CA2478833A1 - Anti-.alpha.v.beta.6 antibodies - Google Patents

Anti-.alpha.v.beta.6 antibodies Download PDF

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Publication number
CA2478833A1
CA2478833A1 CA002478833A CA2478833A CA2478833A1 CA 2478833 A1 CA2478833 A1 CA 2478833A1 CA 002478833 A CA002478833 A CA 002478833A CA 2478833 A CA2478833 A CA 2478833A CA 2478833 A1 CA2478833 A1 CA 2478833A1
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Canada
Prior art keywords
antibody
seq
beta
alpha
light chain
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CA2478833C (en
Inventor
Shelia M. Violette
Paul H. Weinreb
Kenneth J. Simon
Diane R. Leone
Dean Sheppard
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University of California
Biogen MA Inc
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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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.

Description

ANTI-a,.~i6 ANTIBODIES
FIELD OF THE INVENTION
This invention relates generally to the field of molecular biology and specifically to antibodies to c~,~i6 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, a and ~3. The binding specificity of integrins is dictated by the combination of some 1 ~ different a chains with some ~ different ~ chains. The c~,,~6 integrin can bind to a number of ligands including fibronectin, tenascin, vitronectin, and the recently identified latency associated peptide "LAP," a 27~
amino acid peptide synthesized as part of the precursor TGF-(3 protein (Munger et al., Cell 96(3):319-32~ (1999)). LAP is cleaved from the mature form of TGF-~3 as an N-terminal peptide during secretion but remains noncovalently associated with TGF-(3 to maintain its latent state. This complex cannot bind to the TGF-~3 receptor and hence is not biologically active. The a,,(36 integrin can bind directly to an RGD motif contained within LAP, resulting in release of LAP and activation of TGF-(3. Since c~,~36 s binding to LAP may be important in the conversion of TGF-,Q
to its active state, blocking the binding may result in inhibition of a~/36-mediated activation of TGF-(3 and the associated fibrotic pathology.
SUMMARY OF THE INVENTION
This invention is based on the discovery and characterization of high affinity antibodies against «,,~36, 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 c~,~i6; (b) inhibits the binding of a,,,~6 to its ligand such as LAP, fibronectin, vitronectin, and tenascin with an ICS value lower than that of l ODS
(International Patent Application Publication WO 99/07405); (c) blocks activation of TGF-~3;
(d) contains certain amino acid sequences in the CDRs (e.g., those shown in Figs.

and 7B) that provide binding specificity to a,,~i6; (e) specifically binds to the X36 subunit; and/or (f) recognizes c~,~36 in immunostaining procedures, such as immunostaining of paraffin-embedded tissues.
It has been discovered that antibodies that bind to a,,(36 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 a"~i6 (blockers). This class of antibodies can be further divided into subclasses of cation-dependent Mockers 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 a,,(36 and yet does not block binding of a,,~i6 to a ligand (nonblockers).
Accordingly, in some embodiments of this invention, some antibodies of this invention are divalent cation-dependent for binding to a",~6, 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, 6.369, 6.866, 6.2B1, 6.2B10, 6.2A1, 6.2E5, 7.1610, 7.765, 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:l, 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 l, 2 and 3 consist essentially of the sequences of SEQ ID NOs:l l, 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 117 NOs:l2, 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 l, 2 and 3 consist essentially of the sequences of SEQ 117 NOs:50, 52 and 54, respectively, and/or a light chain whose CDRs 1, 2 and 3 consist essentially of the sequences of SEQ B7 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:l9-36 and 61-62.
In some embodiments, the antibodies comprise heavy and light chain variable domain sequences of (1) SEQ m NOs:l9 and 37;
(2) SEQ )D N0:20 or 21, and SEQ )D N0:38;
(3) SEQ >D NOs:22 and 43;
(4) SEQ 1D NOs:23 and 44;
(5) SEQ m NOs:24 and 45;
(6) SEQ )D N0:25 or 26 and SEQ )D N0:42;
(7) SEQ m N0:27, 28, or 29, and SEQ 117 N0:39;
(8) SEQ )D N0:34 or 35, and SEQ >D N0:40;
(9) SEQ )D NOs:36 and 41;
(10) SEQ m NOs:61 and 63; or
(11) SEQ m NOs:62 and 64, respectively.
In some embodiments, the antibodies specifically binds to c~,~i6 but does not inhibit the binding of c~,(36 to latency associated peptide (LAP). At least some of these antibodies are capable of binding to a,,~i6 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 Garner. 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 ration-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 c~,~36, 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 a,,~ib 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, 6.369, 6.866, 6.2B1, 6.2A1, 6.2E5, 7.1610, 7.765, and 7.1C5; isolated nucleic acids comprising a coding sequence for any one of SEQ ID NOs:l9-45 and 61-64;
isolated polypeptides comprising an amino acid sequence of any one of SEQ ID
NOs:l9-45 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., IgAl and IgA2), IgG (e.g., IgGl, 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 ammo 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, 6.369, 6.866, and 6.2B1 were deposited on August 16, 2001, and have accession numbers ATCC PTA-3647, -3648, -3649, -3645, and -3646, respectively.
Hybridoma clones 6.2A1, 6.2E5, 7.1610, 7.765, and 7.1C5 were deposited on December 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 a"/36 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 a,,~i6 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. 1 A and 1 B are bar graphs showing the results of ~a cell capture assay that determined the ability of various anti-c~,(36 monoclonal antibodies ("mAb") to bind (36-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-aV~36 "Fusion 6" monoclonal antibodies to bind soluble recombinant human a,,(36 ("hsc~,(36"). These antibodies were generated by immunizing ~i6-/- mice with soluble human truncated c~,(36.
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-a,,~36 "Fusion 7" monoclonal antibodies to bind soluble recombinant hsc~,~36. These antibodies were generated by immunizing (36-/- mice with ~i6-transfected NIH 3T3 cells (Fusion #7).
Figs. 3A-F are graphs showing the differential cation dependence of the binding of various anti-x,(36 monoclonal antibodies to hsa,,~36.
Figs. 4A and 4B are graphs showing that Fusion #6 and Fusion #7 monoclonal antibodies, respectively, inhibit the binding of biotin-hsa~(36 to LAP.
Figs. SA-E are graphs showing that exemplary monoclonal antibodies of the invention inhibit the binding of (36-transfected FDC-P1 cells to LAP. Figs. SA and SB display the results from Fusion #6 antibodies. Figs. SC-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 c~.~36-mediated activation of TGF-Vii, using a PAI-1 luciferase reporter gene assay to monitor TGF-13 activation.
Fig. 7A depicts the amino acid sequences of the variable domains of the heavy chains of a,,~is monoclonal antibodies 6.1A8, 6.866 (subclones A and B), 7.765, 6.2B1, 6.369, 6.2B10 (subclones A and B), 6.262, 6.2A1, 6.4B4 (subclones A, B and C), 7.1 OH2, 7.9H5, 7.4A3 (subclones A and B), 7.1 CS
(subclones A and B) and 7.1610. Antibodies 6. l A8, 6.866 and 7.765 are cation-dependent in binding to a,,(3~, while antibodies 6.2B1, 6.2A1, 6.369, 6.2B10, 6.4B4, 7.1 C5 and 7.1610 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 c~,~36 monoclonal antibodies 6.1A8, 6.866, 6.4B4, 6.2A1, 7.1C5, 7.1610, 6.2B10, 7.765, 6.2B1 and 6.369.

