 enzymatic activity independently of substrate concentration. The AB0045 antibody binds to the same MMP9 epitope as the AB0041 antibody with an affinity in the 1 x 10-12 molar range, as shown by direct binding and surface plasmon résonance (SPR) assays. Both antibodies are spécifie for MMP9, with no significant non-specific binding observed against other purified protein targets including purified domains and full length forms of MMP enzymes. Both AB0045 and AB0041 antibodies are cross-reactive with native and recombinant human and recombinant rat and cynomolgus monkey MMP9.
[0179] The in vitro binding affinity, inhibition characteristics, and the specificity of the antibodies of AB0045, AB0041 and AB0046 for MMP9 of human and non-human origin were determined using Enzyme-Linked Immunosorbent Assay (ELISA) and an MMP9 enzymatic assay. SPR analysis was also used to generate dissociation constants (Kj) of AB0045 and AB0041.
[0180] In the ELISA assay, the Kd value of AB0045 and AB004I antibodies for human, cynomolgus monkey, and rat MMP9 derived from ELISA were ail found to be <400 pM.
The ELISA data illustrated that both AB0045 and AB0041 antibodies cross-react with MMP9 from ail the relevant toxicology species tested. The AB0046 antibody was shown to be spécifie to mouse MMP9 and therefore could be used as a surrogate antibody in mouse efficacy models. The results showed that the Kj value of the AB0045 antibodies for human
MMP9 was 0.168 ± 0.117 nM and the and 1Q value of the AB0041 antibody was 0.133 ±
0.030 nM. The results on the AB0046 antibodies showed it bound to mouse MMP9 with the Kj value of 0.218 ± 0.097 nM. In the SPR analysis, the results showed that the Kj values of AB0045 and AB004I antibodies for human MMP9 were 8.8 pM and 0.4 pM, respectively.
[0181] The enzymatic inhibitory activities of AB0045, AB0041, and AB0046 antibodies were evaluated in an assay assessing MMP9-mediated cleavage of a fluorogenic peptide substrate Mca-PLGL-Dpa-AR-NH2. Ali three antibodies inhibited MMP9 enzyme activity. The IC50 values of AB0045 (0.691 ± 0.097 nM) and AB0041 (0.569 ± 0.185 nM) for human MMP9 were not statistically different. The ICjo value for the AB0046 inhibition of mouse MMP9 was 0.352 ± 0.03 nM. The value was not adjusted for the concentration of active enzyme that was generated during the préparation. Additional MMP9 enzymatic assay under steady-state conditions was used to détermine IC50 and mode of inhibition. In this assay, the IC50 values of AB0045 ranged from 0.148 nM to 0.I6I nM in a 20-fold range of substrate concentration, and in one exampte is 0.158 nmThe results showed that the MMP9 inhibitory activity of AB0045 was non-competitive.
Table 2B: Binding and Inhibitory Properties of AB0045, AB0041, and surrogate mouse antibody AB0046
AB0045 | AB0041 | AB0046 | |
EL | SA | ||
Human MMP9 Dissociation constant | 0.168 ±0.117 nM | 0.133 ± 0.030 nM | >100 nM |
Cynomolgus monkey MMP9 Dissociation constant | 0.082 ± 0.022 nM | O.i45 ±0.i6nM | >100 nM |
Mouse MMP9 Dissociation constant | >100nM | >100 nM | 0.2i8±0.097nM |
RatMMP9 Dissociation constant | 0.3 ii ±0.017 nM | 0.332 ± 0.022 nM | >100 nM |
SPR | |||
Human MMP9 Dissociation constant | 8.8pM | 0.4pM | ND |
Activity Assay | |||
Human MMP9 ICso | 0.691 ± 0.097 nM | 0.569 ±0.185 nM | >i00nM |
Cynomolgus monkey | 0.194 ±0.048 nM* | 0.189 ± 0.019 nM* | >100 nM |
MMP9 ICso | |||
Rat MMP9 ICso | 8.23 ± 1.24 nM* | 2.78±l.l7nM* | >100 nM |
Mouse MMP9 ICso | >100 nM | >100 nM | 0.352±0.03 nM* |
[0182] The results confirmed that AB0045 and AB0041 hâve équivalent binding and inhibitory properties and that AB0046 can serve as a relevant mouse surrogate antibody, for example, in mouse models of human disease.
