# Patent

## Title
Anti-ανβ6 antibodies

## Abstract

Monoclonal antibodies that specifically bind to M.96. Also included are methods of using these antibodies to treat mammals having or at risk of having 006-mediated diseases, or to diagnose % Qmediated diseases.

## Background
CROSS-REFERENCE TO RELATED APPLICATION(S)
This application is a continuation of U.S. application Ser. No. 13/443,261, filed Apr. 10, 2012, now abandoned, which is a continuation of U.S. application Ser. No. 12/260,510, filed Oct. 29, 2008, issued as U.S. Pat. No. 8,153,126, which is a divisional of application Ser. No. 10/507,662, filed May 20, 2005, issued as U.S. Pat. No. 7,465,449, which is a national phase filing under 35 U.S.C. §371 of international application number PCT/US2003/008048, filed Mar. 13, 2003, which claims priority from U.S. provisional application Nos. 60/426,286, filed Nov. 13, 2002 and 60/364,991, filed Mar. 13, 2002. The entire content of each of the prior applications referenced above are incorporated herein by reference in their entirety.


FIELD OF THE INVENTION
This invention relates generally to the field of molecular biology and specifically to antibodies to αvβ6 integrins.
BACKGROUND OF THE INVENTION
Integrins are a superfamily of cell surface receptors that mediate cell-cell and cell-matrix adhesion. These proteins are known to provide anchorage as well as signals for cellular growth, migration and differentiation during development and tissue repair. Integrins have also been implicated in cell dedifferentiation and invasion, notably where cells lose their specialized form and become metastasizing cancer cells.
Integrins are heterodimeric proteins composed of two noncovalently linked subunits, α and β. The binding specificity of integrins is dictated by the combination of some 18 different α chains with some 8 different β chains. The αvβ6 integrin can bind to a number of ligands including fibronectin, tenascin, vitronectin, and the recently identified latency associated peptide “LAP,” a 278 amino acid peptide synthesized as part of the precursor TGF-β protein (Munger et al., Cell 96(3):319-328 (1999)). LAP is cleaved from the mature form of TGF-β as an N-terminal peptide during secretion but remains noncovalently associated with TGF-β to maintain its latent state. This complex cannot bind to the TGF-β receptor and hence is not biologically active. The αvβ6 integrin can bind directly to an RGD motif contained within LAP, resulting in release of LAP and activation of TGF-β. Since αvβ6's binding to LAP may be important in the conversion of TGF-β to its active state, blocking the binding may result in inhibition of αvβ6-mediated activation of TGF-β and the associated fibrotic pathology.
SUMMARY OF THE INVENTION
This invention is based on the discovery and characterization of high affinity antibodies against αvβ6, including the identification and analysis of key amino acid residues in the complementary determining regions (CDRs) of such antibodies.
This invention embraces a monoclonal antibody that (a) specifically binds to αvβ6; (b) inhibits the binding of αvβ6 to its ligand such as LAP, fibronectin, vitronectin, and tenascin with an IC50 value lower than that of 10D5 (International Patent Application Publication WO 99/07405); (c) blocks activation of TGF-β; (d) contains certain amino acid sequences in the CDRs (e.g., those shown in FIGS. 7A and 7B) that provide binding specificity to αvβ6; (e) specifically binds to the β6 subunit; and/or (f) recognizes αvβ6 in immunostaining procedures, such as immunostaining of paraffin-embedded tissues.
It has been discovered that antibodies that bind to αvβ6 can be grouped into biophysically distinct classes and subclasses. One class of antibodies exhibits the ability to block binding of a ligand (e.g., LAP) to αvβ6 (blockers). This class of antibodies can be further divided into subclasses of cation-dependent blockers and cation-independent blockers. Some of the cation-dependent blockers contain an arginine-glycine-aspartate (RGD) peptide sequence, whereas the cation-independent blockers do not contain an RGD sequence. Another class of antibodies exhibits the ability to bind to αvβ6 and yet does not block binding of αvβ6 to a ligand (nonblockers).
Accordingly, in some embodiments of this invention, some antibodies of this invention are divalent cation-dependent for binding to αvβ6, while others are divalent cation-independent. Exemplary cations are Ca2+, Mg2+ and Mn2+.
In some embodiments, the antibody comprises the same heavy and light chain polypeptide sequences as an antibody produced by hybridoma 6.1A8, 6.3G9, 6.8G6, 6.2B1, 6.2B10, 6.2A1, 6.2E5, 7.1G10, 7.7G5, or 7.1C5.
In some embodiments, the antibodies comprise a heavy chain whose complementarity determining regions (CDR) 1, 2 and 3 consist essentially (i.e., with the exception of some conservative variations) of the sequences of SEQ ID Nos:1, 4 and 7, respectively, and/or a light chain whose CDRs 1, 2 and 3 consist essentially of the sequences of SEQ ID NOs:10, 13 and 15, respectively.
In some embodiments, the antibodies comprise a heavy chain whose CDRs 1, 2 and 3 consist essentially of the sequences of SEQ ID NOs:3, 5 and 8, respectively, and/or a light chain whose CDRs 1, 2 and 3 consist essentially of the sequences of SEQ ID NOs:11, 14 and 17, respectively.
In some embodiments, the antibodies comprise a heavy chain whose CDRs 1, 2 and 3 consist essentially of the sequences of SEQ ID NOs:3, 6 and 9, respectively, and/or a light chain whose CDRs 1, 2 and 3 consist essentially of the sequences of SEQ ID NOs:12, 14 and 18, respectively.
In some embodiments, the antibodies comprise a heavy chain whose CDRs 1, 2 and 3 consist essentially of the sequences of SEQ ID NOs:2, 46 and 47, respectively, and/or a light chain whose CDRs 1, 2 and 3 consist essentially of the sequences of SEQ ID NOs:48, 13 and 16, respectively.
In some embodiments, the antibodies comprise a heavy chain whose CDRs 1, 2 and 3 consist essentially of the sequences of SEQ ID NOs:49, 51 and 53, respectively, and/or a light chain whose CDRs 1, 2 and 3 consist essentially of the sequences of SEQ ID NOs:55, 57 and 59, respectively.
In some embodiments, the antibodies comprise a heavy chain whose CDRs 1, 2 and 3 consist essentially of the sequences of SEQ ID NOs:50, 52 and 54, respectively, and/or a light chain whose CDRs 1, 2 and 3 consist essentially of the sequences of SEQ ID NOs:56, 58 and 60, respectively.
In some embodiments, the antibodies comprise a heavy chain variable domain sequence of any one of SEQ ID NOs:19-36 and 61-62.
In some embodiments, the antibodies comprise heavy and light chain variable domain sequences of
(1) SEQ ID NOs:19 and 37;
(2) SEQ ID NO:20 or 21, and SEQ ID NO:38;
(3) SEQ ID NOs:22 and 43;
(4) SEQ ID NOs:23 and 44;
(5) SEQ ID NOs:24 and 45;
(6) SEQ ID NO:25 or 26 and SEQ ID NO:42;
(7) SEQ ID NO:27, 28, or 29, and SEQ ID NO:39;
(8) SEQ ID NO:34 or 35, and SEQ ID NO:40;
(9) SEQ ID NOs:36 and 41;
(10) SEQ ID NOs:61 and 63; or
(11) SEQ ID NOs:62 and 64,
respectively.
In some embodiments, the antibodies specifically binds to αvβ6 but does not inhibit the binding of αvβ6 to latency associated peptide (LAP). At least some of these antibodies are capable of binding to αvβ6 in paraffin-embedded tissue sections and therefore can be used for diagnostic purposes. Exemplary antibodies include 6.2A1 and 6.2E5.
This invention also embraces antibodies that bind to the same epitope as any of the above-described antibodies.
This invention also embraces compositions comprising one or more antibodies of this invention, and a pharmaceutically acceptable carrier. In some of these compositions, the antibodies are conjugated to a cytotoxic agent (i.e., an agent that impairs the viability and/or the functions of a cell) such as a toxin or a radionuclide. The antibodies in these compositions can be cation-dependent antibodies. The compositions can be administered to a subject (e.g., a mammal such as a human) having or at risk of having a disease mediated by αvβ6, so as to treat (e.g., alleviating, mitigating, reducing, preventing, postponing the onset of) the disease. Examples of such diseases include, but are not limited to: fibrosis (e.g., scleroderma, scarring, liver fibrosis, lung fibrosis, and kidney fibrosis); psoriasis; cancer (e.g., epithelial cancer; oral, skin, cervical, ovarian, pharyngeal, laryngeal, esophageal, lung, breast, kidney, or colorectal cancer); Alport's Syndrome; acute and chronic injuries of the lung, liver, kidney and other internal organs; and sclerosis of the lung, liver, kidney and other internal organs. Risks of having such diseases may result from genetic predisposition; certain lifestyles such as smoking and alcoholism; exposure to environmental pollutants such as asbestos; physiological conditions such as diabetes, hepatitis viral infection (e.g., hepatitis C viral infection), autoimmune diseases; and medical treatments such as radiation therapy.
This invention also embraces methods of detecting αvβ6 in a tissue sample from a mammal (e.g., a human), comprising contacting the tissue sample with the antibody of the invention, such as 6.2A1 and 6.2E5.
This invention also embraces cells of hybridomas 6.1A8, 6.2B10, 6.3G9, 6.8G6, 6.2B1, 6.2A1, 6.2E5, 7.1G10, 7.7G5, and 7.1C5; isolated nucleic acids comprising a coding sequence for any one of SEQ ID NOs:19-45 and 61-64; isolated polypeptides comprising an amino acid sequence of any one of SEQ ID NOs:19-45 and 61-64.
An antibody of this invention refers to a full antibody, e.g., an antibody comprising two heavy chains and two light chains, or to an antigen-binding fragment of a full antibody such as a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment or a F(v) fragment. An antibody of this invention can be a murine antibody or a homolog thereof, or a fully human antibody. An antibody of this invention can also be a humanized antibody, a chimeric antibody or a single-chained antibody. An antibody of this invention can be of any isotype and subtype, for example, IgA (e.g., IgA1 and IgA2), IgG (e.g., IgG1, IgG2, IgG3 and IgG4), IgE, IgD, IgM, wherein the light chains of the immunoglobulin may be of type kappa or lambda.
In some embodiments, the antibody of the invention may comprise a mutation (e.g., deletion, substitution or addition) at one or more (e.g., 2, 3, 4, 5, or 6) of certain positions in the heavy chain such that the effector function of the antibody (e.g., the ability of the antibody to bind to a Fc receptor or a complement factor) is altered without affecting the antibody's antigen-binding ability. In other embodiments, the antibody of this invention may contain a mutation at an amino acid residue that is a site for glycosylation such that the glycosylation site is eliminated. Such an antibody may have clinically beneficial, reduced effector functions or other undesired functions while retaining its antigen-binding affinity. Mutation of a glycosylation site can also be beneficial for process development (e.g., protein expression and purification). In still other embodiments, the heavy or light chains can contain mutations that increase affinity or potency.
Several of the Fusion #6 and Fusion #7 hybridomas were deposited at the American Type Culture Collection (“ATCC”; P.O. Box 1549, Manassas, Va. 20108, USA) under the Budapest Treaty. Hybridoma clones 6.1A8, 6.2B10, 6.3G9, 6.8G6, and 6.2B1 were deposited on Aug. 16, 2001, and have accession numbers ATCC PTA-3647, -3648, -3649, -3645, and -3646, respectively. Hybridoma clones 6.2A1, 6.2E5, 7.1G10, 7.7G5, and 7.1C5 were deposited on Dec. 5, 2001, and have accession numbers ATCC PTA-3896, -3897, -3898, -3899, and -3900, respectively. See Table 1, infra.
The antibodies of the invention are useful for treating any clinically undesirable condition or disease (as discussed herein) that is mediated by binding of αvβ6 to its ligand, such as LAP and fibronectin. These antibodies can be more potent, via higher affinity or avidity, and cation dependency or independency of binding to ligand, than previously known αvβ6 antibodies.
In addition to therapeutic applications of the antibodies of the invention, especially the blockers, the nonblocker class of antibodies can be used for diagnostic purposes, such as in antigen capture assays, enzyme-linked immunosorbent assays (ELISAs), immunohistochemistry, and the like.
Other features and advantages of the invention will be apparent from the following detailed description, drawings, and claims.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B are bar graphs showing the results of a cell capture assay that determined the ability of various anti-αvβ6 monoclonal antibodies (“mAb”) to bind β6-transfected FDC-P1 cells (untransfected cells as control).
FIG. 2A is a graph showing the results of ELISA assays that determined the ability of various purified anti-αvβ6 “Fusion 6” monoclonal antibodies to bind soluble recombinant human αvβ6 (“hsαvβ6”). These antibodies were generated by immunizing β6−/− mice with soluble human truncated αvβ6. The numbers in the legend indicate the clone numbers. For the corresponding clone names, see Table 2.
FIG. 2B is a graph showing the results of ELISA assays that determined the ability of various purified anti-αvβ6 “Fusion 7” monoclonal antibodies to bind soluble recombinant hsαvβ6. These antibodies were generated by immunizing β6−/− mice with β6-transfected NIH 3T3 cells (Fusion #7).
FIGS. 3A-F are graphs showing the differential cation dependence of the binding of various anti-αvβ6 monoclonal antibodies to hsαvβ6.
FIGS. 4A and 4B are graphs showing that Fusion #6 and Fusion #7 monoclonal antibodies, respectively, inhibit the binding of biotin-hsαvβ6 to LAP.
FIGS. 5A-E are graphs showing that exemplary monoclonal antibodies of the invention inhibit the binding of β6-transfected FDC-P1 cells to LAP. FIGS. 5A and 5B display the results from Fusion #6 antibodies. FIGS. 5C-E display the results from Fusion #7 antibodies.
FIGS. 6A and 6B are graphs showing that Fusion #6 and Fusion #7 antibodies, respectively, inhibit the αvβ6-mediated activation of TGF-β, using a PAI-1 luciferase reporter gene assay to monitor TGF-β activation.
FIG. 7A depicts the amino acid sequences of the variable domains of the heavy chains of αvβ6 monoclonal antibodies 6.1A8, 6.8G6 (subclones A and B), 7.7G5, 6.2B1, 6.3G9, 6.2B10 (subclones A and B), 6.2G2, 6.2A1, 6.4B4 (subclones A, B and C), 7.10H2, 7.9H5, 7.4A3 (subclones A and B), 7.1C5 (subclones A and B) and 7.1G10. Antibodies 6.1A8, 6.8G6 and 7.7G5 are cation-dependent in binding to αvβ6, while antibodies 6.2B1, 6.2A1, 6.3G9, 6.2B10, 6.4B4, 7.1C5 and 7.1G10 are cation-independent (infra). The numbers in parentheses denote amino acid residue positions. The CDRs are in the large boxes, while the small boxes containing italicized amino acids represent polymorphism in different clones of a particular antibody.
FIG. 7B depicts the amino acid sequences of the variable domains of the light chains of αvβ6 monoclonal antibodies 6.1A8, 6.8G6, 6.4B4, 6.2A1, 7.1C5, 7.1G10, 6.2B10, 7.7G5, 6.2B1 and 6.3G9.
FIG. 8 is a scatter plot showing the expression of αvβ6 in human breast cancer and human squamous carcinoma tissue sections. Normal human tissues show only negligible expression levels of αvβ6.
FIGS. 9A and 9B are quadratic curve graphs depicting the solution binding affinities of two anti-αvβ6 antibodies, 6.8G6 and 6.3G9, respectively, for soluble αvβ6.
FIGS. 10A and 10B are bar graphs demonstrating the ability of purified monoclonal antibodies to compete with biotinylated 6.3G9 and biotinylated 6.8G6, respectively, for binding to αvβ6.
FIG. 11 is a bar graph showing percent smooth actin staining in kidneys from UUO animals treated with anti-αvβ6 mAb treatment.
FIG. 12 shows αvβ6 expression on tumor cell lines by FACS analysis (right side of figure) and inhibition of tumor cell lines binding to the LAP ligand by mAbs 6.3G9 and 6.4B4 (left side of figure).
FIG. 13 is a bar graph demonstrating inhibition of three tumor cell lines binding to the LAP ligand by anti-αvβ6 mAbs 6.3G9, 6.8G6 and 6.4B4. The mAb binding was compared to total binding without the addition of test mAbs (TB) and nonspecific binding to BSA control alone (NSB).
FIGS. 14A and 14B are graphs showing the effects of anti-αvβ6 mAb 6.3G9 and 6.4B4, respectively, over a 33 day study period on tumors arising from subcutaneously implanted Detroit 562 cells.
FIGS. 15A-C are graphs showing the effects of anti-αvβ6 mAb on bleomycin-induced lung fibrosis. (A) Antibody treatment using 6.3G9 mAb was started on day 0 at the time of bleomycin administration and was monitored over a 30 day period; (B) Antibody treatment using 6.3G9 mAb was started 15 days after bleomycin treatment and was monitored over a 30 day period; (C) Antibody treatment using 6.3G9, 6.8G6 and 6.4B4 mAbs was started 15 days after bleomycin treatment and was monitored over an extended 60 day period. In both FIGS. 15A and 15B, the bar graphs on the left represent μg hydroxyproline/lung while the bar graphs on right show percent increase in hydroxyproline above saline treated mice (no bleomycin). In FIG. 15C, the graph shows hydroxyproline content per lung.



