EP1326892A2 - Zusammensetzungen die die proliferation von krebszellen hemmen - Google Patents

Zusammensetzungen die die proliferation von krebszellen hemmen

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Publication number
EP1326892A2
EP1326892A2 EP01977754A EP01977754A EP1326892A2 EP 1326892 A2 EP1326892 A2 EP 1326892A2 EP 01977754 A EP01977754 A EP 01977754A EP 01977754 A EP01977754 A EP 01977754A EP 1326892 A2 EP1326892 A2 EP 1326892A2
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Prior art keywords
cell
cancer
integrin
cells
protein
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French (fr)
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Hartmut Land
Laurent Deleu
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University of Rochester
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University of Rochester
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Priority to EP10012959A priority Critical patent/EP2336166A1/de
Publication of EP1326892A2 publication Critical patent/EP1326892A2/de
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • C07K14/7055—Integrin beta1-subunit-containing molecules, e.g. CD29, CD49
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it

Definitions

  • Carcinogenesis is caused by multiple cooperating genetic lesions leading to a progressive deregulation of cellular signaling and cell cycle restriction point control.
  • the mutations involved result in oncogene activation or loss of tumor-suppressor gene function.
  • single oncogenes are insufficient to cause malignant transformation because they simultaneously induce signals stimulating and inhibiting cell growth. As a result cell proliferation remains restricted.
  • cooperating oncogenic lesions act in concert to disable such inhibitory signals while reinforcing the growth-promoting stimuli.
  • compositions and methods that show survival of various transformed cell types requires cell-autonomous (autocrine) integrin signaling activity. This activity is induced by cooperating oncogenic lesions and involves induction of integrin receptor and ligand components such as integrin alpha ⁇ and integrin beta4, and Iaminin5-gamma2 chains. Blocking of integrin or the laminin ligand function induces rapid apoptosis of the transformed cells, even when growing in presence of ECM. In contrast, normal cells remain viable when exposed to the same treatment.
  • the disclosed compositions and methods are related to the cooperation of oncogenic lesions controlling the ability of transformed cells to proliferate in the absence of contact with the extra-cellular matrix (ECM).
  • ECM extra-cellular matrix
  • oncogenes cooperate to promote a cell-autonomous (autocrine) integrin signaling loop that proves essential for the survival of various transformed Gell types As this signaling loop is not established in corresponding normal cells, the signaling components of this loop constitute attractive targets for cancer therapy
  • this invention in one aspect, relates to compositions and methods related to integrin mediated cancer cell growth
  • Figure 1 shows a series of schematics representative of the disclosed relationships and compositions
  • mteg ⁇ n receptors signal to suppress programmed cell death (apoptosis) when engaged by appropriate extra-cellular matrix (ECM) ligands
  • ECM extra-cellular matrix
  • Figure 2 shows that oncogene cooperation protects cells from apoptosis.
  • Figure 2A shows control, APCm, Ras, APCm+Ras cells that were detached from collagen IV substrates with
  • FIG. 2B shows control, APCm, Ras, APCm Ras, SW480 cells were detached and maintained in suspension as described in (A).
  • SW480 beta4 dn/Gal4VPER cells express a 40H-tamoxifen-inducible dominant-negative form of the beta4 integrin.
  • FIG. 3 shows alterations of integrin and laminin expression profiles in malignant cell transformation.
  • Figure 3A shows integrin expression and figure 3B shows laminin expression.
  • the indicated cell populations were cultured on collagen IV-coated dishes at 39°C in RPMI 10%
  • RT-PCR laminins, alpha4, gamma2 and GAPDH
  • RNase protection 10 ⁇ g of total RNA was used per reaction. Products were resolved on a 4.5% polyacrylamide/lOM urea gel.
  • Figure 4 shows alpha6/beta4 integrin is engaged by the laminin gamma2 chain to activate She (a src homology domain containing protein).
  • Figure 4A shows cells detached from collagen IV coated dishes with 3mM EDTA (in PBS) were incubated on ice with the indicated antibodies for 1 h. Cells were then plated on 96well dishes coated with gammal or gamma2-specific peptides and were permitted to attach for 30 min in RPMI medium at 39°C After incubation, wells were washed with RPMI.
  • the cells were incubated with betal and beta4 integrin antibodies as well as the laminin gamma2-specific peptide for 40 min. Protein extracts were prepared and subjected to an immuno-precipitation with an anti-She antibody. Phospho-tyrosine was detected with a phopho-tyrosine-specific antibody in IP-western-blots (upper panels). The levels of She protein were monitored with a She-specific antibody reusing the same membrane (lower panels).
  • Figure 5 shows integrin alpha6/beta4 and Laminin gamma2 chain expression is essential for survival of transformed cells.
  • Figure 5A1 shows the indicated cell populations were infected at an MOI of 2 with recombinant retroviruses expressing anti-sense RNA for alpha6 integrin, the gamma2 laminin chain or a beta4 integrin dominant-negative mutant together with a puromycin resistance gene. After 2 weeks of selection with puromycin, colonies were stained with Gie sa and counted.
  • APCm+Ras alpha ⁇ and APCm+Ras p35 cells express the alpha ⁇ integrin chain and the anti-apoptotic baculovirus p35, respectively.
  • APCm+Ras cells were also plated on dishes pre- coated with a laminin gamma2-specific peptide.
  • Figure 5A2 shows APCm+Ras hygro
  • APCm+Ras Bcl2 and APCm+Ras p35 cells express the hygromycin resistance marker, and the anti-apoptotic proteins Bcl2 or p35, respectively.
  • the cells were infected at an MOI of 2 with retroviruses carrying the beta4 integrin dominant-negative mutant and a puromycin resistance gene, or the puromycin resistance gene alone. Cells (10 ⁇ ) were maintained in soft agar at 39°C for two weeks after which macroscopically visible colonies were counted.
  • Figure 5A3 shows the indicated cell populations were infected at an MOI of 2 with recombinant retroviruses, drug- selected and maintained as described in Al .
  • Figure 5 4 shows the indicated cell populations were infected at an MOI of 2 with recombinant retroviruses expressing anti-sense RNA for alpha6 integrin, the beta4 integrin dominant-negative mutant or the puromycin resistance marker.
  • Cells (1 ( ) were maintained in soft agar and evaluated as described in A2.
  • Figure 5B shows the effect of the alpha ⁇ anti-sense RNA expression on the cell surface expression of alpha ⁇ integrin was monitored by FACS analysis using an anti-alpha ⁇ rat monoclonal antibody and a F1TC conjugated goat anti-rat antiserum as the secondary antibody (upper panel). Expression of the beta4 dominant-negative mutant was confirmed by RNAse protection.
  • FIG. 5C shows arf null mouse embryo fibroblasts (MEFs) that were infected with retroviruses expressing the oncogenes Myc and Ras. Cells were then additionally infected with a retroviruses expressing anti-sense RNA for alpha ⁇ integrin or the beta4 integrin dominant-negative mutant as shown in (A). MEFS Ras/Myc/ArfnulI+alpha ⁇ sense express the alpha ⁇ integrin chain.
  • Figure 4D shows SW480 human colon carcinoma cells that were infected as described in (A) using VSV pseudo-typed viruses.
  • Infected cells were selected with puromycin in soft agar. Clones were counted 2 weeks after selection.
  • SW480 beta4 dn/Gal4VPER cells express a 40H-tamoxifen-inducible dominant- negative form of the beta4 integrin. The cells (10->) were maintained in soft agar for 2 weeks in presence or absence of 40H-tamoxifen.
  • Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each ofthe ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
  • Primers are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur.
  • a primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation.
  • Probes are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art.
  • compositions and methods related to integrins and integrin signaling are disclosed. It is shown herein that integrin alpha ⁇ , integrin beta4, and laminin5, through at least up regulation of the beta and gamma chains of laminin5, are upregulated in cancer cells. Integrin alpha ⁇ and integrin beta4 interact to form the integrin receptor, A6B4. The integrin receptor A6B4 specifically interacts with laminin5 and laminin5 specifically interacts with A6B4.
  • compositions and methods that interfere with the function of molecules involved with the signal transduction that is connected to either alpha ⁇ , beta4, laminin5, the laminin gamma2 chain, or A6B4.
  • methods for reducing the proliferation of cancer cells as well as methods of killing cancer cells that involve using the compositions disclosed herein that interfere, reduce, or eliminate the function or the alpha ⁇ , beta4, laminin5, the laminin gamma2 chain, or A6B4 function.
  • the first type of molecule is an integrin receptor, composed of integrins
  • the second type of molecule is a ligand that interacts with the integrin receptor, through the integrins.
  • integrin receptors There are specificities that exist between the integrin receptors and their ligands.
  • One aspect, disclosed herein is that when a cell goes from a non-cancerous state to a cancerous state, there is a co- upregulation of both the ligand (or parts ofthe ligand, such as subunits) and the cognate integrin receptor.
  • the co-upregulation of both types of molecules creates an autocrine loop situation, wherein the signaling pathways controlled by the integrin receptor become autonomously activated, rather than exogenously activated, as would normally occur.
  • the upregulation of both types of molecules creates a more fully transformed cellular phenotype in which cancer cell survival depends on the autocrine loop. Now therapeutic activities can target both points in the autocrine loop.