Fig. 8 is a scatter plot showing the expression of a,,,~b in human breast cancer and human squamous carcinoma tissue sections. Normal human tissues show only negligible expression levels of a,,~3s.
Figs. 9A and 9B are quadratic curve graphs depicting the solution binding affinities of two anti-a,,(36 antibodies, 6.866 and 6.369, respectively, for soluble a"(36.
Figs. l0A and lOB are bar graphs demonstrating the ability of purified monoclonal antibodies to compete with biotinylated 6.369 and biotinylated 6.866, respectively, for binding to a,,(36.
Fig. 11 is a bar graph showing percent smooth actin staining in kidneys from WO animals treated with anti-a"(36 mAb treatment.
Fig.l2 shows a~(3s 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 6.369 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-a,,(36 mAbs 6.369, 6.866 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-a,,(36 mAb 6.369 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-a,,(36 mAb on bleomycin-induced lung fibrosis. (A) Antibody treatment using 6.369 mAb was started on day 0 at the time of bleomycin administration and was monitored over a 30 day period; (B) Antibody treatment using 6.369 mAb was started 15 days after bleomycin treatment and was monitored over a 30 day period; (C) Antibody treatment using 6.369, 6.866 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 p,g 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 a,,(36. At least one class of the antibodies (blockers) are capable of blocking the binding of a~(36 to LAP or preventing the activation of TGF-Vii.
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.
Patents 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, (36 -/- animals (e.g., mice, rats or rabbits) are immunized with purified or crude a,,(~6 preparations, cells transfected with cDNA constructs encoding a,,, (36 or both antigens, cells that constitutively express a~~36, 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-a,,~36 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 c~,(36 (e.g., binding to fib-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 c~,~36 with an ICSO value lower than that of known anti-a,,(36 antibody l ODS, or inhibiting TGF-(3 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 10 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., marine, 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 IgGl 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. Imnzunol. 147:86-95 (1991), or using phage-displayed antibody libraries, as described in, e.g., U.S. Patent 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 a,,(36 and hybridomas are then made from B cells derived therefrom. These methods are described in, e.g., the various GenPharnl/Medarex (Palo Alto, CA) publications/patents concerning transgenic mice containing human Ig miniloci (e.g., Lonberg U.S. Patent 5,789,650); the various Abgenix (Fremont, CA) publications/patents with respect to XENOMICE (e.g., Kucherlapati U.S. Patents 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 Gerzetics 16:133-1443 (1997)).
Humanized Antibodies The monoclonal antibodies of this invention also include humanized versions of cognate anti-c~,~i6 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 marine 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 marine antibody. When necessary, one or more residues in the human framework regions can be changed to residues at the corresponding positions in the marine 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
12 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 EP 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. Patent 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., ~yl 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. Patent 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
13 framework of the cognate marine antibody. Then, the tertiary structure of the marine V region is modeled by computer in order to visualize framework amino acid residues that are likely to interact with the marine CDRs, and these marine 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., l l lIn or 9oY) for killing of cells targeted by the antibodies (see, e.g., U.S. Patent 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.
14 Diseased Conditions And Animal Models The antibodies of the invention are useful in the treatment, including prevention, of c~,(36-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-(3 or blocking the binding of a~,~36 to any other ligands, such as fibronectin, vitronectin, and tenascin. The novelty of this approach includes: (1) it blocks the activation of TGF-~3 rather than the binding of TGF-13 to its receptor, (2) it can inhibit TGF-(3 locally (i.e., at sites of a~~i6 upregulation) rather than systemically, and (3) it inhibits binding of c~,(36 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 a,,~ib monoclonal antibodies demonstrated that a,,~36 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 GY~a6, 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 a,,~iG increases in the epithelial alveolar cells of the lungs. But X36 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. a,,~36 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 a,,~36 expression also increases in a 10 variety of kidney injury models.
Anti-ar,(36 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);
15 Wyckoff et al., Cancer Res. 60:2504 (2000); and Oft et al., Curr. Biol.
8:1243 (1998). Important a,,(36 ligands in cancer may include TGF-13, which is involved in.
metastasis (for review see Akhurst et al., Trends in Cell Biology 11:544-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 (BUl~ 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 "earner" 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
16 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 ~f 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. l 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 aV~i6 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,
17 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. Patent 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 arzd 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; Gezze Transfer Vectors for Mammalian Cells (J.H. Miller and M.P. Calos, Eds.),1987; Imtnunochenzical 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.
18 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 (36 -/- 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, MN). Antibody lODS was purchased from Chemicon (Temecula, CA). 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 carned out using the ISOSTRIP kit (Roche Diagnostics) according to the manufacturer's instructions. The (36-transfected SW480 cell line was prepared as described in Weinacker et al., J. Biol. Chem. 269:6940-6948 ( 1994).
Example 1: Generation of (36-transfected stable cell lines X36-transfected NIH 3T3 and FDC-P1 cells were generated by electroporating parent cell lines with a DNA construct containing full length murine ,Q6 cDNA and a neomycin selectable marker. Stably transfected cells were selected by passaging cells in culture medium containing 6418 for 14 days followed by fluorescent activated cell sorting (FACS) to isolate cells expressing the highest level of surface 136. 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/1 glucose, and 1.0 mM sodium pyruvate, 10% FBS, 2.5% mouse IL-3 culture supplement, and 1.5 mg/ml active 6418. Transfected NIH 3T3 cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, penicillin/streptomycin, and 1 mg/ml active 6418.
Example 2~ Purification of human soluble a"~36 The c~,(36 protein was purified essentially as described in Weinacker, supra. A CHO cell line expressing hsa"~i6 was cultured, and the resultant supernatant collected by centrifugation. The integrin was purified by affinity
19 chromatography using anti-a,, antibody L230. Purified L230 was cross-linked to CNBr-activated Sepharose 4B (Sigma) at a ratio of 4.8 mg antibody/ml resin.
The c~,~i6 supernatant was loaded at 0.5 mg antibodylml 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 NaCI, 1 mM MgCl2; (2) 50 mM Tris-Cl, pH 7.5, 50 mM