DETAILED DESCRIPTION OF THE INVENTION
This invention features classes and subclasses of antibodies that are specific for the integrin αvβ6. At least one class of the antibodies (blockers) are capable of blocking the binding of αvβ6 to LAP or preventing the activation of TGF-β.
The following describes the various methods of making the antibodies of this invention. Methods that are known in the art but not specifically described herein are also within the scope of this invention. For instance, antibodies of this invention can also be identified using phage-displayed antibody libraries, such as those described in Smith, Science 228:1315-7 (1985); U.S. Pat. Nos. 5,565,332, 5,733,743, 6,291,650, and 6,303,313. Additional antibodies of this invention can be made by coupling the heavy chains identified herein with a noncognate light chain, e.g., a light chain identified by phage display technology.
Non-Human Hybridoma Antibodies
The monoclonal antibodies of this invention can be generated by well known hybridoma technology. To do so, β6−/− animals (e.g., mice, rats or rabbits) are immunized with purified or crude αvβ6 preparations, cells transfected with cDNA constructs encoding αv, β6 or both antigens, cells that constitutively express αvβ6, and the like. The antigen can be delivered as purified protein, protein expressed on cells, protein fragment or peptide thereof, or as naked DNA or viral vectors encoding the protein, protein fragment, or peptide. Sera of the immunized animals are then tested for the presence of anti-αvβ6 antibodies. B cells are isolated from animals that test positive, and hybridomas are made with these B cells.
Antibodies secreted by the hybridomas are screened for their ability to bind specifically to αvβ6 (e.g., binding to β6-transfected cells and not to untransfected parent cells) and for any other desired features, e.g., having the desired CDR consensus sequences, inhibiting (or not inhibiting in the case of nonblockers) the binding between LAP and αvβ6 with an IC50 value lower than that of known anti-αvβ6 antibody 10D5, or inhibiting TGF-β activation.
Hybridoma cells that test positive in the screening assays are cultured in a nutrient medium under conditions that allow the cells to secrete the monoclonal antibodies into the culture medium. The conditioned hybridoma culture supernatant is then collected and antibodies contained in the supernatant are purified. Alternatively, the desired antibody may be produced by injecting the hybridoma cells into the peritoneal cavity of an unimmunized animal (e.g., a mouse). The hybridoma cells proliferate in the peritoneal cavity, secreting the antibody which accumulates as ascites fluid. The antibody may then be harvested by withdrawing the ascites fluid from the peritoneal cavity with a syringe.
The monoclonal antibodies can also be generated by isolating the antibody-coding cDNAs from the desired hybridomas, transfecting mammalian host cells (e.g., CHO or NSO cells) with the cDNAs, culturing the transfected host cells, and recovering the antibody from the culture medium.
Chimeric Antibodies
The monoclonal antibodies of this invention can also be generated by engineering a cognate hybridoma (e.g., murine, rat or rabbit) antibody. For instance, a cognate antibody can be altered by recombinant DNA technology such that part or all of the hinge and/or constant regions of the heavy and/or light chains are replaced with the corresponding components of an antibody from another species (e.g., human). Generally, the variable domains of the engineered antibody remain identical or substantially so to the variable domains of the cognate antibody. Such an engineered antibody is called a chimeric antibody and is less antigenic than the cognate antibody when administered to an individual of the species from which the hinge and/or constant region is derived (e.g., a human). Methods of making chimeric antibodies are well known in the art.
The chimeric antibodies embraced in this invention may contain a heavy chain variable domain having a sequence identical (or substantially so) to any one of SEQ ID NOs:19-36 and/or a light chain variable domain having a sequence identical (or substantially so) to any one of SEQ ID NOs:37-45.
Preferred human constant regions include those derived from IgG1 and IgG4.
Fully Human Antibodies
The monoclonal antibodies of this invention also include fully human antibodies. They may be prepared using in vitro-primed human splenocytes, as described by Boerner et al., J. Immunol. 147:86-95 (1991), or using phage-displayed antibody libraries, as described in, e.g., U.S. Pat. No. 6,300,064.
Some other methods for producing fully human antibodies involve the use of non-human animals that have inactivated endogenous Ig loci and are transgenic for un-rearranged human antibody heavy chain and light chain genes. Such transgenic animals can be immunized with αvβ6 and hybridomas are then made from B cells derived therefrom. These methods are described in, e.g., the various GenPharm/Medarex (Palo Alto, Calif.) publications/patents concerning transgenic mice containing human Ig miniloci (e.g., Lonberg U.S. Pat. No. 5,789,650); the various Abgenix (Fremont, Calif.) publications/patents with respect to XENOMICE (e.g., Kucherlapati U.S. Pat. Nos. 6,075,181, 6,150,584 and 6,162,963; Green et al., Nature Genetics 7:13-21 (1994); and Mendez et al., 15(2):146-56 (1997)); and the various Kirin (Japan) publications/patents concerning “transomic” mice (e.g., EP 843 961, and Tomizuka et al., Nature Genetics 16:133-1443 (1997)).
Humanized Antibodies
The monoclonal antibodies of this invention also include humanized versions of cognate anti-αvβ6 antibodies derived from other species. A humanized antibody is an antibody produced by recombinant DNA technology, in which some or all of the amino acids of a human immunoglobulin light or heavy chain that are not required for antigen binding (e.g., the constant regions and the framework regions of the variable domains) are used to substitute for the corresponding amino acids from the light or heavy chain of the cognate, nonhuman antibody. By way of example, a humanized version of a murine antibody to a given antigen has on both of its heavy and light chains (1) constant regions of a human antibody; (2) framework regions from the variable domains of a human antibody; and (3) CDRs from the murine antibody. When necessary, one or more residues in the human framework regions can be changed to residues at the corresponding positions in the murine antibody so as to preserve the binding affinity of the humanized antibody to the antigen. This change is sometimes called “back mutation.” Humanized antibodies generally are less likely to elicit an immune response in humans as compared to chimeric human antibodies because the former contain considerably fewer non-human components.
The methods for making humanized antibodies are described in, e.g., Winter E P 239 400; Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988); Queen et al., Proc. Nat. Acad. Sci. USA 86:10029 (1989); U.S. Pat. No. 6,180,370; and Orlandi et al., Proc. Natl. Acad. Sci. USA 86:3833 (1989). Generally, the transplantation of murine (or other non-human) CDRs onto a human antibody is achieved as follows. The cDNAs encoding heavy and light chain variable domains are isolated from a hybridoma. The DNA sequences of the variable domains, including the CDRs, are determined by sequencing. The DNAs encoding the CDRs are transferred to the corresponding regions of a human antibody heavy or light chain variable domain coding sequence by site directed mutagenesis. Then human constant region gene segments of a desired isotype (e.g., γ1 for CH and k for CL) are added. The humanized heavy and light chain genes are co-expressed in mammalian host cells (e.g., CHO or NSO cells) to produce soluble humanized antibody. To facilitate large scale production of antibodies, it is often desirable to produce such humanized antibodies in bioreactors containing the antibody-expressing cells, or to produce transgenic mammals (e.g., goats, cows, or sheep) that express the antibody in milk (see, e.g., U.S. Pat. No. 5,827,690).
At times, direct transfer of CDRs to a human framework leads to a loss of antigen-binding affinity of the resultant antibody. This is because in some cognate antibodies, certain amino acids within the framework regions interact with the CDRs and thus influence the overall antigen binding affinity of the antibody. In such cases, it would be critical to introduce “back mutations” (supra) in the framework regions of the acceptor antibody in order to retain the antigen-binding activity of the cognate antibody.
The general approach of making back mutations is known in the art. For instance, Queen et al. (supra), Co et al., Proc. Nat. Acad. Sci. USA 88:2869-2873 (1991), and WO 90/07861 (Protein Design Labs Inc.) describe an approach that involves two key steps. First, the human V framework regions are chosen by computer analysis for optimal protein sequence homology to the V region framework of the cognate murine antibody. Then, the tertiary structure of the murine V region is modeled by computer in order to visualize framework amino acid residues that are likely to interact with the murine CDRs, and these murine amino acid residues are then superimposed on the homologous human framework.
Under this two-step approach, there are several criteria for designing humanized antibodies. The first criterion is to use as the human acceptor the framework from a particular human immunoglobulin that is usually homologous to the non-human donor immunoglobulin, or to use a consensus framework from many human antibodies. The second criterion is to use the donor amino acid rather than the acceptor if the human acceptor residue is unusual and the donor residue is typical for human sequences at a specific residue of the framework. The third criterion is to use the donor framework amino acid residue rather than the acceptor at positions immediately adjacent to the CDRs.
One may also use a different approach as described in, e.g., Tempest, Biotechnology 9: 266-271 (1991). Under this approach, the V region frameworks derived from NEWM and REI heavy and light chains, respectively, are used for CDR-grafting without radical introduction of mouse residues. An advantage of using this approach is that the three-dimensional structures of NEWM and REI variable regions are known from X-ray crystallography and thus specific interactions between CDRs and V region framework residues can be readily modeled.
Other Moieties
The monoclonal antibodies of this invention may further comprise other moieties to effect the desired functions. For instance, the antibodies may include a toxin moiety (e.g., tetanus toxoid or ricin) or a radionuclide (e.g., 111In or 90Y) for killing of cells targeted by the antibodies (see, e.g., U.S. Pat. No. 6,307,026) The antibodies may comprise a moiety (e.g., biotin, fluorescent moieties, radioactive moieties, histidine tag or other peptide tags) for easy isolation or detection. The antibodies may also comprise a moiety that can prolong their serum half life, for example, a polyethylene glycol (PEG) moiety.
Diseased Conditions and Animal Models
The antibodies of the invention are useful in the treatment, including prevention, of αvβ6-mediated diseases. For example, these antibodies can be used to treat fibrosis (e.g., lung fibrosis, acute lung injury, kidney fibrosis, liver fibrosis, Alport's Syndrome, and scleroderma) by blocking the activation of TGF-β or blocking the binding of αvβ6 to any other ligands, such as fibronectin, vitronectin, and tenascin. The novelty of this approach includes: (1) it blocks the activation of TGF-β rather than the binding of TGF-β to its receptor, (2) it can inhibit TGF-β locally (i.e., at sites of αvβ6 upregulation) rather than systemically, and (3) it inhibits binding of αvβ6 to a ligand. Other than fibrotic diseases or conditions, the antibodies of the invention are useful in treating cancer or cancer metastasis (including tumor growth and invasion), particularly epithelial cancers. A subset of epithelial cancers is squamous cell carcinoma, e.g., head and neck, oral, breast, lung, prostate, cervical, pharyngeal, colon, pancreatic and ovarian cancers. Our studies using the new αvβ6 monoclonal antibodies demonstrated that αvβ6 is highly expressed in many epithelial cancers, especially on the leading edge of the tumors. The new antibodies can also be used to any other diseases mediated by αvβ6, including psoriasis.