  • Specific examples, of this co-upregulation in cancer cells are disclosed herein. For example, laminin 5 (both the beta and gamma2 chains are upregulated) and integrin receptor alpha ⁇ beta4, laminin 10/1 1 and the integrin receptors alpha ⁇ betal and alpha3betal
  • the integrins mediate cell adhesion primarily by binding to distinct, although overlapping, subsets of ECM proteins Normal cells require contact with serum-derived ECM components for proliferation, differentiation and survival (e g Clark and Brugge, 1995, Lin and Bissell, 1993, Pa ⁇ se et al , 2000), a phenomenon called anchorage-dependence This involves signaling through integrin receptors (Hynes, 1992) Fibronectin and laminin as well as other ECM proteins are known to act as hgands for integrin receptors (Akiyama et al , 1990) Integrins are transmembrane proteins forming alpha-beta chain heterodimers Alpha and beta chain integrins are members of distinct gene families The ligand binding specificity of the hetero dimers is determined by specific combinations of alpha and beta chain gene family members (Hynes, 1992) Ligand binding triggers signaling of mteg ⁇ n receptors through the cytoplasmic tail of the beta chain via interaction with va ⁇ ous signaling components
  • Integrins activate common as well as subgroup-specific signaling pathways (Clark and Brugge, 1995, Giancotti and Ruoslahti, 1999)
  • FAK focal adhesion kinase
  • the ⁇ l ⁇ l, ⁇ 5 ⁇ l, ⁇ v ⁇ 3 and 6 ⁇ 4 integrins are coupled to the Ras- extracellular signal-regulated kinase (ERK) signaling pathway by She (Maimero et al , 1997, Maimero et al , 1995, Wary et al , 1996)
  • She is an SH2-PTB domain adapter protein expressed in three forms, p46, p52 and p66, two of which (p46 and p52) link various tyrosine kinases to Ras by recruiting the Grb2/SOS complex to the plasma membrane (Pawson and Scott, 1997)
  • SOS Upon activation by SOS, Ras stimulates a kinase cascade culminating in the activation of the mitogen- activate
  • cancer cells In contrast with normal cells, cancer cells generally are able to survive and proliferate in the absence of anchorage to ECM (Giancotti and Maimero, 1994), suggesting that tumor cell survival and proliferation have become independent of the engagement of integrin signaling through ECM
  • Proliferation in the absence of anchorage to ECM of secondary rat embryo fibroblasts requires the cooperation of Ras and Myc or Ras and adenovirus E la oncogenes (Land et al , 1983, Ruley, 1983) Similarly, murine colonic epithelial cells require both activated Ras and mutation of the adenomous polyposis coli gene (APCmin) (D'Abaco et al , 1996) in order to proliferate in suspension
  • Integrins are a large family of cell surface receptor molecules that function to mediate interactions between cells and between cells and the extracellular matrix Integrin receptors are heterodimers composed of two subumts, an alpha integrin and a beta integrin The heterodimer forms, is expressed on the cell surface, and acts to transmit signals obtained from interactions with the extracellular matrix or other cells, through the cellular membrane and into the cytosol of the cell The signal transduction that takes place occurs because of ligand interactions with the receptor Integrin receptors can have a number of hgands, including collagens, fibronectms, and lammms
  • Integrin alpha ⁇ has seven ammo-terminal repeating segments that may fold into a seven unit beta-propeller, five n-terminal FG-GAP domains and three divalent cation sites
  • the transmembrane domain is followed by a short cytoplasmic tail, that is alternatively spliced in A and B forms
  • the alpha ⁇ integrin chain also shows alternative splicing between repeat units IE and IV, resulting in the presence or absence of Exon X2 Integrin alpha ⁇ is processed into a heavy and a light chain that are disulphide linked
  • a representative allele of the human alpha ⁇ cDNA is set forth in SEQ ID NO 1 It is understood that the disclosed functional domains as well as the others contained within alpha ⁇ are considered separately disclosed as discreet fragments of the alpha ⁇ protein as well as the nucleic acid that encodes them
  • Integrin beta4 contains a MIDAS-hke motif and four cysteine- ⁇ ch repeats, three EGF like domains in the N-terminal extracellular domain a trans-membrane region and a long cytoplasmic tail containing two pairs of fibronectin Type III repeats The latter are connected by a variable segment that may undergo alternative splicing Integrin beta4 also undergoes proteolytic processing in its cytoplasmic tail, causing the 200kD mature form to be converted to 165 and 130kD fragments
  • a representative allele of the human beta4 cDNA is set forth in SEQ ID NO 5 It is understood that the disclosed functional domains as well as the others contained within beta4 are considered separately disclosed as disci eet fragments of the beta4 protein as well as the nucleic acid that encodes them
  • Lam ⁇ nm5 is composed ofthe laminin chains alpha3, beta3 and gamma2 Lam ⁇ nm5 can may contain either the shorter lammm alpha3A chain or the longer alpha3B chain Lam ⁇ n ⁇ n5 can also be trimmed by proteolytic processing of the N-termmal portion of its alpha3A chain and the N-terminal portion ofthe gamma2 chain
  • the Lammm gamma2 chain contains at least six lammin EGF-hke domains (Domains III and V) with an embedded lammm B domain (Domain IV) withm the N-termmal half
  • the c-termmal tail contains a coiled-coil domain
  • the N-terminal processed portion of the gamma2 chain is sufficient to bind to and activate the integrin alpha ⁇ /beta4 receptor
  • a representative allele of the human Iam ⁇ n ⁇ n5-gamma2 cDNA is set forth in SEQ ID NO 13 It is understood that the disclosed functional domains as well as the others
  • compositions and methods for inhibiting integrin signaling for example, mtegrin signaling dependent on alpha ⁇ and beta4 integrins
  • compositions and methods that inhibit mtegrin receptor signaling from, for example, the alpha ⁇ beta4 integrin receptor are disclosed
  • the integrin receptor signaling can be affected by, for example, interfering with a molecule, such as a ligand for the mtegrin receptor, or a downstream signaling molecule of the mtegrin receptor in a way that prevents the integrin receptor signal from being fully propagated
  • the compositions and methods for inhibition of integrin signaling and function can be any composition or method that ultimately inhibits the cell proliferation in which the integrin is expressed, by for example killing the cell
  • the compositions and methods typically can fall into three basic non-limiting classes of function regulators, which are discussed herein
  • Production regulators is a broad class of integrin function regulators that are directed at the production of the target integrin, by for example, preventing mRNA synthesis or expression of the target mtegrin, or by causing mRNA degradation of the target integrin which inhibits the translation of the target mtegrin
  • production regulators can be any type of molecule targeting any point in the mtegrin production pathway, typically these types of compositions will target either the mRNA expression or the protein expression of the mtegrin
  • beta4 integrin, alpha ⁇ integrin, or the gamma2 subunit of laminmS which has been shown herein to be upregulated in cancer cells and which causes the cancer cell to be able to live in the absence of the ECM, was the target integrin
  • a typical production regulator of beta4 integrin, alpha ⁇ integrin, or the gamma2 subunit of lam ⁇ n ⁇ n5 would be for example, an antisense molecule that targeted the mRNA of beta
  • mtegrin function regulator typically prevents integrins from interacting to form a functional integrin receptor
  • an integrin to mtegrin regulator could be a composition that would interact with beta4 in a way that would prevent beta4 from interacting with alpha ⁇ to form the alpha ⁇ beta4 integrin receptor or it could be a composition that would interact with alpha ⁇ m a way that would prevent alpha ⁇ from interacting with beta4 to form the alpha ⁇ beta4 mtegrin receptor
  • the function regulators of the mtegrin to integrin interaction can affect the signaling pathways dependent on mtegrin receptors containing alph ⁇ or beta4 integrins It is not required that an integrin to integrin regulator prevent an integrin from interacting with all of the possible integrin partners it could interact with, just that it prevent the interaction o the target integrin with another specific
  • the third class of function regulators are the mtegrin to other molecule regulators These compositions are designed to specifically interfere with molecules such as small molecule hgands or other proteins that interact with the integrin or integrin receptor
  • an integrin to other molecule regulator might target a ligand for a particular mtegrin receptor, such as the alpha ⁇ beta4 receptor
  • the ligand for the alpha6beta4 receptor is lam ⁇ n ⁇ n5 or the lammm gamma2 chain Compositions that interact with lam ⁇ n ⁇ n5 such that lamm ⁇ n5 or the lammin gamma2 chain interactions with alpha6beta4 are inhibited or reduced are specifically contemplated herein
  • there are other molecules, such as She molecules, that also interact with integrins, such as the alpha6beta4 receptor Compositions that specifically interact with the She molecules such that they prevent the appropriate interactions between the She molecule and the alpha6beta4 receptor are disclosed
  • Types lust as there are different general classes of molecules that can regulate (such as by inhibition) the function of the disclosed integrins and integrin receptors, so to there are many different types of molecules that perform that regulation
  • any molecule that can perform the regulation of for example, the disclosed integrins, mtegrm receptors, or signaling pathways produced by the disclosed integrins and mtegrin receptors are contemplated
  • antibodies or small molecules which inhibit the disclosed compositions are herein disclosed
  • functional nucleic acids such as ⁇ bozymes or antisense molecules that can inhibit the disclosed mtegrm function in a variety of ways A non- limiting list of exemplary molecules is discussed herein
  • Antibodies can be used to regulate, for example, the function of the disclosed integrins and mtegrin receptors, molecules that interact with the disclosed integrin receptors, and molecules in the signaling pathways of the disclosed mtegrm receptors
  • antibody encompasses, but is not limited to, whole immunoglobulin (l e , an intact antibody) of any class
  • Native antibodies are usually heterotetrame ⁇ c glycoprotems, composed of two identical light (L) chains and two identical heavy (H) chains
  • each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin lsotypes
  • Each heavy and light chain also has regularly spaced lntracham disulfide bridges
  • Each heavy chain has at one end a variable domain (V(H)) followed by a number of constant domains
  • Each light chain has a variable domain at one end (V(L)) and a constant domain at its other end, the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain
  • Particular ammo acid residues are believed to form an interface between the light and heavy chain
  • variable is used herein to describe certain portions ofthe variable domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen
  • CDRs complementarity determining regions
  • FR framework
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a b-sheet configuration, connected by three CDRs, which form loops connecting, and m some cases forming part of, the b-sheet structure
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat E A et al , "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md (19).
