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WO2008127655A1 - Anticorps anti-intégrine alpha 6 bêta 4 et leurs utilisations - Google Patents

Anticorps anti-intégrine alpha 6 bêta 4 et leurs utilisations Download PDF

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Publication number
WO2008127655A1
WO2008127655A1 PCT/US2008/004714 US2008004714W WO2008127655A1 WO 2008127655 A1 WO2008127655 A1 WO 2008127655A1 US 2008004714 W US2008004714 W US 2008004714W WO 2008127655 A1 WO2008127655 A1 WO 2008127655A1
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WIPO (PCT)
Prior art keywords
seq
antibody
fragment
polynucleotide
integrin
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Ceased
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PCT/US2008/004714
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English (en)
Inventor
Karen Mclachlan
Christilyn Graff
Heather Huet
Ling Ling Chen
Xianjun Cao
Beth Browning
Anne Cheung
Rachel Rennard
Steven Miklasz
Veronica Gabarra
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Biogen Inc
Biogen MA Inc
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Biogen Idec Inc
Biogen Idec MA Inc
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Publication of WO2008127655A1 publication Critical patent/WO2008127655A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM

Definitions

  • ⁇ 6 ⁇ 4 integrin is a laminin receptor expressed on basal epithelial cells.
  • the primary function of ⁇ 6 ⁇ 4 integrin is to maintain the integrity of epithelia, especially the epidermis.
  • studies have shown that expression of ⁇ 6 ⁇ 4 also persists in aggressive carcinomas and that its expression is linked to the behavior of these tumors. It is believed to play a pivotal role in functions associated with carcinoma progression and has also been implicated in the formation of carcinomas. Dajee et al., Nature 421:639-643 (2003).
  • ⁇ 6 ⁇ 4 integrins belong to the integrin family of cell surface receptors.
  • the integrin family serve cellular adhesion functions.
  • the receptors form a link between the extracellular matrix and the cytoskeleton through their binding to various extracellular components.
  • Each integrin receptor is a heterodimer composed of an ⁇ and a ⁇ subunit. At least 18 ⁇ chains and eight ⁇ chains have been characterized.
  • the molecular architecture of the ⁇ 6 ⁇ 4 integrin comprises an ⁇ 6 subunit which is composed of a heavy and light chain linked by a disulfide bond and a ⁇ 4 subunit.
  • the distinguishing structural feature of the ⁇ 6 ⁇ 4 integrin is the atypical cytoplasmic domain of the ⁇ 4 subunit.
  • Two pairs of fibronectin type III repeats separated by a connecting segment characterize the ⁇ 4 domain and it is distinct both in size (approximately 1000 amino acids) and structure from any other integrin subunits.
  • ⁇ 6 ⁇ 4 integrin functions primarily as an adhesion receptor in normal epithelia. It mediates the formation on the basal epithelial cell surface of stable adhesive structures known as hemidesmosomes (HDs) that serve an essential mechanical function.
  • the hemidesmosomes link the intermediate filament cytoskeleton with laminins in the basement membrane (BM). Borradori, L. and Sonnenberg, A., J Invest. Dematol. //2:411-418 (1999).
  • BM basement membrane
  • DiPersio et al J. Cell ScL //5:3051-3062 (2000).
  • mutations in the ⁇ 4 subunit also cause epidermal blistering. Vidal et al., Nature Genetics /0:229-234 (1995).
  • ⁇ 6 ⁇ 4 integrins have also been shown to play a role in the formation, migration, invasion and survival of carcinomas.
  • ⁇ 6 ⁇ 4 integrins have been shown to exist in two "functional states," one as an adhesion receptor localized in the HDs and two, as a signaling competent receptor localized with F-actin in lamellipodia and filopodia during carcinoma migration and invasion. It has been shown to cooperate with specific growth factors, such as EGF, to facilitate signaling and promote the migration and survival of carcinoma cells. Without being bound by theory, it is believed that EGF can increase PKC- ⁇ -mediated phosphorylation of the ⁇ 4 cytoplasmic domain.
  • This phosphorylation leads to disruption of the HDs and mobilization of ⁇ 6 ⁇ 4 to lamellipodia and its association with F-actin.
  • the release of ⁇ 6 ⁇ 4 from the HDs activates its signaling capacity, which may occur by its association with growth factor receptors such as erbB2, Met, and Ron or lipid rafts.
  • the key signaling event mediated by ⁇ 6 ⁇ 4 is activation of PI3-K.
  • the ⁇ 6 ⁇ 4-mediated activation of PB-K and its downstream effectors Akt, mTOR and Rac have been shown to have profound consequences on the biology of carcinoma cells.
  • the regulation of mTOR by ⁇ 6 ⁇ 4 in particular, has been shown to influence the translation of growth factors such as VEGF that can function to further amplify PB-K activation, thus sustaining and enhancing ⁇ 6 ⁇ 4-mediated functions.
  • the invention provides an isolated antibody or antigen- binding fragment thereof which specifically binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59- B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
  • the reference monoclonal Fab antibody fragment is M59-B05.
  • the reference monoclonal Fab antibody fragment is M61-C03.
  • the ⁇ 6 ⁇ 4 integrin epitope is contained within amino acids 446-686 of SEQ ID NO:101.
  • the invention provides an isolated antibody or antigen- binding fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the antibody or fragment competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66- H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to ⁇ 6 ⁇ 4 integrin.
  • the invention provides an isolated antibody or antigen- binding fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the antibody or fragment thereof comprises an antigen binding domain identical to that of a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the heavy chain variable region (VH) of the antibody or fragment thereof comprises an amino acid sequence at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87.
  • VH heavy chain variable region
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the light chain variable region (VL) of the antibody or fragment thereof comprises an amino acid sequence at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82 and SEQ ID NO:92.
  • VL light chain variable region
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH of the antibody or fragment thereof comprises an amino acid sequence identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VL of the antibody or fragment thereof comprises an amino acid sequence identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82 and SEQ ID NO:92.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH of the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of: SEQ BD NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ DD NO: 47, SEQ ID NO: 57, SEQ DD NO: 67, SEQ DD NO: 77, and SEQ DD NO: 87.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VL of the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of: SEQ DD NO: 22, SEQ DD NO: 32, SEQ DD NO: 42, SEQ DD NO: 52, SEQ DD NO: 62, SEQ DD NO: 72, SEQ DD NO: 82 and SEQ DD NO:92.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH and VL of the antibody or fragment thereof comprise, respectively, amino acid sequences at least 90% identical to reference amino acid sequences selected from the group consisting of: SEQ DD NO: 17 and SEQ DD NO: 22; SEQ DD NO: 27 and SEQ DD NO: 32; SEQ DD NO: 37 and SEQ DD NO: 42; SEQ DD NO: 47 and SEQ DD NO: 52; SEQ DD NO: 57 and SEQ DD NO: 62; SEQ DD NO: 67 and SEQ DD NO: 72; SEQ DD NO: 77 and SEQ DD NO: 82; and SEQ DD NO: 87 and SEQ DD NO: 92.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH and VL of the antibody or fragment thereof comprise, respectively, amino acid sequences identical, except for 20 or fewer conservative amino acid substitutions each, to reference amino acid sequences selected from the group consisting of: SEQ DD NO: 17 and SEQ DD NO: 22; SEQ DD NO: 27 and SEQ DD NO: 32; SEQ DD NO: 37 and SEQ DD NO: 42; SEQ DD NO: 47 and SEQ DD NO: 52; SEQ DD NO: 57 and SEQ DD NO: 62; SEQ DD NO: 67 and SEQ DD NO: 72; SEQ DD NO: 77 and SEQ DD NO: 82; and SEQ DD NO: 87 and SEQ ID NO: 92.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH and VL of the antibody or fragment thereof comprise, respectively, amino acid sequences selected from the group consisting of: SEQ DD NO: 17 and SEQ ID NO: 22; SEQ DD NO: 27 and SEQ DD NO: 32; SEQ DD NO: 37 and SEQ DD NO: 42; SEQ DD NO: 47 and SEQ DD NO: 52; SEQ DD NO: 57 and SEQ DD NO: 62; SEQ DD NO: 67 and SEQ DD NO: 72; SEQ DD NO: 77 and SEQ DD NO: 82; and SEQ DD NO: 87 and SEQ DD NO: 92.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH of the antibody or fragment thereof comprises a Kabat heavy chain complementarity determining region- 1 (VH-CDRl) amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VH-CDRl amino acid sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO: 58, SEQ ID NO: 68, SEQ ID NO: 78, and SEQ ID NO: 88.
  • the VH-CDRl amino acid sequence is selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO:
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH of the antibody or fragment thereof comprises a Kabat heavy chain complementarity determining region-2 (VH-CDR2) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR2 amino acid sequence selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 79 and SEQ ID NO: 89.
  • the VH-CDR2 amino acid sequence is selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO:
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH of the antibody or fragment thereof comprises a Kabat heavy chain complementarity determining region-3 (VH-CDR3) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR3 amino acid sequence selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, SEQ ED NO: 60, SEQ ID NO: 70, SEQ BD NO: 80, and SEQ ID NO: 90.
  • the VH-CDR3 amino acid sequence is selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, SEQ ID NO:
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VL of the antibody or fragment thereof comprises a Kabat light chain complementarity determining region- 1 (VL-CDRl) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDRl amino acid sequence selected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 83, and SEQ ID NO: 93.
  • the VL-CDRl amino acid sequence is selected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 53, SEQ ID NO:
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VL of the antibody or fragment thereof comprises a Kabat light chain complementarity determining region-2 (VL-CDR2) amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VL-CDR2 amino acid sequence selected from the group consisting of: SEQ ID NO: 24, SEQ BD NO: 34, SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO: 64, SEQ ID NO: 74, SEQ ID NO: 84, and SEQ ID NO: 94.
  • the VL-CDR2 amino acid sequence is selected from the group consisting of: SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO:
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VL of the antibody or fragment thereof comprises a Kabat light chain complementarity determining region-3 (VL-CDR3) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDR3 amino acid sequence selected from the group consisting of: SEQ ID NO: 25, SEQ ID NO: 35, SEQ ID NO: 45, SEQ ID NO: 55, SEQ ID NO: 65, SEQ ID NO: 75, SEQ ID NO: 85, and SEQ ID NO: 95.
  • the VL-CDR3 amino acid sequence is selected from the group consisting of: SEQ ID NO: 25, SEQ ID NO: 35, SEQ ID NO: 45, SEQ ID NO: 55, SEQ ID NO:
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH of the antibody or fragment thereof comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79 and 80; and SEQ ID NOs: 88, 89 and 90, except for one, two, three, or four amino acid substitutions in at least one of said VH-CDRs.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH of the antibody or fragment thereof comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ED NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ BD NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79 and 80; and SEQ ID NOs: 88, 89 and 90.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VL of the antibody or fragment thereof comprises VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 83, 84 and 85; and SEQ ID NOs 93, 94 and 95, except for one, two, three, or four amino acid substitutions in at least one of said VL-CDRs.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VL of the antibody or fragment thereof comprises VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 83, 84 and 85; and SEQ ID NOs 93, 94 and 95.
  • VH framework regions and/or VL framework regions are human, except for five or fewer amino acid substitutions.
  • the above-described antibodies or fragments thereof bind to a linear epitope or a non-linear conformation epitope
  • the above-described antibodies or fragments thereof are multivalent, and comprise at least two heavy chains and at least two light chains.
  • the above-described antibodies or fragments thereof are multispecific. In further embodiments, the above-described antibodies or fragments thereof are bispecific.
  • the heavy and light chain variable domains are fully human, hi further embodiments, the heavy and light chain variable domains are from a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65- Al 1, M66-H09 and M67-F05.
  • the heavy and light chain variable domains are murine.
  • the heavy and light chain variable domains are from a monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
  • the above-described antibodies or fragments thereof are humanized.
  • the above-described antibodies or fragments thereof are chimeric.
  • the above-described antibodies or fragments thereof are primatized.
  • Fab fragments Fab fragments, Fab' fragments, F(ab) 2 fragments, or Fv fragments.
  • the above-described antibodies are single chain antibodies.
  • the above-described antibodies or fragments thereof comprise light chain constant regions selected from the group consisting of a human kappa constant region and a human lambda constant region.
  • the above-described antibodies or fragments thereof comprise a heavy chain constant region or fragment thereof.
  • the heavy chain constant region or fragment thereof is human IgG4.
  • the above-described antibodies or fragments thereof specifically bind to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or an ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) which is less than the K D for said reference monoclonal antibody, hi further embodiments, the dissociation constant (K D ) is no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 ⁇ 3 M, 10 "3 M, 5 x Kr 4 M, 10 "4 M, 5 x 10 "5 M, 10 '5 M, 5 x 10 "6 M, 10 "6 M, 5 x 10 "7 M, 10 "7 M, 5 x 10 "8 M, 10 “8 M, 5 x Kr 9 M, 10 "9 M, 5 x 10 "10 M, 10 “10 M, 5 x 10 "H M, 10 11 M, 5 x 10 "12 M, 10 "12 M, 5 x 10 "
  • the above-described antibodies or fragments thereof preferentially bind to a human ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, relative to a murine ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof .
  • the above described antibodies or fragments thereof bind to ⁇ 6 ⁇ 4 integrin expressed on the surface of a cell.
  • the cell is a malignant cell, a neoplastic cell, a tumor cell, or a metastatic cell.
  • the above described antibodies or fragments thereof block laminin from binding to ⁇ 6 ⁇ 4 integrin.
  • the above described antibodies or fragments thereof inhibit ⁇ 6 ⁇ 4 integrin association with growth factor receptors.
  • the growth factor receptor is selected from the group consisting of erbB2, Met, and Ron.
  • the above described antibodies or fragments thereof inhibit ⁇ 6 ⁇ 4 integrin-mediated PB-K activation.
  • the above described antibodies or fragments thereof inhibit ⁇ 6 ⁇ 4 integrin-mediated activation of the Ras/MAPK signaling pathway.
  • the above described antibodies or fragments thereof inhibit ⁇ 6 ⁇ 4 integrin-mediated cell proliferation or tumor cell growth.
  • the above described antibodies or fragments thereof induce apoptosis.
  • the above described antibodies or fragments thereof further comprise a heterologous polypeptide fused thereto.
  • the above described antibodies or fragments thereof are conjugated to an agent selected from the group consisting of cytotoxic agent, a therapeutic agent, cytostatic agent, a biological toxin, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents
  • the cytotoxic agent is selected from the group consisting of a radionuclide, a biotoxin, an enzymatically active toxin, a cytostatic or cytotoxic therapeutic agent, a prodrugs, an immunologically active ligand, a biological response modifier, or a combination of two or more of any said cytotoxic agents
  • the detectable label is selected from the group consisting of an enzyme, a fluorescent label, a chemi
  • Certain embodiments of the invention include an isolated polynucleotide comprising a nucleic acid which encodes an antibody VH polypeptide, where the amino acid sequence of the VH polypeptide is at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87; and where an antibody or antigen binding fragment thereof comprising the VH polypeptide specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the amino acid sequence of the VH polypeptide is selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87.
  • the nucleotide sequence encoding the VH polypeptide is optimized for increased expression without changing the amino acid sequence of the VH polypeptide.
  • the optimization comprises identification and removal of splice donor and splice acceptor sites and/or optimization of codon usage for the cells expressing the polynucleotide.
  • the nucleic acid comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 36, SEQ ID NO: 46, SEQ ID NO: 56, SEQ ID NO: 66, SEQ ID NO: 76, and SEQ ID NO: 86.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VL polypeptide, where the amino acid sequence of the VL polypeptide is at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ED NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82, and SEQ ID NO: 92; and where an antibody or antigen binding fragment thereof comprising the VL polypeptide specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the amino acid sequence of the VL polypeptide is selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82, and SEQ ID NO: 92.
  • the nucleotide sequence encoding the VL polypeptide is optimized for increased expression without changing the amino acid sequence of said VL polypeptide.
  • the optimization comprises identification and removal of splice donor and splice acceptor sites and/or optimization of codon usage for the cells expressing the polynucleotide
  • the nucleic acid comprises a nucleotide sequence selected from the group consisting of: SEQ DD NO: 21, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 51, SEQ ID NO: 61, SEQ ID NO: 71, SEQ ID NO: 81, and SEQ ID NO: 91.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VH polypeptide, where the amino acid sequence of the VH polypeptide is identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 27, SEQ E) NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87; and where an antibody or antigen binding fragment thereof comprising said VH polypeptide specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VL polypeptide, where the amino acid sequence of the VL polypeptide is identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82 and SEQ ID NO: 92; and wherein an antibody or antigen binding fragment thereof comprising said VL polypeptide specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VH-CDRl amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VH-CDRl amino acid sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO: 58, SEQ ID NO: 68, SEQ ID NO: 78, and SEQ ID NO: 88; and where an antibody or antigen binding fragment thereof comprising the VH-CDRl specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the VH- CDRl amino acid sequence is selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO: 58, SEQ BD NO: 68, SEQ ID NO: 78, and SEQ ID NO: 88.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VH-CDR2 amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR2 amino acid sequence selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 79, and SEQ ID NO: 89; and where an antibody or antigen binding fragment thereof comprising the VH-CDR2 specifically binds to ⁇ 6 ⁇ 4 integrin.
  • VH- CDR2 amino acid sequence is selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 79, and SEQ ID NO: 89.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VH-CDR3 amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR3 amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 80, and SEQ ID NO: 90; and where an antibody or antigen binding fragment thereof comprising the VH-CDR3 specifically binds to ⁇ 6 ⁇ 4 integrin.
  • VH- CDR3 amino acid sequence is selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 80, and SEQ ID NO: 90.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VL-CDRl amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDRl amino acid sequence selected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 83, and SEQ ID NO: 93; and where an antibody or antigen binding fragment thereof comprising the VL-CDRl specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the VL-CDRl amino acid sequence is selected from the group consisting of: SEQ ED NO: 23, SEQ ID NO: 33, SEQ ED NO: 43, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 83, and SEQ ID NO: 93.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VL-CDR2 amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VL-CDR2 amino acid sequence selected from the group consisting of: SEQ ED NO: 24, SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO: 64, SEQ ED NO: 74, SEQ ED NO: 84 and SEQ ED NO: 94; and wherein an antibody or antigen binding fragment thereof comprising said VL-CDR2 specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the VL-CDR2 amino acid sequence is selected from the group consisting of: SEQ ED NO: 24, SEQ ED NO: 34, SEQ ID NO: 44, SEQ ED NO: 54, SEQ ID NO: 64, SEQ ID NO: 74, SEQ ID NO: 84 and SEQ ID NO: 94.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VL-CDR3 amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDR3 amino acid sequence selected from the group consisting of: SEQ ED NO: 25, SEQ ED NO: 35, SEQ ED NO: 45, SEQ ED NO: 55, SEQ ED NO: 65, SEQ ED NO: 75, SEQ ED NO: 85, and SEQ ED NO: 95; and wherein an antibody or antigen binding fragment thereof comprising said VL-CDR3 specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the VL-CDR3 amino acid sequence is selected from the group consisting of: SEQ ED NO: 25, SEQ ED NO: 35, SEQ ED NO: 45, SEQ ED NO: 55, SEQ ED NO: 65, SEQ ED NO: 75, SEQ ED NO: 85, and SEQ ED NO: 95.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VH polypeptide, where the VH polypeptide comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ED NOs: 18, 19, and 20; SEQ ED NOs: 28, 29; and 30; SEQ ED NOs: 38, 39, and 40; SEQ ED NOs: 48, 49, and 50; SEQ ED NOs: 58, 59, and 60; SEQ ED NOs: 68, 69, and 70; SEQ ED NOs: 78, 79, and 80; and SEQ ID NOs: 88, 89 and 90; and where an antibody or antigen binding fragment thereof comprising the VH-CDR3 specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VL polypeptide, wherein said VL polypeptide comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ED NOs: 23, 24, and 25; SEQ ED NOs: 33, 34, and 35; SEQ ED NOs: 43, 44, and 45; SEQ ED NOs: 53, 54, and 55; SEQ ED NOs: 63, 64, and 65; SEQ ED NOs: 73, 74, and 75; SEQ ED NOs: 83, 84, and 85; and SEQ ID NO: 93, 94, and 95; and wherein an antibody or antigen binding fragment thereof comprising said VL-CDR3 specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the above-described polynucleotides further comprise a nucleic acid encoding a signal peptide fused to the antibody VH polypeptide or the antibody VL polypeptide.
  • the above-described polynucleotides further comprise a nucleic acid encoding a heavy chain constant region CHl domain fused to the VH polypeptide, encoding a heavy chain constant region CH2 domain fused to the VH polypeptide, encoding a heavy chain constant region CH3 domain fused to the VH polypeptide, or encoding a heavy chain hinge region fused to said VH polypeptide.
  • the heavy chain constant region is human IgG4.
  • the above-described polynucleotides comprise a nucleic acid encoding a light chain constant region domain fused to said VL polypeptide.
  • the light chain constant region is human kappa.
  • the antibody or antigen-binding fragment thereof comprising a polypeptide encoded by the nucleic acid specifically binds the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
  • the antibody or antigen-binding fragment thereof comprising a polypeptide encoded by the nucleic acid competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66- H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
  • the framework regions of the VH polypeptide or VL polypeptide are human, except for five or fewer amino acid substitutions.
  • the invention provides an antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid, that binds to a linear epitope or a non-linear conformational epitope.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is multivalent, and comprises at least two heavy chains and at least two light chains.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is multispecific. In further embodiments, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is bispecific.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid comprises heavy and light chain variable domains which are fully human.
  • the heavy and light chain variable domains are identical to those of a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid comprises heavy and light chain variable domains which are murine.