The treatments of this invention are effective on both human and animal subjects afflicted with these conditions. Animal subjects to which the invention is applicable extend to both domestic animals and livestock, raised either as pets or for commercial purposes. Examples are dogs, cats, cattle, horses, sheep, hogs and goats.
The efficacy of the antibodies of the invention can be tested in various animal models. Mouse models for lung fibrosis include bleomycin-(Pittet et al., J. Clin. Invest. 107(12):1537-1544 (2001); and Munger et al., supra) and irradiation-inducible lung fibrosis (Franko et al., Rad. Res. 140:347-355 (1994)). In bleomycin-treated mice, the expression of αvβ6 increases in the epithelial alveolar cells of the lungs. But β6 knockout mice are protected from bleomycin-induced injury and fibrosis.
Mouse models for kidney fibrosis include COL4A3−/− mice (see, e.g., Cosgrove et al., Amer. J. Path. 157:1649-1659 (2000), mice with adriamycin-induced injury (Wang et al., Kidney International 58: 1797-1804 (2000); Deman et al., Nephrol Dial Transplant 16: 147-150 (2001)), db/db mice (Ziyadeh et al., PNAS USA 97:8015-8020 (2000)), and mice with unilateral ureteral obstruction (Fogo et al., Lab Investigation 81: 189A (2001); and Fogo et al., Journal of the American Society of Nephrology 12:819A (2001)). In all of these models, the mice develop kidney injury and fibrosis that can progress to renal failure. αvβ6 is upregulated in the epithelial lining of the ascending and descending tubules of the kidneys of the COL4A3−/− mice, adriamycin-treated mice, and mice that undergo unilateral ureteral obstruction. It is likely that αvβ6 expression also increases in a variety of kidney injury models.
Anti-αvβ6 monoclonal antibodies can also be tested for their ability to inhibit tumor growth, progression, and metastasis in such animal models as the standard in vivo tumor growth and metastasis models. See, e.g., Rockwell et al., J. Natl. Cancer Inst. 49:735 (1972); Guy et al., Mol. Cell Biol. 12:954 (1992); Wyckoff et al., Cancer Res. 60:2504 (2000); and Oft et al., Curr. Biol. 8:1243 (1998). Important αvβ6 ligands in cancer may include TGF-β, which is involved in metastasis (for review see Akhurst et al., Trends in Cell Biology 11:S44-S51 (2001)), fibronectin and vitronectin.
The efficacy of the treatments of this invention may be measured by a number of available diagnostic tools, including physical examination, blood tests, proteinuria measurements, creatinine levels and creatinine clearance, pulmonary function tests, plasma blood urea nitrogen (BUN) levels, observation and scoring of scarring or fibrotic lesions, deposition of extracellular matrix such as collagen, smooth muscle actin and fibronectin, kidney function tests, ultrasound, magnetic resonance imaging (MRI), and CT scan.
Pharmaceutical Compositions
The pharmaceutical compositions of this invention comprise one or more antibodies of the present invention, or pharmaceutically acceptable derivatives thereof, optionally with any pharmaceutically acceptable carrier. The term “carrier” as used herein includes known acceptable adjuvants and vehicles.
According to this invention, the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing, wetting, and suspending agents.
The pharmaceutical compositions of this invention may be given orally, topically, intravenously, subcutaneously, intraperitoneally, intramuscularly, intramedullarily, intra-articularly, intra-synovially, intrasternally, intrathecally, intrahepatically, or intracranially as desired, or just locally at sites of inflammation or tumor growth. The pharmaceutical compositions of this invention may also be administered by inhalation through the use of, e.g., a nebulizer, a dry powder inhaler or a metered dose inhaler.
The dosage and dose rate of the antibodies of this invention effective to produce the desired effects will depend on a variety of factors, such as the nature of the disease to be treated, the size of the subject, the goal of the treatment, the specific pharmaceutical composition used, and the judgment of the treating physician. Dosage levels of between about 0.001 and about 100 mg/kg body weight per day, for example between about 0.1 and about 50 mg/kg body weight per day, of the active ingredient compound are useful. For instance, an antibody of the invention will be administered at a dose ranging between about 0.01 mg/kg body weight/day and about 20 mg/kg body weight/day, e.g., ranging between about 0.1 mg/kg body weight/day and about 10 mg/kg body weight/day, and at intervals of every one to fourteen days. In another embodiment, the antibody is administered at a dose of about 0.3 to 1 mg/kg body weight when administered intraperitoneally. In yet another embodiment, the antibody is administered at a dose of about 5 to 12.5 mg/kg body weight when administered intravenously. In one embodiment, an antibody composition is administered in an amount effective to provide a plasma level of antibody of at least 1 mg/ml.
Diagnostic Methods
The antibodies of this invention can be used to diagnose diseased conditions associated with altered αvβ6 expression levels. A tissue sample from a subject, such as a tissue biopsy, body fluid sample or lavage (e.g., alveolar lavage), can be tested in an antigen capture assay, ELISA, immunohistochemistry assay, and the like using the antibodies. A tissue sample from a normal individual is used as control.
Practice of the present invention will employ, unless indicated otherwise, conventional techniques of cell biology, cell culture, molecular biology, microbiology, recombinant DNA, protein chemistry, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning: A Laboratory Manual, 2nd edition (Sambrook et al., Eds.), 1989; Oligonucleotide Synthesis, (M. J. Gait, Ed.), 1984; U.S. Pat. No. 4,683,195 to Mullis et al.; Nucleic Acid Hybridization, (B. D. Hames and S. J. Higgins), 1984; Transcription and Translation, (B. D. Hames and S. J. Higgins), 1984; Culture of Animal Cells (R. I. Freshney, Ed.), 1987; Immobilized Cells and Enzymes, IRL Press, 1986; A Practical Guide to Molecular Cloning (B. Perbal), 1984; Methods in Enzymology, Volumes 154 and 155 (Wu et al., Eds.), Academic Press, New York; Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos, Eds.), 1987; Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, Eds.), 1987; Handbook of Experiment Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, Eds.), 1986; Manipulating the Mouse Embryo, 1986.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting. Throughout this specification, the word “comprise,” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
EXAMPLES
The following examples are meant to illustrate the methods and materials of the present invention. Suitable modifications and adaptations of the described conditions and parameters normally encountered in the antibody art that are obvious to those skilled in the art are within the spirit and scope of the present invention.
In the following examples, the β6−/− mice were generated as described in Huang et al., J. Cell Biol. 133:921 (1996). Recombinant human LAP was purchased from R & D Systems (Minneapolis, Minn.). Antibody 10D5 was purchased from Chemicon (Temecula, Calif.). The L230 hybridoma was purchased from ATCC and the secreted antibody was purified from the supernatant of saturated cultures by affinity chromatography on immobilized protein A. Isotyping of antibodies was carried out using the ISOSTRIP kit (Roche Diagnostics) according to the manufacturer's instructions. The β6-transfected SW480 cell line was prepared as described in Weinacker et al., J. Biol. Chem. 269:6940-6948 (1994).
Example 1: Generation of β6-Transfected Stable Cell Lines
β6-transfected NIH 3T3 and FDC-P1 cells were generated by electroporating parent cell lines with a DNA construct containing full length murine β6 cDNA and a neomycin selectable marker. Stably transfected cells were selected by passaging cells in culture medium containing G418 for 14 days followed by fluorescent activated cell sorting (FACS) to isolate cells expressing the highest level of surface β6. Transfected FDC-P1 cells were cultured in DMEM supplemented with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/l glucose, and 1.0 mM sodium pyruvate, 10% FBS, 2.5% mouse IL-3 culture supplement, and 1.5 mg/ml active G418. Transfected NIH 3T3 cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, penicillin/streptomycin, and 1 mg/ml active G418.
Example 2: Purification of Human Soluble αvβ6 
The αvβ6 protein was purified essentially as described in Weinacker, supra. A CHO cell line expressing hsαvβ6 was cultured, and the resultant supernatant collected by centrifugation. The integrin was purified by affinity chromatography using anti-αv antibody L230. Purified L230 was cross-linked to CNBr-activated Sepharose 4B (Sigma) at a ratio of 4.8 mg antibody/ml resin. The αvβ6 supernatant was loaded at 0.5 mg antibody/ml resin onto the L230 affinity column, and the column was washed with 10 column volumes of each of (1) 50 mM Tris-Cl, pH 7.5, 1 M NaCl, 1 mM MgCl2; (2) 50 mM Tris-Cl, pH 7.5, 50 mM NaCl, 1 mM MgCl2; and (3) 10 mM Na3PO4, pH 7.0. Hsαvβ6 was eluted with 100 mM glycine, pH 2.5 into 1:10 volume of 1 M Na3PO4, pH 8.0. The protein was dialyzed with several changes against phosphate buffered saline (PBS) and stored at −20° C.
Example 3: Immunization of β6−/− Mice
β6−/− mice were immunized by intraperitoneal (IP) injection with 25 μg of purified recombinant human αvβ6 emulsified in complete Freund's adjuvant (CFA) at a volume ratio of 1:1 in a total volume of 200 μl. Alternatively, β6−/− mice were immunized via IP injection with 4×106β6-transfected NIH 3T3 cells resuspended in 100 μl of PBS supplemented with 1 mg/ml of CaCl2 and 1 mg/ml MgCl2, and the same mice were injected at an adjacent site with 100 μl of CFA. Two weeks and four weeks after the initial immunization, the mice were boosted similarly with the same reagents with the exception that incomplete Freund's adjuvant was used in place of CFA. The mice were bled 7 days after the final boost and anti-β6 titers were determined by binding of the serum to purified recombinant human αvβ6 or to β6-transfected cells. In the case of mice immunized with purified recombinant human αvβ6, mice were rested for 3 months and reimmunized with the same antigen mixed with ImmunEasy (Qiagen). Three days prior to isolating spleens for hybridoma fusions, the mice were immunized with 12.5 μg of purified recombinant human αvβ6 protein by both IP and intravenous injection. On day of fusion, the animals were sacrificed, and their spleens were removed and teased into single cell suspensions. The splenocytes were immortalized by fusion to a drug-selectable cell fusion partner.
Example 4: Screening of Hybridomas
Two groups of antibodies were generated through the immunization of β6−/− mice. One set of antibodies were generated through immunization with soluble human truncated αvβ6 (Fusion #6). The other set of antibodies were generated through immunization with murine β6-transfected NIH 3T3 cells (Fusion #7). Screening for anti-αvβ6 antibodies was carried out using both cell-based and cell-free binding and functional assays as described below. Initial selection of positive clones was based on binding to purified hsαvβ6 and β6-transfected human and murine cells (untransfected cells as control). Selected clones were expanded and terminal cultures were re-evaluated for binding to both β6-transfected and untransfected cells in cell capture assays (Example 5b, infra) (representative examples shown in FIGS. 1A and 1B, where the prefixes “6.” or “7.” of the mAb names, which denote Fusion 6 and Fusion 7, respectively, are omitted; see also Table 2 below). Some antibodies bound preferentially to the β6-transfected cells, while others bound to both transfected and untransfected cells, indicating that only a subset of the antibodies had a preference for β6 (FIGS. 1A and 1B). Further selection was based on the ability of the antibodies to block binding of both biotinylated hsαvβ6 and β6-transfected murine cells to LAP. Select clones were subcloned using FACS and stored frozen until use.
Monoclonal antibodies were screened for specificity of binding to αvβ6 based on their ability to bind β6-transfected cells and not untransfected parent cells. Monoclonal antibodies were further confirmed as specific binders of αvβ6 and not of other αv integrins or non-specific integrins (i.e., non-αv integrins that bind to RGD-containing ligands) based on their lack of binding to cell lines expressing αvβ3, αvβ8, α5β1, αvβ1 or α5β1. These included stably transfected cells as well as untransfected JY, K562, SW480, NIH3T3, and FDCP1 cell lines.
Some of the antibodies that have been deposited with the ATCC are listed below in Table 1.