  • antibody or fragments thereof encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab')2, Fab', Fab and the like, including hybrid fragments
  • fragments of the antibodies that retain the ability to bind their specific antigens are provided
  • fragments of antibodies which maintain EphA2 binding activity are included within the meaning ofthe term "antibody or fragment thereof "
  • Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane Antibodies, A Laboratory Manual Cold Spring Harbor Publications, New York, (1988))
  • antibody or fragments thereof conjugates of antibody fragments and antigen binding proteins (single chain antibodies) as described, for example, in U S Pat No 4,704,692, the contents of which are hereby inco ⁇ orated by reference Single chain divalent antibodies are also provided
  • the antibodies are generated in other species and "humanized" for administration in humans
  • Humanized forms of non-human (e g , murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen binding subsequences of antibodies) which contain minimal sequence derived from non human immunoglobulin
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or 5 rabbit having the desired specificity, affinity and capacity
  • CDR complementary determining region
  • donor antibody such as mouse, rat or 5 rabbit having the desired specificity, affinity and capacity
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues
  • Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported C
  • substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al , Nature, 321 522-525 (1986), Riechmann et al , Nature, 332 323-327 (1988), and Presta, Curr Op Struct Biol , 2 593-
  • a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain Humamzation can be essentially 0 performed following the method of Winter and co-workers (Jones et al , Nature, 321 522-525 (1986), Riechmann et al , Nature, 332 323-327 (1988), Verhoeyen et al , Science, 239 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody Accordingly, such "humanized” antibodies are chimeric antibodies (U S Pat No 4,816,567), wherein substantially less than an intact human variable domain has been 5 substituted by the corresponding sequence from a non-human species In practice, humanized antibodies are typically human antibodies in which some CDR residues
  • variable domains both light and heavy
  • sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences
  • the human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody
  • FR human framework
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies 5 of a particular subgroup of light or heavy chains The same framework may be used for several different humanized antibodies (Carter et al , Proc Natl Acad Sci USA, 89 4285 (1992), Presta et al , J Immunol , 151 2623 (1993))
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences
  • Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences Inspection of these displays permits analysis of the likely role ofthe residues in the functioning of the candidate immunoglobulin sequence, l e , the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen
  • FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target anhgen(s), is achieved
  • the CDR residues are directly and most substantially involved in influencing antigen binding (see, WO 94/04679, published 3 March 1994)
  • Transgenic animals that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production
  • J(H) antibody heavy chain joining region
  • the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production
  • Transfer of the human germ-line immunoglobulin gene array in such germ line mutant mice will result in the production of human antibodies upon antigen challenge (see, e g , Jakobovits et al , Proc Natl Acad Sci USA, 90 2551 255 (1993), Jakobovits et al , Nature, 362 255-258 (1993), Bruggemann et al , Year in Immuno , 7 33 (1993))
  • Human antibodies can also be produced in phage display libraries (Hoogenboom et al , J Mol Biol , 227 381 (1991), Marks et al , J Mol
  • the present invention further provides a hybridoma cell that produces the monoclonal antibody of the invention
  • the term "monoclonal antibody' as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, I e , the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts
  • the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies dem ed from a particular species or belonging to a particular antibody class or subclass, while the remainder of the cham(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired activity (See, U S Pat No 4,816,567 and Morrison et al , Proc Natl Acad Sci USA, 81 6851 -6855 ( 1984))
  • Monoclonal antibodies of the invention may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256 495 (1975) or Harlow and Lane Antibodies, A Laboratory Manual Cold Spring Harbor Publications, New York, (1988)
  • a hybridoma method a mouse or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent
  • the lymphocytes may be immunized in vitro
  • the immunizing agent comprises EphA2
  • the generation of monoclonal antibodies has depended on the availability of purified protein or peptides for use as the immunogen
  • DNA based immunizations have shown promise as a way to elicit strong immune responses and generate monoclonal antibodies
  • DNA-based immunization can be used, wherein DNA encoding a portion of EphA2 expressed as a fusion protein with human IgGl is
  • peripheral blood lymphocytes are used m methods of producing monoclonal antibodies if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (God g, "Monoclonal Antibodies Principles and Practice” Academic Press, (1986) pp 59- 103)
  • Immortalized cell lines are usually transformed mammalian cells, including myeloma cells of rodent, bovine, equine, and human origin Usually, rat or mouse myeloma cell lines are employed
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells For example, if the parental cells lack the enzyme hypoxanthme guanine phospho ⁇ bosyl transfera
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against EphA2
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA)
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the clones may be subcloned by limiting dilution or FACS sorting procedures and grown by standard methods Suitable culture media for this pu ⁇ ose include, for example, Dulbecco s Modified Eagle's Medium and RPMI-1640 medium Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, protein G, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U S Pat No 4,816,567
  • DNA encoding the monoclonal antibodies of the invention can 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 hybridoma cells of the invention serve as a preferred source of such DNA
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, plasmacytoma cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U S Pat No 4,816,567)
  • the Fab fragments produced in the antibody digestion also contain the constant domains of the light chain and the first constant domain of the heavy chain Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain domain including one or more cystemes from the antibody hinge region
  • the F(ab')2 fragment is a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge at the hinge region Fab' SH is the designation herein for Fab' in which the cysteine res ⁇ due(s) of the constant domains bear a free thiol group
  • Antibody fragments originally were produced as pairs of Fab' fragments which have hinge cystemes between them Other chemical couplings of antibody fragments are also known
  • An isolated immunogenically specific paratopc or fragment ofthe antibody is also provided.
  • a specific lmmunogenic epitope of the antibody can be isolated from the whole antibody by chemical or mechanical disruption of the molecule The purified fragments thus obtained are tested to determine their immunogenicity and specificity by the methods taught herein.
  • Immunoreactive paratopes ofthe antibody optionally, are synthesized directly.
  • An immunoreactive fragment is defined as an amino acid sequence of at least about two to five consecutive amino acids derived from the antibody amino acid sequence.
  • One method of producing proteins comprising the antibodies of the present invention is to link two or more peptides or polypeptides together by protein chemistry techniques.
  • peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA).
  • Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry Applied Biosystems, Inc., Foster City, CA.
  • a peptide or polypeptide corresponding to the antibody of the present invention for example, can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment.
  • peptide condensation reactions these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof.
  • the peptide or polypeptide is independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides may be linked to form an antibody or fragment thereof via similar peptide condensation reactions.
  • enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen L et al,, Biochemistry, 30:4151 (1991)).
  • native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al. Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)).
  • the first step is the chemoselective reaction of an unprotected synthetic peptide-alpha-thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.
  • IL-8 human interleukin 8
  • unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer, M et al Science, 256 221 (1992))
  • This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton RC et al , Techniques in Protein Chemistry IV Academic Press, New York, pp 257-267 (1992))
  • the invention also provides fragments of antibodies which have bioactivity
  • the polypeptide fragments of the present invention can be recombinant proteins obtained by cloning nucleic acids encoding the polypeptide in an expression system capable of producing the polypeptide fragments thereof, such as an adenovirus or baculovirus expression system
  • an expression system capable of producing the polypeptide fragments thereof, such as an adenovirus or baculovirus expression system
  • an expression system capable of producing the polypeptide fragments thereof such as an adenovirus or baculovirus expression system
  • an expression system capable of producing the polypeptide fragments thereof
  • an expression system capable of producing the polypeptide fragments thereof such as an adenovirus or baculovirus expression system
  • an expression system capable of producing the polypeptide fragments thereof such as an adenovirus or baculovirus expression system
  • ammo acids found to not contribute to either the activity or the binding specificity or affinity of the antibody can be deleted without a loss in the respective
  • an antibody reagent kit comprising containers of the monoclonal antibody or fragment thereof of the invention and one or more reagents for detecting binding of the antibody or fragment thereof to the EphA2 receptor molecule
  • the reagents can include, for example, fluorescent tags, enzymatic tags, or other tags
  • the reagents can also include secondary or tertiary antibodies or reagents for enzymatic reactions, wherein the enzymatic reactions produce a product that can be visualized
  • Functional nucleic acids can also be used to regulate the , for example, the function ofthe disclosed integrins, integrin receptors, molecules that interact with the disclosed integrin receptors, and molecules in the signaling pathways of the disclosed integrin receptors
  • Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction
  • Functional nucleic acid molecules can be divided into the following categories, which are not meant to be limiting
  • functional nucleic acids include antisense molecules, aptamers, ⁇ bozymes, triplex forming molecules, and external guide sequences
  • the functional nucleic acid molecules can act as affectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules
  • Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains
  • functional nucleic acids can interact with the mRNA of beta4 mtegrin for example, or the genomic DNA of alpha ⁇ mtegrm for example, or they can interact with the polypeptide lam ⁇ n ⁇ n5 or the gamma2 subunit of lam ⁇ mn5
  • functional nucleic acids are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule.
  • the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place.
  • Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication.
  • Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule exist. Exemplary methods would be in vitro selection experiments and DNA modification studies using DMS and DEPC. It is preferred that antisense molecules bind the target molecule with a dissociation constant (k d )less than 10 "6 . It is more preferred that antisense molecules bind with a k d less than IO "8 . It is also more preferred that the antisense molecules bind the target molecule with a k d less than 10 "10 . It is also preferred that the antisense molecules bind the target molecule with a k d less than I O"' 2 .
  • k d dissociation constant
  • Aptamers are molecules that interact with a target molecule, preferably in a specific way.
  • aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets.
  • Aptamers can bind small molecules, such as ATP (United States patent 5,631,146) and theophiline (United States patent 5,580,737), as well as large molecules, such as reverse transcriptase (United States patent 5,786,462) and thrombin (United States patent 5,543,293).
  • Aptamers can bind very tightly with k j S from the target molecule of less than IO "12 M.
  • the aptamers bind the target molecule with a k d less than I O "6 . It is more preferred that the aptamers bind the target molecule with a k d less than 10 "8 . It is also more preferred that the aptamers bind the target molecule with a k d less than 10 "10 . It is also preferred that the aptamers bind the target molecule with a k d less than IO " ' 2 . Aptamers can bind the target molecule with a very high degree of specificity.
  • aptamers have been isolated that have greater than a 10000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule (United States patent 5,543,293). It is preferred that the aptamer have a k d with the target molecule at least 10 fold lower than the k d with a background binding molecule. It is more preferred that the aptamer have a k d with the target molecule at least 100 fold lower than the k d with a background binding molecule. It is more preferred that the aptamer have a kj with the target molecule at least 1000 fold lower than the k d with a background binding molecule.
  • the aptamer have a k d with the target molecule at least 10000 fold lower than the k d with a background binding molecule. It is preferred when doing the comparison for a polypeptide for example, that the background molecule be a different polypeptide.
  • the background protein could be bovine serum albumin. Representative examples of how to make and use aptamers to bind a variety of different target molecules can be found in the following non-limiting list of United States patents: 5,476,766, 5,503,978, 5,631,146, 5,731,424 .
  • Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. Ribozymes are thus catalytic nucleic acid. It is preferred that the ribozymes catalyze intermolecular reactions.
  • ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes, (for example, but not limited to the following United States patents: 5,334,711, 5,436,330, 5,616,466, 5,633,133, 5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288, 5,891 ,683, 5,891,684,
  • ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo (for example, but not limited to the following United States patents: 5,580,967, 5,688,670, 5,807,718, and 5,910,408).
  • Preferred ribozymes cleave RNA or DNA substrates, and more preferably cleave RNA substrates.
  • Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non-canonical base pair interactions.
  • ribozymes particularly good candidates for target specific cleavage of nucleic acids because recognition of the target substrate is based on the target substrates sequence.
  • Representative examples of how to make and use ribozymes to catalyze a variety of different reactions can be found in the following non-limiting list of United States patents: 5,646,042, 5,693,535, 5,731 ,295, 5,81 1,300, 5,837,855, 5,869,253, 5,877,021 , 5,877,022, 5,972,699, 5,972,704, 5,989,906, and 6,017,756.
  • Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acid.
  • triplex molecules When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependant on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is preferred that the triplex forming molecules bind the target molecule with a k d less than IO "6 . It is more preferred that the triplex forming molecules bind with a k,, less than IO "8 .
  • the triplex forming molecules bind the target moelcule with a kj less than 10 '10 . It is also preferred that the triplex forming molecules bind the target molecule with a k d less than IO '12 .
  • Representative examples of how to make and use triplex forming molecules to bind a variety of different target molecules can be found in the following non-limiting list of United States patents: 5, 176,996, 5,645,985, 5,650,316, 5,683,874, 5,693,773, 5,834,185, 5,869,246, 5,874,566, and 5,962,426.