NaCI, 1 mM MgCl2; and (3) 10 mM Na3P04, pH 7Ø Hsa,,(36 was eluted with 100 mM glycine, pH 2.5 into 1:10 volume of 1 M Na3P04, pH 8Ø The protein was dialyzed with several changes against phosphate buffered saline (PBS) and stored at -20°C.
Example 3: Immunization of as -/- mice ~i6 -/- mice were immunized by intraperitoneal (IP) injection with 25 p,g of purified recombinant'human ar,~i6 emulsified in complete Freund's adjuvant (CFA) at a volume ratio of 1:1 in a total volume of 200 ,ul. Alternatively, ~i6 -/-mice were immunized via IP injection with 4 X 106 (36-transfected NIH 3T3 cells resuspended in 100 p,l 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 ~,1 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-~i6 titers were determined by binding of the serum to purified recombinant human a,,/36 or to (~6-transfected cells. In the case of mice immunized with purified recombinant human ex,,,~6, 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 pg of purified recombinant human a"(36 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 (36 -l- mice. One set of antibodies were generated through immunization with soluble human truncated a"~36 (Fusion #6). The other set of antibodies were generated through immunization with marine (36-transfected NIH 3T3 cells (Fusion #7). Screening for anti-a,,/36 antibodies was carned 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 hsa,,~i~ and (36-transfected human 5 and marine cells (untransfected cells as control). Selected clones were expanded and terminal cultures were re-evaluated for binding to both ,66-transfected and untransfected cells in cell capture assays (Example Sb, infr'a) (representative examples shown in Figs. lA and 1B, where the prefixes "6." or "7." of the mAb names, which denote Fusion 6 and Fusion 7, respectively, are omitted; see also 10 Table 2 below). Some antibodies bound preferentially to the X36-transfected cells, while others bound to both transfected and untransfected cells, indicating that only a subset of the antibodies had a preference for X36 (Figs. lA and 1B). Further selection was based on the ability of the antibodies to block binding of both biotinylated hsc~,~i6 and (36-transfected marine cells to LAP. Select clones were 15 subcloned using FACS and stored frozen until use.
Monoclonal antibodies were screened for specificity of binding to a"~36 based on their ability to bind (36-transfected cells and not untransfected parent cells. Monoclonal antibodies were further confirmed as specific binders of a,,(36 and not of other a,, integrins or non-specific integrins (i.e., non-a,, integrins that
20 bind to RGD-containing ligands) based on their lack of binding to cell lines expressing c~,,~3, c~,(3s, x5(31, a~(3i or as(31. These included stably transfected cells as well as untransfected JY, I~562, 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 August 16, 2001 6.2B 10 PTA-3648 August 16, 2001 6.369 ~ PTA-3649 ~ August 16, 2001
21 6.866 PTA-3645 August 16, 2001 6.2B1 PTA-3646 August 16, 2001 6.2A1 PTA-3896 December 5, 2001 6.2E5 PTA-3897 December 5, 2001 7.1610 PTA-3898 December 5, 2001 7.765 PTA-3899 December 5, 2001 7.1C5 PTA-3900 December 5, 2001 Example 5' Assays for screening and characterization a. a"[6 ELISA
A 96-well microtiter plate (Corning COSTAR EASY-WASH) was coated with 50 ~1/well of 5 p,g/ml hsc~,~36 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 ~.1/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 ,ul/well). The plate was incubated for 1 hr at 25°C, washed, and then incubated for 1 hr with 50 ~.l/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 cauture assay A 96-well microtiter plate was coated with 50 pl/well of secondary antibody (donkey anti-mouse IgG (Jackson Immunoresearch); 5 ~,g/ml diluted in 50 mM sodium bicarbonate, pH 9.2) at 4°C overnight. Plates were washed twice with 100 ~l/well of assay buffer (RPMI + 2% BSA) and then blocked with 100 p.l/well of the assay buffer at 37°C for 1 hr. For FDC-Pl cells and X36-transfected FDC-P1 cells, plates were blocked with anti-mouse Ig (Jackson ImmunoResearch;
22 20 p.g/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 p.M fluorescent dye (Calcein-AM, Molecular Probes) in the assay buffer at 5 X 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 cells/ml. Following the blocking step, the buffer was discarded by flicking the plate, and 25 p.l/well of supernatant or purified antibody was added to the plate.
Following a 15 min incubation at 37°C, 25 p,l/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 pl/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 (1X PBS, 2% FBS, 0.1% NaN3, 1 mM
CaCl2, and 1 mM MgCl2). 0.2 X 105 cells were then incubated on ice for 1 hr in FACS buffer containing hybridoma supernatant in a total volume of 100 pl.
After incubation, the cells were washed two times with ice cold FACS buffer, resuspended in 100 pl of FACS buffer containing 5 pg/ml donkey anti-mouse IgG
PE (Jackson ImrnunoResearch), and incubated on ice for 30 min. The cells were then washed twice with ice cold FACS buffer and resuspended in 200 p,l of FACS
buffer. Binding of the PE labeled secondary antibody was monitored by flow cytometry.
d. Binding of biotin-hscx~~i6 to LAP
96-well microtiter plates (Corning COSTAR EASY-WASH) were coated with 0.3 p,g/ml recombinant human LAP (R&D Systems, Cat. # 246-LP) diluted in PBS (50 pl/well) at 4°C overnight. After the coating solution was removed, the plates were blocked with 180 pl/well of 3% BSA/TBS at 25°C
for 1 hr. In a separate 96-well round-bottom plate, 60 p,l/well of a 2X stock (0.5 ~.g/ml (1.25 nM) of biotin-c~,(36, 2 mM CaCl2, and 2 mM MgCl2 in TBS containing 1
23 mg/ml BSA) was combined with 60 ul/well of a 2X 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 ~.1 of the antibody-c~,(36 mixture was transferred to the plate, and incubated for 1 hr at 25°C. The plate was washed as above and incubated with 50 pl/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 a6-FDC-P1 cells to LAP
A 96-well microtiter plate was coated with 50 ~1/well of 0.5 pg/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 pl/well) and blocked with 1% BSA in PBS (100 ~1/well) for 1 hr at 25°C. The plate was washed twice with 100 p,l/well of assay buffer (TBS complete plus 1 mM CaCla and 1 mM MgCl2). Next, to the individual wells of the plate were added 25 ~.l of a hybridoma supernatant (or a purified antibody) and 25 ,ul of (36-FDC-P1 cells (SX
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 pl/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-~i bioassay The TGF-(3 bioassay used herein was a variation of the Mink lung epithelial cell (MLEC) PAI-1 luciferase coculture assay described in Abe et al., Anal. Bi~chem. 216:276-284 (1994), in which (36-transfected cells were cocultured with the reporter cells to monitor activation of TGF-~3 by a,,(36 (hunger, supra). It is a quantitative bioassay for TGF-~i 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-~3 (0.2 to > 30 pM) results in a dose-dependent increase in luciferase activity in the cell lysates.
24 To conduct this assay, TMLC (mink lung epithelial cell line My 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 p.g/ml 6418. SW480 cells transfected with an integrin (36 construct ("(36-SW480" or "SW480 (36" 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-(36 cells were plated at 4 X 104 cells/well in the wash buffer. Monoclonal antibodies were diluted in DMEM (serum-free), added to the SW480-(36 cells and pre-incubated for 20 min at room temperature. TMLC cells were then added at 2 X 104 cells/well to a final volume of 100 ~,1. The plates were incubated for 20 hr in a humidified, C02-enriched incubator. Supernatant from the plates was discarded and replaced with 100 ~1 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 NaCI, pH 2.8 into 1:20 volume of 0.5 M
Na3P04, pH 8.6. For the marine IgGI antibodies, the supernatant was adjusted to 1.5 M glycine, 3 M NaCI, pH 8.9 prior to loading, and the column was washed with 25 mM Na3P04, 3 M NaCI, 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