TABLE 1







Deposited Hybridomas











Hybridoma clones
ATCC No.
Deposit Date







6.1A8
PTA-3647
Aug. 16, 2001



6.2B10
PTA-3648
Aug. 16, 2001



6.3G9
PTA-3649
Aug. 16, 2001



6.8G6
PTA-3645
Aug. 16, 2001



6.2B1
PTA-3646
Aug. 16, 2001



6.2A1
PTA-3896
Dec. 5, 2001



6.2E5
PTA-3897
Dec. 5, 2001



7.1G10
PTA-3898
Dec. 5, 2001



7.7G5
PTA-3899
Dec. 5, 2001



7.10C5
PTA-3900
Dec. 5, 2001










Example 5: Assays for Screening and Characterization
a. αvβ6 ELISA
A 96-well microtiter plate (Corning COSTAR EASY-WASH) was coated with 50 μl/well of 5 μg/ml hsαvβ6 at 4° C. overnight. The plate was washed with wash buffer (0.1% TWEEN-20 in PBS) four times in an automated plate washer. Then 180 μl/well of 3% BSA in TBS was added and incubated for 1 hr at 25° C. to block nonspecific binding. The plate was washed as above, and dilutions of either hybridoma supernatant (for screening assays) or purified antibody (for characterization) in TBS containing 1 mg/ml BSA, 1 mM CaCl2, and 1 mM MgCl2 were added (50 μl/well). The plate was incubated for 1 hr at 25° C., washed, and then incubated for 1 hr with 50 μl/well of peroxide-conjugated goat anti-mouse IgG+A+M antibody (Cappel). Bound antibody was detected using 3,3′,5,5′-tetramethylbenzidine (TMB). Binding was indicated by the absorbency measured at 450 nm.
b. Cell Capture Assay
A 96-well microtiter plate was coated with 50 μl/well of secondary antibody (donkey anti-mouse IgG (Jackson Immunoresearch); 5 μg/ml diluted in 50 mM sodium bicarbonate, pH 9.2) at 4° C. overnight. Plates were washed twice with 100 μl/well of assay buffer (RPMI+2% BSA) and then blocked with 100 μl/well of the assay buffer at 37° C. for 1 hr. For FDC-P1 cells and β6-transfected FDC-P1 cells, plates were blocked with anti-mouse Ig (Jackson ImmunoResearch; 20 μg/ml) for 10 min at room temperature to decrease non-specific Fc receptor binding by secondary antibody (omitted for other cell types). While the plates were being blocked, the cells were labeled with 2 μM fluorescent dye (Calcein-AM, Molecular Probes) in the assay buffer at 5×106 cells/ml. The cells were incubated with the dye in the assay buffer with gentle shaking in a 37° C. water bath for 15 min, collected by centrifugation, and resuspended in assay buffer to 5×106 cells/ml. Following the blocking step, the buffer was discarded by flicking the plate, and 25 μl/well of supernatant or purified antibody was added to the plate. Following a 15 mM incubation at 37° C., 25 μl/well of labeled cells were added, and the plate was incubated for 1 hr at 37° C. The plate was washed 3-5 times with the assay buffer (100 μl/well) and fluorescence emitted by captured cells on the plate was recorded. Percent binding was determined by comparing the fluorescence prior to the final wash step (i.e., total cells added) to that after washing (i.e. bound cells).
c. FACS
Cells were harvested by trypsinization, washed one time in PBS, and then resuspended in FACS buffer (1×PBS, 2% FBS, 0.1% NaN3, 1 mM CaCl2, and 1 mM MgCl2). 0.2×105 cells were then incubated on ice for 1 hr in FACS buffer containing hybridoma supernatant in a total volume of 100 μl. After incubation, the cells were washed two times with ice cold FACS buffer, resuspended in 100 μl of FACS buffer containing 5 μg/ml donkey anti-mouse IgG PE (Jackson ImmunoResearch), and incubated on ice for 30 min. The cells were then washed twice with ice cold FACS buffer and resuspended in 200 μl of FACS buffer. Binding of the PE labeled secondary antibody was monitored by flow cytometry.
d. Binding of Biotin-hsαvβ6 to LAP
96-well microtiter plates (Corning COSTAR EASY-WASH) were coated with 0.3 μg/ml recombinant human LAP (R&D Systems, Cat. #246-LP) diluted in PBS (50 μl/well) at 4° C. overnight. After the coating solution was removed, the plates were blocked with 180 μl/well of 3% BSA/TBS at 25° C. for 1 hr. In a separate 96-well round-bottom plate, 60 μl/well of a 2× stock (0.5 μg/ml (1.25 nM) of biotin-αvβ6, 2 mM CaCl2, and 2 mM MgCl2 in TBS containing 1 mg/ml BSA) was combined with 60 μl/well of a 2× stock of either a hybridoma supernatant (for screening) or a purified antibody (also in TBS containing 1 mg/ml BSA) and incubated at 25° C. for 1 hr. After washing the LAP-coated plate with wash buffer (0.1% TWEEN-20 in PBS) 4 times in an automated plate washer, 100 μl of the antibody-αvβ6 mixture was transferred to the plate, and incubated for 1 hr at 25° C. The plate was washed as above and incubated with 50 μl/well of a 1:1000 dilution of extravidin-horseradish peroxidase conjugate (Sigma) in TBS (1 mg/ml BSA) for 1 hr at 25° C. Bound protein was detected using the TMB substrate.
e. Adhesion of β6-FDC-P1 Cells to LAP
A 96-well microtiter plate was coated with 50 μl/well of 0.5 μg/ml recombinant human LAP (R&D Systems) diluted in 50 mM sodium bicarbonate, pH 9.2 at 4° C. overnight. The plate was washed twice with PBS (100 μl/well) and blocked with 1% BSA in PBS (100 μl/well) for 1 hr at 25° C. The plate was washed twice with 100 μl/well of assay buffer (TBS complete plus 1 mM CaCl2 and 1 mM MgCl2). Next, to the individual wells of the plate were added 25 μl of a hybridoma supernatant (or a purified antibody) and 25 μl of β6-FDC-P1 cells (5× 106 cells/ml, labeled with Calcein AM as described above). The plate was incubated at 25° C. for 1 hr, and then washed 4-6 times with the assay buffer (100 μl/well). The fluorescence emitted from cells captured on the plate was recorded. Percentage binding was determined by comparing the fluorescence signal prior to the final wash step (i.e., total cells added) to that after washing (i.e., bound cells).
f. TGF-β Bioassay
The TGF-β bioassay used herein was a variation of the Mink lung epithelial cell (MLEC) PAI-1 luciferase coculture assay described in Abe et al., Anal. Biochem. 216:276-284 (1994), in which β6-transfected cells were cocultured with the reporter cells to monitor activation of TGF-β by αvβ6 (Munger, supra). It is a quantitative bioassay for TGF-β based on its ability to induce the expression of plasminogen activator inhibitor-1 (PAI-1). In this assay, MLEC cells are stably transfected with an expression construct containing a truncated PAI-1 promoter fused to the firefly luciferase reporter gene. Exposure of the transfected MLEC cells to active TGF-β (0.2 to >30 pM) results in a dose-dependent increase in luciferase activity in the cell lysates.
To conduct this assay, TMLC (mink lung epithelial cell line Mv 1 Lu) cells were transfected with the PAI-1-luciferase construct. The transfected cells were grown in DMEM+10% FBS with L-Gln, Pen/Strep and 200 μg/ml G418. SW480 cells transfected with an integrin β6 construct (“β6-SW480” or “SW480 β6” cells) were grown in DMEM+10% FBS with L-Gln and Pen/Strep. Cells were lifted from flasks with PBS+5 mM EDTA, washed in PBS+0.5% BSA, counted by hemocytometer and plated in 96-well plates. SW480-β6 cells were plated at 4×104 cells/well in the wash buffer. Monoclonal antibodies were diluted in DMEM (serum-free), added to the SW480-β6 cells and pre-incubated for 20 min at room temperature. TMLC cells were then added at 2×104 cells/well to a final volume of 100 μl. The plates were incubated for 20 hr in a humidified, CO2-enriched incubator. Supernatant from the plates was discarded and replaced with 100 μl of PBS+1 mM Ca2+ and 1 mM Mg2+. Cells in the plates were then lysed, and the level of luciferase activity was detected with the glow-type reaction Packard LUCLITE kit (#6016911) and TROPIX microplate luminometer.
Example 6: Antibody Purification
Eight hybridoma clones from Fusion #6 (denoted by the prefix “6.”) and fourteen hybridoma clones from Fusion #7 (denoted by the prefix “7.”) were selected for further scale-up and characterization (Table 2).
A small-scale culture (150 ml) of each hybridoma was prepared, and the supernatant was collected by centrifugation. Antibodies were purified from these supernatants using Protein A affinity chromatography. For the IgG2a isotype antibodies, the supernatant was directly loaded onto Protein A Sepharose 4 Fast Flow (Amersham Pharmacia Biotech, AB, Uppsala, Sweden) (1 ml settled bed volume). The column was washed with PBS, and the IgG fraction was eluted using 25 mM phosphoric acid, 100 mM NaCl, pH 2.8 into 1:20 volume of 0.5 M Na3PO4, pH 8.6. For the murine IgG1 antibodies, the supernatant was adjusted to 1.5 M glycine, 3 M NaCl, pH 8.9 prior to loading, and the column was washed with 25 mM Na3PO4, 3 M NaCl, pH 8.6 prior to elution. These preparations were used for the in vitro biochemical characterization described herein.