  • the heavy and light chain variable domains are identical to those of a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D 10.1 and 1.P5B10.2.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is humanized.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is primatized.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is chimeric.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is fully human.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is an Fab fragment, an Fab' fragment, an F(ab) 2 fragment, or an Fv fragment, hi certain embodiments of the above-described polynucleotides, the antibody or antigen- binding fragment thereof comprising the polypeptide encoded by the nucleic acid is a single chain antibody.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid specifically binds to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or an ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 '2 M, 10 ⁇ 2 M, 5 x 10 "3 M, 10 '3 M, 5 x 10 "4 M, 10 "4 M, 5 x 10 "5 M, 10 "5 M, 5 x 10 "6 M, 10 "6 M, 5 x 10 "7 M, 10 "7 M, 5 x 10 "8 M, 10 “8 M, 5 x 10 "9 M, 1(T 9 M, 5 x 10- 10 M, 10- 10 M, 5 x 1(T” M, 1(T 1 M, 5 x 10 "12 M, 1(T 12 M, 5 x 10
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid preferentially binds to a human ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, relative to a murine ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid binds to ⁇ 6 ⁇ 4 integrin expressed on the surface of a cell.
  • the cell is a malignant cell, a neoplastic cell, a tumor cell, or a metastatic cell.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by said nucleic acid blocks laminin from binding to ⁇ 6 ⁇ 4 integrin.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits ⁇ 6 ⁇ 4 integrin -mediated activation of PI3-K.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits activation of the Ras/MAPK signaling pathway. [0091] hi some embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits ⁇ 6 ⁇ 4 integrin association with growth factor receptors.
  • the growth factor receptor is selected from the group consisting of erbB2,
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits ⁇ 6 ⁇ 4 integrin-mediated cell proliferation or tumor cell growth.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid induces apoptosis.
  • the above-described polynucleotides further comprise a nucleic acid encoding a heterologous polypeptide.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is conjugated to an agent selected from the group consisting of cytotoxic agent, a therapeutic agent, cytostatic agent, a biological toxin, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents.
  • an agent selected from the group consisting of cytotoxic agent, a therapeutic agent, cytostatic agent, a biological toxin, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents.
  • the cytotoxic agent is selected from the group consisting of a radionuclide, a biotoxin, an enzymatically active toxin, a cytostatic or cytotoxic therapeutic agent, a prodrugs, an immunologically active ligand, a biological response modifier, or a combination of two or more of any said cytotoxic agents.
  • the detectable label is selected from the group consisting of an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or a combination of two or more of any said detectable labels.
  • the invention provides compositions comprising the above-described polynucleotides.
  • the invention provides vectors comprising the above-described polynucleotides, hi further embodiments, the polynucleotides are operably associated with a promoter. In additional embodiments, the invention provides host cells comprising such vectors. In further embodiments, the invention provides vectors where the polynucleotide is operably associated with a promoter.
  • the invention provides a method of producing an antibody or fragment thereof which specifically binds ⁇ 6 ⁇ 4 integrin, comprising culturing a host cell containing a vector comprising the above-described polynucleotides, and recovering said antibody, or fragment thereof.
  • the invention provides an isolated polypeptide produced by the above-described method.
  • the invention provides isolated polypeptides encoded by the above-described polynucleotides.
  • the antibody or fragment thereof comprising the polypeptide specifically binds to ⁇ 6 ⁇ 4 integrin.
  • Other embodiments include the isolated antibody or fragment thereof comprising the above- described polypeptides.
  • the invention provides a composition comprising an isolated VH encoding polynucleotide and an isolated VL encoding polynucleotide, where the VH encoding polynucleotide and the VL encoding polynucleotide, respectively, comprise nucleic acids encoding amino acid sequences at least 90% identical to reference amino acid sequences selected from the group consisting of: SEQ ID NO: 17 and SEQ ID NO: 22; SEQ ID NO: 27 and SEQ ID NO: 32; SEQ ED NO: 37 and SEQ ID NO: 42; SEQ ED NO: 47 and SEQ ID NO: 52; SEQ ID NO: 57 and SEQ ED NO: 62; SEQ ID NO: 67 and 72; SEQ ID NO: 77 and 82; and SEQ ED NO: 87 and 92; and where an antibody or fragment thereof encoded by the VH and VL encoding polynucleotides specifically binds ⁇ 6 ⁇ 4 integrin
  • the VH encoding polynucleotide and said VL encoding polynucleotide comprise nucleic acids encoding amino acid sequences selected from the group consisting of: SEQ DD NO: 17 and SEQ DD NO: 22; SEQ DD NO: 27 and SEQ DD NO: 32; SEQ DD NO: 37 and SEQ DD NO: 42; SEQ DD NO: 47 and SEQ DD NO: 52; SEQ DD NO: 57 and SEQ DD NO: 62; SEQ DD NO: 67 and 72; SEQ DD NO: 77 and 82; and SEQ DD NO: 87 and 92.
  • the invention provides a composition comprising an isolated VH encoding polynucleotide and an isolated VL encoding polynucleotide, where the VH encoding polynucleotide and the VL encoding polynucleotide, respectively, comprise nucleic acids encoding amino acid sequences identical, except for less than 20 conservative amino acid substitutions, to reference amino acid sequences selected from the group consisting of: SEQ DD NO: 17 and SEQ DD NO: 22; SEQ DD NO: 27 and SEQ DD NO: 32; SEQ DD NO: 37 and SEQ DD NO: 42; SEQ DD NO: 47 and SEQ DD NO: 52; SEQ DD NO: 57 and SEQ DD NO: 62; SEQ DD NO: 67 and 72; SEQ DD NO: 77 and 82; and SEQ DD NO: 87 and 92; and where an antibody or fragment thereof encoded by the VH and VL encoding
  • the VH encoding polynucleotide encodes a VH polypeptide comprising VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ DD NOs: 18, 19, and 20; SEQ DD NOs: 28, 29, and 30; SEQ ED NOs: 38, 39, and 40; SEQ DD NOs: 48, 49, and 50; SEQ BD NOs: 58, 59, and 60; SEQ ED NOs: 68, 69, and 70; SEQ DD NOs: 78, 79 and 80; and SEQ DD NOs: 88, 89, and 90; where the VL encoding polynucleotide encodes a VL polypeptide comprising VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences selected from the group consisting of: SEQ DD NOs: 23, 24, and 25; SEQ DD NOs:
  • the VL encoding polynucleotide further comprises a nucleic acid encoding a signal peptide fused to the antibody VL polypeptide.
  • the VH encoding polynucleotide further comprises a nucleic acid encoding a heavy chain constant region CHl domain fused to the VH polypeptide, further comprises a nucleic acid encoding a heavy chain constant region CH2 domain fused to the VH polypeptide, further comprises a nucleic acid encoding a heavy chain constant region CH3 domain fused to the VH polypeptide, or further comprises a nucleic acid encoding a heavy chain hinge region fused to the VH polypeptide, hi further embodiments, the heavy chain constant region is human IgG4.
  • the VL encoding polynucleotide further comprises a nucleic acid encoding a light chain constant region domain fused to the VL polypeptide.
  • the light chain constant region is human kappa.
  • the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides specifically binds the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66- H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
  • the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to ⁇ 6 ⁇ 4 integrin.
  • the framework regions of the VH and VL polypeptides are human, except for five or fewer amino acid substitutions.
  • the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides binds to a linear epitope or a non-linear conformational epitope.
  • the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides is multivalent, and comprises at least two heavy chains and at least two light chains.
  • the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides is multispecific. hi further embodiments, the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides is bispecific. [0114] hi some embodiments of the above-described compositions, the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides comprises heavy and light chain variable domains which are fully human. In further embodiments, the heavy and light chain variable domains are identical to those of a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03,
  • the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides comprises heavy and light chain variable domains which are murine, hi further embodiments, the heavy and light chain variable domains are identical to those of a monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is humanized. [0117] hi various embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is primatized. [0118] hi various embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is chimeric. [0119] hi some embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is fully human.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is an Fab fragment, an Fab 1 fragment, an F(ab) 2 fragment, or an Fv fragment.
  • the antibody or antigen- binding fragment thereof comprising the polypeptide encoded by the nucleic acid is a single chain antibody.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid specifically binds to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or an ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 "3 M, 10 "3 M, 5 x 10 "4 M, 10 “4 M, 5 x 10 "5 M, 10 "5 M, 5 x 10 "6 M, 10 “6 M, 5 x 10 "7 M, 10 “7 M, 5 x 10 "8 M, 10 “8 M, 5 x 1(T 9 M, 10 "9 M, 5 x 10 "10 M, 10 "10 M, 5 x 10 "11 M, 10 "11 M, 5 x 10 "12 M, 10 "12 M, 5 x 10 "13 M, 10 "13 M, 5
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid preferentially binds to a human ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, relative to a murine ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid binds to ⁇ 6 ⁇ 4 integrin expressed on the surface of a cell.
  • the cell is a malignant cell, a neoplastic cell, a tumor cell, or a metastatic cell.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by said nucleic acid blocks laminin from binding to ⁇ 6 ⁇ 4 integrin.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits ⁇ 6 ⁇ 4 integrin-mediated activation of PI3-K.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits activation of the Ras/MAPK signaling pathway.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits ⁇ 6 ⁇ 4 integrin association with growth factor receptors.
  • the growth factor receptor is selected from the group consisting of erbB2, Met, and Ron.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits ⁇ 6 ⁇ 4 integrin-mediated cell proliferation or tumor cell growth.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid induces apoptosis.
  • the above-described compositions, the VH encoding polynucleotide, the VL encoding polynucleotide, or both the VH and the VL encoding polynucleotides further comprise a nucleic acid encoding a heterologous polypeptide.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is conjugated to an agent selected from the group consisting of cytotoxic agent, a therapeutic agent, cytostatic agent, a biological toxin, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents.
  • an agent selected from the group consisting of cytotoxic agent, a therapeutic agent, cytostatic agent, a biological toxin, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents.
  • the cytotoxic agent is selected from the group consisting of a radionuclide, a biotoxin, an enzymatically active toxin, a cytostatic or cytotoxic therapeutic agent, a prodrugs, an immunologically active ligand, a biological response modifier, or a combination of two or more of any said cytotoxic agents.
  • the detectable label is selected from the group consisting of an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or a combination of two or more of any said detectable labels.
  • the VH encoding polynucleotide is contained on a first vector and the VL encoding polynucleotide is contained on a second vector, hi further embodiments, the VH encoding polynucleotide is operably associated with a first promoter and the VL encoding polynucleotide is operably associated with a second promoter, hi certain other embodiments, the first and second promoters are copies of the same promoter. In further embodiments, the first and second promoters are non-identical.
  • the first vector and the second vector are contained in a single host cell.
  • the first vector and the second vector are contained in separate host cells.
  • the invention provides a method of producing an antibody or fragment thereof which specifically binds ⁇ 6 ⁇ 4 integrin, comprising culturing the above-described host cells, and recovering the antibody, or fragment thereof.
  • the invention provides a method of producing an antibody or fragment thereof which specifically binds ⁇ 6 ⁇ 4 integrin, comprising co-culturing separate host cells, and recovering the antibody, or fragment thereof.
  • the invention provides combining the VH and VL encoding polypeptides, and recovering the antibody, or fragment thereof.
  • the invention provides an antibody or fragment thereof which specifically binds ⁇ 6 ⁇ 4 integrin, produced by the above-described methods.
  • the invention provides compositions, where the VH encoding polynucleotide and the VL encoding polynucleotide are on the same vector, as well as the vectors therein.
  • the VH encoding polynucleotide and the VL encoding polynucleotide are each operably associated with a promoter.
  • the VH encoding polynucleotide and the VL encoding polynucleotide are fused in frame, are co- transcribed from a single promoter operably associated therewith, and are cotranslated into a single chain antibody or antigen-binding fragment thereof.
  • the VH encoding polynucleotide and said VL encoding polynucleotide are co-transcribed from a single promoter operably associated therewith, but are separately translated.
  • the vectors further comprise an IRES sequence disposed between the VH encoding polynucleotide and the VL encoding polynucleotide.
  • the polynucleotide encoding a VH and the polynucleotide encoding a VL are separately transcribed, each being operably associated with a separate promoter.
  • the separate promoters are copies of the same promoter or the separate promoters are non-identical.
  • the invention provides host cells comprising the above- described vectors. [0143] In other embodiments, the invention provides a method of producing an antibody or fragment thereof which specifically binds ⁇ 6 ⁇ 4 integrin, comprising culturing the above-described host cells, and recovering the antibody, or fragment thereof.
  • the invention provides an antibody or fragment thereof which specifically binds ⁇ 6 ⁇ 4 integrin, produced by the above-described methods.
  • the invention provides a method for treating a hyperproliferative disorder in an animal, comprising administering to an animal in need of treatment a composition comprising: a) an isolated antibody or fragment as described above; and b) a pharmaceutically acceptable carrier, hi further embodiments, the hyperproliferative disease or disorder is selected from the group consisting of cancer, a neoplasm, a tumor, a malignancy, or a metastasis thereof.
  • the antibody or fragment thereof specifically binds to ⁇ 6 ⁇ 4 integrin expressed on the surface of a malignant cell, hi further embodiments, the binding of the antibody or fragment thereof to the malignant cell results in growth inhibition of the malignant cell.
  • the antibody or fragment thereof inhibits ⁇ 6 ⁇ 4 integrin phosphorylation or inhibits tumor cell proliferation
  • the tumor cell proliferation is inhibited through the prevention or retardation of metastatic growth.
  • the antibody or fragment thereof inhibits tumor cell migration, hi further embodiments, the tumor cell proliferation is inhibited through the prevention or retardation of tumor spread to adjacent tissues.
  • the hyperproliferative disease or disorder is a neoplasm located in the: prostate, colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, adrenal gland, parathyroid gland, pituitary gland, testicles, ovary, thymus, thyroid, eye, head, neck, central nervous system, peripheral nervous system, lymphatic system, pelvis, skin, soft tissue, spleen, thoracic region, or urogenital tract.
  • the hyperproliferative disease is cancer, said cancer selected from the group consisting of: epithelial squamous cell cancer, melanoma, leukemia, myeloma, stomach cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, renal cancer, prostate cancer, testicular cancer, thyroid cancer, and head and neck cancer.
  • the cancer is selected from the group consisting of stomach cancer, renal cancer, brain cancer, bladder cancer, colon cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and prostate cancer.
  • the animal is a mammal.
  • the mammal is a human.
  • Figure 1 shows the purification of soluble ⁇ 6Fc ⁇ 4 protein.
  • Figure 2 shows the titration of murine monoclonal antibodies against CHO- human- ⁇ 6 ⁇ 4.
  • Figure 3 shows inhibition of murine antibody binding with ⁇ 6Fc ⁇ 4 protein.
  • Figure 4 shows titration of murine antibodies against colorectal and breast tumor cell lines.
  • Figure 5 shows the titration of IgG4P human antibodies against CHO-human- ⁇ 6 ⁇ 4.
  • Figure 6 shows the titration of IgG4P human antibodies against CHO-murine- ⁇ 6 ⁇ 4.
  • Figure 7 shows specificity of human antibody 59B05.
  • Figure 8 shows the inhibition of human antibody binding with ⁇ 6Fc ⁇ 4.
  • Figure 9 shows titration of human antibody 59B05 binding to breast and colorectal tumor cell lines.
  • Figure 10 shows the inhibition of anchorage independent growth in SW620 colorectal cancer cells.
  • Figure 11 shows the inhibition of anchorage independent growth in MDA-MB-
  • Figure 12 shows the inhibition of adhesion in colorectal cancer cells to SCC25
  • Figure 13 shows induction of apoptosis in human tumor cell lines.
  • Figure 14 shows inhibition of Phospho-AKT in breast tumor cells with human ⁇ 6 ⁇ 4 antibodies.
  • Figure 15 shows inhibition of SW480 colorectal tumor cell line adhesion on purified rat laminin-5 using full-length IgG4 versus Fab versions of human antibodies
  • Figure 16 shows inhibition of anchorage independent growth in BT474 breat cancer cells using full-length IgG4 versus Fab versions of human antibodies 59B05 and 61C03.
  • Figure 17 shows the induction of apoptosis in MCF7 breast cancer cells by human antibody 59B05.
  • Figure 18 shows a western blot of CNBr cleavage products from ⁇ 4 digests.
  • a or “an” entity refers to one or more of that entity; for example, “an ⁇ 6 ⁇ 4 integrin antibody,” is understood to represent one or more ⁇ 6 ⁇ 4 integrin antibodies.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • polypeptide is intended to encompass a singular
  • polypeptide as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
  • polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
  • peptides, dipeptides, tripeptides, oligopeptides, "protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms.
  • polypeptide is also intended to refer to the products of post- expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.
  • a polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.
  • a polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids.
  • Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.
  • glycoprotein refers to a protein coupled to at least one carbohydrate moiety that is attached to the protein via an oxygen-containing or a nitrogen-containing side chain of an amino acid residue, e.g., a serine residue or an asparagine residue.
  • an "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required.
  • an isolated polypeptide can be removed from its native or natural environment.
  • Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
  • polypeptides of the present invention are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof.
  • fragments include any polypeptides which retain at least some of the antigen-binding properties of the corresponding native antibody or polypeptide. Fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein.
  • Variants of ⁇ 6 ⁇ 4 integrin antibodies and antibody polypeptides of the present invention include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may occur naturally or be non-naturally occurring Non- naturally occurring variants may be produced using art-known mutagenesis techniques. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of ⁇ 6 ⁇ 4 integrin antibodies and antibody polypeptides of the present invention, are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins.
  • variant polypeptides may also be referred to herein as "polypeptide analogs.”
  • a “derivative" of an ⁇ 6 ⁇ 4 integrin antibody or antibody polypeptide refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group.
  • derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3- methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
  • polynucleotide is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA).
  • mRNA messenger RNA
  • pDNA plasmid DNA
  • a polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).
  • PNA peptide nucleic acids
  • nucleic acid refer to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.
  • isolated nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
  • a recombinant polynucleotide encoding an ⁇ 6 ⁇ 4 integrin antibody contained in a vector is considered isolated for the purposes of the present invention.
  • Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present invention.
  • Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically.
  • polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
  • a "coding region” is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more coding regions of the present invention can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors.
  • any vector may contain a single coding region, or may comprise two or more coding regions, e.g., a single vector may separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region.
  • a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a nucleic acid encoding an ⁇ 6 ⁇ 4 integrin antibody or fragment, variant, or derivative thereof.
  • Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.
  • the polynucleotide or nucleic acid is DNA.
  • a polynucleotide comprising a nucleic acid which encodes a polypeptide normally may include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions.
  • An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s).
  • Two DNA fragments are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
  • a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid.
  • the promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells.
  • transcription control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.
  • Suitable promoters and other transcription control regions are disclosed herein.
  • transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus).
  • Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit ⁇ -globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
  • translation control elements include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
  • a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA).
  • mRNA messenger RNA
  • Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention.
  • proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
  • polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce a secreted or "mature” form of the polypeptide.
  • the native signal peptide e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it.
  • a heterologous mammalian signal peptide, or a functional derivative thereof may be used.
  • the wild- type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse ⁇ -glucuronidase.
  • the present invention is directed to certain ⁇ 6 ⁇ 4 integrin antibodies, or antigen- binding fragments, variants, or derivatives thereof.
  • ⁇ 6 ⁇ 4 integrin antibodies encompasses full-sized antibodies as well as antigen-binding fragments, variants, analogs, or derivatives of such antibodies, e.g., naturally occurring antibody or immunoglobulin molecules or engineered antibody molecules or fragments that bind antigen in a manner similar to antibody molecules.
  • an antigen binding molecule refers in its broadest sense to a molecule that specifically binds an antigenic determinant. It is understood by those of skill in the art that fragments of mature antibodies can bind specifically to an antigen. Accordingly, an antigen binding molecule, as the term is used herein, includes, but is not limited to, fragments of mature antibodies that bind specifically to a target antigen.
  • An ABM of the present invention need not contain a constant region. If one or more constant region(s) is present, in particular embodiments, the constant region is substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical.
  • the ABMs of the present invention may be glycoengineered to enhance antibody dependent cellular cytotoxicity.
  • antibody and “immunoglobulin” are used interchangeably herein.
  • An antibody or immunoglobulin comprises at least the variable domain of a heavy chain, and normally comprises at least the variable domains of a heavy chain and a light chain.
  • Basic immunoglobulin structures in vertebrate systems are relatively well understood. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).
  • immunoglobulin comprises various broad classes of polypeptides that can be distinguished biochemically.
  • heavy chains are classified as gamma, mu, alpha, delta, or epsilon, ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) with some subclasses among them (e.g., ⁇ l- ⁇ 4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA IgG, or IgE, respectively.
  • immunoglobulin subclasses e.g., IgGl, IgG2, IgG3, IgG4, IgAl, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant invention. All immunoglobulin classes are clearly within the scope of the present invention, the following discussion will generally be directed to the IgG class of immunoglobulin molecules.
  • IgG a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000- 70,000. The four chains are typically joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the heavy chains starting at the mouth of the "Y" and continuing through the variable region.
  • Light chains are classified as either kappa or lambda (K, ⁇ ). Each heavy chain class may be bound with either a kappa or lambda light chain, hi general, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells.
  • the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
  • variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity.
  • the constant domains of the light chain (CL) and the heavy chain (CHl, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like.
  • the N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy- terminus of the heavy and light chain, respectively.
  • variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three dimensional antigen binding site.
  • This quaternary antibody structure forms the antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three CDRs on each of the VH and VL chains, hi some instances, e.g., certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, a complete immunoglobulin molecule may consist of heavy chains only, with no light chains. See, e.g., Hamers-Casterman et ai, Nature 563:446-448 (1993).