6.2B 10 10 IgG2a Y

6.3 G9 25 IgG 1 Y

6.4B4 30 IgGl N

6.6B5 46 IgGl N

6.8B4 55 IgGl N

6.866 56 IgGl Y

6.2B1 85 IgGl 7.1 CS 2 IgG2a Y

7.1610 5 IgG2a Y

7.2A1 6 IgG2a Y

7.2F5 11 IgG2a Y

7.282 12 IgG2a Y

7.4A3 17 IgG2a Y

7.765 32 IgGl Y

7.8H12 39 IgG2a . Y

7.9D4 40 IgG2a . N

7.968 41 IgG2a Y

7.9H5 43 IgG2a Y

7.1 OD7 44 IgG2a Y

7.1 OH2 46 IgG2a Y

*: A blocker is defined as an antibody that blocks the binding of a,,(36 to LAP as determined by blocking of ligand binding either to purified hsa,,(36 or to ,Q6-expressing cells.
For use in animal models, hybridoma clones were scaled up to 2L 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 Na3P04, 25 mM NaCI, pH 8.6. The column was washed with 5 column volumes of equilibration buffer, and bound protein was eluted using 25 mM Na3P04, 150 mM NaCI, pH 7.2. The eluted proteins were sterile-filtered (0.45 ~,m) 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 hsa~~36, (2) bind,~6-transfected SW480 and FDC-P1 cells, (3) inhibit binding of biotin-a,,(36 to LAP, (4) inhibit binding of (36-transfected FDC-P1 cells to LAP, and (5) block a~(36-mediated activation of TGF-~3 in the MLEC assay (supra). The relative potency in each of these assays was compared to that of the known a~(36 antibody l ODS (Huang et al, J. Cell Sci. 111:2189 (1998)) and, in some cases, the anti-a~ antibody L230.
For the characterization of Fusion #7 antibodies, the Fusion #6 antibody 6.866 was also used as a positive control.
An initial binding experiment (Example Sa, supra), carned out in the presence of 1 mM Ca2+ and 1 mM Mg2+, indicated that a majority, of the purified antibodies bound to hsa,,(36 (Figs. 2A and 2B). Unexpectedly, however, no binding was observed for lODS and clones 7.2F5 and 7.1OD7. A subsequent experiment established that binding of l ODS (Fig. 3E), 7.2F5, and 7.1 OD7 was supported only weakly by Ca2+/Mga+, but much more strongly by 1 mM MnClz.
Among the new clones, three (6.1A8 (Fig. 3A), 7.765, and 6.866 (Fig. 3C)) showed a requirement for divalent cations, although no difference between the Caa+~ga+ state and the Mnz+-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 (36-transfected NIH 3T3 cells or cells revealed a similar pattern, with the exception that l ODS, in this context, bound equivalently in the Ca2+/Mg2+ and MnZ+ states. The requirements for binding to soluble a,,(36 may differ from those for binding to cell surface-expressed a,,~36 due to a difference in protein conformation or avidity effects.
These results suggest that there are at least 3 different classes of ~i6-blocking antibodies in this group. One of the classes (lODS) distinguishes between the Caa+/Mga+ and Mn2+ conditions. Another class (including 6.1A8, 7.765, and 6.866) requires cation but does not distinguish between Ca2+/Mg~+ and Mnz+.
The last class (including anti-c~, antibody L230, 6.2B10, 6.369 (Fig. 3B), and 6.2B1 (Fig. 3D), 7.1C5, and 7.1610) is cation-independent.
The purified antibodies were next evaluated for their ability to inhibit the c~,a6-LAP interaction. In the cell-free assay of Example Sd, supra, antibodies 6.1A8, 6.2B1, 6.369, and 6.866 showed ICSO values lower than that of lODS (Fig. 4A; Table 3). 6.2B10 showed a higher ICso 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, 7.1610, 7.2A1, 7.4A3, 7.765, 7.968, 7.9H5, and 7.1OH2 showed ICso values lower than that of l ODS (Fig. 4B; Table 3). Antibodies 7.2F5, 7.2H2 and 7.8H12 displayed nearly identical or higher ICSO values and yet still gave complete inhibition (Fig. 4B).
In the cellular assay described in Example Se, supra, a similar trend was observed, with the exceptions of 6.1 A8, 6.2B 10 and 7.9D4, which were much less potent on cells than on purified protein (Figs. SA-E; Table 3).
Collectively, these results indicate that we have successfully generated antibodies that specifically inhibit the interaction of both human and murine c~,(36 with LAP. Some of these antibodies bound to a,,(36 with high affinity (apparent Id's >_0.3 nM, as determined by flow cytometry), inhibited binding of (36-transfected cells to LAP with an ICSO of ?0.05 nM (8 ng/mL), and prevented a,,(36-mediated activation of TGF-(31 with an ICso of ?0.58 nM (87 ng/mL).

Finally, the purified antibodies were evaluated for their ability to block c~,~3s-mediated activation of TGF-~3 in the PAI-1/luciferase reporter gene assay (Example Sf, supra). Once again, 6.369, 6.866, 6.2B1, 7.1610, and 7.765 were able to inhibit c~,~i6-mediated activation of TGF-~i with ICSO values lower than l ODS, while the remaining antibodies appeared to be significantly less potent in this assay (Figs. 6A and 6B; Table 3). Thus, the ability to block a,,(36's interaction with LAP correlates with the ability to inhibit activation of TGF-(3 in vitro.
Table 3 Characterization of hybridoma clones Clone Clonea"(~6-bindingc~,(36-LAP FDCP1-LAP PAI-1 name No. ELISA blocking blocking luciferase EC50 (ng/ml)IC50 (ng/ml)IC50 (ng/ml)ICSO (ng/ml) 6.2B1 85 34.7 225 8 87 6.369 25 76.7 271 17 375 6.866 56 17.5 169 23 312 lODS - - 605 50 2070 6.1A8 2 3.7 179 2520 40,000 6.2B1010 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.

7.16105 4.2 113 30 250 7.765 32 13.0 155 51 700 7.1C5 2 2.5* 80* 83 n.t.

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

lODS - 43* 377 n.t. 2,000 7.4A3 17 5.7 204 67 3,500 7.1OH246 6.6 254 63 3,500 7.2H2 12 9.3 370 106 5,500 7.9H5 43 7.3 230 55 7,000 7.968 41 6.2 ~ 264 ~ 284 ( >20,000 ~ ~

Clone Clonec~,(36-bindingc~,(36-LAP FDCP1-LAP PAI-1 name No. ELISA blocking blocking luciferase EC50 (ng/ml)IC50 (ng/ml)IC50 (ng/ml)IC50 (ng/ml) 7.8H1239 46.0 1140 969 >20,000 7.2F5 11 >5000 529 1490 >20,000 7.9D4 40 1.7 incomplete >10,000 >20,000 7.1OD744 >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 Caz+ and 1 mM Mg2+.
****: Antibodies in boldface are prior art antibodies lODS and L230, and new antibodies of particular high inhibitory potency for a"~ib.
Next, the solution affinities of 6.369 and 6.866 for soluble c~,/36 were determined by using a kinetic exclusion assay (KinExA). A series of dilutions of the soluble integrin (1 X 10-g M to 2.4 X 10-12 M) were incubated with 1X101° 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, Idaho). In the case of 6.866, 1mM CaCl2 and 1mM 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 (Ka) for each interaction. The Id's determined using this method were 15.6 pM for 6.369 and 22.8 pM for 6.866 (Figs. 9A and 9B). Thus, both of these antibodies had very high affinities for a.~(36.
We further identified classes of anti-c~,(36 antibodies that recognized "activated" states of the integrin. There are two potential activation states of c~,~36.
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 MnClz and 1 mM MgCl2 (non-activating cation) by flow cytometry indicated that some of the a,,(36 antibodies described here, including 6.1A8 and 6.6B5, showed significantly enhanced binding in the presence of MnCla.