TABLE 2







Characterization of Hybridoma Clones












Clone Name
Clone No.
Isotype
Blocker*
















6.1A8
2
IgG2a
Y



6.2B10
10
IgG2a
Y



6.3G9
25
IgG1
Y



6.4B4
30
IgG1
N



6.6B5
46
IgG1
N



6.8B4
55
IgG1
N



6.8G6
56
IgG1
Y



6.2B1
85
IgG1
Y



7.105
2
IgG2a
Y



7.1G10
5
IgG2a
Y



7.2A1
6
IgG2a
Y



7.2F5
11
IgG2a
Y



7.2H2
12
IgG2a
Y



7.4A3
17
IgG2a
Y



7.7G5
32
IgG1 
Y



7.8H12
39
IgG2a
Y



7.9D4
40
IgG2a
N



7.9G8
41
IgG2a
Y



7.9H5
43
IgG2a
Y



7.10D7
44
IgG2a
Y



7.10H2
46
IgG2a
Y







*A blocker is defined as an antibody that blocks the binding of ανβ6 to LAP as determined by blocking of ligand binding either to purified hsανβ6 or to β6-expressing cells.






For use in animal models, hybridoma clones were scaled up to 2 L of media and grown for 4 weeks in Lifecell Culture Bags-PL732 (Nexell, Cat. No. R4R2113). Antibodies from the hybridomas were purified first by Protein A affinity chromatography as described above, followed by an ion-exchange step on Q Sepharose (Amersham Pharmacia). The eluate from the Protein A chromatographic step was adjusted to pH 8.6 using 2 M Tris base, diluted 10-fold with water, and loaded onto a Q Sepharose column (20 mg protein/ml resin) that had been equilibrated in 10 mM Na3PO4, 25 mM NaCl, pH 8.6. The column was washed with 5 column volumes of equilibration buffer, and bound protein was eluted using 25 mM Na3PO4, 150 mM NaCl, pH 7.2. The eluted proteins were sterile-filtered (0.45 μm) and stored at −70° C. until use.
Example 7: Characterization of Purified Antibodies
The purified antibodies (Table 2, supra) were characterized quantitatively with respect to their ability to (1) bind hsαvβ6, (2) bind β6-transfected SW480 and FDC-P1 cells, (3) inhibit binding of biotin-αvβ6 to LAP, (4) inhibit binding of β6-transfected FDC-P1 cells to LAP, and (5) block αvβ6-mediated activation of TGF-β in the MLEC assay (supra). The relative potency in each of these assays was compared to that of the known αvβ6 antibody 10D5 (Huang et al, J. Cell Sci. 111:2189 (1998)) and, in some cases, the anti-αv antibody L230. For the characterization of Fusion #7 antibodies, the Fusion #6 antibody 6.8G6 was also used as a positive control.
An initial binding experiment (Example 5a, supra), carried out in the presence of 1 mM Ca2+ and 1 mM Mg2+, indicated that a majority of the purified antibodies bound to hsαvβ6 (FIGS. 2A and 2B). Unexpectedly, however, no binding was observed for 10D5 and clones 7.2F5 and 7.10D7. A subsequent experiment established that binding of 10D5 (FIG. 3E), 7.2F5, and 7.10D7 was supported only weakly by Ca2+/Mg2+, but much more strongly by 1 mM MnCl2. Among the new clones, three (6.1A8 (FIG. 3A), 7.7G5, and 6.8G6 (FIG. 3C)) showed a requirement for divalent cations, although no difference between the Ca2+/Mg2+ state and the Mn2+-bound state was observed.
The remaining clones showed no requirement for divalent cations, i.e. could bind to the antigen in the presence of 10 mM EDTA (FIGS. 3B, 3D and 3F). FACS analysis of antibody binding to β6-transfected NIH 3T3 cells or SW480 cells revealed a similar pattern, with the exception that 10D5, in this context, bound equivalently in the Ca2+/Mg2+ and Mn2+ states. The requirements for binding to soluble αvβ6 may differ from those for binding to cell surface-expressed αvβ6 due to a difference in protein conformation or avidity effects.
These results suggest that there are at least 3 different classes of β6-blocking antibodies in this group. One of the classes (10D5) distinguishes between the Ca2+/Mg2+ and Mn2+ conditions. Another class (including 6.1A8, 7.7G5, and 6.8G6) requires cation but does not distinguish between Ca2+/Mg2+ and Mn2+. The last class (including anti-αv antibody L230, 6.2B10, 6.3G9 (FIG. 3B), and 6.2B1 (FIG. 3D), 7.1C5, and 7.1G10) is cation-independent.
The purified antibodies were next evaluated for their ability to inhibit the αvβ6-LAP interaction. In the cell-free assay of Example 5d, supra, antibodies 6.1A8, 6.2B1, 6.3G9, and 6.8G6 showed IC50 values lower than that of 10D5 (FIG. 4A; Table 3). 6.2B10 showed a higher IC50 but still gave complete inhibition (FIG. 4A). 6.4B4 showed only partial inhibition, whereas 6.6B5 and 6.8B4 showed no inhibition (FIG. 4A). Using the same assay system, antibodies 7.1C5, 7.1G10, 7.2A1, 7.4A3, 7.7G5, 7.9G8, 7.9H5, and 7.10H2 showed IC50 values lower than that of 10D5 (FIG. 4B; Table 3). Antibodies 7.2F5, 7.2H2 and 7.8H12 displayed nearly identical or higher IC50 values and yet still gave complete inhibition (FIG. 4B).
In the cellular assay described in Example 5e, supra, a similar trend was observed, with the exceptions of 6.1A8, 6.2B10 and 7.9D4, which were much less potent on cells than on purified protein (FIGS. 5A-E; Table 3).
Collectively, these results indicate that we have successfully generated antibodies that specifically inhibit the interaction of both human and murine αvβ6 with LAP. Some of these antibodies bound to αvβ6 with high affinity (apparent Kd's≧0.3 nM, as determined by flow cytometry), inhibited binding of β6-transfected cells to LAP with an IC50 of ≧0.05 nM (8 ng/mL), and prevented αvβ6-mediated activation of TGF-β1 with an IC50 of ≧0.58 nM (87 ng/mL).
Finally, the purified antibodies were evaluated for their ability to block αvβ6-mediated activation of TGF-β in the PAI-1/luciferase reporter gene assay (Example 5f, supra). Once again, 6.3G9, 6.8G6, 6.2B1, 7.1G10, and 7.7G5 were able to inhibit αvβ6-mediated activation of TGF-β with IC50 values lower than 10D5, while the remaining antibodies appeared to be significantly less potent in this assay (FIGS. 6A and 6B; Table 3). Thus, the ability to block αvβ6's interaction with LAP correlates with the ability to inhibit activation of TGF-β in vitro.







TABLE 3







Characterization of hybridoma clones














ανβ6-binding 
ανβ6-LAP 
FDCP1-LAP
PAI-1


Clone 
Clone
ELISA
blocking
blocking
luciferase


name
No.
EC50 (ng/ml)
IC50 (ng/ml)
IC50 (ng/ml)
IC50 (ng/ml)















6.2B1
85
34.7
    225
      8
     87


6.3G9
25
76.7
    271
     17
    375


6.8G6
56
17.5
    169
     23
    312


10D5
—
—
    605
     50
   2070


6.1A8
2
 3.7
    179
   2520
~40,000


6.2B10
10
78.9
   1950
>30,000
>40,000


6.4B4
30
25.4
>50,000
>30,000
>40,000


6.6B5
46
17.1
>50,000
>30,000
>40,000


6.8B4
55
94.4
>50,000
>30,000
>40,000


L230
—
27.1
    229
n.t.**
n.t.


7.1G10
5
 4.2
    113
     30
    250


7.7G5
32
13.0
    155
     51
    700


7.1C5
2
  2.5*
      80*
     83
n.t.


7.2A1
6
5*
     300*
    101
n.t.


10D5
—
43* 
    377
n.t.  
  2,000


7.4A3
17
5.7
    204
     67
  3,500


7.10H2
46
6.6
    254
     63
  3,500


7.2H2
12
9.3
    370
    106
  5,500


7.9H5
43
7.3
    230
     55
  7,000


7.9G8
41
6.2
    264
    284
>20,000


7.8H12
39
46.0 
   1140
    969
>20,000


7.2F5
11
>5000      
    529
   1490
>20,000


7.9D4
40
1.7
incomplete
>10,000
>20,000


7.10D7
44
>5000      
    3000*
   1120
n.t.





*Data obtained from separate experiments.


**Not tested.


***All experiments summarized in Table 3 were conducted in the presence of 1 mM Ca2+ and 1 mM Mg2+.


****Antibodies in boldface are prior art antibodies 10D5 and L230, and new antibodies of particular high inhibitory potency for ανβ6.






Next, the solution affinities of 6.3G9 and 6.806 for soluble αvβ6 were determined by using a kinetic exclusion assay (KinExA). A series of dilutions of the soluble integrin (1×10−8 M to 2.4×10−12M) were incubated with 1×10−10 M of the antibody for 3 h. These samples were then passed through polymethylmethacrylate beads coated with the integrin using a KinExA instrument (SapidyneInstruments, Inc., Boise, Id.). In the case of 6.8G6, 1 mM CaCl2 and 1 mM MgCl2 were included in the incubation and assay buffers. The amounts of bound and free antibody were determined using a Cy5-labeled anti-mouse secondary antibody. Quadratic curve fitting was performed using the KinExA software to attain a dissociation constant (Kd) for each interaction. The Kd's determined using this method were 15.6 pM for 6.3G9 and 22.8 pM for 6.806 (FIGS. 9A and 9B). Thus, both of these antibodies had very high affinities for αvβ6.
We further identified classes of anti-αvβ6 antibodies that recognized “activated” states of the integrin. There are two potential activation states of αvβ6. In the first state, the activated integrin is defined as having a higher affinity for its ligand. Antibodies specific for this activated state showed enhanced binding to the integrin in the presence of activating cations such as 1 mM MnCl2. A comparison of the extent of binding in 1 mM MnCl2 and 1 mM MgCl2 (non-activating cation) by flow cytometry indicated that some of the αvβ6 antibodies described here, including 6.1A8 and 6.6B5, showed significantly enhanced binding in the presence of MnCl2.
In a second activated state of αvβ6, the integrin can activate latent TGF-β as described above. A cell line expressing truncated αvβ6 (SW480(β6-770T)) was prepared. The cell line was able to bind LAP but could not activate TGF-β in the TMLC luciferase assay (Munger et al., supra). Antibodies which bind to the full length β6-transfected SW480 cells, but not to the 770T truncated transfected cells, were thus specific to the form of αvβ6 that is able to activate TGF-β. Antibodies 7.8B3 and 7.8C9 met this criteria.
Example 8: Epitope Mapping by Antibody Competition
The purified monoclonal antibodies were also tested for their ability to compete with 6.8G6 for binding to biotinylated αvβ6 in an ELISA format. In this assay, 6.8G6 was coated on an ELISA plate, and a mixture of the competing antibody and biotinylated αvβ6 was added in a buffer containing 1 mM each of Ca2+ and Mg2+. Bound integrin was detected using extravidin-HRP conjugate, and competing antibodies were scored for their ability to block binding. All consensus blockers (Table 2) except 6.2B10 (a weak blocker) were shown to be able to compete with 6.8G6 to various degrees (Table 4). These data confirm that these consensus blockers bind to the same or overlapping epitope as 6.8G6.