  • each antigen binding domain is short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain as the antibody assumes its three dimensional configuration in an aqueous environment.
  • the remainder of the amino acids in the antigen binding domains referred to as "framework” regions, show less inter-molecular variability.
  • the framework regions largely adopt a ⁇ - sheet conformation and the CDRs form loops which connect, and in some cases form part of, the ⁇ -sheet structure.
  • framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.
  • the antigen binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope.
  • the amino acids comprising the CDRs and the framework regions, respectively can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined (see, "Sequences of Proteins of Immunological Interest,” Kabat, E., et al, U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol, /95:901-917 (1987), which are incorporated herein by reference in their entireties).
  • CDR complementarity determining region
  • Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody.
  • Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody.
  • Kabat numbering refers to the numbering system set forth by Kabat et al, U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest” (1983).
  • references to the numbering of specific amino acid residue positions in an ⁇ 6 ⁇ 4 integrin antibody or antigen-binding fragment, variant, or derivative thereof of the present invention are according to the Kabat numbering system.
  • VHH the heavy chain variable region
  • VHH variable regions include (a) more hydrophobic amino acids in the light chain contact surface of VH as compared to the corresponding region in VHH, (b) a longer CDR3 in VHH, and (c) the frequent occurrence of a disulfide bond between CDRl and CDR3 in VHH.
  • an antigen binding molecule of the invention comprises at least one heavy or light chain CDR of an antibody molecule. In another embodiment, an antigen binding molecule of the invention comprises at least two CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least three CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least four CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least five CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least six CDRs from one or more antibody molecules. Exemplary antibody molecules comprising at least one CDR that can be included in the subject antigen binding molecules are known in the art and exemplary molecules are described herein.
  • Antibodies or antigen-binding fragments, variants, or derivatives thereof of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab') 2 , Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to ⁇ 6 ⁇ 4 integrin antibodies disclosed herein).
  • anti-Id antigen-binding fragments, variants, or derivatives thereof of the invention
  • Immunoglobulin or antibody molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • Antibody fragments may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHl, CH2, and CH3 domains. Also included in the invention are antigen- binding fragments also comprising any combination of variable region(s) with a hinge region, CHl, CH2, and CH3 domains.
  • Antibodies or immunospecific fragments thereof of the present invention may be from any animal origin including birds and mammals.
  • the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies, hi another embodiment, the variable region may be condricthoid in origin (e.g., from sharks).
  • human antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al. A human antibody is still “human” even if amino acid substitutions are made in the antibody.
  • heavy chain portion includes amino acid sequences derived from an immunoglobulin heavy chain.
  • a polypeptide comprising a heavy chain portion comprises at least one of: a CHl domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.
  • a binding polypeptide for use in the invention may comprise a polypeptide chain comprising a CHl domain; a polypeptide chain comprising a CHl domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CHl domain and a CH3 domain; a polypeptide chain comprising a CHl domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CHl domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain, hi another embodiment, a polypeptide of the invention comprises a polypeptide chain comprising a CH3 domain.
  • a binding polypeptide for use in the invention may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain).
  • a CH2 domain e.g., all or part of a CH2 domain.
  • the heavy chain portions of one polypeptide chain of a multimer are identical to those on a second polypeptide chain of the multimer.
  • heavy chain portion-containing monomers of the invention are not identical.
  • each monomer may comprise a different target binding site, forming, for example, a bispecific antibody.
  • the heavy chain portions of a binding polypeptide for use in the diagnostic and treatment methods disclosed herein may be derived from different immunoglobulin molecules.
  • a heavy chain portion of a polypeptide may comprise a CHl domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule
  • a heavy chain portion can comprise a hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule
  • a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an IgG4 molecule.
  • the term "light chain portion” includes amino acid sequences derived from an immunoglobulin light chain.
  • the light chain portion comprises at least one of a VL or CL domain.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof disclosed herein may be described or specified in terms of the epitope(s) or portion(s) of an antigen, e.g., a target polypeptide ( ⁇ 6 ⁇ 4 integrin) that they recognize or specifically bind.
  • a target polypeptide ⁇ 6 ⁇ 4 integrin
  • the portion of a target polypeptide which specifically interacts with the antigen binding domain of an antibody is an "epitope," or an "antigenic determinant.”
  • a target polypeptide may comprise a single epitope, but typically comprises at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen.
  • the epitope may include a portion of ⁇ 6 and a portion of ⁇ 4 together, i.e., the epitope can comprise portions of both components in the heterodimer.
  • an "epitope" on a target polypeptide may be or include non-polypeptide elements, e.g., an "epitope may include a carbohydrate side chain.
  • the minimum size of a peptide or polypeptide epitope for an antibody is thought to be about four to five amino acids.
  • Peptide or polypeptide epitopes preferably contain at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids. Since a CDR can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids comprising an epitope need not be contiguous, and in some cases, may not even be on the same peptide chain.
  • peptide or polypeptide epitope recognized by ⁇ 6 ⁇ 4 integrin antibodies of the present invention contains a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or between about 15 to about 30 contiguous or non-contiguous amino acids of ⁇ 6 ⁇ 4 integrin.
  • an antibody binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind” to an epitope when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated epitope.
  • the term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope.
  • antibody “A” may be deemed to have a higher specificity for a given epitope than antibody "B,” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.”
  • preferentially binds it is meant that the antibody specifically binds to an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope.
  • an antibody which "preferentially binds" to a given epitope would more likely bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.
  • an antibody may be considered to bind a first epitope preferentially if it binds said first epitope with a dissociation constant (K D ) that is less than the antibody's K D for the second epitope.
  • K D dissociation constant
  • an antibody may be considered to bind a first antigen preferentially if it binds the first epitope with an affinity that is at least one order of magnitude less than the antibody's K D for the second epitope.
  • an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody's K D for the second epitope.
  • an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an off rate (k(off)) that is less than the antibody's k(off) for the second epitope.
  • an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least one order of magnitude less than the antibody's k(off) for the second epitope.
  • an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody's k(off) for the second epitope.
  • An antibody or antigen-binding fragment, variant, or derivative disclosed herein may be said to bind a target polypeptide disclosed herein or a fragment or variant thereof with an off rate (k(off)) of less than or equal to 5 X 10 "2 sec '1 , 10 ⁇ 2 sec "1 , 5 X 10 '3 sec '1 or 10 ⁇ 3 sec "1 .
  • an antibody of the invention may be said to bind a target polypeptide disclosed herein or a fragment or variant thereof with an off rate (k(off)) less than or equal to 5 X 10 "4 sec “1 , 10 "4 sec “1 , 5 X 10 "5 sec '1 , or 10 '5 sec '1 5 X 10 "6 sec “1 , 10 “6 sec “1 , 5 X 10 "7 sec “1 or 10 "7 sec “1 .
  • off rate k(off)
  • An antibody or antigen-binding fragment, variant, or derivative disclosed herein may be said to bind a target polypeptide disclosed herein or a fragment or variant thereof with an on rate (k(on)) of greater than or equal to 10 3 M "1 sec “1 , 5 X 10 3 M “1 sec “1 , 10 4 M “1 sec “1 or 5 X 10 4 M “1 sec “1 .
  • an antibody of the invention may be said to bind a target polypeptide disclosed herein or a fragment or variant thereof with an on rate (k(on)) greater than or equal to 10 5 M “1 sec “1 , 5 X 10 5 M “1 sec “1 , 10 6 M “1 sec “1 , or 5 X 10 6 M “1 sec “1 or 10 7 M “1 sec “1 .
  • An antibody is said to competitively inhibit binding of a reference antibody to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody to the epitope.
  • Competitive inhibition may be determined by any method known in the art, for example, competition ELISA assays.
  • An antibody may be said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
  • the term "affinity” refers to a measure of the strength of the binding of an individual epitope with the CDR of an immunoglobulin molecule. See, e.g., Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) at pages 27-28.
  • the term “avidity” refers to the overall stability of the complex between a population of immunoglobulins and an antigen, that is, the functional combining strength of an immunoglobulin mixture with the antigen. See, e.g. , Harlow at pages 29-34.
  • Avidity is related to both the affinity of individual immunoglobulin molecules in the population with specific epitopes, and also the valencies of the immunoglobulins and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity.
  • ⁇ 6 ⁇ 4 integrin antibodies or antigen-binding fragments, variants or derivatives thereof of the invention may also be described or specified in terms of their cross- reactivity.
  • cross-reactivity refers to the ability of an antibody, specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances.
  • an antibody is cross reactive if it binds to an epitope other than the one that induced its formation.
  • the cross reactive epitope generally contains many of the same complementary structural features as the inducing epitope, and in some cases, may actually fit better than the original.
  • certain antibodies have some degree of cross-reactivity, in that they bind related, but non-identical epitopes, e.g., epitopes with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a reference epitope.
  • epitopes e.g., epitopes with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a reference epitope.
  • An antibody may be said to have little or no cross-reactivity if it does not bind epitopes with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a reference epitope.
  • An antibody may be deemed "highly specific" for a certain epitope, if it does not bind any other analog, ortholog, or homolog of that epitope.
  • binding affinities include those with a dissociation constant or Kd less than 5 x 10 "2 M, 10 "2 M, 5 x 10 "3 M, 10 “3 M, 5 x 10 "4 M, 10 "4 M, 5 x 10 "5 M, 10 "5 M, 5 x 10 "6 M, 10 "6 M, 5 x 10 "7 M, 10 "7 M, 5 x 10 "8 M, 10 "8 M, 5 x 10 "9 M, 10 "9 M, 5 x 10 "10 M, 10 “10 M, 5 x 10 "11 M, 10 "11 M, 5 x 10 "12 M, 10 “12 M, 5 x 10 "13 M, 10 "13 M, 5 x 10 "14 M, 10 “14 M, 5 x 10 "15 M, or 10 "15 M
  • ⁇ 6 ⁇ 4 integrin antibodies or antigen-binding fragments, variants or derivatives thereof of the invention may be "multispecif ⁇ c," e.g., bispecific, trispecific or of greater multispecificity, meaning that it recognizes and binds to two or more different epitopes present on one or more different antigens (e.g., proteins) at the same time.
  • an ⁇ 6 ⁇ 4 integrin antibody is "monospecific” or “multispecif ⁇ c,” e.g., "bispecific,” refers to the number of different epitopes with which a binding polypeptide reacts.
  • Multispecific antibodies may be specific for different epitopes of a target polypeptide described herein or may be specific for a target polypeptide as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material.
  • valency refers to the number of potential binding domains, e.g., antigen binding domains, present in an ⁇ 6 ⁇ 4 integrin antibody, binding polypeptide or antibody. Each binding domain specifically binds one epitope.
  • each binding domain may specifically bind the same epitope, for an antibody with two binding domains, termed “bivalent monospecific,” or to different epitopes, for an antibody with two binding domains, termed “bivalent bispecific.”
  • An antibody may also be bispecific and bivalent for each specificity (termed “bispecific tetravalent antibodies").
  • tetravalent minibodies or domain deleted antibodies can be made.
  • Bispecific bivalent antibodies, and methods of making them are described, for instance in U.S. Patent Nos. 5,731,168; 5,807,706; 5,821,333; and U.S. Appl. Publ. Nos. 2003/020734 and 2002/0155537, the disclosures of all of which are incorporated by reference herein.
  • Bispecific tetravalent antibodies, and methods of making them are described, for instance, in WO 02/096948 and WO 00/44788, the disclosures of both of which are incorporated by reference herein. See generally, PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., J.
  • VH domain includes the amino terminal variable domain of an immunoglobulin heavy chain
  • CHl domain includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain.
  • the CHl domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.
  • CH2 domain includes the portion of a heavy chain molecule that extends, e.g., from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat EA et al. op. cit.
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 residues.
  • Hinge region includes the portion of a heavy chain molecule that joins the CHl domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al, J. Immunol. 757:4083 (1998)).
  • disulfide bond includes the covalent bond formed between two sulfur atoms.
  • the amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group.
  • the CHl and CL regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).
  • chimeric antibody will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified in accordance with the instant invention) is obtained from a second species.
  • the target binding region or site will be from a non-human source (e.g. mouse or primate) and the constant region is human.
  • the term “engineered antibody” refers to an antibody in which the variable domain in either the heavy and light chain or both is altered by at least partial replacement of one or more CDRs from an antibody of known specificity and, if necessary, by partial framework region replacement and sequence changing.
  • the CDRs may be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class and preferably from an antibody from a different species.
  • An engineered antibody in which one or more "donor" CDRs from a non-human antibody of known specificity is grafted into a human heavy or light chain framework region is referred to herein as a "humanized antibody.” It may not be necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another. Rather, it may only be necessary to transfer those residues that are necessary to maintain the activity of the target binding site. Given the explanations set forth in, e.g., U. S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, it will be well within the competence of those skilled in the art, either by carrying out routine experimentation or by trial and error testing to obtain a functional engineered or humanized antibody.
  • polypeptides [0222] As used herein the term "properly folded polypeptide” includes polypeptides
  • a properly folded polypeptide comprises polypeptide chains linked by at least one disulfide bond and, conversely, an improperly folded polypeptide comprises polypeptide chains not linked by at least one disulfide bond.
  • engineered includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g. by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of these techniques).
  • an "in-frame fusion” refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct translational reading frame of the original ORFs.
  • ORFs polynucleotide open reading frames
  • a recombinant fusion protein is a single protein containing two ore more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments may be physically or spatially separated by, for example, in-frame linker sequence.
  • polynucleotides encoding the CDRs of an immunoglobulin variable region may be fused, in-frame, but be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, as long as the "fused" CDRs are co-translated as part of a continuous polypeptide.
  • a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.
  • the term "expression" as used herein refers to a process by which a gene produces a biochemical, for example, an RNA or polypeptide.
  • the process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • shRNA small hairpin RNA
  • siRNA small interfering RNA
  • a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript.
  • Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.
  • additive effect is one in which the effect of two molecules acting simultaneously is the simple sum of the effects that they would have if acting alone.
  • the term “synergistic effect” is one in which the effect of two molecules is greater than the effect of each molecule individually, or the sum of the individual effects.
  • the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • subject or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
  • Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.
  • phrases such as "a subject that would benefit from administration of a binding molecule” and "an animal in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of a binding molecule used, e.g., for detection of an antigen recognized by a binding molecule (e.g., for a diagnostic procedure) and/or from treatment, i.e., palliation or prevention of a disease such as cancer, with a binding molecule which specifically binds a given target protein.
  • the binding molecule can be used in unconjugated form or can be conjugated, e.g., to a drug, prodrug, or an isotope.
  • hyperproliferative disease or disorder all neoplastic cell growth and proliferation, whether malignant or benign, including all transformed cells and tissues and all cancerous cells and tissues.
  • Hyperproliferative diseases or disorders include, but are not limited to, precancerous lesions, abnormal cell growths, benign tumors, malignant tumors, and "cancer.”
  • the hyperproliferative disease or disorder e.g., the precancerous lesion, abnormal cell growth, benign tumor, malignant tumor, or "cancer” comprises cells which express, over-express, or abnormally express ⁇ 6 ⁇ 4 integrin.
  • hyperproliferative diseases, disorders, and/or conditions include, but are not limited to neoplasms, whether benign or malignant, located in the: prostate, colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital tract.
  • neoplasms in certain embodiments, express, over-express, or abnormally express ⁇ 6 ⁇ 4 integrin.
  • hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
  • the diseases involve cells which express, over-express, or abnormally express ⁇ 6 ⁇ 4 integrin.
  • tumor or tumor tissue refer to an abnormal mass of tissue that results from excessive cell division, in certain cases tissue comprising cells which express, over-express, or abnormally express ⁇ 6 ⁇ 4 integrin.
  • a tumor or tumor tissue comprises "tumor cells” which are neoplastic cells with abnormal growth properties and no useful bodily function. Tumors, tumor tissue and tumor cells may be benign or malignant.
  • a tumor or tumor tissue may also comprise "tumor-associated non- tumor cells", e.g., vascular cells which form blood vessels to supply the tumor or tumor tissue. Non-tumor cells may be induced to replicate and develop by tumor cells, for example, the induction of angiogenesis in a tumor or tumor tissue.
  • malignancy refers to a non-benign tumor or a cancer.
  • cancer connotes a type of hyperproliferative disease which includes a malignancy characterized by deregulated or uncontrolled cell growth.
  • examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers are noted below and include: squamous cell cancer (e.g.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer
  • cancer includes primary malignant cells or tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignancy or tumor) and secondary malignant cells or tumors (e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor).
  • primary malignant cells or tumors e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignancy or tumor
  • secondary malignant cells or tumors e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor.
  • Cancers conducive to treatment methods of the present invention involves cells which express, over-express, or abnormally express ⁇ 6 ⁇ 4 integrin.
  • cancers or malignancies include, but are not limited to: Acute
  • the method of the present invention may be used to treat premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above.
  • Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where nonneoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976).
  • Such conditions in which cells begin to express, over-express, or abnormally express ⁇ 6 ⁇ 4 integrin are particularly treatable by the methods of the present invention.
  • Hyperplasia is a form of controlled cell proliferation, involving an increase in cell number in a tissue or organ, without significant alteration in structure or function.
  • Hyperplastic disorders which can be treated by the method of the invention include, but are not limited to, angiofollicular mediastinal lymph node hyperplasia, angiolymphoid hyperplasia with eosinophilia, atypical melanocyte hyperplasia, basal cell hyperplasia, benign giant lymph node hyperplasia, cementum hyperplasia, congenital adrenal hyperplasia, congenital sebaceous hyperplasia, cystic hyperplasia, cystic hyperplasia of the breast, denture hyperplasia, ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia, focal epithelial hyperplasia, gingival hyperplasia, inflammatory fibrous hyperplasia, inflammatory papillary hyperplasia, intravascular papillary endothelial hyperp
  • Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell.
  • Metaplastic disorders which can be treated by the method of the invention include, but are not limited to, agnogenic myeloid metaplasia, apocrine metaplasia, atypical metaplasia, autoparenchymatous metaplasia, connective tissue metaplasia, epithelial metaplasia, intestinal metaplasia, metaplastic anemia, metaplastic ossification, metaplastic polyps, myeloid metaplasia, primary myeloid metaplasia, secondary myeloid metaplasia, squamous metaplasia, squamous metaplasia of amnion, and symptomatic myeloid metaplasia.
  • Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation.
  • Dysplastic disorders which can be treated by the method of the invention include, but are not limited to, anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex, dysplasia epiphysiali
  • Additional pre-neoplastic disorders which can be treated by the method of the invention include, but are not limited to, benign dysproliferative disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps, colon polyps, and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar keratosis.
  • benign dysproliferative disorders e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps, colon polyps, and esophageal dysplasia
  • leukoplakia keratoses
  • Bowen's disease keratoses
  • Farmer's Skin Farmer's Skin
  • solar cheilitis solar keratosis
  • the method of the invention is used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.
  • Additional hyperproliferative diseases, disorders, and/or conditions include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythro leukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, lipos
  • Naturally occurring alpha6/beta4 integrin ( ⁇ 6 ⁇ 4 integrin) is a heterodimer single- pass type I membrane protein composed of one alpha and one beta subunit that are non- covalently linked together.
  • Full-length ⁇ 6 consists of a signal sequence, an extracellular domain, a transmembrane domain and a cytoplasmic domain.
  • An integrin ⁇ 6 heavy chain and an integrin ⁇ 6 light chain are also present in the protein.
  • ⁇ 6 is also known in the art by the names ITGA6, VLA-6 and CD49f antigen. Six isoforms of human ⁇ 6 have been reported, however, two of the isoforms are dominant.
  • VYVYMNQQGR 361 WNNVKPiRLN GTKDSMFGIA VKNIGDINQD GYPDIAVGAP YDDLGKVF ⁇ Y
  • VYVYMNQQGR 361 WNNVKPiRLN GTKDSMFGIA VKNIGDINQD GYPDIAVGAP YDDLGKVF ⁇ Y
  • HGSANGINTK 421 PTQVLKGISP YFGYSIAGNM DLDRNSYPDV AVGSLSDSVT IFRSRPVINI
  • FCDGRLRGHEKFGSCQQGv AATFTKDFHY ⁇ VFGAPGTYNWKGLLFLTSVSYTDPDQFV YKTRPPREQPDTFPDVMMNSYLGFSLDSGKG ⁇ VSKDEITFVSGAPRANHSGAWLLKRD MKSAHLLPEH ⁇ FDGEGLASSFGYDVAWDLNKDGWQD ⁇ VIGAPQYFDRDGEVGGAVY VYMNQQGRWNNVKPIRLNGTKDSMFGIA VKNIGDINQDGYPDIAVGAP YDDLGKVFIY HGSANGiNTKPTQVLKGisPYFGYSiAGNMDLDRNSYPDv AVGSLSDSVTIFRSRPV ⁇ NIQ KTiTVTPNRiDLRQKTACGAPSGiCLQ VKSCFEYT ANP AGYNPSIS ⁇ VGTLEAEKERRKSG
  • mouse ⁇ 6 polypeptide has been reported with the following sequences in
  • FASTA Q61739
  • ⁇ 6 polypeptide domain designations used herein are defined as follows:
  • Full-length ⁇ 4 consists of a signal sequence, an extracellular domain, a transmembrane domain and a cytoplasmic domain.
  • a von Willebrand factor type A (VWFA) domain exists in the extracellular domain and four fibronectin type-Ill domains and one Calx-beta domain exist in the cytoplasmic domain.
  • ⁇ 4 is also known in the art by the names ITGB4, GP150 and CD104 antigen. Five isoforms of human ⁇ 4 exist, however 3 isoforms are dominant.
  • isoform 4 A or isoform 3 sequence and has the accession number AAC51632 in Genbank.
