WO2004007530A2 - Proteines modifiees stabilisees dans une conformation voulue et procedes de production de celles-ci - Google Patents

Proteines modifiees stabilisees dans une conformation voulue et procedes de production de celles-ci Download PDF

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WO2004007530A2
WO2004007530A2 PCT/US2003/022301 US0322301W WO2004007530A2 WO 2004007530 A2 WO2004007530 A2 WO 2004007530A2 US 0322301 W US0322301 W US 0322301W WO 2004007530 A2 WO2004007530 A2 WO 2004007530A2
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protein
integrin
subunit
domain
ofclaim
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PCT/US2003/022301
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WO2004007530A3 (fr
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Timothy A. Springer
Junichi Takagi
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The Center For Blood Research, Inc.
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Publication of WO2004007530A3 publication Critical patent/WO2004007530A3/fr

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    • 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

Definitions

  • the integrin family of adhesion molecules are noncovalently-associated ⁇ / ⁇ heterodimers. To date, at least fourteen different integrin ⁇ subunits and eight different ⁇ subunits have been reported (Hynes, RO (1992) Cell 69:1-25). Lymphocyte function-associated antigen- 1 (LFA-1) is a member of the leukocyte integrin subfamily. Members of the leukocyte integrin subfamily share the common ⁇ 2 subunit (CD18) but have distinct ⁇ subunits, ⁇ L (CDl la), ⁇ M (CDl lb), ⁇ X
  • 83 is largely prominent amongst the adhesion receptors and is essential for platelet aggregation.
  • the ligands for cd ⁇ bj83 are the multivalent adhesive proteins fibrinogen and von Willebrand factor.
  • cdIb/33 In resting platelets, cdIb/33 is normally in a low activation state, unable to interact with soluble fibrinogen. Stimulation of platelets with various agonists will induce a conformational change in cd ⁇ b/33 (inside-out signaling), which is then able to bind soluble fibrinogen resulting in the onset of platelet aggregation.
  • Vascular integrin oN ⁇ 3 interacts with RGD (Arg-Gly-Asp) sequence- containing proteins in the extracellular matrix.
  • RGD Arg-Gly-Asp sequence- containing proteins in the extracellular matrix.
  • the distribution of ⁇ V/33 is highly restricted, with expression on activated endothelium, activated vascular smooth muscle, tumors, and osteoclasts.
  • Expression of cN ⁇ 3 may contribute to a malignant phenotype by supporting the growth and persistence of small blood vessels that nourish the primary and metastatic tumors and increasing invasive potential.
  • Inhibition of ⁇ Vj(33 can modulate tumor-induced angiogenesis and can increase apoptosis of tumor-associated small blood vessels. It might also help control humoral hypercalcemia of malignancy through direct or indirect activity on the osteoclast.
  • the N-terminal region of the integrin ⁇ subunits contains seven repeats of about 60 amino acids each, and has been predicted to fold into a 7-bladed ⁇ -propeller domain (Springer, TA (1997) Proc Natl Acad Sci USA 94:65-72).
  • Some integrin ⁇ subunits e.g., the ⁇ l, ⁇ 2, ⁇ lO, al l, and ⁇ E subunits, contain an inserted domain or I- domain of about 200 amino acids (Larson, RS et al (1989) J Cell Biol 108:703-712; Takada, Y et al. (1989) EMBO J :1361-1368; Briesewitz, R et al.
  • the I domain is inserted into the ⁇ - subunit ⁇ -propeller domain.
  • Figure 13 schematically depicts the orientation of the ⁇ propeller and I domains of the integrin subunit. Other important structural and/or functional domains of the a subunit are also depicted.
  • the three dimensional structure of the ⁇ M, ⁇ L, ⁇ l and ⁇ 2 I-domains has been solved and shows that it adopts the dinucleotide-binding fold with a unique divalent cation coordination site designated the metal ion-dependent adhesion site (MIDAS) (Lee, J-O, et al (1995) Structure 3:1333-1340; Lee, J-O, et al.
  • MIDAS metal ion-dependent adhesion site
  • the integrin ⁇ subunits contain a conserved domain of about 250 amino acids in the N-terminal portion, and a cysteine-rich region in the C-terminal portion.
  • the ⁇ conserved domain, or I-like domain has been predicted to have an "I-domain-like" fold (Puzon-McLaughlin, W and Takada, Y (1996) JBiol Chem 271:20438-20443; Tuckwell, DS and Humphries, MJ (1997) FEBSLett 400: 297-303; Huang, C et al (2000) JBiol Chem 275:21514-24).
  • the ⁇ -subunit I-like domain is inserted, i.e., connected at both its N- and C-termini, to the ⁇ subunit hybrid (Hy) domain.
  • Hy ⁇ subunit hybrid
  • Figure 13 schematically depicts the I-like and Hy domains of the integrin ⁇ subunit as well as other important structural and/or functional domains.