In a second activated state of av(36, the integrin can activate latent TGF-(3 as described above. A cell line expressing truncated av~36 (SW480(~36-770T)) was prepared. The cell line was able to bind LAP but could not activate TGF-(3 in the TMLC luciferase assay (Munger et al., supra). Antibodies which 5 bind to the full length ~i6-transfected SW480 cells, but not to the 770T
truncated transfected cells, were thus specific to the form of a,,(36 that is able to activate TGF-(3. Antibodies 7.8B3 and 7.8C9 met this criteria.
Example 8' Epit~e mappin~by antibody competition The purified monoclonal antibodies were also tested for their ability 10 to compete with 6.866 for binding to biotinylated a"(36 in an ELISA format.
In this assay, 6.866 was coated on an ELISA plate, and a mixture of the competing antibody and biotinylated a,,(36 was added in a buffer containing 1 mM each of Caz+
and MgZ+. Bound integrin was detected using extravidin-HRP conjugate, and competing antibodies were scored for their ability to block binding. All consensus 15 blockers (Table 2) except 6.2B10 (a weak blocker) were shown to be able to compete with 6.866 to various degrees (Table 4). These data confirm that these consensus blockers bind to the same or overlapping epitope as 6.866.
Table 4 Epitope Mapping by Antibody Competition Clone Consensus Competition with Blocker? 6.866 6.2A1 N -6.4B4 N -6.6B5 N -6.8B4 N -7.9D4 Y/N -1ODS Y ++

L230 Y ++

6.1A8 Y ++

6.2B 10 Y (weak) -6.3G9 Y +

6.866 Y ++

6.2B 1 Y ++

7.1C5 Y

7.1 G10 Y +++

7.2A1 Y ++

7.2F5 Y ++

7.2H2 Y ++

7.4A3 Y

7.765 Y ++

7.8H12 Y ++

7.968 Y ++

7.9H5 Y

7.1OD7 Y +

7.1OH2 Y ++

The purified monoclonal antibodies were tested for their ability to compete with biotinylated 6.369 or biotinylated 6.866 for binding to a,,(36 in ELISA. In this assay, unlabeled a,,(36 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 7.1610) competed with both 6.369 and 6.866 for binding to a,,(36 (Table 4.1, and Figs. l0A and l OB).
Antibodies 6.1A8 and 7.765 showed less competition, probably due to their lower affinity for a"(36. None of the non-blocking antibodies or the anti-a,, antibody L230 showed any competition with 6.369 or 6.866 in this assay. These results indicate that the a,,(36-specific blocking antibodies bind to the same or overlapping epitopes on a"(3s.
Table 4.1 Epitope Mapping by Antibody Competition Clone Consensus Competition Competition Blocker? with biotin-with biotin-ylated 6.866ylated 6.369 6.3 Y +++ +++

6.2B Y +++ +++

6.866 Y +++ ++

7.1 Y +++ +++
CS

7.1610 Y +++ +++ I

7.765 Y ++ +

6.1 Y ++ +

6.2A1 N - -6.2E5 N - -6.2G2 N - -6.4B4 N - -7.8B3 N - -7.8C9 N - -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 Irnmunology, 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 6.866, 6.1A8, 6.2B1, 6.369 and 6.2A1 and of nonbloclcer 6.262 are compared as follows (dashes indicate gaps).
CDRl 6.866 SYTFTDYAMH (SEQ ID NO:1) 6.1A8 SYTFTDYTMH (SEQ ID N0:2) 6.2B1 GFTFSRYVMS (SEQ ID N0:3) 6.369 GFTFSRYVMS (SEQ ID N0:3) 6.2A1 GYDFNNDLIE (SEQ ID N0:49) 6.262 GYAFTNYLIE (SEQ ID N0:50) 6.866 VISTYYGNTNYNQKFKG (SEQ N0:4) ID

6.1A8 VIDTYYGKTNYNQKFEG (SEQ N0:46) ID

6.2B1 SISSG-GSTYYPDSVKG (SEQ N0:5) ID

6.369 SISSG-GRMYYPDTVKG (SEQ N0:6) ID

6.2A1 VINPGSGRTNYNEKFKG (SEQ N0:51) ID

6.262 VISPGSGIINYNEKFKG (SEQ N0:52) ID

6.866 GGLRRGDRPSLRYAMDY (SEQ N0:7) ID

6.1A8 GGFRRGDRPSLRYAMDS (SEQ N0:47) ID

6.2B1 GAIYDG-----YYVFAY (SEQ N0:8) ID

6.369 GSIYDG-----YYVFPY (SEQ N0:9) ID

6.2A1 IYYGPH-----SYAMDY (SEQ N0:53) ID

6.262 ID-YSG-----PYAVDD (SEQ N0:54) ID

In SEQ D7 N0:7, 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 6.262 are compared as follows.
CDRl 6.866 RASQSVSTSS-YSYMY (SEQ ID N0:10) 6.1A8 RASQSVSIST-YSYIH (SEQ ID N0:48) 6.2B1 SASSSVSSS----YLY (SEQ ID N0:11) 6.369 SANSSVSSS----YLY (SEQ ID N0:12) 6.2A1 KASLDVRTAVA (SEQ ID N0:55) 6.262 KASQAVNTAVA (SEQ ID N0:56) 6.866 YASNLES (SEQ ID N0:13) 6.1A8 YASNLES (SEQ ID N0:13) 6.2B1 STSNLAS (SEQ ID N0:14) 6.369 STSNLAS (SEQ ID N0:14) 6.2A1 SASYRYT (SEQ ID N0:57) 6.262 SASYQYT (SEQ ID N0:58) 6.866 QHNWEIPFT (SEQ ID N0:15) 6.1A8 QHSWEIPYT (SEQ ID N0:16) 6.2B1 HQWSSYPPT (SEQ ID N0:17) 6.369 HQWSTYPPT (SEQ ID N0:18) 6.2A1 QQHYGIPWT (SEQ ID N0:59) 6.262 QHHYGVPWT (SEQ ID N0:60) As shown in Figs. 7A and 7B, the mAbs that fall into the divalent cation-dependent class (e.g., 6.1A8 and 6.866) seem to contain very similar amino acid sequences within the CDRs, while the divalent cation-independent mAbs (e.g., 6.2B 1 and 6.369) contain another set of motifs in their CDRs.

The potency and specificity of anti-ar,~36 monoclonal antibodies may be governed by subtly different amino acid residues. In the case of 6.1A8 and 6.866, 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, c~,(36.
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 confernng binding specificity to the integrin-ligand interaction. In 6.1A8 and 6.866, 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 6.866 include those at positions 33 (CDRl); positions 52, 57, and 65 (CDR2); and position 115 (CDR3). Another difference in the heavy chain lies at position 4 in framework l, 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 a~~6 binding. The three remaining differences between 6.1A8 and 6.866 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 6.866 (i.e., 6.2B1, 6.369, 7.1OH2, 7.9H5, 7.1C5, 7.1610, and 7.4A3); and those that do not (i.e., 6.2A1, 6.2B10 and 6.4B4).
The 6.866-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 aV~i6.
Additionally, this FXY-containing class of antibodies probably bind to an epitope on a,.(36 that is overlapping with, yet distinct from, the RGD-binding pocket. Antibodies 6.2B