  • APEGGFDAIL 241 QTAVCTRDIG WRPDSTHLLV FSTESAFHYE ADGANVLAGI MSRNDERCHL
  • SDHLDTPMLR 781 SGNLKGRDVV RWKVTNNMQR PGFATHAASI NPTELVPYGL SLRLARLCTE
  • PRSAKPALLK 901 LTEKQVEQRA FHDLKVAPGY YTLTADQDAR GMVEFQEGVE LVDVRVPLFI
  • APEGGFDAIL 241 QTAVCTRDIG WRPDSTHLLV FSTESAFHYE ADGANVLAGI MSRNDERCHL
  • SDHLDTPMLR 781 SGNLKGRDW RWKVTNNMQR PGF ATHAASI NPTELVPYGL SLRLARLCTE
  • PRSAKPALLK 901 LTEKQVEQRA FHDLKVAPGY YTLTADQDAR GMVEFQEGVE LVDVRVPLFI
  • HLVNGRMDFA 1381 FPGSTNSLHR MTTTSAAAYG THLSPHVPHR VLSTSSTLTR DYNSLTRSEH
  • RAGLFQHPLQ 1741 SEYSSISTTH TSATEPFLVD GPTLGAQHLE AGGSLTRHVT QEFVSRTLTT
  • APEGGFDAIL 241 QTAVCTRDIG WRPDSTHLLV FSTESAFHYE ADGANVLAGI MSRNDERCHL
  • SDHLDTPMLR 781 SGNLKGRDVV RWKVTNNMQR PGFATHAASI NPTELVPYGL SLRLARLCTE
  • PRSAKPALLK 901 LTEKQVEQRA FHDLKVAPGY YTLTADQDAR GMVEFQEGVE LVDVRVPLFI
  • FASTA sp_vs
  • mouse ⁇ 4 polypeptide (isoform 1) has been reported with the following and has the accession number NP_001005608 in Genbank.
  • Full-Length mouse ⁇ 4 (isoform 1) (SEQ ID NO: 15):
  • VHLRAVEDID 421 GTHVCQLAKE DQGGNIHLKP SFSDGLRMDA SVICDVCPCE LQKEVRSARC
  • ICNGRGYCEC 601 GRCHCNQQSL YTDTTCEINY SAIRLGLCED LRSCVQCQAW GTGEKKGRAC
  • VNGRMDFAYP 1381 GSANSLHRMT AANVAYGTHL SPHLSHRVLS TSSTLTRDYH SLTRTEHSHS
  • ⁇ 4 polypeptide domain designations used herein are defined as follows:
  • the present invention is also directed to ⁇ 6 ⁇ 4 integrin antibodies, or antigen- binding fragments, variants, or derivatives thereof which bind specifically, preferentially, or competitively to non-human ⁇ 6 ⁇ 4 integrin proteins, e.g., ⁇ 6 ⁇ 4 integrin from rodents or non-human primates.
  • ⁇ 6 ⁇ 4 integrin is expressed in a large number of tumor cells, including, but not limited to certain of the following: bladder tumors (Oncol. Rep. 7:13-16 (2000)); breast tumors (Breast Cancer Res. 9:203 (2007) and Exp. Cell Res. 312:3822-3834 (2006)); colon tumors, (Exp. Cell Res. 266:1-10 (2001)); gastric tumors (Am. J. Pathol. 149:781- 793 (1996)); lung tumors (J. Cell Biochem. 55:409-418 (1994)); ovarian tumors (Hum. Pathol. 34:803-808 (2003)); pancreatic tumors,(J. Cell Sci.
  • the present invention is directed to ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof.
  • the present invention includes at least the antigen-binding domains of certain monoclonal antibodies, and fragments, variants, and derivatives thereof shown in Tables 4 and 5 .
  • Table 4 lists human anti-human ⁇ 6 ⁇ 4 integrin Fab regions identified from a phage display library.
  • Table 5 lists murine anti-human ⁇ 6 ⁇ 4 integrin monoclonal antibodies identified by hybridoma technology.
  • the term "antigen binding domain” includes a site that specifically binds an epitope on an antigen (e.g., an epitope of ⁇ 6 ⁇ 4 integrin).
  • the antigen binding domain of an antibody typically includes at least a portion of an immunoglobulin heavy chain variable region and at least a portion of an immunoglobulin light chain variable region. The binding site formed by these variable regions determines the specificity of the antibody.
  • the present invention is more specifically directed to an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant or derivatives thereof, where the ⁇ 6 ⁇ 4 integrin antibody specifically binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61- C03, M65-A11, M66-H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to ⁇ 6 ⁇ 4 integrin.
  • a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61- C03, M65-A11, M66-H09 and M67-F05
  • a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B
  • the invention is further drawn to an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant or derivatives thereof, where the ⁇ 6 ⁇ 4 integrin antibody competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to ⁇ 6 ⁇ 4 integrin from binding to ⁇ 6 ⁇ 4 integrin.
  • a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05
  • a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to
  • the invention is also drawn to an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant or derivatives thereof, where the ⁇ 6 ⁇ 4 integrin antibody comprises an antigen binding domain identical to that of a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to ⁇ 6 ⁇ 4 integrin.
  • a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05
  • a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to ⁇ 6 ⁇ 4 integrin
  • determining which amino acids, or epitope, of ⁇ 6 ⁇ 4 integrin to which the antibody or antigen binding fragment binds can be determined by epitope mapping protocols as described herein as well as methods known in the art (e.g. double antibody-sandwich ELISA as described in "Chapter 11 - Immunology,” Current Protocols in Molecular Biology, Ed. Ausubel et ai, v.2, John Wiley & Sons, Inc. (1996)). Additional epitope mapping protocols may be found in Morris, G.
  • Epitope Mapping Protocols New Jersey: Humana Press (1996), which are both incorporated herein by reference in their entireties. Epitope mapping can also be performed by commercially available means (i.e. ProtoPROBE, Inc. (Milwaukee, Wisconsin)).
  • antibodies produced which bind to any portion of ⁇ 6 ⁇ 4 integrin can then be screened for their ability to act as an antagonist of ⁇ 6 ⁇ 4 integrin for example, to inhibit binding of laminin to ⁇ 6 ⁇ 4 integrin, to inhibit activation of the PI3-K/Akt pathway including but not limited to Rac, mTOR and Akt, to inhibit activation of the Ras/MAPK signaling pathway, to inhibit the interaction of ⁇ 6 ⁇ 4 with growth factor receptors including but not limited to erbB2, Met, and Ron, to inhibit integrin clustering in lipid rafts, to inhibit the interaction with cytoskeletal components, to induce apoptosis, to inhibit receptor tyrosine kinase (RTK) activity, to promote caspase cleavage, to induce integrin internalization or to inhibit tumor cell growth, proliferation, motility or metastasis.
  • Antibodies can be screened for these and other properties according to methods described in detail in the Examples. Other functions of antibodies of the present invention can be
  • the present invention includes an antibody, or antigen- binding fragment, variant, or derivative thereof which specifically or preferentially binds to at least one epitope of ⁇ 6 ⁇ 4 integrin, where the epitope comprises, consists essentially of, or consists of at least about four to five amino acids of SEQ ID NOs: 1-15, at least seven, at least nine, or between at least about 15 to about 30 amino acids of SEQ ID NO:1-15.
  • amino acids of a given epitope of SEQ ID NOs:l-15 as described may be, but need not be contiguous or linear, hi certain embodiments, at least one epitope of ⁇ 6 ⁇ 4 integrin comprises, consists essentially of, or consists of a non-linear epitope formed by the extracellular domain of ⁇ 6 or ⁇ 4 integrin as expressed on the surface of a cell or as a soluble fragment, e.g., fused to an IgG Fc region.
  • At least one epitope of ⁇ 6 ⁇ 4 integrin comprises, consists essentially of, or consists of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguous amino acids of SEQ ID NOs: 1-15, where non-contiguous amino acids form an epitope through protein folding.
  • the present invention includes an antibody, or antigen- binding fragment, variant, or derivative thereof which specifically or preferentially binds to at least one epitope of ⁇ 6 ⁇ 4 integrin, where the epitope comprises, consists essentially of, or consists of a portion of ⁇ 6 and a portion of ⁇ 4.
  • At least one epitope of ⁇ 6 ⁇ 4 integrin comprises, consists essentially of, or consists of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguous amino acids of an ⁇ 6 sequence selected from the group consisting of SEQ ID NOs: 1-8 and at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguous amino acids of a ⁇ 4 sequence selected from the group consisting of SEQ ID NOs: 1
  • the present invention includes an antibody, or antigen- binding fragment, variant, or derivative thereof which specifically or preferentially binds to at least one epitope of ⁇ 6 ⁇ 4 integrin, where the epitope comprises, consists essentially of, or consists of, in addition to one, two, three, four, five, six or more contiguous or noncontiguous amino acids of SEQ ID NOs:l-15 as described above, and an additional moiety which modifies the protein, e.g., a carbohydrate moiety may be included such that the ⁇ 6 ⁇ 4 integrin antibody binds with higher affinity to modified target protein than it does to an unmodified version of the protein. Alternatively, the oc6 ⁇ 4 integrin antibody does not bind the unmodified version of the target protein at all.
  • the present invention is directed to an antibody, or antigen- binding fragment, variant, or derivative thereof which specifically binds to a ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or an ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) which is less than the K D for a given reference monoclonal antibody.
  • K D dissociation constant
  • an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention binds specifically to at least one epitope of ⁇ 6 ⁇ 4 integrin or fragment or variant described above, i.e., binds to such an epitope more readily than it would bind to an unrelated, or random epitope; binds preferentially to at least one epitope of ⁇ 6 ⁇ 4 integrin or fragment or variant described above, i.e., binds to such an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope; competitively inhibits binding of a reference antibody which itself binds specifically or preferentially to a certain epitope of ⁇ 6 ⁇ 4 integrin or fragment or variant described above; or binds to at least one epitope of ⁇ 6 ⁇ 4 integrin or fragment or variant described above with an affinity characterized by a dissociation constant K D of less than about 5 x 10 "2 M, about 10
  • the antibody or fragment thereof preferentially binds to a human ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, relative to a murine ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, hi another particular aspect, the antibody or fragment thereof preterentially binds to one or more ⁇ 6 ⁇ 4 integrin polypeptides or fragments thereof, e.g., one or more mammalian ⁇ 6 ⁇ 4 integrin polypeptides.
  • an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention binds ⁇ 6 ⁇ 4 integrin polypeptides or fragments or variants thereof with an off rate (k(off)) of less than or equal to 5 X 10 "2 sec “1 , 10 "2 sec “1 , 5 X 10 "3 sec “1 or 10 '3 sec “1 .
  • an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention binds ⁇ 6 ⁇ 4 integrin polypeptides or fragments or variants thereof with an off rate (k(off)) of less than or equal to 5 X 10 "4 sec “1 , 10 "4 sec “1 , 5 X 10 "5 sec “1 , or 10 "5 sec “1 5 X 10 "6 sec “1 , 10 “6 sec “1 , 5 X 10 "7 sec “1 or 10 "7 sec “1 .
  • an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention binds ⁇ 6 ⁇ 4 integrin polypeptides or fragments or variants thereof with an on rate (k(on)) of greater than or equal to 10 3 M "1 sec “1 , 5 X 10 3 M “1 sec “1 , 10 4 M “1 sec “1 or 5 X 10 4 M “1 sec “1 .
  • an antibody, or antigen- binding fragment, variant, or derivative thereof of the invention binds ⁇ 6 ⁇ 4 integrin polypeptides or fragments or variants thereof with an on rate (k(on)) greater than or equal to 10 5 M "1 sec “1 , 5 X 10 5 M “1 sec “1 , 10 6 M “1 sec “1 , or 5 X 106 M “1 sec “1 or 10 7 M “1 sec “1 .
  • an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof as described herein is an antagonist of ⁇ 6 ⁇ 4 integrin activity.
  • binding of an antagonist ⁇ 6 ⁇ 4 integrin antibody to ⁇ 6 ⁇ 4 integrin as expressed on a tumor cell inhibits binding of laminin to ⁇ 6 ⁇ 4 integrin, inhibits activation of the PB-K/ Akt pathway, or inhibits tumor cell proliferation, motility or metastasis.
  • an ⁇ 6 ⁇ 4 integrin antibody e.g., an antibody of the invention is a bispecific ⁇ 6 ⁇ 4 integrin antibody, e.g., a bispecific antibody, minibody, domain deleted antiDody, or fusion protein having binding specificity for more than one epitope, e.g., more than one antigen or more than one epitope on the same antigen.
  • a bispecific ⁇ 6 ⁇ 4 integrin antibody has at least one binding domain specific for at least one epitope on a target polypeptide disclosed herein, e.g., ⁇ 6 ⁇ 4 integrin.
  • a bispecific ⁇ 6 ⁇ 4 integrin antibody has at least one binding domain specific for an epitope on a target polypeptide and at least one target binding domain specific for a drug or toxin.
  • a bispecific ⁇ 6 ⁇ 4 integrin antibody has at least one binding domain specific for an epitope on a target polypeptide disclosed herein, and at least one binding domain specific for a prodrug.
  • a bispecific ⁇ 6 ⁇ 4 integrin antibody may be a tetravalent antibody that has two target binding domains specific for an epitope of a target polypeptide disclosed herein and two target binding domains specific for a second target.
  • a tetravalent bispecific ⁇ 6 ⁇ 4 integrin antibody may be bivalent for each specificity.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can comprise a constant region which mediates one or more effector functions.
  • binding of the Cl component of complement to an antibody constant region may activate the complement system.
  • Activation of complement is important in the opsonisation and lysis of cell pathogens.
  • the activation of complement also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity.
  • antibodies bind to receptors on various cells via the Fc region, with a Fc receptor binding site on the antibody Fc region binding to a Fc receptor (FcR) on a cell.
  • FcR Fc receptor
  • Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • certain embodiments of the invention include an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof, in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as reduced effector functions, the ability to non-covalently dimerize, increased ability to localize at the site of a tumor, reduced serum half-life, or increased serum half-life when compared with a whole, unaltered antibody of approximately the same immunogenicity.
  • certain antibodies for use in the diagnostic and treatment methods described herein are domain deleted antibodies which comprise a polypeptide chain similar to an immunoglobulin heavy chain, but which lack at least a portion of one or more heavy chain .domains.
  • one entire domain of the constant region of the modified antibody will be deleted, for example, all or part of the CH2 domain will be deleted.
  • certain antibodies for use in the diagnostic and treatment methods described herein have a constant region, e.g., an IgG4 heavy chain constant region, which is altered to eliminate glycosylation, referred to elsewhere herein as "agly" antibodies. While not being bound by theory, it is believed that "agly" antibodies may have an improved safety and stability profile in vivo. Methods of producing aglycosylated antibodies, having desired effector function are found for example in WO 2005018572, which is incorporated by reference in its entirety.
  • the Fc portion may be mutated to decrease effector function using techniques known in the art.
  • the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified antibody thereby increasing tumor localization.
  • constant region modifications consistent with the instant invention moderate complement binding and thus reduce the serum half life and nonspecific association of a conjugated cytotoxin.
  • modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility.
  • the resulting physiological profile, bioavailability and other biochemical effects of the modifications such as tumor localization, biodistribution and serum half- life, may easily be measured and quantified using well know immunological techniques without undue experimentation.
  • Modified forms of ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be made from whole precursor or parent antibodies using techniques known in the art. Exemplary techniques are discussed in more detail herein.
  • both the variable and constant regions of ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof are fully human.
  • Fully human antibodies can be made using techniques that are known in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make such antibodies are described in US patents: 6,150,584; 6,458,592; 6,420,140. Other techniques are known in the art. Fully human antibodies can likewise be produced by various display technologies, e.g., phage display or other viral display systems, as described in more detail elsewhere herein.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be made or manufactured using techniques that are known in the art.
  • antibody molecules or fragments thereof are "recombinantly produced," i.e., are produced using recombinant DNA technology. Exemplary techniques for making antibody molecules or fragments thereof are discussed in more detail elsewhere herein.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention also include derivatives that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from specifically binding to its cognate epitope.
  • the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention will not elicit a deleterious immune response in the animal to be treated, e.g., in a human.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention are modified to reduce their immunogenicity using art-recognized techniques.
  • antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be made.
  • Non-human antibodies are derived from a non-human antibody, typically a murine or primate antibody, that retains or substantially retains the antigen- binding properties of the parent antibody, but which is less immunogenic in humans.
  • This may be achieved by various methods, including (a) grafting the entire non-human variable domains onto human constant regions to generate chimeric antibodies; (b) grafting at least a part of one or more of the non-human complementarity determining regions (CDRs) into a human framework and constant regions with or without retention of critical framework residues; or (c) transplanting the entire non-human variable domains, but "cloaking" them with a human-like section by replacement of surface residues.
  • CDRs complementarity determining regions
  • De-immunization can also be used to decrease the immunogenicity of an antibody.
  • the term "de-immunization” includes alteration of an antibody to modify T cell epitopes ⁇ see, e.g., WO9852976A1, WO0034317A2).
  • VH and VL sequences from the starting antibody are analyzed and a human T cell epitope "map" from each V region showing the location of epitopes in relation to complementarity-determining regions (CDRs) and other key residues within the sequence.
  • CDRs complementarity-determining regions
  • VH and VL sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof for use in the diagnostic and treatment methods disclosed herein, which are then tested for function.
  • binding polypeptides e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof for use in the diagnostic and treatment methods disclosed herein, which are then tested for function.
  • Typically, between 12 and 24 variant antibodies are generated and tested.
  • Complete heavy and light chain genes comprising modified V and human C regions are then cloned into expression vectors and the subsequent plasmids introduced into cell lines for the production of whole antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be generated by any suitable method known in the art.
  • Polyclonal antibodies to an antigen of interest can be produced by various procedures well known in the art.
  • an ⁇ 6 ⁇ 4 integrin antibody e.g., a binding polypeptide, e.g., an ⁇ 6 ⁇ 4 integrin-specific antibody or immunospecific fragment thereof can be administered to various host animals including, but not limited to, rabbits, mice, rats, chickens, hamsters, goats, donkeys, etc., to induce the production of sera containing polyclonal antibodies specific for the antigen.
  • adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988); Hammerling et al, in: Monoclonal Antibodies and T-CeIl Hybridomas Elsevier, N.Y., 563-681 (1981) (said references incorporated by reference in their entireties).
  • the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Thus, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology.
  • Monoclonal antibodies can be prepared using ⁇ 6 ⁇ 4 integrin knockout mice to increase the regions of epitope recognition.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma and recombinant and phage display technology as described elsewhere herein.
  • antibodies are raised in mammals by multiple subcutaneous or intraperitoneal injections of the relevant antigen ⁇ e.g., purified ⁇ 6 ⁇ 4 integrin or cells or cellular extracts comprising ⁇ 6 ⁇ 4 integrin) and an adjuvant.
  • This immunization typically elicits an immune response that comprises production of antigen-reactive antibodies from activated splenocytes or lymphocytes.
  • the resulting antibodies may be harvested from the serum of the animal to provide polyclonal preparations, it is often desirable to isolate individual lymphocytes from the spleen, lymph nodes or peripheral blood to provide homogenous preparations of monoclonal antibodies (MAbs).
  • the lymphocytes are obtained from the spleen.
  • lymphocytes from a mammal which has been injected with antigen are fused with an immortal tumor cell line (e.g., a myeloma cell line), thus, producing hybrid cells or "hybridomas" which are both immortal and capable of producing the genetically coded antibody of the B cell.
  • an immortal tumor cell line e.g., a myeloma cell line
  • hybrid cells or "hybridomas" which are both immortal and capable of producing the genetically coded antibody of the B cell.
  • the resulting hybrids are segregated into single genetic strains by selection, dilution, and regrowth with each individual strain comprising specific genes for the formation of a single antibody. They produce antibodies, which are homogeneous against a desired antigen and, in reference to their pure genetic parentage, are termed "monoclonal.”
  • Hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • reagents, cell lines and media for the formation, selection and growth of hybridomas are commercially available from a number of sources and standardized protocols are well established.
  • culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies against the desired antigen.
  • the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the monoclonal antibodies secreted by the subclones may be separated from culture medium, ascites fluid or serum by conventional purification procedures such as, for example, protein-A, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
  • Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab') 2 fragments may be produced recombinantly or by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments). F(ab') 2 fragments contain the variable region, the light chain constant region and the CHl domain of the heavy chain. [0303] Those skilled in the art will also appreciate that DNA encoding antibodies or antibody fragments (e.g., antigen binding sites) may also be derived from antibody libraries, such as phage display libraries.
  • such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and M 13 binding domains expressed from phage with Fab, Fv OE DAB (individual Fv region from light or heavy chains) or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
  • Ribosomal display can be used to replace bacteriophage as the display platform (see, e.g., Hanes et al, Nat. Biotechnol. 75:1287 (2000); Wilson et al, Proc. Natl. Acad. Sci. USA 98:3750 (2001); or Irving et al, J. Immunol. Methods 248:31 (2001)).
  • cell surface libraries can be screened for antibodies (Boder et al, Proc. Natl. Acad. Sci. USA 97: 10701 (2000); Daugherty et al, J. Immunol Methods 243:211 (2000)).
  • Such procedures provide alternatives to traditional hybridoma techniques for the isolation and subsequent cloning of monoclonal antibodies.
  • phage display methods functional antibody domains are displayed on the surface of phage particles, which carry the polynucleotide sequences encoding them.
  • DNA sequences encoding VH and VL regions are amplified or otherwise isolated from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues) or synthetic cDNA libraries.
  • the DNA encoding the VH and VL regions are joined together by an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS).
  • the vector is electroporated in E. coli and the E.
  • Phage used in these methods are typically filamentous phage including fd and M 13 and the VH or VL regions are usually recombinantly fused to either the phage gene III or gene VIII.