  • the C-terminal Cys-rich region of the ⁇ subunit appears to be important in the regulation of integrin function, as a number of activating antibodies to the ⁇ l, ⁇ 2 and ⁇ 3 subunits bind to this region (Petruzzelli, L et al. (1995) J Immunol 155:854-866; Robinson, MK et al. (1992) J Immunol 148:1080-1085; Faull, RJ et al. (1996) JBiol Chem 271:25099-25106; Shih, DT et al. (1993) J Cell Biol 122:1361-1371; Du, X et al. (1993) JBiol Chem 268:23087-23092).
  • Electron microscopic images of integrms reveal that the N-terminal portions of the ⁇ and ⁇ subunits fold into a globular head that is connected to the membrane by two rod-like tails about 16 nm long corresponding to the C-terminal portions of the ⁇ and ⁇ extracellular domains (Nermut, MV et al. (1988), EMBO Jl. -4093-4099; Weisel, JW et al. (1992) JBiol Chem 267:16637-16643; Wippler, J et al. (1994) J Biol Chem 269: 8754-8761) (see, e.g., Figure 15).
  • ⁇ and ⁇ subunits both contain a headpiece domain and a tailpiece domain.
  • the ⁇ subunit headpiece domain comprises the ⁇ -propeller domain and the "thigh" domain.
  • the ⁇ tailpiece domain comprises the "calf-1" and "calf-2" domains.
  • the ⁇ headpiece domain comprises the I-like domain, the Hy domain, the plexin/semiphorix integrin (PSI) domain, and the I- EGF1 domain.
  • the ⁇ tailpiece domain comprises the I-EGF2, 1-EGF3, 1-EGF4, and ⁇ -tail domains. Jjitegrins use bi-directional signaling to integrate the intracellular and extracellular environments, hi outside-in signaling, ligand binding activates intracellular signaling pathways (Giancotti and Ruoslahti, 1999; Schwartz and Ginsberg, 2002).
  • signals received by other receptors activate intracellular signaling pathways that impinge on integrin cytoplasmic domains, and make the extracellular domain competent for ligand binding on a time-scale of less than 1 s (Hughes and Pfaff, 1998; Shimaoka et al, 2002). These signaling pathways expose activation-dependent or ligand-induced binding site (LIBS) epitopes in integrin extracellular domains, and may also regulate integrin clustering (Bazzoni and Hemler, 1998).
  • LIBS activation-dependent or ligand-induced binding site
  • the present invention is based, at least in part, on the principle that computational design can be used to introduce a modification (e.g., a disulfide bond, a glycosylation site, a chemical modification site, an internal deletion, an insertion) into a protein or polypeptide, e.g., an integrin, such that the molecule has an altered conformational preference, e.g., relative to a wild-type protein.
  • a modification e.g., a disulfide bond, a glycosylation site, a chemical modification site, an internal deletion, an insertion
  • the invention features an isolated or recombinant protein including extracellular domains of integrin ⁇ and ⁇ subunits, wherein at least one of the subunits includes one or more mutations that cause a difference in conformational preference of the protein between bent and extended conformational states, the difference being relative to the conformational preference of an otherwise identical protein without the one or more mutations.
  • the difference is at least 0.5, 2, 4, 10, 100, 10 3 , or 10 4 fold.
  • Conformational preference can be measured, e.g., by evaluating the number of molecules in a population that are in the particular conformational state relative to the other conformational state, e.g., under physiological conditions.
  • the invention features an isolated or recombinant protein including extracellular domains of integrin ⁇ and ⁇ subunits, wherein at least one of the subunits includes one or more mutations that enable the protein to bind to a cognate ligand in the absence of an activating metal ion, an activating RGD- containing ligand or functional substitute thereof (e.g., an activating antibody).
  • the invention features an isolated or recombinant protein including extracellular domains of integrin ⁇ and ⁇ subunits, wherein at least one of the subunits includes one or more mutations that alters the average distance between the ⁇ subunit I like domain and the ⁇ subunit ⁇ TD domain by at least 5, 10, 20, or 50 Angstroms.
  • the one or more mutations alter the average distance between the ⁇ carbon of an amino acid at a position corresponding to amino acid 332 of human ⁇ 3 subunit and an amino acid at a position corresponding to amino acid 674 of human ⁇ 3 subunit.
  • the invention features an isolated or recombinant protein including extracellular domains of integrin ⁇ and ⁇ subunits, wherein the subunits are covalently linked by an non-naturally occurring disulfide bond.
  • a protein described herein can include one or more of the following features: at least two mutations are cysteine mutations in different domains of the alpha and/or beta subunits; one of the cysteine mutations is in the alpha subunit, one of the cysteine mutations is in the beta subunit, and the cysteines can form an inter-subunit disulfide bond; the cysteine mutations are in the beta subunit and can form an intra-subunit disulfide bond between different domains of the beta subunit; the one or more mutations include a cysteine mutations in the beta subunit that can form an intra- subunit disulfide bond; the one or more mutations (e.g., mutations causative of a particular property) are in a region other than the alpha-subunit I domain; and no more than one the mutations (e.g., mutations causative of a particular property) is in the alpha-subunit I domain.