TABLE 4







Epitope Mapping by Antibody Competition












Consensus
Competition with



Clone
Blocker?
6.8G6







6.2A1
N
−



6.4B4
N
−



6.6B5
N
−



6.8B4
N
−



7.9D4
Y/N
−



10D5
Y
++



L230
Y
++



6.1A8
Y
++



6.2B10
Y (weak)
−



6.3G9
Y
+



6.8G6
Y
++



6.2B1
Y
++



7.1C5
Y
+++



7.1G10
Y
+++



7.2A1
Y
++



7.2F5
Y
++



7.2H2
Y
++



7.4A3
Y
++



7.7G5
Y
++



7.8H12
Y
++



7.9G8
Y
++



7.9H5
Y
++



7.10D7
Y
+



7.10H2
Y
++










The purified monoclonal antibodies were tested for their ability to compete with biotinylated 6.3G9 or biotinylated 6.8G6 for binding to αvβ6 in ELISA. In this assay, unlabeled αvβ6 was coated on an ELISA plate, and a mixture of the competing antibody and the biotinylated antibody was added in a buffer containing 1 mM each of Ca2+ and Mg2+. Bound biotinylated antibody was detected by using neutravidin-HRP conjugate. The data showed that the most potent blocking antibodies (e.g., 6.2B1, 7.1C5, and 7.1G10) competed with both 6.3G9 and 6.8G6 for binding to αvβ6 (Table 4.1, and FIGS. 10A and 10B). Antibodies 6.1A8 and 7.7G5 showed less competition, probably due to their lower affinity for αvβ6. None of the non-blocking antibodies or the anti-αv antibody L230 showed any competition with 6.3G9 or 6.8G6 in this assay. These results indicate that the αvβ6-specific blocking antibodies bind to the same or overlapping epitopes on αvβ6.







TABLE 4.1







Epitope Mapping by Antibody Competition














Competition
Competition




Consensus
with biotin-
with biotin-



Clone
Blocker?
ylated 6.8G6
ylated 6.3G9







6.3G9
Y
+++
+++



6.2B1
Y
+++
+++



6.8G6
Y
+++
++



7.1C5
Y
+++
+++



7.1G10
Y
+++
+++



7.7G5
Y
++
+



6.1A8
Y
++
+



6.2A1
N
−
−



6.2E5
N
−
−



6.2G2
N
−
−



6.4B4
N
−
−



7.8B3
N
−
−



7.8C9
N
−
−



L230
Y
−
−










Example 9: CDR Sequences
The cDNAs for some of the purified monoclonal antibodies were isolated and sequenced using standard techniques as described in Coligan et al. (eds), Current Protocols in Immunology, Wiley, Media, Pa. (2001). The deduced amino acid sequences are shown in FIGS. 7A and 7B.
Amino acid sequences of the heavy chain CDRs of high affinity binders 6.8G6, 6.1A8, 6.2B1, 6.3G9 and 6.2A1 and of nonblocker 6.2G2 are compared as follows (dashes indicate gaps).












CDR1










6.8G6
SYTFTDYAMH (SEQ ID NO: 1)



 



6.1A8
SYTFTDYTMH (SEQ ID NO: 2)



 



6.2B1
GFTFSRYVMS (SEQ ID NO: 3)



 



6.3G9
GFTFSRYVMS (SEQ ID NO: 3)



 



6.2A1
GYDFNNDLIE (SEQ ID NO: 49)



 



6.2G2
GYAFTNYLIE (SEQ ID NO: 50)



 







CDR2










6.8G6
VISTYYGNTNYNQKFKG (SEQ ID NO: 4)



 



6.1A8
VIDTYYGKTNYNQKFEG (SEQ ID NO: 46)



 



6.2B1
SISSG-GSTYYPDSVKG (SEQ ID NO: 5)



 



6.3G9
SISSG-GRMYYPDTVKG (SEQ ID NO: 6)



 



6.2A1
VINPGSGRTNYNEKFKG (SEQ ID NO: 51)



 



6.2G2
VISPGSGIINYNEKFKG (SEQ ID NO: 52)



 



 







CDR3










6.8G6
GGLRRGDRPSLRYAMDY (SEQ ID NO: 7)



 



6.1A8
GGFRRGDRPSLRYAMDS (SEQ ID NO: 47)



 



6.2B1
GAIYDG-----YYVFAY (SEQ ID NO: 8)



 



6.3G9
GSIYDG-----YYVFPY (SEQ ID NO: 9)



 



6.2A1
IYYGPH-----SYAMDY (SEQ ID NO: 53)



 



6.2G2
ID-YSG-----PYAVDD (SEQ ID NO: 54)










In SEQ ID NO:7, the “R” in boldface (the twelfth residue) indicates that it is subject to polymorphism and can be, for example, a Q.
Amino acid sequences of the light chain CDRs of these four high affinity binders and of nonblocker 6.2G2 are compared as follows.












CDR1










6.8G6
RASQSVSTSS-YSYMY (SEQ ID NO: 10)



 



6.1A8
RASQSVSIST-YSYIH (SEQ ID NO: 48)



 



6.2B1
SASSSVSSS----YLY (SEQ ID NO: 11)



 



6.3G9
SANSSVSSS----YLY (SEQ ID NO: 12)



 



6.2A1
KASLDVRTAVA (SEQ ID NO: 55)



 



6.2G2
KASQAVNTAVA (SEQ ID NO: 56)



 







CDR2










6.8G6
YASNLES (SEQ ID NO: 13)



 



6.1A8
YASNLES (SEQ ID NO: 13)



 



6.2B1
STSNLAS (SEQ ID NO: 14)



 



6.3G9
STSNLAS (SEQ ID NO: 14)



 



6.2A1
SASYRYT (SEQ ID NO: 57)



 



6.2G2
SASYQYT (SEQ ID NO: 58)



 







CDR3










6.8G6
QHNWEIPFT (SEQ ID NO: 15)



 



6.1A8
QHSWEIPYT (SEQ ID NO: 16)



 



6.2B1
HQWSSYPPT (SEQ ID NO: 17)



 



6.3G9
HQWSTYPPT (SEQ ID NO: 18)



 



6.2A1
QQHYGIPWT (SEQ ID NO: 59)



 



6.2G2
QHHYGVPWT (SEQ ID NO: 60)










As shown in FIGS. 7A and 7B, the mAbs that fall into the divalent cation-dependent class (e.g., 6.1A8 and 6.8G6) seem to contain very similar amino acid sequences within the CDRs, while the divalent cation-independent mAbs (e.g., 6.2B1 and 6.3G9) contain another set of motifs in their CDRs.
The potency and specificity of anti-αvβ6 monoclonal antibodies may be governed by subtly different amino acid residues. In the case of 6.1A8 and 6.8G6, the amino acid sequences of the variable domains are very similar, containing 10 amino acid differences in the heavy chain, three of which are conservative, and 11 amino acid differences in the light chain. Yet these antibodies have a roughly 100-fold difference in activity in in vitro assays. The amino acid differences are dispersed throughout the variable domains of the polypeptide chains and these residues may function alone or synergistically with residues on the same chain or the partner chain to affect the potency of the antibodies. In the heavy chain, seven residues are located such that they are likely to be in close proximity to, or play an active role in binding to, αvβ6.
An RGD motif is found in a number of integrin-binding proteins (ligands). This motif has been shown to mediate their interaction with integrins by directly contacting the binding pocket on the integrin. Because RGD itself is fairly common among integrin-binding proteins, flanking residues outside the motif must play a role in conferring binding specificity to the integrin-ligand interaction. In 6.1A8 and 6.8G6, one such flanking residue is at position 101 in the heavy chain, within CDR3. This amino acid residue flanks the RGD motif and may be located at the site of antigen recognition, contributing to binding potency and specificity.
Other different residues within the same heavy chain CDRs of 6.1A8 and 6.8G6 include those at positions 33 (CDR1); positions 52, 57, and 65 (CDR2); and position 115 (CDR3). Another difference in the heavy chain lies at position 4 in framework 1, which is near the N-terminus. This residue is predicted by crystallographic models to fold close to the CDRs of the antibody and may play an important role in αvβ6 binding. The three remaining differences between 6.1A8 and 6.8G6 are conservative differences at positions 20 (framework 1), 44 (framework 2) and 82 (framework 3).
The amino acid sequences of the cation-independent antibodies are also highly homologous. They can be divided into two classes: those that compete with the RGD-containing antibody 6.8G6 (i.e., 6.2B1, 6.3G9, 7.10H2, 7.9H5, 7.1C5, 7.1G10, and 7.4A3); and those that do not (i.e., 6.2A1, 6.2B10 and 6.4B4). The 6.8G6-competing class contains a FXY motif in CDR3 of the heavy chain, whereas the noncompeting class does not. This difference suggests that the FXY motif is important for mediating cation-independent binding to αvβ6. Additionally, this FXY-containing class of antibodies probably bind to an epitope on αvβ6 that is overlapping with, yet distinct from, the RGD-binding pocket. Antibodies 6.2B10 and 6.4B4 do not contain an FXY motif and are poor αvβ6 blockers. They were shown to bind to the αvβ6 I-domain-like portion and define yet another epitope to which anti-αvβ6 antibodies bind. Interestingly, the monoclonal antibody 6.2A1 belongs to the cation-independent class but does not contain the RGD sequence, as with other cation-independent mAbs.
Monoclonal antibody 7.7G5 belongs to the cation-dependent class. However, the light chain sequence of 7.7G5 is highly homologous to the cation-independent, I-domain binding antibody 6.2B10. The heavy chain of 7.7G5 is also similar to cation-independent antibodies in CDR1. Yet its CDR2 and CDR3 are more similar to those of the cation-dependent class. This observation suggests that specific CDRs confer specificity to an antibody. This is particularly true for CDR3 of the heavy chain, presumably due to the high degree of variability within this part of the antibody. In fact, two out of three cation-dependent and seven out of nine cation-independent antibodies contain heavy chain CDR3 sequences that are likely to play an important role in αvβ6 recognition. Of note, 7.7G5 lacks an RGD motif but contains an XGD motif in its heavy chain CDR2. This XGD motif may function in a similar fashion to RGD and confer binding affinity/specificity to 7.7G5.
The above sequence observations and inferences made therefrom provide a basis for rational design of specific variable region amino acid sequences that confer specific binding properties.
Example 10: Diagnostic Antibodies
Antibodies that can detect αvβ6 expression in paraffin-embedded tissue sections or other tissue samples may be useful as diagnostics. These diagnostic tools can be used to, e.g., detect upregulated αvβ6 in tissue sections for such indications as cancer or fibrosis.
In order to identify antibodies that detect αvβ6 in paraffin-embedded tissues, we first screened a panel of antibodies for binding to HPLC-purified β6 subunit. Antibodies which bind this subunit are likely recognizing linear peptide epitopes, and were therefore expected to have a greater likelihood for success in paraffin-embedded tissues. Binding to purified β6 subunit was carried out using an ELISA format identical to that described for measuring αvβ6 binding (supra), except with the purified β6 integrin, rather than the αvβ6 protein, immobilized on the plate. Using this method, a number of Fusion 6 antibodies capable of binding both the purified αvβ6 protein and the purified β6 subunit were identified. See Table 5, infra, where the prefix “6.” in the clone names is omitted.