and 6.4B4 do not contain an FXY motif and are poor cx,,(36 blockers. They were shown to bind to the c~,(36 I-domain-like portion and define yet another epitope to which anti-a~,(36 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.
10 Monoclonal antibody 7.765 belongs to the cation-dependent class.
However, the light chain sequence of 7.765 is highly homologous to the cation-independent, I-domain binding antibody 6.2B 10. The heavy chain of 7.765 is also similar to cation-independent antibodies in CDRl. Yet its CDR2 and CDR3 are more similar to those of the cation-dependent class. This observation suggests that 15 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 c~,(36 recognition. Of note, 7.765 lacks an RGD
motif 20 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 7.765.
The above sequence observations and inferences made therefrom provide a basis for rational design of specific variable region amino acid sequences
25 that confer specific binding properties.
Example 10' Diagnostic antibodies Antibodies that can detect a,,~36 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 a,.(3~ in tissue sections for 30 such indications as cancer or fibrosis.
In order to identify antibodies that detect a,,~ib in paraffin-embedded tissues, we first screened a panel of antibodies for binding to HPLC-purified ~i6 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 (36 subunit was carried out using an ELISA format identical to that described for measuring a"~3G binding (sz~pra), except with the purified (36 integrin, rather than the a,,(36 protein, immobilized on the plate. Using this method, a number of Fusion 6 antibodies capable of binding both the purified c~,(36 protein and the purified fib subunit were identified.
See Table 5, infra, where the prefix "6." in the clone names is omitted.
Table 5 Antibody Binding to Purified a"~36 or Purified (36 Subunit Clone name Binding to av~6 Binding to HPLC-nurified !36 subunit lAl + 6 lA8 + _ lAll + +

lEl + +

lE6 + +

1H10 +/-2A1 + +

ZB8 - _ 2B 10 + -2C4 + +

2C7 + +

2E5 + +

2G3 + _ 3A6 + -3B1 + _ 3B2 + +

3B11 + , +

3C2 + +

3D5 + -3D10 + +

3F1 + -3G5 + -3G9 + -3H2 + _ 3H11 + -4A4 +/- +

Clone name Binding to av~6 Binding to HPLC-purified ~6 subunit 4B2 + _ 4B4 + _ 4B10 +

4D2 + ' 4E4 + +

4G3 + ' 4G4 + +

4H4 + +

4H12 + ' SA2 + ' SB6 + +

SD6 + +

SD8 - ' SG9 + +

SG10 + +

SH3 + +

6B1 + +

6B5 + +

6C4 + ' 6D12 + +

6E6 + ' 6E10 + ' 6G3 +~- ' _ 7C7 + +

7E5 + _ 7F8 + _ 8B4 + +

8G6 + _ 9B5 + +

9B7 + +

9B9 + _ 9B10 + -9D 11 + +

9E12 + +

9F5 + +

9G1 + _ 9H11 + -10A2 + -10A3 +~-10A4 + -10A8 + -Clone name Binding to av~6 Binding to HPLC-purified X36 subunit 1 OA9 + -1OB10 +

1 OD3 + -lODl l +

10E4 + +

lOFl +

1OF12 +

lOGl +

lOG2 - -lOHll + +

lA7 + _ 1D6 +

1D9 +

1F6 +

2B1 + _ 2D9 + +

2G2 + +

3D9 + +

3E5 +

4C 12 +

4E6 + +

4G5 +

SA3 + _ SB3 +

SB8 + +

SB9 + _ SF7 + _ 6C10 + +

6D8 + _ 6F4 +

6G9. + _ 6H8 + +

6H9 + -7A5 + +

7Al 1 +

7E6 + +

7G9 - +

7H3 + _ 9A2 - _ 9A3 + +

9A9 + -Clone name Binding to av,~6 Binding to HPLC-nurified ~6 subunit 9F1 +

1 OBS + -lOB7 + _ l OB9 +/- -1 OCS +/- -lOC7 +/- -1 OD6 +/- -1 OE 12 +/- -lOF7 - -As shown above, some antibodies bound to purified (36 subunit.
They will have a high likelihood to bind to denatured c~,~i6 and thus can be useful in detecting c~,(36 in paraffin-embedded tissue sections. Other antibodies bound to soluble a,,(36 but not the (36 subunit. Both types of antibodies were used to stain denatured paraffin-embedded (36-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% H202 for 1 S 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, CA), 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 ~.1 of solution A
(1:50) and 20 ~,1 of solution B (1:50) from Vector Kit PK-6102; and the mixture 5 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 ~.1/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 10 were rinsed again as described above. Then the substrate (Vector SK-4100), ~,1 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 H20, 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 H20z Solution, mix well. Then the slides were 15 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 20 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.
25 The results suggested that Fusion 6 antibodies lAl, 2C4, 3B2, 3B11, SD6, SG9, SH3, 6D12, 7C7, 9B5, 9B7, 9D11, 9F5, 10E4, lOHll, 6H8, 7A5, 7G9, 9A3, 2A1, 2E5, 4E4, 4H4, 8B4, 2G2, and 4E6, all of which could bind to purified ~i6 subunit (Table 5), indeed stained paraffin-embedded (3~-transfected SW480 cells strongly, while not staining untransfected parent cells (Table 6).

Table 6 Antibody Binding to Paraffin-Embedded SW480 Cells ID# Clone name Binding to Binding to SW480/,86+ SW480 1 lAl +++

2 lA8 + +

3 lAl1 +-~ -4 lEl +

lE6 ++ +

7 2A1 +++

11 2C4 +++

13 2E5 +++ -17 3B2 +++

18 3B 11 ++ -30 4B4 + +

33 4E4 +++ -35 4G4 . - -36 4H4 +++ -39 SB6 + -40 SD6 +++ -42 SG9 +++ -44 SH3 +++ -48 6D12 +++

52 7C7 ++ -55 8B4 +++ -57 9B5 ++ -58 9B7 ++

61 9D11 ++ -62 9E12 + -63 9F5 -f--~ -75 10E4 -E--~+ -80 1 OH11 ~++ -85 2B 1 + +

87 2G2 +-~ -91 4E6 -E-~ -ID# Clone name Binding to Binding to SW4801~6+ SW480 98 6C10 +