  • EGSs External guide sequences
  • RNase P RNase P
  • EGSs can be designed to specifically target a RNA molecule of choice.
  • RNAse P aids in processing transfer RNA (tRNA) within a cell.
  • Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 by Yale, and Forster and Altman, Science 238:407- 409 (1990)).
  • RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukaryotic cells.
  • compositions can be used as targets for any combinatorial technique to identify molecules or macromolecular molecules that interact with the disclosed compositions, such as beta4 mtegrm, alpha ⁇ mtegrin, or the gamma2 subunit of lam ⁇ nm5, in a desired way
  • the nucleic acids, peptides, and related molecules disclosed herein can be used as targets for the combinatorial approaches
  • compositions that are identified through combinatorial techniques or screening techniques in which the herein disclosed compositions, for example set forth in SEQ ID NOS 1-19 or portions thereof, are used as the target or reagent in a combinatorial or screening protocol
  • compositions such as macromolecular molecules
  • molecules such as macromolecular molecules
  • the molecules identified and isolated when using the disclosed compositions are also disclosed
  • the products produced using the combinatorial or screening approaches that involve the disclosed compositions such as, beta4 mtegrm, alpha6 integrin, or the gamma2 subunit of lamm ⁇ n5
  • Combinatorial chemistry includes but is not limited to all methods for isolating small molecules or macromolecules that are capable of binding either a small molecule or another macromolecule, typically in an iterative process
  • Proteins, oligonucleotides, and sugars are examples of macromolecules
  • phage display libraries have been used to isolate numerous peptides that interact with a specific target (See for example, United States Patent No 6,031,071, 5,824,520, 5,596,079, and 5,565,332 which are herein inco ⁇ orated by reference at least for their material related to phage display and methods relate to combinatorial chemistry)
  • RNA molecule is generated in which a puromycin molecule is covalently attached to the 3'-end ofthe RNA molecule An in vtti o translation of this modified RNA molecule causes the correct protein, encoded by the RNA to be translated In addition, because of the attachment of the puromycin, a peptdyl acceptor which cannot be extended, the growing peptide chain is attached to the puromycin which is attached to the RNA Thus, the protein molecule is attached to the genetic material that encodes it Normal in vitio selection procedures can now be done to isolate functional peptides Once the selection procedure for peptide function is complete traditional nucleic acid manipulation procedures are performed to amplify the
  • FN3 fibronectm type III domain
  • FN3 can be used at least as a scaffold for engineering novel binding proteins (Koide A, Bailey CW, Huang X, Koide S , "The fibronectm type III domain as a scaffold for novel binding proteins "J Mol Biol 1998 284,1141- 1151 which is herein inco ⁇ orated by reference at least for material related to the fibronectm based novel binding proteins) Using methodology well known to those of skill in the art,
  • Combinatorial libraries can be made from a wide array of molecules using a number of different synthetic techniques.
  • libraries containing fused 2,4-pyrimidinediones (United States patent 6,025,371) dihydrobenzopyrans (United States Patent 6,017,768and 5,821 , 130), amide alcohols (United States Patent 5,976,894), hydroxy-amino acid amides (United States Patent 5,972,719) carbohydrates (United States patent 5,965,719), l ,4-benzodiazepin-2,5- diones (United States patent 5,962,337), cyclics (United States patent 5,958,792), biaryl amino acid amides (United States patent 5,948,696), thiophenes (United States patent 5,942,387), tricyclic Tetrahydroquinolines (United States patent 5,925,527), benzofurans (United States patent 5,919,955), isoquinolines (
  • Screening molecules similar to alpha ⁇ for inhibition of alpha6beta4 formation is a method of identifying and isolating desired compounds that can inhibit the formation of A6B4 receptor.
  • the disclosed compositions such as alpha ⁇ integrin or beta4 integrin can be used as targets in a selection scheme disclosed herein, and then the counter part integrin could be used as a competitive inhibitor to isolate the desired molecules.
  • a library of molecules could be incubated with beta4 integrin, which is bound to a solid support. The solid support can be washed to remove the unbound molecules and then the solid support can be incubated with, for example, alpha ⁇ integrin at a concentration that will saturate all beta4 binding sites.
  • the molecules which are collected in the flowthrough after washing the solid support will be enriched for molecules that interact with beta4 integrin in a way that is competitive to the alpha ⁇ -beta4 interaction.
  • the solid support, bound with a target integrin, or more preferably a target integrin receptor, such as alpha ⁇ beta4 receptor could also be washed, with for example, laminin5 or the gamma2 subunit of lamininS at a concentration that will saturate all of the gamma2 binding sites on the beta4 integrin. Collection of the wash under these conditions will yield a population of molecules enriched for molecules that competitively interact with beta4 integrin at the beta4- gamma2 site.
  • alpha ⁇ beta4 and/or beta4 are equally applicable to alpha ⁇ , as well as other alpha ⁇ betax receptors, such alpha ⁇ betal
  • Also disclosed are methods of isolating molecules that bind with a target molecule selected from the group consisting, B4 integrin, alpha ⁇ mtegrm, and the gamma2 subunit of lam ⁇ n ⁇ n5 comprising 1) contacting a library of molecules with the target molecule and 2) collecting molecules that bind the target molecule producing an enriched population of molecules
  • combinatorial methods and libraries included traditional screening methods and libraries as well as methods and libraries used in iterative processes
  • compositions can be used as targets for any molecular modeling technique to identify either the structure of the disclosed compositions or to identify potential or actual molecules, such as small molecules, which interact in a desired way with the disclosed compositions
  • nucleic acids, peptides, and related molecules disclosed herein can be used as targets in any molecular modeling program or approach
  • Examples of molecular modeling systems are the CHARMm and QUANTA programs, Polygen Co ⁇ oration, Waltham, MA CHARMm performs the energy minimization and molecular dynamics functions QUANTA performs the construction, graphic modeling and analysis of 5 molecular structure QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other
  • Nucleic acids there are a variety of molecules disclosed herein that are nucleic acid based, including for 5 example the nucle acids that encode, for example beta4 and the gamma2 subunit of lamm ⁇ n5, as well as various functional nucleic acids
  • the disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes Non-limiting examples of these and other molecules are discussed herein It is understood that for example, when a vector is expressed in a cell, that the expressed mRNA will typically be made up of A, C, G, and U Likewise, it is 0 understood that if, for example, an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery, it is advantageous that the antisense molecule be
  • a nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an mternucleoside linkage
  • the base moiety of a nucleotide can be adenm-9 yl (A), cytosm- 1 -yl (C), guan ⁇ n-9-yl (G), uracil- 1 -yl (U), and thym ⁇ n-1-yl (T)
  • the sugar moiety of a nucleotide is a ⁇ bose or a deoxy ⁇ bose
  • the phosphate moiety of a nucleotide is pentavalent phosphate
  • An non-limiting example of a nucleotide would be 3'-AMP (3'-adenos ⁇ ne monophosphate) or 5'-GMP (5'-guanos ⁇ ne
  • a nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties Modifications to the base moiety would include natural and synthetic modifications of A, C, G, and T/U as well as different purine or pyrimidine bases, such as urac ⁇ l-5-yl ( psi ), hypoxanth ⁇ n-9-yl (I), and 2-ammoadenm-9-yl
  • a modified base includes but is not limited to 5-methylcytos ⁇ ne (5-me-C), 5-hydroxymethyl cytosme, xanthme, hypoxanthme, 2-ammoademne, 6-mefhyl and other alkyl derivatives of adenine and guamne, 2-propyl and other alkyl derivatives of adenine and guanme, 2-th ⁇ ourac ⁇ l, 2-fh ⁇ othymme and 2-th ⁇ ocytosme, 5- halouracil and cytosine,
  • Nucleotide analogs can also include modifications of the sugar moiety Modifications to the sugar moiety would include natural modifications of the ⁇ bose and deoxy ⁇ bose as well as synthetic modifications Sugar modifications include but are not limited to the following modifications at the 2' position OH-, F-, 0-, S-, or N-alkyl, 0-, S-, or -alkenyl, 0-, S- or N- alkynyl, or O alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C, to C
  • modifications at the 2' position include but are not limited to C, to C )0 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O alkaryl or O aralkyl, SH, SCH 3 , OCN, CI, Br, CN, CF 3 , OCF 3 , SOCII 3 , S0 2 CH 3 , ON0 2 , N0 2 , N 3 , NH, heterocycloalkyl, heterocycloalkaryl, ammoalkylamino, polyalkylammo, substituted silyl, an RNA cleaving group, a reporter group, an mtercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties Similar modifications may also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in
  • Nucleotide analogs can also be modified at the phosphate moiety
  • Modified phosphate moieties include but are not limited to those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphot ⁇ ester, aminoalkylphosphot ⁇ ester, methyl and other alkyl phosphonates including 3 - alkylene phosphonate and chiral phosphonates, phosphmates, phosphoramidates including 3'- ammo phosphoramidate and ammoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates It is understood that these phosphate or modified phosphate linkage between two nucleotides can be through a 3'-5' linkage or a 2'-5' linkage, and the linkage can
  • nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA)
  • PNA peptide nucleic acid
  • Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety
  • Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid
  • Nucleotide substitutes are nucleotides or nucleotide analogs that have had the phosphate moiety and/or sugar moieties replaced Nucleotide substitutes do not contain a standard phosphorus atom
  • Substitutes for the phosphate can be for example, shoit chain alkyl or cycloalkyl lnternucleoside linkages, mixed heteroatom and alkyl or cycloalkyl mternucleoside linkages, or one or more short chain heteroatomic or heterocyclic mternucleoside linkages These include those having mo ⁇ hohno linkages (formed in part from the sugar portion of a nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones ; formacetyl and thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene containing backbones, sulfamate backbone
  • conjugates can be chemically linked to the nucleotide or nucleotide analogs.
  • conjugates include but are not limited to lipid moieties such as a cholesterol moiety (Letsmger et al , Proc Natl Acad Sci USA, 1989,
  • an oligonucleotide can be made from any combination of nucleotides, nucleotide analogs, or nucleotide substitutes disclosed herein or related molecules not specifically recited herein.
  • a Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute.
  • the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl, and C6 positions of a purine based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.
  • a Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA.
  • the Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides
  • compositions including primers and probes, which are capable of interacting with the for example, the alpha ⁇ gene or mRNA, beta4 gene or mRNA, or gamma2 subunit ofthe laminin5 ligand gene as disclosed herein or mRNA as wells as primers or probes for any of the sequences or fragments of the sequences, set forth in SEQ ID NOs: l, 3,5, 7,9,1 1, and 13.
  • the primers are used to support DNA amplification reactions.
  • the primers will be capable of being extended in a sequence specific manner.
  • Extension of a primer in a sequence specific manner includes any methods wherein the sequence and/or composition o the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer.