TABLE 5







Antibody Binding to Purified ανβ6 or Purified β6 Subunit










Binding to 
Binding to HPLC-


Clone name
ανβ6
purified β6 subunit





1A1
+
6


1A8
+
−


1A11
+
+


1E1
+
+


1E6
+
+


1H10
+/−
−


2A1
+
+


2A10
−
−


2B8
−
−


2B10
+
−


2C4
+
+


2C7
+
+


2E5
+
+


2G3
+
−


3A6
+
−


3B1
+
−


3B2
+
+


3B11
+
+


3C2
+
+


3D5
+
−


3D10
+
+


3F1
+
−


3G3
−
−


3G5
+
−


3G9
+
−


3H2
+
−


3H11
+
−


4A4
+/−
+


4B2
+
−


4B4
+
−


4B10
+
−


4D2
+
−


4E4
+
+


4G3
+
−


4G4
+
+


4H4
+
+


4H12
+
−


5A2
+
−


5B6
+
+


5D6
+
+


5D8
−
−


5G9
+
+


5G10
+
+


5H3
+
+


6B1
+
+


6B5
+
+


6C4
+
−


6D12
+
+


6E6
+
−


6E10
+
−


6G3
+/−
−


7C7
+
+


7E5
+
−


7F8
+
−


8B4
+
+


8G6
+
−


9B5
+
+


9B7
+
+


9B9
+
−


9B10
+
−


9D11
+
+


9E12
+
+


9F5
+
+


9F7
−
−


9G1
+
−


9H11
+
−


10A2
+
−


10A3
+/−
−


10A4
+
−


10A8
+
−


10A9
+
−


10B10
+
−


10D3
+
−


10D11
+
−


10E4
+
+


10F1
+
−


10F12
+
−


10G1
+
−


10G2
−
−


10H11
+
+


1A7
+
−


1D6
+
−


1D9
+
−


1F6
+
−


2B1
+
−


2D9
+
+


2G2
+
+


3D9
+
+


3E5
+
−


4C12
+
−


4E6
+
+


4G5
+
−


5A3
+
−


5B3
+
−


5B8
+
+


5B9
+
−


5F7
+
−


6C10
+
+


6D8
+
−


6F4
+
−


6G9
+
−


6H8
+
+


6H9
+
−


7A5
+
+


7A11
+
−


7E6
+
+


7G9
−
+


7H3
+
−


9A2
−
−


9A3
+
+


9A4
−
−


9A9
+
−


9D2
−
−


9D3
−
−


9E4
−
−


9F1
+
−


9F12
−
−


9G11
−
−


10B5
+
−


10B7
+
−


10B9
+/−
−


10C5
+/−
−


10C7
+/−
−


10D6
+/−
−


10E12
+/−
−


10F7
−
−









As shown above, some antibodies bound to purified β6 subunit. They will have a high likelihood to bind to denatured αvβ6 and thus can be useful in detecting αvβ6 in paraffin-embedded tissue sections. Other antibodies bound to soluble αvβ6 but not the β6 subunit. Both types of antibodies were used to stain denatured paraffin-embedded β6-transfected SW480 cells and untransfected parent cells, and the data are shown in Table 6.
To stain paraffin-embedded tissues or cells, the sample slides were first de-paraffinized by incubation in the following solutions: (1) Xylene, 5 min, twice; (2) 100% ethanol, 2 min, twice; (3) 95% ethanol, 2 min, twice; (4) 50% ethanol, 2 min, once; and (5) distilled water, 2 min, once. The slides were then incubated in a solution consisting of 200 ml of methanol and 3 ml of 30% H2O2 for 15 min to block endogenous peroxidase. The slides were rinsed twice in PBS for 2 min each time. The paraffin sections on the slides were then unmasked with pepsin (Zymed 00-3009) for 5 min at 37° C. The slides were rinsed again twice in PBS for 2 min each time. Next, the slides were blocked with avidin and then biotin (Vector SP-2001; Vector Laboratories, Burlingame, Calif.), 10 min each at room temperature, with washing between each incubation as described above. After the blocking solution was drained off the slides, the primary antibody (hybridoma culture supernatant) diluted in PBS/0.1% BSA was applied to the slides and incubated overnight at 4° C.
The next day, the slides were rinsed in PBS as described above. Meanwhile, the avidin-biotin complex-horseradish peroxidase solution (ABC reagent) was prepared as follows: 1 ml of PBS was mixed with 20 μl of solution A (1:50) and 20 μl of solution B (1:50) from Vector Kit PK-6102; and the mixture was incubated for 30 min at room temperature before use. During this time, the slides were incubated for 30 min at room temperature with anti-mouse-biotinylated antibody (1:200) from the Vector Kit with 15 μl/ml normal serum. The slides were then rinsed twice in PBS, 2 min each time. Then the above-described ABC reagent was applied to the slides and incubated for 30 min at room temperature. The slides were rinsed again as described above. Then the substrate (Vector SK-4100), 100 μl of DAB (3,3′-diaminobenzidine), were applied to the slides and incubated for 5 min at room temperature. DAB was prepared as follows: to 5 ml of H2O, add 2 drops of Buffer Stock Solution, mix well; then add 4 drops of DAB Stock solution, mix well; and then add 2 drops of H2O2 Solution, mix well. Then the slides were rinsed in running water for 2 min. Next, the DAB signal was enhanced for all slides as follows: rinse the paraffin sections in 0.05 M sodium bicarbonate, pH 9.6, for 10 min; blot excess buffer; apply the DAB Enhancing Solution 15 seconds; and then quickly rinse with water for 1 min to stop reaction. The slides were then stained in Mayer's Hematoxylin (a nuclear counterstain) for 1 min. The slides were rinsed in running water for 1 min, and then submerged in PBS for 1 min so that the hematoxylin turned blue. The slides were then rinsed again in running water for 1 min and dehydrated and cleared as follows: submerge in (1) 95% ethanol for 1 min, twice; (2) 100% ethanol for 1 min, twice; and (3) Xylene for 2 min, twice. Coverslips were then applied to the slides using permount.
The results suggested that Fusion 6 antibodies 1A1, 2C4, 3B2, 3B11, 5D6, 5G9, 5H3, 6D12, 7C7, 9B5, 9B7, 9D11, 9F5, 10E4, 10H11, 6H8, 7A5, 7G9, 9A3, 2A1, 2E5, 4E4, 4H4, 8B4, 2G2, and 4E6, all of which could bind to purified β6 subunit (Table 5), indeed stained paraffin-embedded β6-transfected SW480 cells strongly, while not staining untransfected parent cells (Table 6).







TABLE 6







Antibody Binding to Paraffin-Embedded SW480 Cells














Binding to
Binding to



ID#
Clone name
SW480/β6+
SW480
















1
1A1
+++
−



2
1A8
+
+



3
1A11
++
−



4
1E1
+
−



5
1E6
++
+



7
2A1
+++
−



10
2B10
−
−



11
2C4
+++
−



12
2C7
−
−



13
2E5
+++
−



17
3B2
+++
−



18
3B11
++
−



25
3G9
−
−



30
4B4
+
+



33
4E4
+++
−



35
4G4
−
−



36
4H4
+++
−



39
5B6
+
−



40
5D6
+++
−



42
5G9
+++
−



43
5G10
−
−



44
5H3
+++
−



46
6B5
−
−



48
6D12
+++
−



52
7C7
++
−



54
7F8
−
−



55
8B4
+++
−



56
8G6
−
−



57
9B5
++
−



58
9B7
++
−



61
9D11
++
−



62
9E12
+
−



63
9F5
++
−



68
10A3
−
−



75
10E4
+++
−



80
10H11
++
−



85
2B1
+
+



87
2G2
+++
−



91
4E6
+++
−



95
5B8
−
−



98
6C10
+
−



102
6H8
++
−



104
7A5
+++
−



107
7G9
+++
−



110
9A3
+++
−










Example 11: Diagnosis of Cancer
αvβ6 is normally expressed at negligible to low levels in healthy adult tissues. However, αvβ6 expression is upregulated in injury, fibrosis, and cancer (see, e.g., Thomas et al. J. Invest. Dermatology 117:67-73 (2001); Brunton et al., Neoplasia 3: 215-226 (2001); Agrez et al., Int. J. Cancer 81:90-97 (1999); Breuss, J. Cell Science 108:2241-2251 (1995)). Thus, antibodies that bind specifically to αvβ6 expressed on paraffin-embedded tissues can be used in standard immunohistochemistry techniques to detect αvβ6 expression for diagnosis of fibrosis, cancer and any other diseases in which αvβ6 is upregulated.
As described above, certain antibodies of the present invention bind to HPLC-purified β6 integrin and paraffin-embedded and fixed β6-transfected cells. These antibodies were also shown to bind to representative squamous and breast cancer tissues in immunostaining. See, e.g., FIG. 8, where monoclonal antibody 6.2A1 was used to show relative staining of paraffin-embedded breast and squamous carcinomas. Thus, these new antibodies are useful as diagnostic tools.
Example 12: Effects of Anti-αvβ6 Blocking mAbs in Alport Mice
The collagen 4A3 (COL4A3) knockout (Alport) mice have been established as an in vivo model for kidney fibrosis and used to test the therapeutic effects of pharmacological agents (supra). We tested mAb 6.8G6 (cation-dependent) and 6.3G9 (cation-independent) in Alport mice to determine if they would inhibit the fibrosis normally observed in seven week old Alport mice. As shown above, these two antibodies were found to inhibit αvβ6 binding to LAP and to inhibit activation of TGF-β in a bioassay. Antibody 1E6 was used as a negative control.
Three week old Alport mice were given intraperitoneal injections 3 times a week with one of the following antibodies: (1) 6.8G6, 4 mg/kg (7 mice); (2) 6.3G9, 4 mg/kg (4 mice); and (3) 1E6, 1 mg/kg (6 mice). The injections were continued for 4 weeks. The mice were then sacrificed, and their kidneys retrieved.
Paraffin-embedded sections of the kidneys were made as described above, and then stained to detect smooth muscle actin, a marker for myoblasts and matrix deposition in kidney fibrosis. We found a significant decrease in smooth muscle actin staining in both the interstitial and glomerular regions of the kidney from the Alport mice treated with mAb 6.8G6 or 6.3G9, as compared to mice treated with 1E6.
FIGS. 11A and 11B show a dot plot of smooth muscle actin staining in the glomerular and interstitial regions of the Alport kidney. There was significantly reduced smooth muscle actin staining in the kidneys of the Alport mice treated with 6.8G6 and 6.3G9, as compared to negative control 1E6-treated mice.
Example 13: Effectiveness of Anti-αvβ6 mAbs in Preventing Unilateral Ureteral Obstruction-Induced Nephrosclerosis
We used another mouse model for renal fibrotic progression to test the antifibrotic efficacy of 6.8G6 and 6.3G9. In this mouse model, a ureter of the animal is ligated, resulting in unilateral ureteral obstruction (UUO). UUO causes progressive nephrosclerosis without near-term renal failure in mice because the unobstructed kidney can maintain relatively normal renal function. While the obstructed kidney undergoes rapid global fibrosis, the unobstructed kidney undergoes adaptive hypertrophy.
This study quantitated morphometrically the impact of anti-αvβ6 treatment on UUO-induced renal fibrosis. Male, 8-12 week old, viral antigen-free C57BL mice of 25.5±0.2 g in weight (Jackson Laboratories, Bar Harbor, Me.) were used. The mice were allowed to accommodate for seven days prior to beginning the study. The mice had ad libitum access to irradiated standard mouse chow and sterile water throughout the accommodation and experimental period. Body weight was measured at intervals as part of animal health monitoring. The results showed that age-matched unoperated mice gained about 10% body weight over the two-week study period. UUO mice lost about 9% body weight by day 2 but gradually regained the lost body weight by day 14. This weight change pattern occurred irrespective of therapeutic treatment.
To induce renal fibrosis, the left ureter was aseptically isolated via a left-of-midline laparotomy under ketamine:xylazine (100:10 mg/kg s.c.) anesthesia. Two tight, occlusive 6-0 silk ligatures were placed on the ureter at the level of the lower pole of the kidney, and the ureter cut between the ligatures. The abdominal wall was closed with 4-0 Vicryl suture and the skin closed with 4-0 nylon. The animals were allowed to recover on a heating pad and given 0.05 mg/kg s.c. buprenorphine twice daily on days 0 and 1. The procedure was adapted from Ma et al., Kidney Int. 53 (4):937-944 (1998).
The mice were then divided into the following study groups:















Dose 



Group
Treatment
(mg/kg, i.p.)
n*


















1
6.8G6 (cation dependent anti-ανβ6 mAb)
4
9


2
6.3G9 (cation-independent anti-ανβ6 mAb) 
4
8


3
1E6 (negative Control mAb)
4
10


4
PBS Vehicle Control
(100 μl)
8


5
Unoperated, Untreated Control
—
8





*Number of animals.






All animals except those in Group 5 were dosed twice weekly beginning on the day before surgery.