  • Phage expressing an antigen binding domain that binds to an antigen of interest i.e., an ⁇ 6 ⁇ 4 integrin polypeptide or a fragment thereof
  • an antigen of interest i.e., an ⁇ 6 ⁇ 4 integrin polypeptide or a fragment thereof
  • can be selected or identified with antigen e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • phage display methods that can be used to make the antibodies include those disclosed in Brinkman et al, J. Immunol. Methods 752:41-50 (1995); Ames et al, J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol 24:952-958 (1994); Persic et al, Gene 187:9-18 (1997); Burton et al, Advances in Immunology 57:191-280 (1994); PCT Application No.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
  • Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al, BioTechniques 4:214 (1986); Gillies et al, J. Immunol. Methods /25:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entireties.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, e.g., humanized antibodies.
  • Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al, U.S. Pat. No.
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR- grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5) :489-498 (1991); Studnicka et al, Protein Engineering 7 ⁇ :805-814 (1994); Roguska. et al, PNAS 97:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).
  • Human antibodies are particularly desirable for therapeutic treatment of human patients.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
  • Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. Ln particular, homozygous deletion of the JH region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
  • the chimeric mice are then bred to produce homozygous offspring that express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a desired target polypeptide.
  • Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B-cell differentiation, and subsequently undergo class switching and somatic mutation.
  • Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection.” hi this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al, Bio/Technology 72:899-903 (1988). See also, U.S. Patent No. 5,565,332.) [0312] Further, antibodies to target polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" target polypeptides using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J.
  • antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate antiidiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand.
  • antiidiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand.
  • anti-idiotypic antibodies can be used to bind a desired target polypeptide and/or to bind its ligands/receptors, and thereby block its biological activity.
  • DNA encoding desired monoclonal antibodies may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the isolated and subcloned hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells such as, but not limited to, E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulins.
  • the isolated DNA (which may be synthetic as described herein) may be used to clone constant and variable region sequences for the manufacture antibodies as described in Newman et al, U.S. Pat. No. 5,658,570, filed January 25, 1995, which is incorporated by reference herein. Essentially, this entails extraction of RNA from the selected cells, conversion to cDNA, and amplification by PCR using Ig specific primers. Suitable primers for this purpose are also described in U.S. Pat. No. 5,658,570. As will be discussed in more detail below, transformed cells expressing the desired antibody may be grown up in relatively large quantities to provide clinical and commercial supplies of the immunoglobulin.
  • an ⁇ 6 ⁇ 4 integrin antibody of the invention comprises at least one heavy or light chain CDR of an antibody molecule.
  • an ⁇ 6 ⁇ 4 integrin antibody of the invention comprises at least two CDRs from one or more antibody molecules.
  • an ⁇ 6 ⁇ 4 integrin antibody of the invention comprises at least three CDRs from one or more antibody molecules.
  • an ⁇ 6 ⁇ 4 integrin antibody of the invention comprises at least four CDRs from one or more antibody molecules.
  • an ⁇ 6 ⁇ 4 integrin antibody of the invention comprises at least five CDRs from one or more antibody molecules.
  • the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
  • CDRs complementarity determining regions
  • one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody.
  • the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. MoI. Biol.
  • the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds to at least one epitope of a desired polypeptide, e.g., ⁇ 6 ⁇ 4 integrin.
  • a desired polypeptide e.g., ⁇ 6 ⁇ 4 integrin.
  • one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
  • Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain antibody. Techniques for the assembly of functional Fv fragments in E coli may also be used (Skerra et al., Science 242:1038-1041 (1988)).
  • Yet other embodiments of the present invention comprise the generation of human or substantially human antibodies in transgenic animals (e.g., mice) that are incapable of endogenous immunoglobulin production (see e.g., U.S. Pat. Nos. 6,075,181, 5,939,598, 5,591,669 and 5,589,369 each of which is incorporated herein by reference).
  • transgenic animals e.g., mice
  • the homozygous deletion of the antibody heavy- chain joining region in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of a human immunoglobulin gene array to such germ line mutant mice will result in the production of human antibodies upon antigen challenge.
  • Another preferred means of generating human antibodies using SCDD mice is disclosed in U.S. Pat. No. 5,811,524 which is incorporated herein by reference. It will be appreciated that the genetic material associated with these human antibodies may also be isolated and manipulated as described herein.
  • lymphocytes can be selected by micromanipulation and the variable genes isolated.
  • peripheral blood mononuclear cells can be isolated from an immunized mammal and cultured for about 7 days in vitro. The cultures can be screened for specific IgGs that meet the screening criteria. Cells from positive wells can be isolated.
  • Individual Ig-producing B cells can be isolated by FACS or by identifying them in a complement-mediated hemolytic plaque assay.
  • Ig-producing B cells can be micromanipulated into a tube and the VH and VL genes can be amplified using, e.g. , RT-PCR.
  • the VH and VL genes can be cloned into an antibody expression vector and transfected into cells (e.g. , eukaryotic or prokaryotic cells) for expression.
  • antibody-producing cell lines may be selected and cultured using techniques well known to the skilled artisan. Such techniques are described in a variety of laboratory manuals and primary publications. In this respect, techniques suitable for use in the invention as described below are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991) which is herein incorporated by reference in its entirety, including supplements.
  • Antibodies of the present invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques as described herein.
  • an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof of the invention comprises a synthetic constant region wherein one or more domains are partially or entirely deleted ("domain-deleted antibodies").
  • compatible modified antibodies will comprise domain deleted constructs or variants wherein the entire CH2 domain has been removed ( ⁇ CH2 constructs).
  • ⁇ CH2 constructs domain deleted constructs or variants wherein the entire CH2 domain has been removed
  • a short connecting peptide may be substituted for the deleted domain to provide flexibility and freedom of movement for the variable region.
  • Domain deleted constructs can be derived using a vector encoding an IgGi human constant domain (see, e.g., WO 02/060955 A2 and WO02/096948A2). This vector is engineered to delete the CH2 domain and provide a synthetic vector expressing a domain deleted IgGj constant region.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention are minibodies.
  • Minibodies can be made using methods described in the art (see, e.g., US patent 5,837,821 or WO 94/09817A1).
  • an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof of the invention comprises an immunoglobulin heavy chain having deletion or substitution of a few or even a single amino acid as long as it permits association between the monomelic subunits.
  • the mutation of a single amino acid in selected areas of the CH2 domain may be enough to substantially reduce Fc binding and thereby increase tumor localization.
  • Such partial deletions of the constant regions may improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject constant region domain intact.
  • the constant regions of the disclosed antibodies may be synthetic through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this respect it may be possible to disrupt the activity provided by a conserved binding site (e.g. Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified antibody.
  • Yet other embodiments comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as effector function or provide for more cytotoxin or carbohydrate attachment. In such embodiments it may be desirable to insert or replicate specific sequences derived from selected constant region domains.
  • the present invention also provides antibodies that comprise, consist essentially of, or consist of, variants (including derivatives) of antibody molecules (e.g., the VH regions and/or VL regions) described herein, which antibodies or fragments thereof immunospecifically bind to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment or variant thereof.
  • Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding an ⁇ 6 ⁇ 4 integrin antibody, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions.
  • the variants encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference VH region, VH-CDRl, VH-CDR2, VH-CDR3, VL region, VL-CDRl, VL-CDR2, or VL-CDR3.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge.
  • Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains ( e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e
  • mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind an ⁇ 6 ⁇ 4 integrin polypeptide).
  • mutations can be introduced only in framework regions or only in CDR regions of an antibody molecule. Introduced mutations may be silent or neutral missense mutations, i.e., have no, or little, effect on an antibody's ability to bind antigen, indeed some such mutations do not alter the amino acid sequence whatsoever. These types of mutations may be useful to optimize codon usage, or improve a hybridoma's antibody production.
  • Codon-optimized coding regions encoding ⁇ 6 ⁇ 4 integrin antibodies of the present invention are disclosed elsewhere herein.
  • non-neutral missense mutations may alter an antibody's ability to bind antigen.
  • the location of most silent and neutral missense mutations is likely to be in the framework regions, while the location of most non-neutral missense mutations is likely to be in CDR, though this is not an absolute requirement.
  • One of skill in the art would be able to design and test mutant molecules with desired properties such as no alteration in antigen binding activity or alteration in binding activity (e.g., improvements in antigen binding activity or change in antibody specificity).
  • the encoded protein may routinely be expressed and the functional and/or biological activity of the encoded protein, (e.g., ability to immunospecif ⁇ cally bind at least one epitope of an ⁇ 6 ⁇ 4 integrin polypeptide) can be determined using techniques described herein or by routinely modifying techniques known in the art.
  • the present invention also provides for nucleic acid molecules encoding ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention.
  • the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH), where at least one of the CDRs of the heavy chain variable region or at least two of the VH-CDRs of the heavy chain variable region are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDRl, VH-CDR2, or VH-CDR3 amino acid sequences from monoclonal ⁇ 6 ⁇ 4 integrin antibodies disclosed herein.
  • VH immunoglobulin heavy chain variable region
  • VH-CDRl, VH-CDR2, and VH- CDR3 regions of the VH are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences from monoclonal ⁇ 6 ⁇ 4 integrin antibodies disclosed herein.
  • a heavy chain variable region of the invention has VH-CDRl, VH-CDR2, or VH-CDR3 polypeptide sequences related to the polypeptide sequences shown in Table 6.
  • the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL), where at least one of the VL-CDRs of the light chain variable region or at least two of the VL-CDRs of the light chain variable region are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDRl, VL-CDR2, or VL-CDR3 amino acid sequences from monoclonal ⁇ 6 ⁇ 4 integrin antibodies disclosed herein.
  • VL immunoglobulin light chain variable region
  • VL-CDRl, VL-CDR2, and VL-CDR3 regions of the VL are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences from monoclonal ⁇ 6 ⁇ 4 integrin antibodies disclosed herein.
  • a light chain variable region of the invention has VL-CDRl, VL-CDR2, or VL-CDR3 polypeptide sequences related to the polypeptide sequences shown in Table 6.
  • N nucleotide sequence
  • P polypeptide sequence
  • sequence identity between two polypeptides or two polynucleotides is determined by comparing the amino acid or nucleic acid sequence of one polypeptide or polynucleotide to the sequence of a second polypeptide or polynucleotide.
  • sequence identity is determined by comparing the amino acid or nucleic acid sequence of one polypeptide or polynucleotide to the sequence of a second polypeptide or polynucleotide.
  • whether any particular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711).
  • BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences.
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.
  • an antibody or antigen-binding fragment comprising the
  • VH encoded by the polynucleotide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the nucleotide sequence encoding the VH polypeptide is altered without altering the amino acid sequence encoded thereby.
  • the sequence may be altered for improved codon usage in a given species, to remove splice sites, or the remove restriction enzyme sites. Sequence optimizations such as these are described in the examples and are well known and routinely carried out by those of ordinary skill in the art.
  • the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2, and VH-CDR3 regions have polypeptide sequences which are identical to the VH-CDRl, VH-CDR2, and VH-CDR3 groups shown in Table 6.
  • VH immunoglobulin heavy chain variable region
  • an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VH polypeptide, where the VH polypeptide comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ED NOs: 78, 79, and 80; and SEQ ID NOs: 88, 89 and 90; and where an antibody or antigen binding fragment thereof comprising the VH-CDR3 specifically binds to ⁇ 6 ⁇ 4 integrin.
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to ⁇ 6 ⁇ 4 integrin.
  • a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybrid
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or a ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 "3 M, 10 "3 M, 5 x 10 "4 M, 10 “4 M, 5 x 10 "5 M, 10 "5 M, 5 x 10 "6 M, 10 "6 M, 5 x 10 "7 M, 10 "7 M, 5 x 10 "8 M, 10 “8 M, 5 x 10 "9 M, 10 "9 M, 5 x 10 "10 M, 10 "10 M, 5 x 10 "11 M, 10 '11 M, 5 x 10- 12 M, 10- 12 M,
  • an antibody or antigen-binding fragment comprising the
  • VL encoded by the polynucleotide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL) in which the VL-CDRl, VL-CDR2, and VL-CDR3 regions have polypeptide sequences which are identical to the VL-CDRl, VL- CDR2, and VL-CDR3 groups shown in Table 6.
  • VL immunoglobulin light chain variable region
  • an antibody or antigen-binding fragment comprising the VL encoded by the polynucleotide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL) in which the VL-CDRl, VL-CDR2, and VL-CDR3 regions are encoded by nucleotide sequences which are identical to the nucleotide sequences which encode the VL-CDRl, VL-CDR2, and VL-CDR3 groups shown in Table 6.
  • an antibody or antigen-binding fragment comprising the VL encoded by the polynucleotide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VL polypeptide, wherein said VL polypeptide comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs:83, 84, and 85; and SEQ ID NOs:93, 94 and 95; and wherein an antibody or antigen binding fragment thereof comprising said VL-CDR3 specifically binds to ⁇ 6 ⁇ 4 integrin.
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to ⁇ 6 ⁇ 4 integrin.
  • a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybrid
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or a ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 "3 M, 10 3 M, 5 x 10 "4 M, lO "4 M, 5 x 10 "5 M, W 5 M, 5 x 10 "6 M, 10 "6 M, 5 x 10 ⁇ 7 M, 10 "7 M, 5 x Kr 8 M, 1(T 8 M, 5 x ICT 9 M, 10 '9 M, 5 x 10 "10 M, lO '10 M, 5 x lO '11 M, 1(T 11 M, 5
  • K D dissociation constant
  • the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a VH at least 80%, 85%, 90% 95% or 100% identical to a reference VH polypeptide sequence selected from the group consisting of SEQ ID NOs: 17, 27, 37, 47, 57, 67, 77 and 87.
  • an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VH having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 17, 27, 37, 47, 57, 67, 77 and 87.
  • an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a VH-encoding nucleic acid at least 80%, 85%, 90% 95% or 100% identical to a reference nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16, 26, 36, 46, 56, 66, 76 and 86.
  • an antibody or antigen-binding fragment comprising the VH encoded by such polynucleotides specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VH of the invention, where the amino acid sequence of the VH is selected from the group consisting of SEQ ED NOs: 17, 27, 37, 47, 57, 67, 77 and 87.
  • the present invention further includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VH of the invention, where the sequence of the nucleic acid is selected from the group consisting of SEQ ID NOs: 16, 26, 36, 46, 56, 66, 76 and 86.
  • an antibody or antigen-binding fragment comprising the VH encoded by such polynucleotides specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to ⁇ 6 ⁇ 4 integrin.
  • a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybrid
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or a ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 "3 M, 1(T 3 M, 5 x 10 "4 M, 10 "4 M, 5 x 10 "5 M, 10 "5 M, 5 x 10 "6 M, 10 “6 M, 5 x 1(T 7 M, 10 "7 M, 5 x 10 "8 M, 10 “8 M, 5 x 10 “9 M, 1(T 9 M, 5 x 10 "10 M, 10 "10 M, 5 x 10 "11 M, 1(T 11 M, 5 x 10 "12 M
  • the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a VL at least 80%, 85%, 90% 95% or 100% identical to a reference VL polypeptide sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 32, 42, 52, 62, 72, 82 and 92.
  • the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a VL- encoding nucleic acid at least 80%, 85%, 90% 95% or 100% identical to a reference nucleic acid sequence selected from the group consisting of SEQ ID NOs: 21, 31, 41, 51, 61, 71, 81 and 91.
  • an antibody or antigen-binding fragment comprising the VL encoded by such polynucleotides specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VL having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 22, 32, 42, 52, 62, 72, 82 and 92.
  • the present invention further includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VL of the invention, where the sequence of the nucleic acid is selected from the group consisting of SEQ ID NOs: 21, 31, 41, 51, 61, 71, 81 and 91.
  • an antibody or antigen-binding fragment comprising the VL encoded by such polynucleotides specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to ⁇ 6 ⁇ 4 integrin.
  • a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybrid
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or a ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 "3 M, 10 '3 M, 5 x 10 "4 M, 10 "4 M, 5 x 10 "5 M, 10 "5 M, 5 x l( ⁇ 6 M, 10 "6 M, 5 x 10 "7 M, 10 "7 M, 5 x 10 "8 M, 10 “8 M, 5 x 10 "9 M, 10 "9 M, 5 x 10 "10 M, 10 "10 M, 5 x 10 "11 M, 10 "11 M, 5 x 10 " 12 M,
  • any of the polynucleotides described above may further include additional nucleic acids, encoding, e.g., a signal peptide to direct secretion of the encoded polypeptide, antibody constant regions as described herein, or other heterologous polypeptides as described herein.
  • the present invention includes compositions comprising the polynucleotides comprising one or more of the polynucleotides described above.
  • the invention includes compositions comprising a first polynucleotide and second polynucleotide wherein said first polynucleotide encodes a VH polypeptide as described herein and wherein said second polynucleotide encodes a VL polypeptide as described herein.
  • composition which comprises, consists essentially of, or consists of a VH polynucleotide, and a VL polynucleotide, wherein the VH polynucleotide and the VL polynucleotide encode polypeptides, respectively at least 80%, 85%, 90% 95% or 100% identical to reference VH and VL polypeptide amino acid sequences selected from the group consisting of SEQ ID NOs: 17 and 22, 27 and 32, 37 and 42, 47 and 52, 57 and 62, 67 and 72, 77 and 82, and 87 and 92.
  • compositions which comprises, consists essentially of, or consists of a VH polynucleotide, and a VL polynucleotide at least 80%, 85%, 90% 95% or 100% identical, respectively, to reference VL and VL nucleic acid sequences selected from the group consisting of SEQ ID NOs: 16 and 21, 26 and 31, 36 and 41, 46 and 51, 56 and 61, 66 and 71, 76 and 81, and 86 and 91.
  • an antibody or antigen-binding fragment comprising the VH and VL encoded by the polynucleotides in such compositions specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention also includes fragments of the polynucleotides of the invention, as described elsewhere. Additionally polynucleotides which encode fusion polynucleotides, Fab fragments, and other derivatives, as described herein, are also contemplated by the invention.
  • the polynucleotides may be produced or manufactured by any method known in the art.
  • a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 77:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • a polynucleotide encoding an ⁇ 6 ⁇ 4 integrin antibody, or antigen- binding fragment, variant, or derivative thereof may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the antibody may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+RNA, isolated from, any tissue or cells expressing the antibody or other ⁇ 6 ⁇ 4 integrin antibody, such as hybridoma cells selected to express an antibody) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g.
  • a cDNA clone from a cDNA library that encodes the antibody or other ⁇ 6 ⁇ 4 integrin antibody may then be cloned into replicable cloning vectors using any method well known in the art.
  • nucleotide sequence and corresponding amino acid sequence of the ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.
  • a polynucleotide encoding an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • a polynucleotide encoding ⁇ 6 ⁇ 4 integrin antibody, or antigen- binding fragment, variant, or derivative thereof can be composed of single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • a polynucleotide encoding an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • a polynucleotide encoding an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • “Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
  • An isolated polynucleotide encoding a non-natural variant of a polypeptide derived from an immunoglobulin ⁇ e.g., an immunoglobulin heavy chain portion or light chain portion) can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues.
  • the present invention is further directed to isolated polypeptides which make up ⁇ 6 ⁇ 4 integrin antibodies, and polynucleotides encoding such polypeptides.
  • ⁇ 6 ⁇ 4 integrin antibodies of the present invention comprise polypeptides, e.g., amino acid sequences encoding ⁇ 6 ⁇ 4 integrin-specific antigen binding regions derived from immunoglobulin molecules.
  • a polypeptide or amino acid sequence "derived from" a designated protein refers to the origin of the polypeptide having a certain amino acid sequence.
  • the polypeptide or amino acid sequence which is derived from a particular starting polypeptide or amino acid sequence has an amino acid sequence that is essentially identical to that of the starting sequence, or a portion thereof, wherein the portion consists of at least 10-20 amino acids, at least 20-30 amino acids, at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting sequence.
  • the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH), where at least one of VH-CDRs of the heavy chain variable region or at least two of the VH-CDRs of the heavy chain variable region are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDRl, VH-CDR2 or VH-CDR3 amino acid sequences from monoclonal ⁇ 6 ⁇ 4 integrin antibodies disclosed herein.
  • VH immunoglobulin heavy chain variable region
  • VH-CDRl, VH-CDR2 and VH-CDR3 regions of the VH are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDRl, VH-CDR2 and VH-CDR3 amino acid sequences from monoclonal ⁇ 6 ⁇ 4 integrin antibodies disclosed herein.
  • a heavy chain variable region of the invention has VH-CDRl, VH-CDR2 and VH-CDR3 polypeptide sequences related to the groups shown in Table 6, supra. While Table 6 shows VH-CDRs defined by the Kabat system, other CDR definitions, e.g., VH-CDRs defined by the Chothia system, are also included in the present invention.
  • an antibody or antigen- binding fragment comprising the VH specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2 and VH-CDR3 regions have polypeptide sequences which are identical to the VH-CDRl, VH-CDR2 and VH-CDR3 groups shown in Table 6.
  • VH immunoglobulin heavy chain variable region
  • an antibody or antigen-binding fragment comprising the VH specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2 and VH-CDR3 regions have polypeptide sequences which are identical to the VH-CDRl, VH-CDR2 and VH-CDR3 groups shown in Table 6, except for one, two, three, four, five, or six amino acid substitutions in any one VH-CDR.
  • VH immunoglobulin heavy chain variable region
  • CDRs e.g., VH-CDR-3
  • additional substitutions may be made in the CDR, as long as the a VH comprising the VH-CDR specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the amino acid substitutions are conservative.