  • Extension ofthe primer in a sequence specific manner therefore includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence specific manner are preferred.
  • the primers are used for the DNA amplification reactions, such as PCR or direct sequencing.
  • the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner.
  • the disclosed primers hybridize with the beta4 gene, alpha ⁇ gene, or Iaminin5-gamma2 subunit gene, for example, or region ofthe beta4 gene, alpha ⁇ gene, or Iaminin5-gamma2 subunit gene, for example, or they hybridize with the complement of the beta4 gene, alpha6 gene, or Iaminin5-gamma2 subunit gene, for example, or complement of a region of the beta4 gene, alpha ⁇ gene, or Iaminin5-gamma2 subunit gene, or any ofthe sequences or fragments ofthe sequences, set forth in SEQ ID NOs: 1 ,3,5,7,9,1 1, and 13, for example.
  • Iaminin5-gamma2 subunit gene for example, in certain embodiments can be any size that supports the desired enzymatic manipulation of the primer, such as DNA amplification or the simple hybridization of the probe or primer.
  • a typical primer or probe for beta4 gene, alpha ⁇ gene, or Iam ⁇ n ⁇ n5-gamma2 subunit gene, for example, or primer or probe for any of the sequences or fragments of the sequences, set forth in SEQ ID NOs 1 ,3,5,7,9, 1 1 , and 13 would be at least about 5 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 1 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67,
  • a primer or probe for beta4 gene, alpha ⁇ gene, or Iaminin5-gamma2 subunit gene for example, primer or probe or a primer or probe for any of the sequences or fragments ofthe sequences, set forth in SEQ ID NOs: 1 ,3,5,7,9,11, and 13 can be less than or equal to about 6, 7, 8, 9, 10, 1 1, 12 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 15 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
  • the size of the product will be such that the size can be accurately determined to within 3, or 2 or 1 nucleotides
  • this product is at least about 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29,
  • the product is less than or equal to about 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 1 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 35 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700,
  • variants of genes and proteins herein disclosed typically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence
  • the homology can be calculated after aligning the two sequences so that the homology is at its highest level
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv Appl Math 2 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J MoL Biol 48 443 (1970), by the search for similarity method of Pearson and Lipman, Proc Natl Acad Sci U S A 85 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr , Madison, WI), or by inspection
  • nucleic acids can be obtained by for example the algorithms disclosed in Zuker, M Science 244 48-52, 1989, Jaeger et al P/ oc Natl Acad Sci USA 86 7706-7710, 1989, Jaeger et al Methods Enzymol 183 281 -306, 1989 which are herein inco ⁇ orated by reference for at least mate ⁇ al related to nucleic acid alignment It is understood that any of the methods typically can be used and that m certain instances the results of these various methods may differ, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein For example, as used herein, a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above.
  • a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other calculation methods
  • a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any ofthe other calculation methods.
  • a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated homology percentages).
  • integrins have domains that interact with the other integrins. It may be advantageous in certain embodiments to utilize just the integrin binding domain fragment of, for example, the beta4 integrin, in a selection protocol disclosed herein. By using this domain of the beta4 integrin as the selection target, for example, the selection protocol will be biased for molecules binding this domain of beta4 integrin.
  • hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene.
  • Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide specific manner. For example, G interacting with C or A interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face o the nucleotide.
  • the hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature o the reaction all affect whether two nucleic acid molecules will hybridize.
  • selective hybridization conditions can be defined as stringent hybridization conditions
  • stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps
  • the conditions of hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12-25°C below the Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5°C to 20°C below the Tm
  • Tm the melting temperature at which half of the molecules dissociate from their hybridization partners
  • the temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies
  • Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA hybridizations
  • the conditions can be used as described above to achieve stringency
  • selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid is bound to the non-hmitmg nucleic acid
  • the non-limiting primer is in for example, 10 or 100 or 1000 fold excess This type of assay can be performed at under conditions where both the limiting and non-limiting primer are for example, 10 fold or 100 fold or 1000 fold below their kj, or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above
  • selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72 73, 74, 75, 76 77, 78, 79, 80, 81 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer is enzymatically manipulated under conditions which promote the enzymatic manipulation, for example if the enzymatic manipulation is DNA extension, then selective hybridization conditions would be when at least about 60, 65, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90,
  • compositions and methods often entail delivery of the compositions to cells.
  • antisense molecules directed to alpha ⁇ mRNA or gamma2 mRNA can be delivered to cells via any method.
  • a number of exemplary methods are disclosed herein.
  • non-nucleic acid molecules will be and can be delivered to cells, for example an antibody to beta4 integrin, alpha ⁇ integrin or, gamma2, or a small molecule, or a peptide. Delivery of these molecules can occur by any means, and exemplary compositions and methods for such delivery are disclosed herein.
  • compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems.
  • the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes.
  • Transfer vectors can be any nucleotide construction used to deliver genes into cells (e g , a plasmid), or as part of a general strategy to deliver genes, e g , as part of recombinant retrovirus or adenovirus (Ram et al Cancer Res 53 83-88, (1993))
  • plasmid or viral vectors are agents that transport the disclosed nucleic acids, such as gamma2 antisense producing molecules into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered
  • the delivery vectors are derived from either a virus or a retrovirus
  • Viral vectors are, for example, Adenovirus, Adeno-associated virus, He ⁇ es virus, Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus, Smdbis and other RNA viruses, including these viruses with the HIV backbone
  • Retroviruses include Murine Moloney Leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector Retroviral vectors are able to carry a larger genetic payload, l e , a transgene or marker gene, than other viral vectors, and for this reason are a commonly used
  • Viral vectors can have higher transaction (ability to introduce genes) abilities than chemical or physical methods to introduce genes into cells
  • viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome
  • viruses typically have one or more of the early genes removed and a gene or gene/promoter cassette is inserted into the viral genome in place of the removed viral DNA Constructs of this type can carry up to about 8 kb of foreign genetic material
  • the necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans
  • Retroviral Vectors A retrovirus is an animal virus belonging to the virus family of Retroviridae, including any types, subfamilies, genus, or tropisms.
  • Retroviral vectors in general, are described by Verma, I.M., Retroviral vectors for gene transfer. In Microbiology- 1985, American Society for Microbiology, pp. 229-232, Washington, (1985), which is inco ⁇ orated by reference herein. Examples of methods for using retroviral vectors for gene therapy are described in U.S. Patent Nos. 4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136; and Mulligan, (Science 260:926-932 (1993)); the teachings of which are inco ⁇ orated herein by reference.
  • a retrovirus is essentially a package which has packed into it nucleic acid cargo.
  • the nucleic acid cargo carries with it a packaging signal, which ensures that the replicated daughter molecules will be efficiently packaged within the package coat.
  • a packaging signal In addition to the package signal, there are a number of molecules which are needed in cis, for the replication, and packaging ofthe replicated virus.
  • a retroviral genome contains the gag, pol, and env genes which are involved in the making ofthe protein coat. It is the gag, pol, and env genes which are typically replaced by the foreign DNA that it is to be transferred to the target cell.
  • Retrovirus vectors typically contain a packaging signal for inco ⁇ oration into the package coat, a sequence which signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich sequence 5' to the 3' LTR that serve as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the LTRs that enable the insertion of the DNA state ofthe retrovirus to insert into the host genome.
  • a packaging signal for inco ⁇ oration into the package coat a sequence which signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich sequence 5' to the 3' LTR that serve as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequence
  • gag, pol, and env genes allow for about 8 kb of foreign sequence to be inserted into the viral genome, become reverse transcribed, and upon replication be packaged into a new retroviral particle. This amount of nucleic acid is sufficient for the delivery of a one to many genes depending on the size of each transcript. It is preferable to include either positive or negative selectable markers along with other genes in the insert.
  • a packaging cell line is a cell line which has been transfected or transformed with a retrovirus that contains the replication and packaging machinery, but lacks any packaging signal.
  • the vector carrying the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.
  • AAV adeno-associated virus
  • AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19 Vectors which contain this site specific integration property are preferred
  • An especially preferred embodiment of this type of vector is the P4 1 C vector produced by Avigen, San Francisco, CA, which can contain the he ⁇ es simplex virus fhymidine kinase gene, HSV-tk, and/or a marker gene, such as the gene encoding the green fluorescent protein, GFP
  • the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene
  • ITRs inverted terminal repeats
  • the vectors of the present invention thus provide DNA molecules which are capable of integration into a mammalian chromosome without substantial toxicity
  • a promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site
  • a promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements
  • compositions can be delivered to the target cells in a variety of ways
  • the compositions can be delivered through electroporation, or through lipofection, or through calcium phosphate precipitation
  • the delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for example in vivo or in vitro.
  • compositions can comprise, in addition to the disclosed nucleic acids and proteins or vectors for example, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes.
  • liposomes can further comprise proteins to facilitate targeting a particular cell, if desired.
  • Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract.
  • liposomes see, e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol.
  • the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.
  • delivery of the compositions to cells can be via a variety of mechanisms.
  • delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTESF, LIPOFECTAMTNE (G ⁇ BCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art.
  • nucleic acid or vector of this invention can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Co ⁇ ., Arlington, AZ).
  • the materials may be in solution or suspension (for example, inco ⁇ orated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor Hgands.
  • the following references are examples o the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconiugate Chem.. 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer. 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer. 58:700-703, (1988); Senter, et al., Bioconiugate Chem,.
  • Vehicles include "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced These receptors cluster m clath ⁇ n- coated pits, enter the cell via clath ⁇ n-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation Many receptors follow more than one intracellular pathway, depending on the cell type, receptor
  • Nucleic acids that are delivered to cells which are to be integrated into the host cell genome typically contain integration sequences These sequences are often viral related sequences, particularly when viral based systems are used These viral integration systems can also be inco ⁇ orated into nucleic acids which are to be delivered using a non-nucleic acid based system of deliver, such as a liposome, so that the nucleic acid contained m the delivery system can be come integrated into the host genome
  • compositions can be administered in a pharmaceutically acceptable carrier and can be delivered to the subject ⁇ s cells m vivo and/or e ⁇ vivo by a variety of mechanisms well known in the art (e g , uptake of naked DNA, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis and the like)
  • cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art
  • the compositions can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection or proteohposomes
  • the transduced cells can then be infused (e g , in a pharmaceutically acceptable carrier) or homotopically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.
  • constructs which produce an integrin signal transduction inhibitor are driven by inducible promoters, rather than constitutive promoters.
  • inducible systems provide certain advantages, to the expression of the disclosed constructs. Any inducible system can be used. Also disclosed are cells containing the inducible systems, described herein, and in the Examples. These cells, can be used as model systems in a wide variety of assays, as well as in vivo settings.
  • the nucleic acids that are delivered to cells typically contain expression controlling systems.