102 6H8 ++

104 7A5 +++ -107 7G9 +++

110 9A3 +++ -Example 11: Diagnnosis of cancer c~,(36 is normally expressed at negligible to low levels in healthy adult tissues. However, a,,~ib 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 c~,(36 expressed on paraffin-embedded tissues can be used in standard immunohistochemistry techniques to detect a~(36 expression for diagnosis of fibrosis, cancer and any other diseases in which a~(36 is upregulated.
As described above, certain antibodies of the present invention bind to HPLC-purified (36 integrin and paraffin-embedded and fixed X36-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-a~~36 blockin~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 6.866 (cation-dependent) and 6.369 (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 a~,(36 binding to LAP
and to inhibit activation of TGF-!3 in a bioassay. Antibody lE6 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) 6.866, 4mg/kg (7 mice);
(2) 6.369, 4 mg/kg (4 mice); and (3) 1E6, lmg/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 6.866 or 6.369, 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 6.866 and 6.369, as compared to negative control lE6-treated mice.
Example 13: Effectiveness of anti-a~~i6 mAbs in preventing unilateral ureteral obstruction-induced nephrosclerosis We used another mouse model for renal fibrotic progression to test the antifibrotic efficacy of 6.866 and 6.369. 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-oc~(36 treatment on WO-induced renal fibrosis. Male, 8-12 week old, viral antigen-free C57BL mice of 25.50.2 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:
Group Treatment Dose (mg/kg, i.p.) n*
1 6.866 (cation dependent anti-a,,(36 mAb) 4 9 2 6.369 (cation-independent anti-a,,[36 mAb) 4 8 3 lE6 (negative Control mAb) 4 10 4 PBS Vehicle Control (100 ~,1) 8 5 Unoperated, Untreated Control --*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 10 within the measured fields. These included all cortical and medullary tissue from the section except the renal papilla.
In conclusion, mice treated with 6.369 and 6.~G6 show a significant reduction in fibrosis.
Example 14: Effectiveness of anti-a~r~6 blocking_mAbs against bleomycin-induced 15 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 S-15 days following 20 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 6.369 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 25 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 ~l of sterile saline. The animals were sacrificed at day 30 and the extent of lung fibrosis was assessed.
Antibody lE6 was used as a negative control.
Lungs were harvested from each animal and hydroxyproline content 30 was measured as an index of lung collagen deposition, as described in Munger et al., supra. As shown in Fig. 15A, treatment with 6.369 beginning at day 0 significantly inhibited the bleomycin-induced increase in lung hydroxyproline content. Importantly, the 6.369 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 6.369, cation-dependent 6.866, 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 6.866 significantly inhibited bleomycin-induced fibrosis (a more than 70% reduction in hyrdroxyproline content compared to animals treated with bleomycin and saline). Treatment with 6.369 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-av(36 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 a"~is in human~soriasis lesions In order to determine whether a"(36 is involved in psoriasis, a~[36 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 a,,~i6 expression in psoriatic lesions, as compared to nonlesional skin from psoriatic patients and normal controls. Thus, the upregulation of a,,~36 in psoriatic lesions suggests both diagnostic and therapeutic implication for the use of anti-a,,(36 antibodies.
Example 16: Upre~lation of a"~36 in mouse and human liver with biliary duct disease As previously discussed, a,,(36 expression has been implicated in tissue injury. In this study, expression of a,,(36 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 JPathol 156:11 S-24 (2000). Using mAb 6.262, we found that expression of a,,(36 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 a~(36, as determined by immunohistochemistry using the mAb 6.262. Elevated expression of a,,(36 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-a"(36 antibodies are useful diagnostic and therapeutic tools for liver diseases.
Example 17' Upre~ulation of a" .ij~6 in various human cancers Integrin c~,(36 is normally expressed at negligible to low levels in healthy adult tissues. A variety of human tumor tissues was evaluated for a,.(36 expression using the antibody 6.2A1 and methods generally described herein.
The results showed that a,,(36 integrin expression was significantly upregulated in several human epithelial cancers. Notably, immunohistology showed that a,,(36 was expressed especially prominently on the edges of tumor islands in many of the epithelial cancers. To further study expression of a"~36 in epithelial cancer cells, Detroit 562 cells (pharynx carcinoma) and SCC-14 cells (tongue squamous cell carcinoma) as well as SW480~i6 cells (supra) were stained with 6.369 and 6.4B4 and analyzed by flow cytometry.
The right side of Fig. 12 shows a,,(36 expression on the different tumor cell lines as indicated by 6.369 binding in fluorescence activated cell sorting (FACS). The solid peak represents 6.369 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 6.369 or 6.4B4. 6.4B4 was a significantly less potent inhibitor of a,,(36 binding to LAP, compared to 6.369 (> 10-fold ICso for Detroit 562, > 30-fold ICSO for SW480136, and > 100-fold ICSO for SCC-14). This is consistent with previous in vitro results indicating that 6.369 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-a~(36 mAbs. Both 6.369 and 6.866 displayed equivalent inhibiting activity (consistent with all previous data) while 6.4B4 was a significantly less potent inhibitor of a,,(36 binding to LAP.
Example 18: Effects of anti-ay~i6 blocking mAbs in a human tumor xeno~raft 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-av(36 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 a"(36 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 5X106 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) 6.369, 1 mg/kg, 10 mice; (2) 6.369, 4 mg/kg, 10 mice; (3) 6.369, 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/lcg 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, a~(36 expression assessed by immunohistology, and serum anti- a"(36 levels measured.
Immunohistological staining showed that the implanted tumor cells strongly expressed a~(3s in vivo. Tumor weight data further showed that blocking mAb 6.369 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-a~(36 antibody did not inhibit tumor growth.
Example 19: a"~36 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-a,.(36 ' antibodies to be internalized was studied in SW480(36 (supra) and SCC-14 cells.
Cells were split 1:5 and plated onto 4-chambered glass slides for overnight incubation at 37°C, 5% C02. The next day, mAbs 6.866, 6.1A8, 6.369, 7.1C5, 6.4B4, lODS, and 8B3 were diluted to a final concentration of 20 ~tg/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, S, 10 and min, and l, 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 25 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 a~(3s 30 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 63x (1.32 numerical aperture, ail immersion) obj ective (Leica) with a 2x 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-5 containing ligand mimetics) such as 6.866 and 6.1A8. No internalization was observed for cation-independent mAbs such as 6:369, 7.1C5, and 6.4B4.

Claims (59)