The animals were then euthanized with carbon dioxide at day 10 after ligation and dissected. In UUO mice, the renal pelvis and ureter were markedly swollen and fluid-filled above the obstructing ligature. The degree of swelling and extent of remaining renal tissue mass varied among treatment groups. Group 2 showed about half as much swelling as negative control groups. Ligated kidneys were pale in color. Contralateral kidneys were bright red and enlarged by about one third.
Next, both kidneys (left ligated, right unligated) of the animals were removed and halved transversely through the center of the renal pelvis. One half of each kidney was placed in 10% neutral buffered formalin for fixed-tissue staining. The other half of each kidney was placed in 15% sucrose, followed by 30% sucrose, for immunohistochemical staining.
Formalin-fixed kidney sections were immunostained for myofibroblasts (smooth muscle actin), a marker of fibrosis. Images were captured using standardized lighting conditions and digital cameral exposure settings, corrected for background, and calibrated to distance standards. Images of contiguous fields covering the entire left kidney section were taken from each animal for quantitation.
Smooth muscle actin was expressed as a percent of total tissue area within the measured fields. These included all cortical and medullary tissue from the section except the renal papilla.
In conclusion, mice treated with 6.3G9 and 6.8G6 show a significant reduction in fibrosis.
Example 14: Effectiveness of Anti-αvβ6 Blocking mAbs Against Bleomycin-Induced Lung Fibrosis in Mice
Bleomycin-induced lung fibrosis in mice has been established as an in vivo model for lung fibrosis and used to test the therapeutic effects of pharmacological agents. Inflammation is normally evident 5-15 days following bleomycin treatment. In 129 strain mice, the degree of pulmonary fibrosis progressively increases for up to 60 days after bleomycin treatment. Matrix accumulation usually becomes detectable around day 15. In this example, mAb 6.3G9 was injected intraperitoneally at a concentration of 4 mg/kg/dose into mice with bleomycin-induced lung fibrosis starting at day 0 or day 15, three times weekly. Lung fibrosis was induced at day 0 by administering a single intratracheal dose of bleomycin at a concentration of 0.03 units/kg in 50 μL of sterile saline. The animals were sacrificed at day 30 and the extent of lung fibrosis was assessed. Antibody 1E6 was used as a negative control.
Lungs were harvested from each animal and hydroxyproline content was measured as an index of lung collagen deposition, as described in Munger et al., supra. As shown in FIG. 15A, treatment with 6.3G9 beginning at day 0 significantly inhibited the bleomycin-induced increase in lung hydroxyproline content. Importantly, the 6.3G9 treatment was at least as effective when it began 15 days after bleomycin administration, a time when collagen deposition had already begun.
We also examined the effects of 6.3G9, cation-dependent 6.8G6, and the non-blocking antibody 6.4B4 in inhibiting more substantial degrees of lung fibrosis in an extended bleomycin-induced fibrosis protocol (lasting 60 days). To do this, we started the antibody treatments 15 days after bleomycin administration (day 15). Lungs were then harvested at day 60 to determine hydroxyproline content. As shown by FIG. 15C, treatment with 6.8G6 significantly inhibited bleomycin-induced fibrosis (a more than 70% reduction in hyrdroxyproline content compared to animals treated with bleomycin and saline). Treatment with 6.3G9 also showed a trend toward protection, but these results did not reach statistical significance (FIG. 15C).
In conclusion, both cation-dependent and cation-independent anti-αVβ6 blocking mAbs reduced lung fibrosis in mice treated with bleomycin. Furthermore, this intervention was effective even when antibody treatment was not initiated until after the initial onset of fibrosis.
Example 15: Upregulation of αvβ6 in Human Psoriasis Lesions
In order to determine whether αvβ6 is involved in psoriasis, αvβ6 expression was examined on lesional and nonlesional skin biopsies from five psoriasis patients and four normal individuals. Using mAb 6.2A1 immunostaining, we found a significant increase in αvβ6 expression in psoriatic lesions, as compared to nonlesional skin from psoriatic patients and normal controls. Thus, the upregulation of αvβ6 in psoriatic lesions suggests both diagnostic and therapeutic implication for the use of anti-αvβ6 antibodies.
Example 16: Upregulation in Mouse and Human Liver with Biliary Duct Disease
As previously discussed, αvβ6 expression has been implicated in tissue injury. In this study, expression of αvβ6 was investigated in mouse and human liver injured by biliary duct disease.
Hepatic injury in mice was induced by ligation of the biliary duct. See, e.g., George et al., PNAS 96:12719-24 (1999); George et al., Am J Pathol 156:115-24 (2000). Using mAb 6.2G2, we found that expression of αvβ6 was significantly elevated at days 9, 14 and 16 following biliary duct ligation.
Similarly, human liver sections from patients with biliary duct disease displayed upregulated expression of αvβ6, as determined by immunohistochemistry using the mAb 6.2G2. Elevated expression of αvβ6 was observed for example, in liver samples from a 44 year old male with acute cholestasis, a post-transplant 59 year old male with acute bile duct obstruction, a 22 year old male with biliary atresia, and a 24 year old male with chronic bile duct obstruction.
In sum, the new anti-αvβ6 antibodies are useful diagnostic and therapeutic tools for liver diseases.
Example 17: Upregulation of αvβ6 in Various Human Cancers
Integrin αvβ6 is normally expressed at negligible to low levels in healthy adult tissues. A variety of human tumor tissues was evaluated for αvβ6 expression using the antibody 6.2A1 and methods generally described herein. The results showed that αvβ6 integrin expression was significantly upregulated in several human epithelial cancers. Notably, immunohistology showed that αvβ6 was expressed especially prominently on the edges of tumor islands in many of the epithelial cancers. To further study expression of αvβ6 in epithelial cancer cells, Detroit 562 cells (pharynx carcinoma) and SCC-14 cells (tongue squamous cell carcinoma) as well as SW480β6 cells (supra) were stained with 6.3G9 and 6.4B4 and analyzed by flow cytometry.
The right side of FIG. 12 shows αvβ6 expression on the different tumor cell lines as indicated by 6.3G9 binding in fluorescence activated cell sorting (FACS). The solid peak represents 6.3G9 binding while the open peak represents background binding of the secondary mAb alone. The line graph on the left side of FIG. 12 shows inhibition of the tumor cell lines' binding to the LAP ligand by increasing concentrations of 6.3G9 or 6.4B4. 6.4B4 was a significantly less potent inhibitor of αvβ6 binding to LAP, compared to 6.3G9 (>10-fold IC50 for Detroit 562, >30-fold IC50 for SW480136, and >100-fold IC50 for SCC-14). This is consistent with previous in vitro results indicating that 6.3G9 is a potent blocking mAb and 6.4B4 is a weak blocking mAb. This data is also consistent with negligible inhibitory activity of 6.4B4 in the Detroit xenograft model (FIG. 14B). FIG. 13 further shows the relative inhibition of tumor cell lines' binding to LAP by various anti-αvβ6 mAbs. Both 6.3G9 and 6.8G6 displayed equivalent inhibiting activity (consistent with all previous data) while 6.4B4 was a significantly less potent inhibitor of αvβ6 binding to LAP.
Example 18: Effects of Anti-αvβ6 Blocking mAbs in a Human Tumor Xenograft Model
Immunodeficient animals (e.g., nude mice and SCID mice) transplanted with human tumor xenografts have been established as a useful in vivo model system to test the therapeutic effects of anti-cancer agents (see, e.g., van Weerden et al., Prostate 43(4):263-71 (2000); Bankert et al., Front Biosci 7:c44-62 (2002)). Thus, the blocking anti-αvβ6 monoclonal antibodies of the present invention can be tested in vivo in a xenograft model for their ability to inhibit tumor growth. In this experiment, we tested the ability of some of the new αvβ6 antibodies to inhibit tumor growth in athymic nude female mice transplanted with cancerous human pharyngeal (Detroit 562 cell line) xenograft.
To do this, Detroit 562 cells (ATCC) were passed in vitro in Minimum essential medium (Eagle) with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids, and 1.0 mM sodium pyruvate, and 10% fetal bovine serum, without antibiotics. About 5×106 cells/0.2 ml media (without serum) were implanted subcutaneously into nude mice on the right flank. Three to four days later, tumor size measurements were started and continued until the tumors were about 5 mm (length) by 5 mm (width). The mice were randomized and injected intraperitoneally with the test antibodies or control solutions on day 1, followed by three injections weekly for a period of 33 days. The test antibodies and control solutions were: (1) 6.3G9, 1 mg/kg, 10 mice; (2) 6.3G9, 4 mg/kg, 10 mice; (3) 6.3G9, 10 mg/kg, 10 mice; (4) 6.4B4, 1 mg/kg, 10 mice; (5) 6.4B4, 4 mg/kg, 10 mice; (6) 6.4B4, 10 mg/kg, 10 mice; and (7) vehicle control (PBS), 0.2 ml/per mouse, 30 mice. In addition, cis-platinum was injected into 10 mice subcutaneously at 2 mg/kg as a chemotherapeutical control. The cis-platinum injections were done on day 1 and then every 2 days for a total of six treatments. At the end of the 33 day period, animal weights and tumor sizes were measured, αvβ6 expression assessed by immunohistology, and serum anti-αvβ6 levels measured.
Immunohistological staining showed that the implanted tumor cells strongly expressed αvβ6 in vivo. Tumor weight data further showed that blocking mAb 6.3G9 effectively inhibited tumor growth at all three concentrations tested (FIG. 14A). In contrast, weak blocking mAb 6.4B4 did not inhibit tumor growth (FIG. 14B).
In sum, blocking antibodies inhibited tumor growth by 40-50% in the treated mice. In contrast, a weak blocking anti-αvβ6 antibody did not inhibit tumor growth.
Example 19: αvβ6 Antibody Internalization
Antibodies that are internalized by cells offer an advantage for certain clinical indications such as cancer, because the antibodies can then be conjugated with toxins, radioactive compounds or other anti-cancer agents to selectively target and inhibit growth of cancer cells. The ability of anti-αvβ6 antibodies to be internalized was studied in SW48006 (supra) and SCC-14 cells.
Cells were split 1:5 and plated onto 4-chambered glass slides for overnight incubation at 37° C., 5% CO2. The next day, mAbs 6.8G6, 6.1A8, 6.3G9, 7.1C5, 6.4B4, 10D5, and 8B3 were diluted to a final concentration of 20 μg/mL. The mAbs or medium alone were added to appropriate wells. A time course of internalization was run from 0 h to 48 h. Time points included were 0, 5, 10 and 30 min, and 1, 4, 24 and 48 h. A secondary antibody (anti-murine-Alexa 594) was added as a negative control. Internalization was stopped at each time point by removing the antibody and washing the cell layer with buffer. Wheat Germ Agglutinin-Alexa-488 was added for 20 min at 18° C. to stain the outer edge of the cells with green fluorescence. After the cells were washed, Cytofix/Cytoperm solution was added for 20 min at 18° C. to fix and permeabilize the cells. The cells were washed again and the secondary anti-mouse-Alexa 594 (red fluorescence) was added for 20 min at 18° C. to label the bound or internalized murine αvβ6 antibody. The cells were then washed and fixed by addition of 2% paraformaldehyde and examined by confocal microscopy. Images were then taken with a Leitz Plan-Apochromatic 63× (1.32 numerical aperture, ail immersion) objective (Leica) with a 2× digital zoom. Each frame represented a single optical section from the middle section of the cells observed for internalization under all conditions. There was no staining observed in the nucleus.
Internalization was observed for cation-dependent mAbs (RGD-containing ligand mimetics) such as 6.8G6 and 6.1A8. No internalization was observed for cation-independent mAbs such as 6.3G9, 7.1C5, and 6.4B4.

## Claims

1. A DNA encoding a heavy chain of an anti-αvβ6 antibody or antigen-binding fragment thereof, the humanized heavy chain comprising a heavy chain variable region comprising complementarity determining regions (CDRs) 1, 2, and 3 consisting of the amino acid sequences set forth in SEQ ID NOs.: 3, 6, and 9, respectively.

2. A DNA encoding a light chain of an anti-αvβ6 antibody or antigen-binding fragment thereof, the light chain comprising a light chain variable region comprising complementarity determining regions (CDRs) 1, 2, and 3 consisting of the amino acid sequences set forth in SEQ ID NOs.: 12, 14, and 18, respectively.

3. A host cell producing an anti-αvβ6 antibody or antigen-binding fragment thereof, comprising:
a first DNA encoding a heavy chain of the anti-αvβ6 antibody or antigen-binding fragment thereof, the heavy chain comprising a heavy chain variable region comprising complementarity determining regions (CDRs) 1, 2, and 3 consisting of the amino acid sequences set forth in SEQ ID NOs.: 3, 6, and 9, respectively; and
a second DNA encoding a light chain of the anti-αvβ6 antibody or antigen-binding fragment thereof, the light chain comprising a light chain variable region comprising CDRs 1, 2, and 3 consisting of the amino acid sequences set forth in SEQ ID NOs.: 12, 14, and 18, respectively.

4. The host cell of claim 3, wherein the host cell is a CHO cell.

5. The host cell of claim 3, wherein the host cell is an NSO cell.

6. A method of producing a soluble anti-αvβ6 antibody or antigen-binding fragment thereof, the method comprising culturing the host cell of claim 3 under conditions that produce the soluble anti-αvβ6 antibody or antigen-binding fragment thereof.

7. The method of claim 6, wherein the host cell is a CHO cell.

8. The method of claim 6, wherein the host cell is an NSO cell.