  • the inserted genes in viral and retroviral systems usually contain promoters, and/or enhancers to help control the expression of the desired gene product.
  • a promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site.
  • a promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.
  • Viral Promoters and Enhancers Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter.
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature. 273: 113 (1978)).
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment (Greenway, P.J. e al, Gene 18: 355-360 (1982)).
  • promoters from the host cell or related species also are useful herein.
  • Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci, 78: 993 (1981)) or 3' (Lusky, M.L, et al., Mol. Cell Bio. 3: 1 108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J.L. et al.. Cell 33 : 729 (1983)) as well as within the coding sequence itself (Osbome, T.F., et al., Mol. Cell Bio. 4: 1293 (1984)).
  • Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene.
  • enhancer sequences are now known from mammalian genes (globm, elastase, albumin, ot fetoprotein and insulin), typically one will use an enhancet from a eukaryotic cell virus for general expression
  • Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers
  • the promoter and/or enhancer may be specifically activated either by light or specific chemical events which trigger their function
  • Systems can be regulated by reagents such as tetracyclme and dexamethasone
  • the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize expression ofthe region of the transcription unit to be transcribed
  • the promoter and/or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time
  • a preferred promoter of this type is the CMV promoter (650 bases)
  • Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTF
  • GFAP ghal fib ⁇ llary acetic protein
  • the viral vectors can include nucleic acid sequence encoding a marker product This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed.
  • Preferred marker genes are the E. Coli lacZ gene, which encodes ⁇ - galactosidase, and green fluorescent protein.
  • the marker may be a selectable marker.
  • suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin.
  • DHFR dihydrofolate reductase
  • thymidine kinase thymidine kinase
  • neomycin neomycin analog G418, hydromycin
  • puromycin puromycin.
  • selectable markers When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure.
  • These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media.
  • An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.
  • the second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg. P.. J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al.. Mol. Cell. Biol. 5: 410-413 (1985)).
  • the three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puromycin. h) Peptides
  • beta4 integrin protein As discussed herein there are numerous variants of the beta4 integrin protein, alpha ⁇ integrin protein, and gamma2 laminin5 protein, for example, that are known and herein contemplated.
  • known functional homologue variants there are derivatives of the beta4, alpha ⁇ , and gamma2, and other disclosed proteins which also function in the disclosed methods and compositions. Protein variants and derivatives are well understood to those of skill in the art and in can involve amino acid sequence modifications.
  • amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants
  • Insertions include amino and/or carboxyl terminal fusions as well as mtrasequence insertions of single or multiple ammo acid residues Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues
  • Immunogenic fusion protein derivatives are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linkmg m vitro or by recombinant cell culture transformed with DNA encoding the fusion
  • Deletions are characterized by the removal of one or more amino acid residues from the protein sequence Typically, no more than about from 2 to 6 residues are deleted at any one site withm the protein molecule
  • These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA
  • substitutions that are less conservative than those in Table 2, I e , selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain
  • substitutions which m general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophihc residue, e g seryl or threonyl, is substituted for (or by) a hydrophobic residue, e g leucyl, isoleucyl, phenylalanyl, valyl or alanyl, (b) a cysteine or prohne is substituted for (or by) any other residue, (c) a residue having an electropositive side chain, e g , lysyl, argmyl, or his
  • substitutions include combinations such as, for example Gly, Ala Val, He, Leu, Asp, Glu, Asn, Gin Ser, Thr, Lys, Arg, and Phe, Tyr
  • substitutions include combinations such as, for example Gly, Ala Val, He, Leu, Asp, Glu, Asn, Gin Ser, Thr, Lys, Arg, and Phe, Tyr
  • Substitutional or deletional mutagenesis can be employed to insert sites for N- glycosylation (Asn-X Thr/Ser) or O-glycosylation (Ser or Thr)
  • Deletions of cysteine or other labile residues also may be desirable Deletions or substitutions of potential proteolysis sites, e g Arg, is accomplished for example by deleting one o the basic residues or substituting one by glutaminyl or histidyl residues
  • Certain post translational denvatizations are the result of the action of recombinant host cells on the expressed polypeptide Glutaminyl and asparaginyl residues are frequently post- translationally deamidated to the corresponding glutamyl and asparyl residues Alternatively, these residues are deamidated under mildly acidic conditions
  • Other post-translational modifications include hydroxylation of prohne and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation ofthe o-ammo groups of lysine, arginine, and histidine side chains (T E Creighton, Proteins Structure and Molecular Properties, W H Freeman & Co , San Francisco pp 79-86 [1983]), acetylation of the N-termmal amine and, in some instances, amidation of the C-terminal carboxyl
  • variants and derivatives of the disclosed proteins herein are through defining the variants and derivatives in terms of homology/identity to specific known sequences
  • SEQ ID NO SEQ ID NO 5 sets forth a particular sequence of beta4 mtegrm cDNA
  • SEQ ID NO 6 sets forth a particular sequence of a beta4 integrm protein
  • variants of these and other proteins herein disclosed which have at least, 70% or 75% or 80% or 85% or 90% or 95%> homology to the stated sequence
  • the homology can be calculated after aligning the two sequences so that the homology is at its highest level
  • Another way of calculating homology can be performed by published algorithms
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv Appl Math 2 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J MoL Biol 48 443 (1970), by the search for
  • nucleic acids can be obtained by for example the algorithms disclosed in Zuker, M Science 244 48 52 1989, Jaeger et al Pi oc Natl Acad Sci USA 86 7706-7710, 1989, Jaeger et al Methods Enzymol 183 281-306, 1989 which are herein inco ⁇ orated by reference for at least material related to nucleic acid alignment.
  • nucleic acid sequences that can encode the protein sequence set forth in SEQ ID NO:6 is set forth in SEQ ID NO:5.
  • Another nucleic acid sequence that encodes the same protein sequence set forth in SEQ ID NO:6 is set forth in SEQ ID NO: 16.
  • a disclosed conservative derivative of SEQ ID NO:2 is shown in SEQ ID NO: 17, where the valine (V) at position 34 is changed to a isoleucine (I).
  • nucleic acid sequences that encode this particular derivative of the beta4 integrin are also disclosed including for example SEQ ID NO: 18 and SEQ ID NO: 19 which set forth two of the degenerate nucleic acid sequences that encode the particular polypeptide set forth in SEQ ID NO: 17. It is also understood that while no amino acid sequence indicates what particular DNA sequence encodes that protein within an organism, where particular variants of a disclosed protein are disclosed herein, the known nucleic acid sequence that encodes that protein in the particular organism from which that protein arises is also known and herein disclosed and described. i) Pharmaceutical carriers/Delivery of pharmaceutical products
  • compositions for example, compositions that inhibit alpha ⁇ function, beta4 function, or gamma2 function can also be administered in vivo in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extraco ⁇ oreally, topically or the like, although topical mtranasal administration or administration by inhalant is typically preferred
  • topical mtranasal administration means delivery ofthe compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosohzation of the nucleic acid or vector The latter may be effective when a large number of animals is to be treated simultaneously
  • Administration ofthe compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism Delivery can also be directly to any area of the respiratory system (e g , lungs) via intubation
  • the exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition o the subject, the seventy of the allergic disorder being treated, the particular nu
  • the materials may be in solution, suspension (for example, inco ⁇ orated into microparticles, liposomes, or cells) These may be targeted to a particular cell type via antibodies, receptors, or receptor hgands
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al , Bioconiugate Chem , 2 447-451 , (1991), Bagshawe, K D , Br J Cancer. 60 275-281, (1989), Bagshawe, et al , Br J Cancer. 58 700-703, (1988), Senter, et al , Bioconiugate Chem . 4 3-9, (1993), Battelh, et al , Cancer Immunol Immunother .
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to Golonic carcinoma), receptor mediated targeting of DNA through cell specific hgands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine ghoma cells in vivo
  • stealth and other antibody conjugated liposomes (including lipid mediated drug targeting to Golonic carcinoma), receptor mediated targeting of DNA through cell specific hgands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine ghoma cells in vivo
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced These receptors cluster in clath ⁇ n-coated pits, enter the cell via clathrm-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).
  • compositions including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
  • compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.
  • the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • the disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid, glyco
  • the dosage ranges for the administration ofthe compositions are those large enough to produce the desired effect in which the symptoms disorder are effected.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent ofthe disease in the patient and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counterindications, Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
  • Chips and micro arrays Disclosed are chips where at least one address is the sequences or part of the sequences set forth in any of the nucleic acid sequences disclosed herein. Also disclosed are chips where at least one address is the sequences or portion of sequences set forth in any of the peptide sequences disclosed herein.
  • chips where at least one address is a variant of the sequences or part of the sequences set forth in any of the nucleic acid sequences disclosed herein. Also disclosed are chips where at least one address is a variant of the sequences or portion of sequences set forth in any of the peptide sequences disclosed herein k) Computer readable mediums It is understood that the disclosed nucleic acids and proteins can be represented as a sequence consisting of the nucleotides of amino acids There are a variety of ways to display these sequences, for example the nucleotide guanosme can be represented by G or g Likewise the ammo acid valme can be represented by Val or V Those of skill in the art understand how to display and express any nucleic acid or protein sequence in any of the variety of ways that exist, each of which is considered herein disclosed Specifically contemplated herein is the display of these sequences on computer readable mediums, such as, commercially available floppy disks, tapes, chips, hard drives, compact disks, and video disks, or
  • kits that are drawn to reagents that can be used m practicing the methods disclosed herein
  • the kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice ofthe disclosed methods
  • the kits could include primers to perform the amplification reactions discussed in certain embodiments of the methods, as well as the buffers and enzymes required to use the primers as intended
  • a kit for assessing a subject's risk for acquiring cancer, such as colon cancer comprising the primers or probes that hybridize to the sequences set forth in SEQ ID NOs 1 , 3, 5, 7, 9, 1 1, and 13, for example m) Compositions with similar functions
  • the compositions disclosed herein have certain functions, such as inhibiting gamma2 function or binding alpha ⁇ mtegrm or inhibiting beta4 function
  • Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures which can perform the same function which
  • compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted.
  • the nucleic acids such as, the oligonucleotides to be used as primers can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al, Molecular Cloning: A Laboratoiy
  • Protein nucleic acid molecules can be made using known methods such as those described by Nielsen et al, Bioconjug. Chem. 5:3-7 (1994).
  • Peptide synthesis One method of producing the disclosed proteins, or fragments of the disclosed proteins, such as a fragment of SEQ ID NO:6, is to link two or more peptides or polypeptides together by protein chemistry techniques.
  • peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9- fluorenylmefhyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry. (Applied Biosystems, 5 Inc., Foster City, CA).