  • the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2 and VH-CDR3 regions have polypeptide sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79 and 80; and SEQ ID NOs: 88, 89 and 90, except for one, two, three, four, five or six amino acid substitutions in at least one of said VH-CDRs.
  • VH immunoglobulin heavy chain variable region
  • the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2 and VH-CDR3 regions have polypeptide sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79 and 80; and SEQ ID NOs: 88, 89 and 90.
  • VH immunoglobulin heavy chain variable region
  • the present invention includes an isolated polypeptide comprising, consisting essentially of, or consisting of a VH polypeptide at least 80%, 85%, 90% 95% or 100% identical to a reference VH polypeptide amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 27, 37, 47, 57, 67, 77 and 87.
  • an antibody or antigen-binding fragment comprising the VH polypeptide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention includes an isolated polypeptide comprising, consisting essentially of, or consisting of a VH polypeptide selected from the group consisting of SEQ ID NOs: 17, 27, 37, 47, 57 67, 77 and 87.
  • an antibody or antigen-binding fragment comprising the VH polypeptide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a one or more of the VH polypeptides described above specifically or preferentially binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to ⁇ 6 ⁇ 4 integrin.
  • a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of one or more of the VH polypeptides described above specifically or preferentially binds to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or a ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 '2 M, 10 "2 M, 5 x 10 "3 M, 10 "3 M, 5 x 10 "4 M, 10 "4 M, 5 x 10 '5 M, 10 "5 M, 5 x 10 '6 M, 10 "6 M, 5 x 10 "7 M, 10 "7 M, 5 x 10 "8 M, 10 " 8 M, 5 x 10 '9 M, 10 "9 M, 5 x 10 "10 M, 10 "10 M, 5 x 10 "11 M, 10 "11 M, 5 x 10 "12 M, 10 "12 M, 5
  • the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL), where at least one of the VL-CDRs of the light chain variable region or at least two of the VL-CDRs of the light chain variable region are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDRl, VL-CDR2 or VL-CDR3 amino acid sequences from monoclonal ⁇ 6 ⁇ 4 integrin antibodies disclosed herein.
  • VL immunoglobulin light chain variable region
  • VL-CDRl, VL-CDR2 and VL-CDR3 regions of the VL are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDRl, VL-CDR2 and VL- CDR3 amino acid sequences from monoclonal ⁇ 6 ⁇ 4 integrin antibodies disclosed herein.
  • a light chain variable region of the invention has VL- CDRl, VL-CDR2 and VL-CDR3 polypeptide sequences related to the polypeptides shown in Table 6, supra.
  • VL-CDRs defined by the Kabat system
  • other CDR definitions e.g., VL-CDRs defined by the Chothia system
  • an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL) in which the VL-CDRl, VL-CDR2 and VL-CDR3 regions have polypeptide sequences which are identical to the VL-CDRl, VL-CDR2 and VL-CDR3 groups shown in Table 6.
  • VL immunoglobulin light chain variable region
  • an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VL) in which the VL-CDRl, VL-CDR2 and VL-CDR3 regions have polypeptide sequences which are identical to the VL-CDRl, VL-CDR2 and VL-CDR3 groups shown in Table 6, except for one, two, three, four, five, or six amino acid substitutions in any one VL-CDR.
  • additional substitutions may be made in the VL-CDR, as long as the a VL comprising the VL-CDR specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the amino acid substitutions are conservative, hi certain embodiments, an antibody or antigen-binding fragment comprising the VL specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VL) in which the VL-CDRl, VL-CDR2 and VL-CDR3 regions have polypeptide sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ED NOs: 83, 84 and 85; and SEQ ID NOs: 93, 94 and 95, except for one, two, three, four, five or six amino acid substitutions in at least one of said VL-CDRs.
  • VL immunoglobulin heavy chain variable region
  • the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VL) in which the VL-CDRl, VL-CDR2 and VL-CDR3 regions have polypeptide sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ED NOs: 33, 34, and 35; SEQ ED NOs: 43, 44, and 45; SEQ ED NOs: 53, 54, and 55; SEQ ED NOs: 63, 64, and 65; SEQ ED NOs: 73, 74, and 75; SEQ ED NOs: 83, 84 and 85; and SEQ ED NOs: 93, 94 and 95.
  • VL immunoglobulin heavy chain variable region
  • the present invention includes an isolated polypeptide comprising, consisting essentially of, or consisting of a VL polypeptide at least 80%, 85%, 90% 95% or 100% identical to a reference VL polypeptide sequence selected from the group consisting of SEQ ED NOs: 22, 32, 42, 52, 62, 72, 82 and 92.
  • an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention includes an isolated polypeptide comprising, consisting essentially of, or consisting of a VL polypeptide selected from the group consisting of SEQ ED NOs: 22, 32, 42, 52, 62, 72, 82 and 92.
  • an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, one or more of the VL polypeptides described above specifically or preferentially binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61- C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to ⁇ 6 ⁇ 4 integrin.
  • a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61- C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a one or more of the VL polypeptides described above specifically or preferentially binds to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or a ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 ⁇ 3 M, 10 "3 M, 5 x 10 "4 M, 10 "4 M, 5 x 10 "5 M, 10 "5 M, 5 x 10 6 M, 10 "6 M, 5 x 10 "7 M, 10 '7 M, 5 x 10 '8 M, 10 "8 M, 5 x 10 "9 M, 10 '9 M, 5 x 10 '10 M, 10 "10 M, 5 x lO "11 M, 10 " ⁇ M, 5 x 10 " 12 M, 10 "12
  • an antibody or antigen-binding fragment thereof comprises, consists essentially of or consists of a VH polypeptide, and a VL polypeptide, where the VH polypeptide and the VL polypeptide, respectively are at least 80%, 85%, 90% 95% or 100% identical to reference VL and VL polypeptide amino acid sequences selected from the group consisting of SEQ ID NOs: 17 and 22, 27 and 32, 37 and 42, 47 and 52, 57 and 62, 67 and 72, 77 and 82, and 87 and 92.
  • an antibody or antigen-binding fragment comprising these VH and VL polypeptides specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • polypeptides described above may further include additional polypeptides, e.g., a signal peptide to direct secretion of the encoded polypeptide, antibody constant regions as described herein, or other heterologous polypeptides as described herein.
  • polypeptides of the invention include polypeptide fragments as described elsewhere.
  • polypeptides of the invention include fusion polypeptide, Fab fragments, and other derivatives, as described herein.
  • compositions comprising the polypeptides described above.
  • ⁇ 6 ⁇ 4 integrin antibody polypeptides as disclosed herein may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived.
  • a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the starting sequence.
  • nucleotide or amino acid substitutions, deletions, or insertions leading to conservative substitutions or changes at "non-essential" amino acid regions may be made.
  • a polypeptide or amino acid sequence derived from a designated protein may be identical to the starting sequence except for one or more individual amino acid substitutions, insertions, or deletions, e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more individual amino acid substitutions, insertions, or deletions
  • a polypeptide or amino acid sequence derived from a designated protein may be identical to the starting sequence except for one or more individual amino acid substitutions, insertions, or deletions, e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more individual amino acid substitutions, insertions, or deletions.
  • a polypeptide or amino acid sequence derived from a designated protein may be identical to the starting sequence except for two or fewer, three or fewer, four or fewer, five or fewer, six or fewer, seven or fewer, eight or fewer, nine or fewer, ten or fewer, fifteen or fewer, or twenty or fewer individual amino acid substitutions, insertions, or deletions.
  • a polypeptide or amino acid sequence derived from a designated protein has one to five, one to ten, one to fifteen, or one to twenty individual amino acid substitutions, insertions, or deletions relative to the starting sequence.
  • Certain ⁇ 6 ⁇ 4 integrin antibody polypeptides of the present invention comprise, consist essentially of, or consist of an amino acid sequence derived from a human amino acid sequence.
  • certain ⁇ 6 ⁇ 4 integrin antibody polypeptides comprise one or more contiguous amino acids derived from another mammalian species.
  • an ⁇ 6 ⁇ 4 integrin antibody of the present invention may include a primate heavy chain portion, hinge portion, or antigen binding region, hi another example, one or more murine-derived amino acids may be present in a non-murine antibody polypeptide, e.g.
  • ⁇ 6 ⁇ 4 integrin-specific antibodies, or antigen-binding fragments, variants, or analogs thereof are designed so as to not be immunogenic in the animal to which the antibody is administered.
  • an ⁇ 6 ⁇ 4 integrin antibody polypeptide comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below.
  • a single-chain fv antibody fragment of the invention may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).
  • An ⁇ 6 ⁇ 4 integrin antibody polypeptide of the invention may comprise, consist essentially of, or consist of a fusion protein.
  • Fusion proteins are chimeric molecules which comprise, for example, an immunoglobulin antigen-binding domain with at least one target binding site, and at least one heterologous portion, i.e., a portion with which it is not naturally linked in nature.
  • the amino acid sequences may normally exist in separate proteins that are brought together in the fusion polypeptide or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. Fusion proteins may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
  • heterologous as applied to a polynucleotide or a polypeptide, means that the polynucleotide or polypeptide is derived from a distinct entity from that of the rest of the entity to which it is being compared.
  • a “heterologous polypeptide” to be fused to an ⁇ 6 ⁇ 4 integrin antibody, or an antigen- binding fragment, variant, or analog thereof is derived from a non-immunoglobulin polypeptide of the same species, or an immunoglobulin or non-immunoglobulin polypeptide of a different species.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.
  • glycine asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family.
  • a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
  • mutations may be introduced randomly along all or part of the immunoglobulin coding sequence, such as by saturation mutagenesis, and the resultant mutants can be incorporated into ⁇ 6 ⁇ 4 integrin antibodies for use in the diagnostic and treatment methods disclosed herein and screened for their ability to bind to the desired antigen, e.g., ⁇ 6 ⁇ 4 integrin.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen- binding fragments, variants, or derivatives thereof of the invention may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions.
  • ⁇ 6 ⁇ 4 integrin-specif ⁇ c ⁇ 6 ⁇ 4 integrin antibodies may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody binding ⁇ 6 ⁇ 4 integrin.
  • the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
  • ⁇ 6 ⁇ 4 integrin-specfic antibodies may be modified by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.
  • Modifications can occur anywhere in the ⁇ 6 ⁇ 4 integrin-specific antibody, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini, or on moieties such as carbohydrates. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given ⁇ 6 ⁇ 4 integrin-specific antibody. Also, a given ⁇ 6 ⁇ 4 integrin-specific antibody may contain many types of modifications. ⁇ 6 ⁇ 4 integrin-specific antibodies may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching.
  • Cyclic, branched, and branched cyclic ⁇ 6 ⁇ 4 integrin-specific antibodies may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selen
  • the present invention also provides for fusion proteins comprising an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof, and a heterologous polypeptide.
  • a fusion protein of the invention comprises, consists essentially of, or consists of, a polypeptide having the amino acid sequence of any one or more of the VH regions of an antibody of the invention or the amino acid sequence of any one or more of the VL regions of an antibody of the invention or fragments or variants thereof, and a heterologous polypeptide sequence.
  • a fusion protein for use in the diagnostic and treatment methods disclosed herein comprises, consists essentially of, or consists of a polypeptide having the amino acid sequence of any one, two, three of the VH-CDRs of an ⁇ 6 ⁇ 4 integrin-specif ⁇ c antibody, or fragments, variants, or derivatives thereof, or the amino acid sequence of any one, two, three of the VL-CDRs of an ⁇ 6 ⁇ 4 integrin-specific antibody, or fragments, variants, or derivatives thereof, and a heterologous polypeptide sequence.
  • the fusion protein comprises a polypeptide having the amino acid sequence of a VH-CDR3 of an ⁇ 6 ⁇ 4 integrin-specific antibody of the present invention, or fragment, derivative, or variant thereof, and a heterologous polypeptide sequence, which fusion protein specifically binds to at least one epitope of ⁇ 6 ⁇ 4 integrin.
  • a fusion protein comprises a polypeptide having the amino acid sequence of at least one VH region of an ⁇ 6 ⁇ 4 integrin-specific antibody of the invention and the amino acid sequence of at least one VL region of an ⁇ 6 ⁇ 4 integrin-specific antibody of the invention or fragments, derivatives or variants thereof, and a heterologous polypeptide sequence.
  • a fusion protein for use in the diagnostic and treatment methods disclosed herein comprises a polypeptide having the amino acid sequence of any one, two, three or more of the VH CDRs of an ⁇ 6 ⁇ 4 integrin-specific antibody and the amino acid sequence of any one, two, three or more of the VL CDRs of an ⁇ 6 ⁇ 4 integrin-specific antibody, or fragments or variants thereof, and a heterologous polypeptide sequence.
  • VH-CDR(s) or VL-CDR(s) correspond to single source antibody (or scFv or Fab fragment) of the invention.
  • Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention.
  • Exemplary fusion proteins reported in the literature include fusions of the T cell receptor (Gascoigne et al, Proc. Natl. Acad. Sci. USA 54:2936-2940 (1987)); CD4 (Capon et al., Nature 537:525-531 (1989); Traunecker et al, Nature 339:68-70 (1989); Zettmeissl et al., DNA Cell Biol.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be fused to heterologous polypeptides to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art.
  • PEG can be conjugated to the ⁇ 6 ⁇ 4 integrin antibodies of the invention to increase their half- life in vivo. Leong, S.R., et al, Cytokine /(5:106 (2001); Adv. in Drug Deliv. Rev. 54:531 (2002); or Weir et al, Biochem. Soc. Transactions 30:512 (2002).
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be fused to marker sequences, such as a peptide to facilitate their purification or detection.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • Other peptide tags useful for purification include, but are not limited to, the "HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al, Cell 37:161 (1984)) and the "flag" tag.
  • Fusion proteins can be prepared using methods that are well known in the art (see for example US Patent Nos. 5,116,964 and 5,225,538). The precise site at which the fusion is made may be selected empirically to optimize the secretion or binding characteristics of the fusion protein. DNA encoding the fusion protein is then transfected into a host cell for expression.
  • ⁇ 6 ⁇ 4 integrin antibodies of the present invention may be used in non-conjugated form or may be conjugated to at least one of a variety of molecules, e.g., to improve the therapeutic properties of the molecule, to facilitate target detection, or for imaging or therapy of the patient.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be labeled or conjugated either before or after purification, when purification is performed.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
  • conjugates may also be assembled using a variety of techniques depending on the selected agent to be conjugated.
  • conjugates with biotin are prepared e.g. by reacting a binding polypeptide with an activated ester of biotin such as the biotin N-hydroxysuccinimide ester.
  • conjugates with a fluorescent marker may be prepared in the presence of a coupling agent, e.g. those listed herein, or by reaction with an isothiocyanate, preferably fluorescein-isothiocyanate.
  • Conjugates of the ⁇ 6 ⁇ 4 integrin antibodies, or antigen- binding fragments, variants, or derivatives thereof of the invention are prepared in an analogous manner.
  • the present invention further encompasses ⁇ 6 ⁇ 4 integrin antibodies, or antigen- binding fragments, variants, or derivatives thereof of the invention conjugated to a diagnostic or therapeutic agent.
  • the ⁇ 6 ⁇ 4 integrin antibodies can be used diagnostically to, for example, monitor the development or progression of a neurological disease as part of a clinical testing procedure to, e.g. , determine the efficacy of a given treatment and/or prevention regimen. Detection can be facilitated by coupling the ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, e.g., U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin;
  • suitable radioactive material include 125 1, 131 1, 111 In or 99 Tc.
  • An ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged ⁇ 6 ⁇ 4 integrin antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof can be detectably labeled is by linking the same to an enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)" Microbiological Associates Quarterly Publication, Walkersville, Md., Diagnostic Horizons 2:1-7 (1978)); Voller et al., J. Clin. Pathol. 37:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981); Maggio, E.
  • EIA enzyme immunoassay
  • the enzyme which is bound to the ⁇ 6 ⁇ 4 integrin antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
  • Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Detection may also be accomplished using any of a variety of other immunoassays.
  • a radioimmunoassay ⁇ see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, (March, 1986)), which is incorporated by reference herein).
  • the radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.
  • An ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • binding molecules e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin- specific antibodies or immunospecific fragments thereof for use in the diagnostic and treatment methods disclosed herein may be conjugated to cytotoxins (such as radioisotopes, cytotoxic drugs, or toxins) therapeutic agents, cytostatic agents, biological toxins, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, immunologically active ligands (e.g., lymphokines or other antibodies wherein the resulting molecule binds to both the neoplastic cell and an effector cell such as a T cell), or PEG.
  • cytotoxins such as radioisotopes, cytotoxic drugs, or toxins
  • therapeutic agents such as radioisotopes, cytotoxic drugs, or toxins
  • cytostatic agents such as radioisotopes, cytotoxic drugs, or toxins
  • biological toxins such as radioisotopes, cytotoxic drugs
  • a binding molecule e.g., a binding polypeptide, e.g., an ⁇ 6 ⁇ 4 integrin-specific antibody or immunospecific fragment thereof for use in the diagnostic and treatment methods disclosed herein can be conjugated to a molecule that decreases vascularization of tumors.
  • the disclosed compositions may comprise binding molecules, e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof coupled to drugs or prodrugs.
  • binding molecules e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof conjugated to specific biotoxins or their cytotoxic fragments such as ricin, gelonin, pseudomonas exotoxin or diphtheria toxin.
  • binding molecules e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof conjugated to specific biotoxins or their cytotoxic fragments such as ricin, gelonin, pseudomonas exotoxin or diphtheria toxin.
  • adjunct treatment e.g., chemotherapy or external radiation
  • radioisotopes include: 90 Y, 125 I, 131 I, 123 I, 111 In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 1 86 Re and 188 Re.
  • the radionuclides act by producing ionizing radiation which causes multiple strand breaks in nuclear DNA, leading to cell death.
  • the isotopes used to produce therapeutic conjugates typically produce high energy ⁇ - or ⁇ -particles which have a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells. Radionuclides are essentially non-immunogenic.
  • binding molecules e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof may be directly labeled (such as through iodination) or may be labeled indirectly through the use of a chelating agent.
  • the phrases "indirect labeling” and “indirect labeling approach” both mean that a chelating agent is covalently attached to a binding molecule and at least one radionuclide is associated with the chelating agent.
  • Such chelating agents are typically referred to as bifunctional chelating agents as they bind both the polypeptide and the radioisotope.
  • Particularly preferred chelating agents comprise l-isothiocycmatobenzyl-3- methyldiothelene triaminepentaacetic acid ("MX-DTPA”) and cyclohexyl diethylenetriamine pentaacetic acid (“CHX-DTPA”) derivatives.
  • Other chelating agents comprise P-DOTA and EDTA derivatives.
  • Particularly preferred radionuclides for indirect labeling include 111 In and 90 Y.
  • direct labeling and “direct labeling approach” both mean that a radionuclide is covalently attached directly to a polypeptide (typically via an amino acid residue). More specifically, these linking technologies include random labeling and site-directed labeling. In the latter case, the labeling is directed at specific sites on the polypeptide, such as the N-linked sugar residues present only on the Fc portion of the conjugates. Further, various direct labeling techniques and protocols are compatible with the instant invention.
  • Technetium-99 labeled polypeptides may be prepared by ligand exchange processes, by reducing pertechnate (TcO 4 " ) with stannous ion solution, chelating the reduced technetium onto a Sephadex column and applying the binding polypeptides to this column, or by batch labeling techniques, e.g. by incubating pertechnate, a reducing agent such as SnCl 2 , a buffer solution such as a sodium-potassium phthalate-solution, and the antibodies.
  • a reducing agent such as SnCl 2
  • a buffer solution such as a sodium-potassium phthalate-solution
  • preferred radionuclides for directly labeling antibodies are well known in the art and a particularly preferred radionuclide for direct labeling is 131 I covalently attached via tyrosine residues.
  • Binding molecules e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecif ⁇ c fragments thereof for use in the diagnostic and treatment methods disclosed herein may be derived, for example, with radioactive sodium or potassium iodide and a chemical oxidizing agent, such as sodium hypochlorite, chloramine T or the like, or an enzymatic oxidizing agent, such as lactoperoxidase, glucose oxidase and glucose.
  • a chemical oxidizing agent such as sodium hypochlorite, chloramine T or the like
  • an enzymatic oxidizing agent such as lactoperoxidase, glucose oxidase and glucose.
  • Patents relating to chelators and chelator conjugates are known in the art.
  • U.S. Patent No. 4,831,175 of Gansow is directed to polysubstituted diethylenetriaminepentaacetic acid chelates and protein conjugates containing the same, and methods for their preparation.
  • U.S. Patent Nos. 5,099,069, 5,246,692, 5,286,850, 5,434,287 and 5,124,471 of Gansow also relate to polysubstituted DTPA chelates. These patents are incorporated herein by reference in their entireties.
  • compatible metal chelators are ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA), 1,4,8,11-tetraazatetradecane, 1,4,8,11- tetraazatetradecane- 1 ,4,8, 11 -tetraacetic acid, 1 -oxa-4,7, 12, 15-tetraazaheptadecane- 4,7,12,15-tetraacetic acid, or the like. Cyclohexyl-DTPA or CHX-DTPA is particularly preferred and is exemplified extensively below. Still other compatible chelators, including those yet to be discovered, may easily be discerned by a skilled artisan and are clearly within the scope of the present invention.
  • Compatible chelators including the specific bifunctional chelator used to facilitate chelation U.S. Patent Nos. 6,682,134, 6,399,061, and 5,843,439, incorporated herein by reference in their entireties, are preferably selected to provide high affinity for trivalent metals, exhibit increased tumor-to-non-tumor ratios and decreased bone uptake as well as greater in vivo retention of radionuclide at target sites, i.e., B-cell lymphoma tumor sites.
  • target sites i.e., B-cell lymphoma tumor sites.
  • other bifunctional chelators that may or may not possess all of these characteristics are known in the art and may also be beneficial in tumor therapy.