Claims (17)
1. An isolated antibody or fragment thereof, that binds to matrix Metalloproteinase 9, comprising: a heavy chain variable (VH) région having a heavy chain complementary determining région (CDR) with an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.
2. The antibody or fragment of claim 1, wherein the VH région has a heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 14, and a heavy chain CDR3 with the amino acid sequence of SEQ IDNO: 15.
3. An isolated antibody or fragment thereof, comprising: a light chain variable (VL) région having a light chain complementary determining région (CDR) with an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ IDNO: 18.
4. The antibody or fragment of claim 3, wherein the VL région has a light chain CDR1 with the amino acid sequence of SEQ ID NO: 16, a light chain CDR2 with the amino acid sequence of SEQ ID NO: 17, and a light chain CDR3 with the amino acid sequence of SEQ IDNO: 18.
5. The antibody or fragment of claim 3 or claim 4, wherein the antibody further comprises a VH région having a heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 14, and a heavy chain CDR3 with the amino acid sequence of SEQ ID NO: 15.
6. The antibody or fragment of any of daims 1,2, and 5, wherein the VH région has the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
7. The antibody or fragment of any of claims 3-6 wherein the VL région has the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: II, or SEQ IDNO: 12.
5
8. The antibody or fragment of claim 5, wherein the VH région has the amino acid sequence set forth in SEQ ID NO: 7 and the VL région has the amino acid sequence set forthinSEQ IDNO: 12.
9. An isolated antibody or fragment thereof that competes for binding to MMP9
10 with an antibody having a VH région with the amino acid sequence set forth în SEQ ID NO:
7 and a VL région with the amino acid sequence set forth in SEQ ID NO: 12.
10. An isolated antibody or fragment thereof that specifically binds to an epitope of MMP9, wherein the epitope comprises an amino acid residue within a région of MMP9,
15 the région consisting of residues 104-119, residues 159-166, or residues 191-202 ofSEQ ID NO: 27or wherein the epitopecomprises ElII, DI 13,R162, or 1198 ofSEQ IDNO: 27.
11. The antibody or fragment of any of claims 1-10, wherein the antibody or fragment inhibits the enzymatic activity of MMP9, and/or wherein the antibody or fragment
20 inhibits the enzymatic activity of MMP9 and the enzymatic activity is non-competiti ve, and/or which is human or humanized.
12. An isolated nucleic acid, comprising: a nucléotide sequence encoding a heavy chain polypeptide comprising CDRs with the amino acid sequences set forth in SEQ ID NOs:
25 13-15 and/or 1,3 and 5-8 or a light chain polypeptide comprising CDRs with the amino acid sequences set forth in SEQ ID NOs: 16-18 and/or 2,4 and 9-12.
13. The isolated nucleic acid of claim 12, wherein the nucléotide sequence comprises a sequence selected from the group consisting ofSEQ ID NOs: 19-26.
14. A pharmaceutical composition, comprising the antibody or fragment thereof of any of claims 1-11.
15. A method of detecting MMP9 expression in a test sample from a subject, the method comprising:
(a) contacting the test sample with an antibody or fragment of any of daims I -11 ; and
5 (b) detecting binding ofthe antibody or fragment to protein in the sample, thereby detecting the presence of MMP9.
16. An isolated polypeptide, having an amino acid sequence consisting essentialiy of residues 111-198 of SEQ ID NO: 27.
17. An isolated mutant MMP9 polypeptide, comprising an amino acid sequence containing residues 111-198 of SEQ ID NO: 27 with an amino acid substitution at residue 111,113,162, or 198.
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