  • Fmoc 9- fluorenylmefhyloxycarbonyl
  • Boc tert -butyloxycarbonoyl
  • a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a peptide or protein can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally 0 blocked on the other fragment.
  • peptide condensation reactions these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof.
  • peptide or polypeptide is independently synthesized / ' ;; vivo as described herein. Once isolated, these independent peptides or polypeptides may be linked to form a peptide or fragment thereof via similar peptide condensation reactions
  • enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen L et al , Biochemistry, 30 4151 (1991))
  • native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments This method consists of a two step chemical reaction (Dawson et al Synthesis of Proteins by Native Chemical Ligation Science, 266 776-779 (1994))
  • the first step is the chemoselective reaction of an unprotected synthetic peptide— thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester linked intermediate as the initial covalent product Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site
  • compositions such as that for SEQ ID NO 6, beta4 mtegrin, can be made using any traditional recombinant biotechnology method Examples of such methods can be found in Sambrook et al which is herein inco ⁇ orated by reference at least for material related to production of proteins and antibodies c) Processes for making the compositions
  • compositions as well as making the intermediates leading to the compositions
  • nucleic acids in SEQ ID NOs 1, 3, 5, 7, 9, 1 1 , and 13
  • va ⁇ ety of methods that can be used for making these compositions, such as synthetic chemical methods and standard molecular biology methods It is understood that the methods of making these and the other disclosed compositions are specifically disclosed
  • nucleic acid molecules produced by the process comprising linking in an operative way a nucleic add molecule comprising the sequence set forth in SEQ ID Nos 1 , 3, 5, 7, 9, 1 1 , or 13 or a fragment thereof, and a sequence controlling the expression of the nucleiG acid
  • nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence having at least 80% identity to a sequence set forth in SEQ ID Nos: l, 3, 5, 7, 9, 1 1 , or 13 or a fragment thereof, and a sequence controlling the expression of the nucleic acid.
  • nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence that hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the sequence set forth in SEQ ID Nos:l , 3, 5, 7, 9, 1 1, or 13 or a fragment thereof, and a sequence controlling the expression of the nucleic acid.
  • nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence encoding a peptide set forth in SEQ ID Nos:2, 4, 6, 8, 10, 12, or 14 or a fragment thereof, and a sequence controlling an expression of the nucleic acid molecule.
  • nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence encoding a peptide having 80% identity to a peptide set forth in SEQ ID Nos:2, 4, 6, 8, 10, 12, or 14 or a fragment thereof and a sequence controlling an expression ofthe nucleic acid molecule.
  • nucleic acids produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence encoding a peptide having 80% identity to a peptide set forth in SEQ ID Nos:2, 4, 6, 8, 10, 12, or 14 or a fragment thereof, wherein any change from the sequences set forth in SEQ ID Nos:2, 4, 6, 8, 10, 12, or 14 or a fragment thereof are conservative changes and a sequence controlling an expression of the nucleic acid molecule.
  • animals produced by the process of transfecting a cell within the animal any of the nucleic acid molecules disclosed herein wherein the animal is a mammal. Also disclosed are animals produced by the process of transfecting a cell within the animal any of the nucleic acid molecules disclosed herein, wherein the mammal is mouse, rat, rabbit, cow, sheep, pig, or primate.
  • animals produced by the process of adding to the animal any ofthe cells disclosed herein.
  • Products produced from selection protocols Also disclosed are methods of obtaining molecules that act as functional regulators of integrin function, integrin receptor function, and functional regulators of signaling pathways related to integrin receptors, in particular integrin alpha ⁇ beta4.
  • Disclosed are methods for isolating molecules that interact with the proteins set forth in SEQ ID Nos:2, 4, 6, 8, 10, 12, or 14 or a fragment thereof comprising, interacting a library of molecules with the proteins set forth in SEQ ID Nos:2, 4, 6, 8, 10, 12, or 14 or a fragment thereof, removing the unbound molecules, and collecting the molecules that are bound to at least one of the proteins set forth in SEQ ID Nos:2, 4, 6, 8, 10, 12, or 14 or a fragment thereof.
  • compositions can be used for example as targets in combinatorial chemistry protocols or other screening protocols to isolate molecules that possess desired functional properties related to A6B4 and A6B 1 signaling pathways.
  • the disclosed compositions can also be used diagnostic tools related to diseases such as cancer, such as colon cancer,
  • the disclosed compositions can be used as discussed herein as either reagents in micro arrays or as reagents to probe or analyze existing microarrays.
  • the disclosed compositions can be used in any known method for isolating or identifying single nucleotide polymo ⁇ hisms.
  • the compositions can also be used in any method for determining allelic analysis of for example, beta4 alleles having varying function, particularly allelic analysis as it relates to beta4 signaling and functions.
  • the compositions can also be used in any known method of screening assays, related to chip/micro arrays.
  • compositions can also be used in any known way of using the computer readable embodiments ofthe disclosed compositions, for example, to study relatedness or to perform molecular modeling analysis related to the disclosed compositions.
  • Methods for affecting cancer The disclosed compositions can be used to affect the growth of cancer cells because as disclosed herein, the disclosed relationships are fundamental to the ability of cancer cells to continue growing.
  • the disclosed compositions such as antisense constructs that will inhibit the production of either alpha ⁇ , beta4, or the gamma2 chain of laminin5 reduce the proliferation of cancer cells. In fact, the compositions cannot only reduce the proliferation of the cancer cells, but the compositions can kill the cancer cells, as shown herein.
  • cancer is caused by a variety of cellular events, of which certain events related to alpha ⁇ integrin (up regulated), beta4 integrin (upregulated) and gamma2 (upregulated) allow the continued viability of cancer cells, and that interference of these upregulated molecules inhibits the growth and kills the cancer cells.
  • alpha ⁇ integrin up regulated
  • beta4 integrin upregulated
  • gamma2 upregulated
  • the disclosed anti-cancer compositions can be used in combination with other anti-cancer compositions.
  • Great benefits can be obtained from using anti-cancer compositions that target different molecules, in the same signal transduction pathway as well as, or in addition to, targeting molecules in different signal transduction pathways than those disclosed herein.
  • the disclosed compositions which can affect the growth of cancer cells indeed kill cancer cells, can be used in conjunction with any other chemotherapy, radiation, or any other anticancer therapy.
  • Also disclosed are methods of selectively reducing the proliferation of cancer cells which comprises reducing integrins from interacting with one another, integrins from clustering, or integrins from interacting with other proteins associated with cancer cells.
  • Disclosed are methods of reducing the proliferation of a cancer cell which comprises reducing the production of an integrin by the cancer cell.
  • Also disclosed are methods of reducing the proliferation of a cancer cell which comprises reducing the production of an integrin receptor ligand by the cancer cell.
  • methods of selectively killing or reducing the proliferation of cancer cells which comprises inhibiting ligand binding to mtegrin receptors on the cancer cells, wherein the ligand comprises a lam ⁇ n ⁇ n5
  • Also disclosed are methods of selectively killing or reducing the proliferation of cancer cells which comprises inhibiting ligand binding to mtegrm receptors on the cancer cells, wherein the ligand comprises the gamma2 subunit of the lammin L5
  • Disclosed are methods of selectively killing or reducing the proliferation of cancer cells which comprises inhibiting ligand binding to integrm receptors on the cancer cells, wherein the integrin receptor comprises an alpha ⁇ mtegrin
  • methods of selectively killing or reducing the proliferation of cancer cells which comprises preventing mtegrin receptor subumts from interacting with one another, preventing integrin clustering, or preventing mtegrin receptor subumts from interacting with other proteins on cancer cells, wherein the mtegrm receptor comprises a B4 mtegrin
  • Disclosed are methods of selectively killing or reducing the proliferation of cancer cells which comprises reducing the production of lammin by the cancer cells, wherein the laminin comp ⁇ ses lamm ⁇ n5, and/or any of the subumts of lam ⁇ nm5, such as gamma2
  • a method of selectively killing or reducing the proliferation of cancer cells which comprises interfering with an integrm signaling pathway, wherein the integrin signaling pathway comprises a B4 integrm or an alpha ⁇ integrin or a betal mtegrin or a lammin or a Iamm ⁇ n5 or the gamma2 subunit of lam ⁇ n ⁇ n5
  • the integrin receptor comprises mtegrin B4 and/or wherein the mtegrm receptor comp ⁇ ses integrin A6, and/or wherein the ligand that binds to the mtegrin receptor is lam ⁇ mn5, and/or wherein the integrm receptor is A6B4, and/or wherein the ligand comprises lammm5, and/or wherein the ligand comprises the gamma-2 subunit of Iam ⁇ nm5, and/or wherein inhibiting ligand binding to an integrin receptor does not occur by using an antisense molecule to
  • the mtegrm comp ⁇ ses an A6 mtegrm, and/or wherein the mtegrm comprises a B4 integrm, and/or further comprising reducing a lammrn5 -integrm interaction, and/or further comprising reducing a lam ⁇ mn5 gamma2 integrm interaction, and/or wherein the cancer cell comprises normal p53, and/or wherein the proliferation ofthe cancer cells is not dependent on AKT/PKB, and/or wherein reducing the proliferation of the cancer cells is selective, and/or wherein the cancer cell is not an MDA-MB-435 cell, and/or wherein the cancer cell is not an HMT-3522 cell, and/or wherein the cancer cell is not an RKO colon carcinoma line, and/or wherein the cancer cell does not express exogenous B4 mtegrin, and/or further comprising contacting the cancer cells with a small molecule, peptide, peptide mimetic, or
  • Also disclosed are methods of reducing the proliferation of a cancer cell in a patient which comprises administering to the patient a composition which reduces integrin- integrm interaction, integrm receptor clustering interaction, or integrin-non-integrin protein interaction
  • Disclosed are methods of reducing the proliferation of a cancer cell in a patient which comprises administering to a patient a vector comprising coding sequence for a protein or peptide which inhibits ligand binding to integrin receptors on cancer cells wherein the coding sequence is under the control of a promoter which functions in mammalian cells
  • Also disclosed are methods of reducing the proliferation of a cancer cell in a patient which comprises administering to a patient a vector comprising coding sequence for a protein or peptide which prevents mtegrin receptor subu ts from interacting with one another, prevents integrm receptor clustering interaction, or prevents integrin receptor subumts from interacting with other proteins on cancer cells wherein the coding sequence is under the control of a promoter which functions in mammalian cells