  • binding molecules may be conjugated to different radiolabels for diagnostic and therapeutic purposes.
  • U.S. Patent Nos. 6,682,134, 6,399,061, and 5,843,439 disclose radiolabeled therapeutic conjugates for diagnostic "imaging" of tumors before administration of therapeutic antibody.
  • “In2B8" conjugate comprises a murine monoclonal antibody, 2B8, specific to human CD20 antigen, that is attached to 1 11 In via a bifunctional chelator, i.e., MX-DTPA (diethylenetriaminepentaacetic acid), which comprises a 1 :1 mixture of l-isothiocyanatobenzyl-3-methyl-DTPA and 1-methyl- 3-isothiocyanatobenzyl-DTPA.
  • MX-DTPA diethylenetriaminepentaacetic acid
  • 111 In is particularly preferred as a diagnostic radionuclide because between about 1 to about 10 mCi can be safely administered without detectable toxicity; and the imaging data is generally predictive of subsequent 90 Y-labeled antibody distribution.
  • 131 I is a well known radionuclide used for targeted immunotherapy.
  • the clinical usefulness of 131 I can be limited by several factors including: eight-day physical half-life; dehalogenation of iodinated antibody both in the blood and at tumor sites; and emission characteristics (e.g., large gamma component) which can be suboptimal for localized dose deposition in tumor.
  • emission characteristics e.g., large gamma component
  • 90 Y provides several benefits for utilization in radioimmunotherapeutic on applications: the 64 hour half- life of Y is long enough to allow antibody accumulation by tumor and, unlike e.g., 131 I, 90 Y is a pure beta emitter of high energy with no accompanying gamma irradiation in its decay, with a range in tissue of 100 to 1,000 cell diameters. Furthermore, the minimal amount of penetrating radiation allows for outpatient administration of 90 Y-labeled antibodies. Additionally, internalization of labeled antibody is not required for cell killing, and the local emission of ionizing radiation should be lethal for adjacent tumor cells lacking the target molecule.
  • cytotoxic drugs particularly those which are used for cancer therapy.
  • a cytotoxin or cytotoxic agent means any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit or destroy a cell or malignancy.
  • cytotoxins include, but are not limited to, radionuclides, biotoxins, enzymatically active toxins, cytostatic or cytotoxic therapeutic agents, prodrugs, immunologically active ligands and biological response modifiers such as cytokines. Any cytotoxin that acts to retard or slow the growth of immunoreactive cells or malignant cells is within the scope of the present invention.
  • Exemplary cytotoxins include, in general, cytostatic agents, alkylating agents, anti-metabolites, antiproliferative agents, tubulin binding agents, hormones and hormone antagonists, and the like.
  • Exemplary cytostatics that are compatible with the present invention include alkylating substances, such as mechlorethamine, triethylenephosphoramide, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan or triaziquone, also nitrosourea compounds, such as carmustine, lomustine, or semustine.
  • alkylating substances such as mechlorethamine, triethylenephosphoramide, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan or triaziquone
  • nitrosourea compounds such as carmustine, lomustine, or semustine.
  • Other preferred classes of cytotoxic agents include, for example, the maytansinoid family of
  • cytotoxic agents include, for example, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, and the podophyllotoxins.
  • Particularly useful members of those classes include, for example, adriamycin, carminomycin, daunorubicin (daunomycin), doxorubicin, aminopterin, methotrexate, methopterin, mithramycin, streptonigrin, dichloromethotrexate, mitomycin C, actinomycin-D, porfiromycin, 5-fluorouracil, floxuridine, ftorafur, 6-mercaptopurine, cytarabine, cytosine arabinoside, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine and the like.
  • cytotoxins that are compatible with the teachings herein include taxol, taxane, cytochalasin B, gramicidin D, ethidium bromide, emetine, tenoposide, colchicin, dihydroxy anthracin dione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Hormones and hormone antagonists such as corticosteroids, e.g. prednisone, progestins, e.g. hydroxyprogesterone or medroprogesterone, estrogens, e.g. diethylstilbestrol, antiestrogens, e.g.
  • tamoxifen, androgens e.g. testosterone
  • aromatase inhibitors e.g. aminogluthetimide
  • One skilled in the art may make chemical modifications to the desired compound in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention.
  • cytotoxins comprise members or derivatives of the enediyne family of anti-tumor antibiotics, including calicheamicin, esperamicins or dynemicins. These toxins are extremely potent and act by cleaving nuclear DNA, leading to cell death. Unlike protein toxins which can be cleaved in vivo to give many inactive but immunogenic polypeptide fragments, toxins such as calicheamicin, esperamicins and other enediynes are small molecules which are essentially non-immunogenic. These non-peptide toxins are chemically-linked to the dimers or tetramers by techniques which have been previously used to label monoclonal antibodies and other molecules. These linking technologies include site-specific linkage via the N-linked sugar residues present only on the Fc portion of the constructs. Such site-directed linking methods have the advantage of reducing the possible effects of linkage on the binding properties of the constructs.
  • compatible cytotoxins for preparation of conjugates may comprise a prodrug.
  • prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form.
  • Prodrugs compatible with the invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate containing prodrugs, peptide containing prodrugs, ⁇ -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted to the more active cytotoxic free drug.
  • Further examples of cytotoxic drugs that can be derivatized into a prodrug form for use in the present invention comprise those chemotherapeutic agents described above.
  • binding molecules e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof disclosed herein can also be associated with or conjugated to a biotoxin such as ricin subunit A, abrin, diptheria toxin, botulinum, cyanginosins, saxitoxin, shigatoxin, tetanus, tetrodotoxin, trichothecene, verrucologen or a toxic enzyme.
  • a biotoxin such as ricin subunit A, abrin, diptheria toxin, botulinum, cyanginosins, saxitoxin, shigatoxin, tetanus, tetrodotoxin, trichothecene, verrucologen or a toxic enzyme.
  • a biotoxin such as ricin subunit A, abrin, diptheria toxin, botulin
  • binding molecules e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin- specific antibodies or immunospecific fragments thereof disclosed herein comprise cytokines such as lymphokines and interferons.
  • cytokines such as lymphokines and interferons.
  • Another class of compatible cytotoxins that may be used in association with or conjugated to the disclosed binding molecules, e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof, are radiosensitizing drugs that may be effectively directed to tumor or immunoreactive cells. Such drugs enhance the sensitivity to ionizing radiation, thereby increasing the efficacy of radiotherapy.
  • An antibody conjugate internalized by the tumor cell would deliver the radiosensitizer nearer the nucleus where radiosensitization would be maximal.
  • the unbound radiosensitizer linked binding molecules of the invention would be cleared quickly from the blood, localizing the remaining radiosensitization agent in the target tumor and providing minimal uptake in normal tissues.
  • adjunct radiotherapy would be administered in one of three ways: 1.) external beam radiation directed specifically to the tumor, 2.) radioactivity directly implanted in the tumor or 3.) systemic radioimmunotherapy with the same targeting antibody.
  • a potentially attractive variation of this approach would be the attachment of a therapeutic radioisotope to the radiosensitized immunoconjugate, thereby providing the convenience of administering to the patient a single drug.
  • a moiety that enhances the stability or efficacy of a binding molecule e.g., a binding polypeptide, e.g., an ⁇ 6 ⁇ 4 integrin -specific antibody or immunospecific fragment thereof can be conjugated.
  • a binding polypeptide e.g., an ⁇ 6 ⁇ 4 integrin -specific antibody or immunospecific fragment thereof
  • PEG can be conjugated to the binding molecules of the invention to increase their half- life in vivo. Leong, S.R., et al., Cytokine 75:106 (2001); Adv. in DrugDeliv. Rev. 54:531 (2002); or Weir et al., Biochem. Soc. Transactions 30:512 (2002).
  • the present invention further encompasses the use of binding molecules, e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments conjugated to a diagnostic or therapeutic agent.
  • the binding molecules can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment and/or prevention regimen. Detection can be facilitated by coupling the binding molecule, e.g., binding polypeptide, e.g., ⁇ 6 ⁇ 4 integrin-specific antibody or immunospecific fragment thereof to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin;
  • suitable radioactive material include 125 1, 131 1, 111 In or 99 Tc.
  • a binding molecule e.g., a binding polypeptide, e.g., an ⁇ 6 ⁇ 4 integrin-specific antibody or immunospecific fragment thereof also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged binding molecule is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a binding molecule e.g., a binding polypeptide, e.g., an ⁇ 6 ⁇ 4 integrin-specific antibody or immunospecific fragment thereof can be detectably labeled is by linking the same to an enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)" Microbiological Associates Quarterly Publication, Walkersville, Md., Diagnostic Horizons 2:1-7 (1978)); Voller et ah, J. Clin. Pathol. 57:507-520 (1978); Butler, J. E., Meth. Enrymol. 75:482-523 (1981); Maggio, E.
  • EIA enzyme immunoassay
  • the enzyme which is bound to the binding molecule will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
  • Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5 -steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose- 6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Detection may also be accomplished using any of a variety of other immunoassays.
  • the binding molecule e.g., binding polypeptide, e.g., ⁇ 6 ⁇ 4 integrin-specific antibody or immunospecific fragment thereof
  • a radioimmunoassay RIA
  • the radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.
  • a binding molecule e.g., a binding polypeptide, e.g., an ⁇ 6 ⁇ 4 integrin- specific antibody or immunospecific fragment thereof can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • a binding molecule e.g., a binding polypeptide, e.g., an ⁇ 6 ⁇ 4 integrin-specific antibody or immunospecific fragment thereof are well known, see, e.g., Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp.
  • RNA may be isolated from the original hybridoma cells or from other transformed cells by standard techniques, such as guanidinium isothiocyanate extraction and precipitation followed by centrifugation or chromatography. Where desirable, mRNA may be isolated from total RNA by standard techniques such as chromatography on oligo dT cellulose. Suitable techniques are familiar in the art.
  • cDNAs that encode the light and the heavy chains of the antibody may be made, either simultaneously or separately, using reverse transcriptase and DNA polymerase in accordance with well known methods.
  • PCR may be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences.
  • PCR also may be used to isolate DNA clones encoding the antibody light and heavy chains. In this case the libraries may be screened by consensus primers or larger homologous probes, such as mouse constant region probes.
  • DNA typically plasmid DNA
  • DNA may be isolated from the cells using techniques known in the art, restriction mapped and sequenced in accordance with standard, well known techniques set forth in detail, e.g., in the foregoing references relating to recombinant DNA techniques.
  • the DNA may be synthetic according to the present invention at any point during the isolation process or subsequent analysis.
  • the polynucleotides encoding the ⁇ 6 ⁇ 4 integrin antibodies are typically inserted in an expression vector for introduction into host cells that may be used to produce the desired quantity of ⁇ 6 ⁇ 4 integrin antibody.
  • an antibody, or fragment, derivative or analog thereof e.g., a heavy or light chain of an antibody which binds to a target molecule described herein, e.g., ⁇ 6 ⁇ 4 integrin
  • an expression vector containing a polynucleotide that encodes the antibody Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
  • the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes both heavy and light chain polypeptides.
  • the light chain is advantageously placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. ScL USA 77:2197 (1980)).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • vectors used in accordance with the present invention as a vehicle for introducing into and expressing a desired gene in a host cell.
  • vectors may easily be selected from the group consisting of plasmids, phages, viruses and retroviruses.
  • vectors compatible with the instant invention will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.
  • vector systems may be employed.
  • one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus.
  • Others involve the use of polycistronic systems with internal ribosome binding sites.
  • cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells. The marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper.
  • the selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals. [0436] In particularly preferred embodiments the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (preferably human) synthetic as discussed above, hi one embodiment, this is effected using a proprietary expression vector of Biogen EDEC, Inc., referred to as NEOSPLA (disclosed in U.S. patent 6,159,730).
  • This vector contains the cytomegalovirus promoter/enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, the dihydrofolate reductase gene and leader sequence.
  • This vector has been found to result in very high level expression of antibodies upon incorporation of variable and constant region genes, transfection in CHO cells, followed by selection in G418 containing medium and methotrexate amplification.
  • any expression vector which is capable of eliciting expression in eukaryotic cells may be used in the present invention.
  • Suitable vectors include, but are not limited to plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEFl/His, pIND/GS, pRc/HCMV2, PSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAXl, and pZeoSV2 (available from Invitrogen, San Diego, CA), and plasmid pCI (available from Promega, Madison, WI).
  • screening large numbers of transformed cells for those which express suitably high levels if immunoglobulin heavy and light chains is routine experimentation which can be carried out, for example, by robotic systems. Vector systems are also taught in U.S. Pat. Nos.
  • the ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be expressed using polycistronic constructs such as those disclosed in United States Patent Application Publication No. 2003-0157641 Al, filed November 18, 2002 and incorporated herein in its entirety.
  • polycistronic constructs such as those disclosed in United States Patent Application Publication No. 2003-0157641 Al, filed November 18, 2002 and incorporated herein in its entirety.
  • multiple gene products of interest such as heavy and light chains of antibodies may be produced from a single polycistronic construct.
  • IRES internal ribosome entry site
  • the expression vector may be introduced into an appropriate host cell.
  • Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See, Ridgway, A. A. G. "Mammalian Expression Vectors" Vectors, Rodriguez and Denhardt, Eds., Butterworths, Boston, Mass., Chapter 24.2, pp. 470-472 (1988).
  • plasmid introduction into the host is via electroporation.
  • the host cells harboring the expression construct are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy and/or light chain protein synthesis.
  • Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorter analysis (FACS), immunohistochemistry and the like.
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody for use in the methods described herein.
  • the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • host cells refers to cells which harbor vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene.
  • the terms “cell” and “cell culture” are used interchangeably to denote the source of antibody unless it is clearly specified otherwise.
  • recovery of polypeptide from the “cells” may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.
  • host-expression vector systems may be utilized to express antibody molecules for use in the methods described herein.
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichi ⁇ ) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mamm
  • bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al, Bio/Technology 8:2 (1990)).
  • the host cell line used for protein expression is often of mammalian origin; those skilled in the art are credited with ability to preferentially determine particular host cell lines which are best suited for the desired gene product to be expressed therein.
  • Exemplary host cell lines include, but are not limited to, CHO (Chinese Hamster Ovary), DG44 and DUXBI l (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with S V40 T antigen), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/0 (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-IcIBPT (bovine endothelial cells), RAJI (human
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • cell lines which stably express the antibody molecule may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which stably express the antibody molecule.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al, Cell 22:817 1980) genes can be employed in tk-, hgprt- or aprt-cells, respectively.
  • anti-metabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sd. USA 77:357 (1980); O'Hare et al, Proc. Natl. Acad. Sd. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci.
  • the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987)).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987)).
  • a marker in the vector system expressing antibody is amplifiable
  • increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al, MoI. Cell. Biol. 3:257 (1983)).
  • the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose or (immuno-)affinity chromatography, e.g., after preferential biosynthesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein.
  • customary chromatography methods for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose or (immuno-)affinity chromatography, e.g., after preferential biosynthesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein.
  • Genes encoding ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can also be expressed non-mammalian cells such as bacteria or insect or yeast or plant cells.
  • Bacteria which readily take up nucleic acids include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that, when expressed in bacteria, the heterologous polypeptides typically become part of inclusion bodies. The heterologous polypeptides must be isolated, purified and then assembled into functional molecules. Where tetravalent forms of antibodies are desired, the subunits will then self- assemble into tetravalent antibodies (WO02/096948A2).
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al, EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione- agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • eukaryotic microbes may also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms although a number of other strains are commonly available, e.g. , Pichia pastoris.
  • the plasmid YRp7 for example, (Stinchcomb et al., Nature 282:39 (1979); Kingsman et al, Gene 7:141 (1979); Tschemper et al., Gene 10:157 (1980)) is commonly used.
  • This plasmid already contains the TRPl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)).
  • the presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is typically used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody coding sequence may be cloned individually into nonessential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • an antibody molecule of the invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography ⁇ e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • differential solubility e.g., differential solubility
  • One embodiment of the present invention provides methods for treating a hyperproliferative disease or disorder, e.g. , cancer, a malignancy, a tumor, or a metastasis thereof, in an animal suffering from such disease or predisposed to contract such disease, the method comprising, consisting essentially of, or consisting of administering to the animal an effective amount of an antibody or immunospecific fragment thereof, that binds to ⁇ 6 ⁇ 4 integrin or a variant of ⁇ 6 ⁇ 4 integrin.
  • Suitable antibodies include all antibodies and antigen-specific fragments thereof described herein.
  • Examples include, but are not limited to, an isolated antibody or antigen-binding fragment thereof which specifically binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66- H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, an isolated antibody or antigen- binding fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the antibody or fragment thereof competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66- H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2
  • an antibody of the present invention which specifically binds to ⁇ 6 ⁇ 4 integrin or a variant thereof inhibits laminin from binding to ⁇ 6 ⁇ 4 integrin.
  • an antibody of the present invention which specifically binds to ⁇ 6 ⁇ 4 integrin or a variant thereof expressed on a cell, in particular a tumor cell inhibits activation of downstream signal transduction molecules involved in cell proliferation, motility and/or metastasis.
  • Such molecules include, but are not limited to PI3 -K, Akt, mTOR and Rac.
  • an antibody of the present invention which specifically binds to ⁇ 6 ⁇ 4 integrin or a variant thereof expressed on a cell, in particular a tumor cell, inhibits activation of the Ras/MAPK signaling pathway.
  • an antibody of the present invention which specifically binds to ⁇ 6 ⁇ 4 integrin or a variant thereof expressed on a cell, in particular a tumor cell, inhibits the interaction of ⁇ 6 ⁇ 4 with growth factor receptors such as erbB2, Met, and Ron.
  • an antibody of the present invention which specifically binds to ⁇ 6 ⁇ 4 integrin or a variant thereof expressed on a cell, in particular a tumor cell inhibits integrin clustering in lipid rafts.
  • an antibody of the present invention which specifically binds to ⁇ 6 ⁇ 4 integrin or a variant thereof expressed on a cell, in particular a tumor cell, inhibits the interaction with cytoskeletal components, induces apoptosis, and induces integrin internalization.
  • an antibody of the present invention which specifically binds to ⁇ 6 ⁇ 4 integrin or a variant thereof expressed on a cell, in particular a tumor cell, inhibits cell proliferation, motility, and/or metastasis.
  • An antibody of the present invention which specifically binds to ⁇ 6 ⁇ 4 integrin or a variant thereof, to be used in treatment methods disclosed herein can be prepared and used as a therapeutic agent that stops, reduces, prevents, or inhibits cellular activities involved in cellular hyperproliferation, e.g., cellular activities that induce the altered or abnormal pattern of vascularization that is often associated with hyperproliferative diseases or disorders.
  • Antibodies or immunospecific fragments thereof of the present invention include, but are not limited to monoclonal, chimeric or humanized antibodies, and fragments of antibodies that bind specifically to tumor-associated proteins such as ⁇ 6 ⁇ 4 integrin.
  • the antibodies may be monovalent, bivalent, polyvalent, or bifunctional antibodies, and the antibody fragments include Fab F(ab') 2 , and Fv.
  • Therapeutic antibodies according to the invention can be used in unlabeled or unconjugated form, or can be coupled or linked to cytotoxic moieties such as radiolabels and biochemical cytotoxins to produce agents that exert therapeutic effects.
  • an antibody, or immunospecific fragment thereof of the invention includes an antigen binding domain.
  • An antigen binding domain is formed by antibody variable regions that vary from one antibody to another. Naturally occurring antibodies comprise at least two antigen binding domains, i.e., they are at least bivalent.
  • the term "antigen binding domain” includes a site that specifically binds an epitope on an antigen (e.g., a cell surface or soluble antigen).
  • the antigen binding domain of an antibody typically includes at least a portion of an immunoglobulin heavy chain variable region and at least a portion of an immunoglobulin light chain variable region. The binding site formed by these variable regions determines the specificity of the antibody.
  • the present invention provides methods for treating various hyperproliferative disorders, e.g., by inhibiting tumor growth, in a mammal, comprising, consisting essentially of, or consisting of administering to the mammal an effective amount of a antibody or antigen-binding fragment thereof which specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin, e.g., human ⁇ 6 ⁇ 4 integrin.
  • the present invention is more specifically directed to a method of treating a hyperproliferative disease, e.g., inhibiting or preventing tumor formation, tumor growth, tumor invasiveness, and/or metastasis formation, in an animal, e.g., a mammal, e.g., a human, comprising, consisting essentially of, or consisting of administering to an animal in need thereof an effective amount of a an antibody or immunospecific fragment thereof, which specifically or preferentially binds to one or more epitopes of ⁇ 6 ⁇ 4 integrin.
  • a hyperproliferative disease e.g., inhibiting or preventing tumor formation, tumor growth, tumor invasiveness, and/or metastasis formation
  • an animal e.g., a mammal, e.g., a human
  • an effective amount of a an antibody or immunospecific fragment thereof which specifically or preferentially binds to one or more epitopes of ⁇ 6 ⁇ 4 integrin.
  • the present invention includes a method for treating a hyperproliferative disease, e.g., inhibiting tumor formation, tumor growth, tumor invasiveness, and/or metastasis formation in an animal, e.g., a human patient, where the method comprises administering to an animal in need of such treatment an effective amount of a composition comprising, consisting essentially of, or consisting of, in addition to a pharmaceutically acceptable carrier, an antibody, or immunospecific fragment thereof, which specifically binds to at least one epitope of ⁇ 6 ⁇ 4 integrin, where the epitope comprises, consists essentially of, or consists of at least about four to five amino acids amino acids of SEQ ED NOs: 1-15, at least seven, at least nine, or between at least about 15 to about 30 amino acids of SEQ ED NOs:l-15.