What is Claimed is:
1. A monoclonal antibody that (a) specifically binds to .alpha.v.beta.6; and (b) inhibits the binding of .alpha.v.beta.6 to latency associated peptide (LAP) with an IC50 value lower than that of 10D5.
2. The antibody of claim 1, wherein the antibody comprises the same heavy and light chain polypeptide sequences as an antibody produced by hybridoma 6.1A8 (ATCC accession number PTA-3647).
3. The antibody of claim 1, wherein the antibody comprises the same heavy and light chain polypeptide sequences as an antibody produced by hybridoma 6.3G9 (ATCC accession number PTA-3649).
4. The antibody of claim 1, wherein the antibody comprises the same heavy and light chain polypeptide sequences as an antibody produced by hybridoma 6.8G6 (ATCC accession number PTA-3645).
5. The antibody of claim 1, wherein the antibody comprises the same heavy and light chain polypeptide sequences as an antibody produced by hybridoma 6.2B1 (ATCC accession number PTA-3646).
6. The antibody of claim 1, wherein the antibody comprises the same heavy and light chain polypeptide sequences as an antibody produced by hybridoma 7.1G10 (ATCC accession number PTA-3898).
7. The antibody of claim 1, wherein the antibody comprises the same heavy and light chain polypeptide sequences as an antibody produced by hybridoma 7.7G5 (ATCC accession number PTA-3899).
8. The antibody of claim 1, wherein the antibody comprises the same heavy and light chain polypeptide sequences as an antibody produced by hybridoma 7.1C5 (ATCC accession number PTA-3900).
9. The antibody of claim 1, wherein the binding between the antibody and .alpha.v.beta.6 is divalent cation-dependent.
10. The antibody of claim 9, wherein the divalent cation is Ca2+, Mg2+ or Mn2+.
11. The antibody of claim 1, wherein the binding between the antibody and .alpha.v.beta.6 is divalent cation-independent.
12. An anti-.alpha.v.beta.6 antibody whose heavy chain complementarity determining regions (CDR) 1, 2 and 3 consist essentially of the sequences of SEQ ID NOs:1, and 7, respectively, and whose light chain CDRs optionally consist essentially of the sequences of SEQ ID NOs:10, 13 and 15, respectively.
13. An anti-.alpha.v.beta.6 antibody whose heavy chain complementarity determining regions (CDR) 1, 2 and 3 consist essentially of the sequences of SEQ ID NOs:3, and 8, respectively, and whose light chain CDRs optionally consist essentially of the sequences of SEQ ID NOs:11, 14 and 17, respectively.
14. An anti-.alpha.v.beta.6 antibody whose heavy chain complementarity determining regions (CDR) 1, 2 and 3 consist essentially of the sequences of SEQ ID NOs:3, and 9, respectively, and whose light chain CDRs optionally consist essentially of the sequences of SEQ ID NOs:12, 14 and 18, respectively.
15. An anti-.alpha.v.beta.6 antibody whose heavy chain complementarity determining regions (CDR) 1, 2 and 3 consist essentially of the sequences of SEQ ID NOs:2, and 47, respectively, and whose light chain CDRs optionally consist essentially of the sequences of SEQ ID NOs:48, 13 and 16, respectively.
16. An anti-.alpha.v.beta.6 antibody whose heavy chain complementarity determining regions (CDR) 1, 2 and 3 consist essentially of the sequences of SEQ ID
NOs:49, 51, and 53, respectively, and whose light chain CDRs optionally consist essentially of the sequences of SEQ ID NOs:55, 57 and 59, respectively.
17. An anti-.alpha.v.beta.6 antibody whose heavy chain complementarity determining regions (CDR) 1, 2 and 3 consist essentially of the sequences of SEQ ID
NOs:50, 52, and 54, respectively, and whose light chain CDRs optionally consist essentially of the sequences of SEQ ID NOs:56, 58 and 60, respectively.
18. An anti-.alpha.v.beta.6 antibody comprising a heavy chain variable domain sequence of any one of SEQ ID NOs:19-36 and 61-62.
19. An anti-.alpha.v.beta.6 antibody comprising a heavy chain variable domain sequence of SEQ ID NO:19 and a light chain variable domain sequence of SEQ ID NO:37.
20. An anti-.alpha.v.beta.6 antibody comprising a heavy chain variable domain sequence of SEQ ID NO:20 or 21, and a light chain variable domain sequence of SEQ ID
NO:38.
21. An anti-.alpha.v.beta.6 antibody comprising a heavy chain variable domain sequence of SEQ ID NO:22 and a light chain variable domain sequence of SEQ ID NO:43.
22. An anti-.alpha.v.beta.6 antibody comprising a heavy chain variable domain sequence of SEQ ID NO:23 and a light chain variable domain sequence of SEQ ID NO:44.
23. An anti-.alpha.v.beta.6 antibody comprising a heavy chain variable domain sequence of SEQ ID NO:24 and a light chain variable domain sequence of SEQ ID NO:45.
24. An anti-.alpha.v.beta.6 antibody comprising a heavy chain variable domain sequence of SEQ ID NO:25 or 26 and a light chain variable domain sequence of SEQ ID
NO:42.
25. An anti-.alpha.v.beta.6 antibody comprising a heavy chain variable domain sequence of SEQ ID NO:27, 28, or 29, and a light chain variable domain sequence of SEQ
ID NO:39.
26. An anti-.alpha.v.beta.6 antibody comprising a heavy chain variable domain sequence of SEQ ID NO:34 or 35, and a light chain variable domain sequence of SEQ ID
NO:40.
27. An anti-.alpha.v.beta.6 antibody comprising a heavy chain variable domain sequence of SEQ ID NO:36, and a light chain variable domain sequence of SEQ ID NO:41.
28. An anti-.alpha.v.beta.6 antibody comprising a heavy chain variable domain sequence of SEQ ID NO:61, and a light chain variable domain sequence of SEQ ID NO:63.
29. An anti-.alpha.v.beta.6 antibody comprising a heavy chain variable domain sequence of SEQ ID NO:62, and a light chain variable domain sequence of SEQ ID NO:64.
30. A monoclonal antibody that specifically binds to .alpha.v.beta.6 but does not inhibit the binding of .alpha.v.beta.6 to latency associated peptide (LAP).
31. The antibody of claim 30, wherein the antibody comprises the same heavy and light chain polypeptide sequences as an antibody produced by hybridoma 6.2A1 (ATCC accession number PTA-3896).
32. The antibody of claim 30, wherein the antibody comprises the same heavy and light chain polypeptide sequences as an antibody produced by hybridoma 6.2E5 (ATCC accession number PTA-3897).
33. A composition for preventing or treating a disease mediated by .alpha.v.beta.6 in a mammal, comprising the antibody of any one of claims 1-32, and a pharmaceutically acceptable carrier.
34. The composition of claim 33, wherein the antibody is conjugated to a cytotoxic agent.
35. The composition of claim 33, wherein the antibody is a cation-dependent antibody.
36. A method for treating a subject having or at risk of having a disease mediated by .alpha.v.beta.6, comprising administering to the subject the composition of claim 32, thereby alleviating, or postponing the onset of, the disease.
37. The method of claim 36, wherein the subject is a human.
38. The method of claim 36, wherein the disease is fibrosis.
39. The method of claim 38, wherein the fibrosis is scleroderma, scarring, liver fibrosis, kidney fibrosis, or lung fibrosis.
40. The method of claim 36, wherein the disease is psoriasis.
41. The method of claim 36, wherein the disease is cancer.
42. The method of claim 41, wherein the cancer is epithelial cancer.
43. The method of claim 41, wherein the cancer is oral, skin, cervical, ovarian, pharyngeal, laryngeal, esophageal, lung, breast, kidney, or colorectal cancer.
44. The method of claim 36, wherein the disease is Alport's Syndrome.
45. A method of detecting .alpha.v.beta.6 in a tissue sample from a mammal, comprising contacting the tissue sample with the antibody of claim 1 or 30.
46. The method of claim 45, wherein the antibody is selected from the group consisting of 6.2A1 (ATCC accession number PTA 3896) and 6.2E5 (ATCC
accession number PTA 3897).
47. A cell of hybridoma 6.1A8 (ATCC accession number PTA-3647).
48. A cell of hybridoma 6.2B10 (ATCC accession number PTA-3648).
49. A cell of hybridoma 6.3G9 (ATCC accession number PTA-3649).
50. A cell of hybridoma 6.8G6 (ATCC accession number PTA-3645).
51. A cell of hybridoma 6.2B1 (ATCC accession number PTA-3646).
52. A cell of hybridoma 6.2A1 (ATCC accession number PTA-3896).
53. A cell of hybridoma 6.2E5 (ATCC accession number PTA-3897).
54. A cell of hybridoma 7.1G10 (ATCC accession number PTA-3898).
55. A cell of hybridoma 7.7G5 (ATCC accession number PTA-3899).
56. A cell of hybridoma 7.1C5 (ATCC accession number PTA-3900).
57. An isolated nucleic acid comprising a coding sequence for any one of SEQ
ID NOs:19-45 and 61-64.
58. An isolated polypeptide comprising an amino acid sequence of any one of SEQ ID NOs:l9-45 and 61-64.
59. A monoclonal antibody comprising the same heavy and light chain polypeptide sequences as an antibody produced by hybridoma 6.2B10 (ATCC
accession number PTA-3648).
CA2478833A 2002-03-13 2003-03-13 Anti-.alpha.v.beta.6 antibodies Expired - Lifetime CA2478833C (en)

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