  • methods of reducing the proliferation of a cancer cell in a patient which comprises administering to a patient a vector comprising coding sequence for a protein or peptide which prevents integrin receptor subumts from interacting with one another, prevents mtegrin receptor clustering interaction, or prevents integrin receptor subumts from interacting with other proteins in cancer cells wherein the coding sequence is under the control of a promoter which functions in mammalian cells
  • Also disclosed are method of reducing the proliferation of a cancer cell in a patient which comprises administering to a patient a vector comprising coding sequence for a protein or peptide which interferes with integrin subunit or lammin production wherein the coding sequence is under the control of a promoter which functions m mammalian cells
  • methods of reducing the proliferation of a cancer cell in a patient which comprises administering to a patient a vector comprising coding sequence for a protein or peptide which interferes with an integrin signaling pathway of cells wherein the coding sequence is under the control of a promoter which functions m mammalian cells
  • One of the preferred characteristics of performing homologous recombination m mammalian cells is that the cells should be able to be cultured, because the desired recombination event occur at a low frequency
  • an animal can be produced from this cell through either stem cell technology or cloning technology
  • stem cell technology For example, if the cell into which the nucleic acid was transfected was a stem cell for the organism, then this cell, after transfection and culturmg, can be used to produce an organism which will contain the gene modification or disruption in germ line cells, which can then in turn be used to produce another animal that possesses the gene modification or disruption m all of its cells
  • cloning technologies can be used These technologies generally take the nucleus of the transfected cell and either through fusion or replacement fuse the transfected nucleus with an oocyte which can then be manipulated to produce an animal
  • the advantage of procedures that use cloning instead of ES technology is that cells other than ES cells can be transfected
  • a fibroblast cell which is very easy to culture can be used as the cell which is transfected and has a gene modification or disruption event take place
  • Disclosed are methods of assessing a subject's risk of acquiring cancer comprising determining the amount of B4 present in a target cell obtained from the subject, wherein a determination of increased levels of B4 correlates with an increased risk of cancer
  • Disclosed are methods of assessing a subject's risk of acquiring cancer comprising determining the amount of lamm ⁇ n5 present m a target cell obtained from the subject, wherein a determination of increased levels of lam ⁇ nm5 correlates with an increased risk of cancer
  • Disclosed are methods of assessing a subject's risk of acquiring cancer comprising determining the amount of gamma2 subunit present in a target cell obtained from the subject, wherein a determination of increased levels of gamma2 subunit correlates with an increased risk of cancer
  • Example 1 Colonic epithelial cells dependent on alpha6beta4 receptor signal transduction for growth in the absence of ECM Integrin alpha ⁇ and integrm beta4 and laminm gamma2 chains are essential for tumor cell survival in vivo
  • the data disclosed herein indicate that interfering with tegrm alpha ⁇ -mediated signaling, integrm beta 4s ⁇ gnahng, and laminin gamma2 signaling can constitute an effective approach to induce programmed cell death in cancer cells without damaging normal cells Ablation of tegrin alpha ⁇ -dependent signaling, integrin beta4-dependent signaling, and lammin gamma2- dependent signaling in human cancer cell lines supports this
  • control cells control
  • cells containing an activated ras oncogene Ras
  • APCmin a deletion in the APC gene
  • both alterations together Ras+APCmin
  • the cells were derived from transgenic mice containing a temperature-sensitive allele of SV40 large T under the control of a gamma interferon-inducible promoter permitting conditional immortalization (Jat et al , 1991) All experiments were carried out with the cells being kept at non-permissive temperature in the absence of gamma interferon As shown previously (D'Abaco et al , 1996), only the cells carrying activated Ras and the APCmin mutation were able to form colonies in soft agar in the absence of anchorage to a substratum All other cell populations did not give rise to colonies under these conditions
  • Alpha ⁇ mtegrm can form functional receptors together with either betal or beta4 integrins (Clark and Brugge, 1995), leading to signaling through downstream effectors such as focal adhesion kinase (FAK) or the SH2 domain adapter She (Giancotti and Ruoslahti, 1999)
  • beta4 integrin only binds to alpha ⁇ tegrin (Clark and Brugge, 1995)
  • alpha ⁇ integrin functions in conjunction with mtegrm beta4 and is engaged by the laminm gamma2 chain to activate She (see Fig 4)
  • Ras+APCmm cells can bind a laminm gamma2 specific peptide in an alpha6 and beta4 lnteg ⁇ n-specific manner
  • RNAs specific for alpha ⁇ integrm and gamma2 lammin, as well as dominant- negative beta4 mtegrin, were expressed and results indicated that mtegrin alpha ⁇ , mtegrm beta4, and lammm gamma2 expression are relevant for the survival of Ras+ APCmin cells m
  • Ras+APCmm, Ras, APC and control cells All three constructs efficiently inhibit the growth of Ras+APCmin cells in soft agar (not shown) and when attached to plastic (Fig 5A1) In contrast, the proliferation of wt, Ras and APCmin cells is not affected by this treatment (Fig 5A1) Importantly, the colony formation of Ras+APC cells exposed to any of the three inhibitory constructs can be rescued by co-expression of baculovirus p35, a potent inhibitor of caspase activity and of apoptosis (Res coff e.
  • Ras- APCmin cells which lack alpha6/beta4 mtegrin receptor signaling activity, show high levels of caspase 8 activity when kept in suspension, while at the same time caspase 9 activity cannot be detected (Fig 2D)
  • caspase inhibitor baculovirus p35 Fig. 5 Al , A2 and text above
  • cell death induced by expression of dominant- negative beta4 integrin cannot be prevented by expression ofthe survival factor Bcl2 (see Fig. 5 A2).
  • Bcl2 binds to and neutralizes BH3-domain killer proteins that cause mitochondrial damage, cytochrome C release and caspase 9 activation (Luo et al, 1998).
  • AKT has been described to promote cell survival via phosphorylation and inactivation of Bad, a BH3-domain killer protein (Datta e/ -./., 1999).
  • Example 2 Other cancer cells dependent on alpha ⁇ beta4 receptor signal transduction for growth in the absence of ECM
  • the human colon cancer cell line SW480 carrying multiple oncogenic mutations such as activated Ras (Fujita et al., 1988), amplified c-Myc (Cherif et al, 1988), a mutated APC allele (Munemitsu et al, 1995) and a p53 mutation (Abarzua et al, 1995) is effectively killed by anti-sense integrin alpha ⁇ and laminin gamma2 RNAs as well as dominant-negative beta4 integrin (Fig. 4D). Similarly, integrin alpha ⁇ ablation even leads to apoptosis of Ras/APCmin cells in the presence of active SV40 large T (not shown).
  • Oncogenic mutations co-operate to engage autocrine integrin signaling.
  • this signaling mechanism involving alpha ⁇ integrin, beta4 integrin and the laminin gamma2 chain, becomes an essential component of the survival mechanism in transformed colonic epithelial cells.
  • fibroblasts and human colon cancer cells also rely on integrin alpha ⁇ for survival.
  • normal and partially transformed cells that express alpha ⁇ integrin at low levels do not require this polypeptide for survival.
  • integrin signaling inhibition can lead to selective killing of cancer cells.
  • the laminin-integrin receptor signaling loop is also relevant to the survival of cells transformed by other combinations of oncogenic lesions.
  • DNp53/neo cells were able to grow in the absence of ECM contact in soft agar (not shown). Moreover, Ras and DNp53 cooperate in the suppression of caspase 3 activity in suspension (Fig. 2B), and in the induction of alpha ⁇ integrin expression (Fig. 4A). In Ras/DNp53 cells passaged through a single round of soft agar growth, alpha ⁇ integrin is expressed at even higher levels, demonstrating a correlation between the ability of the cells to survive in the absence of ECM contact and alpha ⁇ integrin expression. In Ras/DNp53 cells beta4 integrin expression levels are also increased, when compared to controls (Fig. 4A). As expected expression of the laminin gamma2 chain is induced in Ras/bleo and Ras/DNp53 cells (Fig. 4B).
  • Anti-sense RNAs specific for alpha ⁇ integrin and gamma2 laminin, as well as dominant- negative beta4 integrin were expressed in Ras/DNp53, Ras/bleo, DNp53/neo and neo bleo cells. All three constructs efficiently inhibit the growth of Ras/DNp53 cells when attached to plastic. In contrast, the proliferation of neo/bleo, Ras/bleo and DNp53/neo cells is not affected by this treatment (Fig.5A3). Anti-sense alpha ⁇ integrin or dominant-negative beta4 integrin also inhibit the growth of Ras/DNp53 cells in soft agar (Fig. 5A4).
  • Integrin alpha ⁇ expression can also be induced by activated Ras and DNp53 in primary murine colon crypt epithelial cells (not shown). Furthermore introduction of Ras and Myc oncoproteins into our control colon epithelial cells also leads to an induction of alpha ⁇ expression (not shown), suggesting that induction of alpha ⁇ integrin expression may be an integral component of distinct oncogene cooperation paradigms a) Cell types to be tested
  • dox dox- ⁇ nduc ⁇ ble reverse tetracyclme transactivator
  • rtTA doxycychne- ⁇ nduc ⁇ ble reverse tetracyclme transactivator
  • tTS tetracyclme controlled transcriptional silencer
  • I e tTS effectively eliminates leaky baseline expression without altering the lnducibility of rtTA-regulated genes
  • optimal "off/on" regulation of gene expression can be accomplished with the combined use of tTS and rtTA
  • CLONTECH This system can be used for the preparation of all inducible cell lines
  • inducible cell lines are first stably transfected with the anti-sense or dominant negative constructs coupled to the regulatable promoters into the four test cell lines Subsequently, the activator is introduced and the repressor, such as rtTA and tTS (see above) via infection using recombinant retroviruses with different selectable markers
  • Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway E nbo J, 16, 2783-93
  • Integrin binding specificity of laminin- 10/1 1 lam ⁇ n ⁇ n-10/1 1 are recognized by alpha 3 beta 1 , alpha 6 beta
  • Niessen, C M Hogervorst, F , Jaspars, L II , de Melker, A A , Delwel, G O , Hulsman, E H ,
  • alpha 6 beta 4 integrin is a receptor for both laminin and kalinin Exp Cell Res, 211, 360-7 Pa ⁇ se, L V , Lee, J and Juhano, R L (2000) New aspects of mtegrin signaling in cancer Semin
  • SEQ ID NO:5 Human integrin beta4 subunit cds (acc# 6453379): abggcagggccacgccccagcccabgggccaggcbgcbccbggcagccbbgabcagcgbcagccbcbgggaccbbgaac cgcbgcaagaaggccccagbgaagagcbgcacggagbgbgbccgbgbggabaaggacbgcgccbacbgcacagacgagabg btcagggaccggcgcbgcaacacccaggcggagcbgcbggccgcgggcbgccagcgggagagcabcgbggbcabggagagc
  • SEQ ID NO:6 Human beta4 integrin protein sequence (acc# CAB61345):
  • PVEGELLFHP GETWKE ⁇ QVK Ii E QEVDSL bRGRQVRRFQVQLSNPKFGA RLGQPSTTTV ILDETDRSItl
  • SEQ ID NO:17 Human beta4 integrin protein sequence Variant V at 34 to Ii

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