  • the amino acids of a given epitope of SEQ ED NOs:l-15 as described may be, but need not be contiguous.
  • the at least one epitope of ⁇ 6 ⁇ 4 integrin comprises, consists essentially of, or consists of a non-linear epitope formed by the extracellular domain of ⁇ 6 ⁇ 4. integrin as expressed on the surface of a cell.
  • the at least one epitope of ⁇ 6 ⁇ 4 integrin comprises, consists essentially of, or consists of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguous amino acids of SEQ ED NOs: 1-15, where non-contiguous amino acids form an epitope through protein folding.
  • the present invention includes an antibody, or antigen- binding fragment, variant, or derivative thereof which specifically or preferentially binds to at least one epitope of ⁇ 6 ⁇ 4 integrin, where the epitope comprises, consists essentially of, or consists of a portion of ⁇ 6 and a portion of ⁇ 4.
  • At least one epitope of ⁇ 6 ⁇ 4 integrin comprises, consists essentially of, or consists of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or noncontiguous amino acids of an ⁇ 6 sequence selected from the group consisting of SEQ ID NOs: 1-8 and at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguous amino acids of a ⁇ 4 sequence selected from the group consisting of SEQ ID NOs: 1-8
  • the present invention includes a method for treating a hyperproliferative disease, e.g., inhibiting tumor formation, tumor growth, tumor invasiveness, and/or metastasis formation in an animal, e.g., a human patient, where the method comprises administering to an animal in need of such treatment an effective amount of a composition comprising, consisting essentially of, or consisting of, in addition to a pharmaceutically acceptable carrier, an antibody, or immunospecific fragment thereof, which specifically binds to at least one epitope of ⁇ 6 ⁇ 4 integrin, where the epitope comprises, consists essentially of, or consists of, in addition to one, two, three, four, five, six or more contiguous or non-contiguous amino acids of SEQ ID NOs:l-15 as described above, and an additional moiety which modifies the protein, e.g., a carbohydrate moiety may be included such that the binding molecule binds with higher affinity to modified target protein than it does to an unmodified version
  • the present invention provides a method of treating cancer in a human, comprising administering to a human in need of treatment a composition comprising an effective amount of an ⁇ 6 ⁇ 4 integrin-specific antibody or immunospecific fragment thereof, and a pharmaceutically acceptable carrier.
  • Types of cancer to be treated include, but are not limited to, stomach cancer, renal cancer, brain cancer, bladder cancer, colon cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and prostate cancer.
  • an antibody or fragment thereof binds specifically to at least one epitope of ⁇ 6 ⁇ 4 integral or fragment or variant described above, i.e., binds to such an epitope more readily than it would bind to an unrelated, or random epitope; binds preferentially to at least one epitope of ⁇ 6 ⁇ 4 integrin or fragment or variant described above, i.e., binds to such an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope; competitively inhibits binding of a reference antibody which itself binds specifically or preferentially to a certain epitope of ⁇ 6 ⁇ 4 integrin or fragment or variant described above; or binds to at least one epitope of ⁇ 6 ⁇ 4 integrin or fragment or variant described above with an affinity characterized by a dissociation constant K D of less than about 5 x 10 "2 M, about 10 "2 M, about 5 x 10 "3 M, about 10 "3
  • antibodies and fragments thereof of the present invention cross-react with ⁇ 6 ⁇ 4 integrin proteins of other species from which they were raised, e.g., an antibody or fragment thereof which specifically binds to human ⁇ 6 ⁇ 4 integrin also binds to murine ⁇ 6 ⁇ 4 integrin.
  • suitable antibodies or fragments thereof of the present invention include those that are highly species specific.
  • antibodies or immunospecific fragments thereof disclosed herein bind ⁇ 6 ⁇ 4 integrin polypeptides or fragments or variants thereof with an off rate (k(off)) of less than or equal to 5 X 10 "2 sec “1 , 10 "2 sec “1 , 5 X 10 "3 sec “1 or 10 "3 sec “1 .
  • antibodies or immunospecific fragments thereof disclosed herein bind ⁇ 6 ⁇ 4 integrin polypeptides or fragments or variants thereof with an off rate (k(off)) of less than or equal to 5 X 10 "4 sec “1 , 10 "4 sec “1 , 5 X 10 5 sec “1 , or 10 "5 sec “1 5 X 10 "6 sec “1 , 10 “6 sec “1 , 5 X lO- 7 SeC- 1 Or IO- 7 SeC “1 .
  • off rate k(off)
  • antibodies or immunospecific fragments thereof disclosed herein bind ⁇ 6 ⁇ 4 integrin polypeptides or fragments or variants thereof with an on rate (k(on)) of greater than or equal to 10 3 M '1 sec “1 , 5 X 10 3 M “1 sec “1 , 10 4 M “1 sec “1 or 5 X 10 4 M “1 sec “1 .
  • antibodies or immunospecific fragments thereof for use in the diagnostic and treatment methods disclosed herein bind ⁇ 6 ⁇ 4 integrin polypeptides or fragments or variants thereof with an on rate (k(on)) greater than or equal to 10 5 M "1 sec “1 , 5 X 10 5 M “1 sec “1 , 10 6 M “1 sec '1 , or 5 X 10 6 M “1 sec “1 or 10 7 M “1 sec “1 .
  • one or more binding molecules as described above is an antagonist of ⁇ 6 ⁇ 4 integrin activity, for example, binding of an antagonist ⁇ 6 ⁇ 4 integrin antibody to ⁇ 6 ⁇ 4 integrin as expressed on a tumor cell inhibits binding of laminin, inhibits activation of molecules downstream in the signal transduction pathway, e.g., PI3- K, Akt, mTOR and RAC , or inhibits tumor cell proliferation, motility or metastasis.
  • ⁇ 6 ⁇ 4 integrin activity for example, binding of an antagonist ⁇ 6 ⁇ 4 integrin antibody to ⁇ 6 ⁇ 4 integrin as expressed on a tumor cell inhibits binding of laminin, inhibits activation of molecules downstream in the signal transduction pathway, e.g., PI3- K, Akt, mTOR and RAC , or inhibits tumor cell proliferation, motility or metastasis.
  • ⁇ 6 ⁇ 4 integrin-specific antibodies, or fragments, derivatives, or analogs thereof can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of ⁇ 6 ⁇ 4 integrin.
  • ⁇ 6 ⁇ 4 integrin expression is increased in tumor tissue and other neoplastic conditions.
  • ⁇ 6 ⁇ 4 integrin-specific antibodies or fragments thereof are useful for diagnosis, treatment, prevention and/or prognosis of hyperproliferative disorders in mammals, preferably humans.
  • disorders include, but are not limited to, cancer, neoplasms, tumors and/or as described under elsewhere herein, especially ⁇ 6 ⁇ 4 integrin-associated cancers such as stomach cancer, brain cancer, bladder cancer, colon cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and prostate cancer.
  • ⁇ 6 ⁇ 4 integrin expression is associated with at least stomach, brain, bladder, colon, lung, breast, pancreatic, ovarian, and prostate tumor tissues. Accordingly, antibodies (and antibody fragments) directed against ⁇ 6 ⁇ 4 integrin may be used to detect particular tissues expressing increased levels of ⁇ 6 ⁇ 4 integrin. These diagnostic assays may be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.
  • the invention provides a diagnostic method useful during diagnosis of a cancers and other hyperproliferative disorders, which involves measuring the expression level of ⁇ 6 ⁇ 4 integrin protein or transcript in tissue or other cells or body fluid from an individual and comparing the measured expression level with a standard ⁇ 6 ⁇ 4 integrin expression levels in normal tissue or body fluid, whereby an increase in the expression level compared to the standard is indicative of a disorder.
  • One embodiment provides a method of detecting the presence of abnormal hyperproliferative cells, e.g., precancerous or cancerous cells, in a fluid or tissue sample, comprising assaying for the expression of ⁇ 6 ⁇ 4 integrin in tissue or body fluid samples of an individual and comparing the presence or level of ⁇ 6 ⁇ 4 integrin expression in the sample with the presence or level of ⁇ 6 ⁇ 4 integrin expression in a panel of standard tissue or body fluid samples, where detection of ⁇ 6 ⁇ 4 integrin expression or an increase in ⁇ 6 ⁇ 4 integrin expression over the standards is indicative of aberrant hyperproliferative cell growth.
  • abnormal hyperproliferative cells e.g., precancerous or cancerous cells
  • the present invention provides a method of detecting the presence of abnormal hyperproliferative cells in a body fluid or tissue sample, comprising (a) assaying for the expression of ⁇ 6 ⁇ 4 integrin in tissue or body fluid samples of an individual using ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof of the present invention, and (b) comparing the presence or level of ⁇ 6 ⁇ 4 integrin expression in the sample with a the presence or level of ⁇ 6 ⁇ 4 integrin expression in a panel of standard tissue or body fluid samples, whereby detection of ⁇ 6 ⁇ 4 integrin expression or an increase in ⁇ 6 ⁇ 4 integrin expression over the standards is indicative of aberrant hyperproliferative cell growth.
  • the presence of a relatively high amount of ⁇ 6 ⁇ 4 integrin protein in biopsied tissue from an individual may indicate the presence of a tumor or other malignant growth, may indicate a predisposition for the development of such malignancies or tumors, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
  • ⁇ 6 ⁇ 4 integrin-specific antibodies of the present invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al, J. Cell Biol. 707:976-985 (1985); Jalkanen, et al, J. Cell Biol. 705:3087-3096 (1987)).
  • Other antibody-based methods useful for detecting protein expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • Suitable antibody assay labels include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. Suitable assays are described in more detail elsewhere herein.
  • One aspect of the invention is a method for the in vivo detection or diagnosis of a hyperproliferative disease or disorder associated with aberrant expression of ⁇ 6 ⁇ 4 integrin in an animal, preferably a mammal and most preferably a human.
  • diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled antibody or fragment thereof of the present invention, which specifically binds to ⁇ 6 ⁇ 4 integrin; b) waiting for a time interval following the administering for permitting the labeled binding molecule to preferentially concentrate at sites in the subject where ⁇ 6 ⁇ 4 integrin is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of ⁇ 6 ⁇ 4 integrin.
  • Background level can be determined by various methods including comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
  • the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
  • the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of, e.g., 99 Tc.
  • the labeled binding molecule e.g., antibody or antibody fragment, will then preferentially accumulate at the location of cells which contain the specific protein.
  • In vivo tumor imaging is described in S. W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W.
  • the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours, hi another embodiment the time interval following administration is 5 to 20 days or 7 to 10 days.
  • Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
  • CT computed tomography
  • PET position emission tomography
  • MRI magnetic resonance imaging
  • sonography sonography
  • the binding molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050).
  • the binding molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument
  • the binding molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography
  • the binding molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • Antibody labels or markers for in vivo imaging of ⁇ 6 ⁇ 4 integrin expression include those detectable by X-radiography, nuclear magnetic resonance imaging (NMR), MRI, CAT-scans or electron spin resonance imaging (ESR).
  • suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
  • suitable markers for NMR and ESR. include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
  • monitoring of an already diagnosed disease or disorder is carried out by repeating any one of the methods for diagnosing the disease or disorder, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
  • detection methods as disclosed herein are useful as a prognostic indicator, whereby patients continuing to exhibiting enhanced ⁇ 6 ⁇ 4 integrin expression will experience a worse clinical outcome relative to patients whose expression level decreases nearer the standard level.
  • ⁇ 6 ⁇ 4 integrin polypeptide expression level in the first biological sample is measured or estimated and compared to a standard ⁇ 6 ⁇ 4 integrin polypeptide level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder.
  • a standard ⁇ 6 ⁇ 4 integrin polypeptide level is known, it can be used repeatedly as a standard for comparison.
  • biological sample any biological sample obtained from an individual, cell line, tissue culture, or other source of cells potentially expressing ⁇ 6 ⁇ 4 integrin.
  • biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid), and other tissue sources which contain cells potentially expressing ⁇ 6 ⁇ 4 integrin. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.
  • antibodies, or immunospecific fragments of antibodies directed to a conformational epitope of ⁇ 6 ⁇ 4 integrin may be used to quantitatively or qualitatively detect the presence of ⁇ 6 ⁇ 4 integrin gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluoresence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
  • Cancers that may be diagnosed, and/or prognosed using the methods described above include but are not limited to, stomach cancer, renal cancer, brain cancer, bladder cancer, colon cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and prostate cancer.
  • ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof disclosed herein may be assayed for immunospecific binding by any method known in the art.
  • the immunoassays which can be used include but are not limited to competitive and noncompetitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
  • Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors ⁇ e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4.degree.
  • a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol
  • protein phosphatase and/or protease inhibitors ⁇ e.g.
  • Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32p or 1251) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen
  • ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
  • ELISAs e.g., Ausubel et al., eds, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, Vol. 1 (1994) at 11.2.1.
  • the binding affinity of an antibody to an antigen and the off-rate of an antibody- antigen interaction can be determined by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen ⁇ e.g., 3 H or 125 I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
  • the affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by Scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays.
  • the antigen is incubated with antibody of interest is conjugated to a labeled compound (e.g., 3H or 125 I) in the presence of increasing amounts of an unlabeled second antibody.
  • ⁇ 6 ⁇ 4 integrin-specific antibodies may, additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of cancer antigen gene products or conserved variants or peptide fragments thereof.
  • In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled ⁇ 6 ⁇ 4 integrin-specific antibody or fragment thereof, preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • a labeled ⁇ 6 ⁇ 4 integrin-specific antibody or fragment thereof preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • Immunoassays and non-immunoassays for ⁇ 6 ⁇ 4 integrin gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectably labeled antibody capable of binding to ⁇ 6 ⁇ 4 integrin or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.
  • the biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • the support may then be washed with suitable buffers followed by treatment with the detectably labeled ⁇ 6 ⁇ 4 integrin-specific antibody.
  • the solid phase support may then be washed with the buffer a second time to remove unbound antibody.
  • the antibody is subsequently labeled.
  • the amount of bound label on solid support may then be detected by conventional means.
  • solid phase support or carrier any support capable of binding an antigen or an antibody.
  • supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
  • the binding activity of a given lot of ⁇ 6 ⁇ 4 integrin-specific antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • SPR Surface plasmon reasonance as performed on BIAcore offers a number of advantages over conventional methods of measuring the affinity of antibody-antigen interactions: (i) no requirement to label either antibody or antigen; (ii) antibodies do not need to be purified in advance, cell culture supernatant can be used directly; (iii) real-time measurements, allowing rapid semi-quantitative comparison of different monoclonal antibody interactions, are enabled and are sufficient for many evaluation purposes; (iv) biospecific surface can be regenerated so that a series of different monoclonal antibodies can easily be compared under identical conditions; (v) analytical procedures are fully automated, and extensive series of measurements can be performed without user intervention.
  • BIAapplications Handbook version AB (reprinted 1998), BIACORE code No. BR-1001-86; BIAtechnology Handbook, version AB (reprinted 1998), BIACORE code No. BR-1001-84.
  • SPR based binding studies require that one member of a binding pair be immobilized on a sensor surface.
  • the binding partner immobilized is referred to as the ligand.
  • the binding partner in solution is referred to as the analyte.
  • the ligand is attached indirectly to the surface through binding to another immobilized molecule, which is referred as the capturing molecule.
  • SPR response reflects a change in mass concentration at the detector surface as analytes bind or dissociate.
  • the resulting sensorgram can be divided into three essential phases: (i) Association of analyte with ligand during sample injection; (ii) Equilibrium or steady state during sample injection, where the rate of analyte binding is balanced by dissociation from the complex; (iii) Dissociation of analyte from the surface during buffer flow.
  • the equilibrium phase provides information on the affinity of the analyte- ligand interaction (K D ).
  • BIAevaluation software provides comprehensive facilities for curve fitting using both numerical integration and global fitting algorithms. With suitable analysis of the data, separate rate and affinity constants for interaction can be obtained from simple BIAcore investigations. The range of affinities measurable by this technique is very broad ranging from mM to pM.
  • Epitope specificity is an important characteristic of a monoclonal antibody.

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Abstract

Cette invention a trait à des anticorps qui se fixent à l'intégrine alpha 6 bêta 4 (intégrine α6β4) et à leurs utilisations, en particulier dans le diagnostic et le traitement du cancer. L'invention concerne des anticorps humains et murins spécifiques qui inhibent les voies de prolifération tumorale et de pro-survie associées à l'intégrine α6β4, et leurs variants, fragments, et dérivés. L'invention a également trait à des anticorps humains et murins spécifiques qui bloquent la capacité du ligand, de la laminine à se fixer à l'intégrine α6β4, ainsi qu'aux fragments, aux variants et aux dérivés de ces anticorps. L'invention concerne par ailleurs des polynucléotides codant lesdits anticorps ci-dessus ou leurs fragments, variants ou dérivés, ainsi que des vecteurs et des cellules hôtes comprenant ces polynucléotides. L'invention comprend en outre des procédés de diagnostic et de traitement du cancer utilisant lesdits anticorps de l'invention.
PCT/US2008/004714 2007-04-13 2008-04-11 Anticorps anti-intégrine alpha 6 bêta 4 et leurs utilisations Ceased WO2008127655A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US91182707P 2007-04-13 2007-04-13
US60/911,827 2007-04-13
US2663108P 2008-02-06 2008-02-06
US61/026,631 2008-02-06

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WO2008127655A1 true WO2008127655A1 (fr) 2008-10-23

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010098166A1 (fr) * 2009-02-24 2010-09-02 国立大学法人宮崎大学 Inhibiteur d'adhérence cellulaire et son utilisation
US20100284918A1 (en) * 2007-10-03 2010-11-11 Christian Rohlff Protein
WO2014081831A1 (fr) 2012-11-20 2014-05-30 The University Of North Carolina At Chapel Hill Procédés et compositions pour protéines facteur ix
WO2015034052A1 (fr) 2013-09-05 2015-03-12 国立大学法人 宮崎大学 Anticorps qui réagit spécifiquement avec l'intégrine humaine a6b4
WO2015070179A1 (fr) * 2013-11-08 2015-05-14 Children's Hospital Los Angeles Méthodes de traitement d'hémopathies malignes
CN109906232A (zh) * 2016-09-23 2019-06-18 埃尔斯塔治疗公司 包含λ轻链和κ轻链的多特异性抗体分子
US11773176B2 (en) 2020-01-24 2023-10-03 Aprilbio Co., Ltd. Multispecific antibodies, compositions comprising the same, and vectors and uses thereof
WO2024208354A1 (fr) * 2023-04-07 2024-10-10 Cstone Pharmaceuticals (Suzhou) Co., Ltd. Conjugués anticorps-médicament
WO2024244006A1 (fr) * 2023-06-02 2024-12-05 昱言科技(北京)有限公司 Anticorps ciblant itga2, conjugué anticorps-médicament correspondant, et utilisation
EP4337698A4 (fr) * 2021-05-11 2025-11-19 Pasithea Therapeutics Corp Agents de liaison à l'intégrine alpha 5 bêta 1 et leurs utilisations

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KADOYA ET AL.: "Antibodies against domain E3 of laminin-1 and integrin alpha 6 subunit perturb branching epithelial morphogenesis of submandibular gland, but by different modes", J. CELL BIOL., vol. 129, no. 2, 1995, pages 521 - 534 *
LEE ET AL., J. CELL BIOL., vol. 117, no. 3, 1992, pages 671 - 678 *
TOZEREN ET AL.: "Integrin alpha 6 beta 4 mediates dynamic interactions with laminin", J. CELL SCI., vol. 107, no. 11, 1994, pages 3153 - 3163 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100284918A1 (en) * 2007-10-03 2010-11-11 Christian Rohlff Protein
WO2010098166A1 (fr) * 2009-02-24 2010-09-02 国立大学法人宮崎大学 Inhibiteur d'adhérence cellulaire et son utilisation
WO2014081831A1 (fr) 2012-11-20 2014-05-30 The University Of North Carolina At Chapel Hill Procédés et compositions pour protéines facteur ix
EP3042956A4 (fr) * 2013-09-05 2017-03-15 University of Miyazaki Anticorps qui réagit spécifiquement avec l'intégrine humaine a6b4
JPWO2015034052A1 (ja) * 2013-09-05 2017-03-02 国立大学法人 宮崎大学 ヒトインテグリンａ６ｂ４と特異的に反応する抗体
WO2015034052A1 (fr) 2013-09-05 2015-03-12 国立大学法人 宮崎大学 Anticorps qui réagit spécifiquement avec l'intégrine humaine a6b4
US10030071B2 (en) 2013-09-05 2018-07-24 University Of Miyazaki Antibody which specifically reacts with human integrin A6B4
WO2015070179A1 (fr) * 2013-11-08 2015-05-14 Children's Hospital Los Angeles Méthodes de traitement d'hémopathies malignes
CN109906232A (zh) * 2016-09-23 2019-06-18 埃尔斯塔治疗公司 包含λ轻链和κ轻链的多特异性抗体分子
CN109906232B (zh) * 2016-09-23 2023-11-07 马伦戈治疗公司 包含λ轻链和κ轻链的多特异性抗体分子
US11773176B2 (en) 2020-01-24 2023-10-03 Aprilbio Co., Ltd. Multispecific antibodies, compositions comprising the same, and vectors and uses thereof
EP4337698A4 (fr) * 2021-05-11 2025-11-19 Pasithea Therapeutics Corp Agents de liaison à l'intégrine alpha 5 bêta 1 et leurs utilisations
WO2024208354A1 (fr) * 2023-04-07 2024-10-10 Cstone Pharmaceuticals (Suzhou) Co., Ltd. Conjugués anticorps-médicament
WO2024244006A1 (fr) * 2023-06-02 2024-12-05 昱言科技(北京)有限公司 Anticorps ciblant itga2, conjugué anticorps-médicament correspondant, et utilisation

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