  • a protein described herein can also include one or more of the following features: the cysteine mutation in the alpha subunit is in the 3-propeller domain of the subunit; the cysteine mutation in the beta subunit is an EGF domain of the beta subunit; the disulfide bond is in the /3-propeller domain of the subunit and within the EGF4 domain of the ⁇ 3 subunit; the cysteine mutation in the alpha subunit is at an amino acid position that is within 10, 7, 5, 3, or 2 amino acids of a residue that corresponds to Gly307 of the ⁇ V subunit and the cysteine mutation in the beta subunit is at an amino acid position that is within 10, 7, 5, 3, or 2 amino acids of a residue that corresponds to Arg563 or Asp565 of the ⁇ 3 subunit; the cysteine mutation in the alpha subunit is at an amino acid position that corresponds to a residue that is within 20, 10, 5, or 3 Angstroms of Gly307 in the PDB model 1JV2 of the oV subunit and the cyste
  • a protein can also include one or more of the following features: one of the cysteine mutations is in an EGF domain or in a ⁇ TD domain; one of the cysteine mutations in an I-like domain; the cysteine mutation in the I-like domain is within 10, 7, 5, 3, or 2 amino acid residues of an amino acid residue that corresponds to Valine 332 of the human ⁇ 3 subunit; the cysteine mutation in the I-like domain is within 10, 7, 5, 3, or 2 amino acid residues of an amino acid residue that corresponds to Valine 332 of the human ⁇ 3 subunit; the disulfide bond is introduced between a cysteine residue substituted at an amino acid residue corresponding to Val332 and a cysteme residue substituted at an amino acid residue corresponding to Ser674 of the human ⁇ 3 subunit; the disulfide bond is introduced between a cysteine residue substituted for Val332 and a cysteine residue substituted for residue Ser674 of the human ⁇ 3 subunit; and the disulfide bond is introduced between a
  • the invention features an isolated or recombinant protein including extracellular domains of integrin ⁇ and ⁇ subunits, wherein at least one of the subunits includes an altered surface feature such that the protein has (1) a difference in conformational preference of the protein between bent and extended states, the difference being relative to the conformation preference of an otherwise identical protein without the altered surface feature, or (2) a difference in affinity for a cognate ligand under one or more reaction conditions, the difference being relative to the affinity of an otherwise identical protein without the altered surface feature.
  • the protein has at least a 0.5, 2, 4, 10, 100, 10 3 , or 10 4 fold difference in conformational preference of the protein between bent and extended states, the difference being relative to the conformation preference of an otherwise identical protein without the altered surface feature
  • the protein has at least a 1 0.5, 2, 4, 10, 100, 10 3 , or
  • the protein can include one or more of the following features: the surface feature is due, at least in part, to mutation of a partially solvent-accessible amino acid residue; the surface feature is due to the mutation of the partially solvent-accessible amino acid residue alone; the surface feature is due, at least in part, to glycosylation of a site that is not glycosylated in a corresponding wild-type protein; the surface feature is due, at least in part, to chemical modification of a site that is not modified in a corresponding wild-type protein; the surface feature includes a covalent bond that links the subunits (e.g., a disulfide bond).
  • a covalent bond that links the subunits (e.g., a disulfide bond).
  • the invention features an isolated or recombinant protein including extracellular domains of integrin all and ⁇ 3 subunits, wherein the protein includes at least one amino acid substitution relative to a wild-type ⁇ T ⁇ b/33 but adheres to the fibrinogen ligand at a lower concentration of fibrinogen as compared to the wild-type (e,g. at least 0.5, 2, 4, 10, 100, 10 3 , or 10 4 fold lower).
  • the invention features an isolated or recombinant protein including extracellular domains of integrin ⁇ and ⁇ subunits, wherein the protein includes at least one amino acid substitution relative to a wild-type integrin, wherein the protein exposes a ligand-induced binding site (LIBS) in the absence of the inducing ligand.
  • LIBS ligand-induced binding site
  • the protein exposes the LJBS in the absence of the inducing ligand (e.g., an RGD peptide) and the presence of calcium or has an increased amount of exposure on the order of 0.5, 2, 4, or 10 fold.
  • the invention features an isolated or recombinant protein including extracellular domains of integrin ⁇ and ⁇ subunits, wherein at least one of the subunits includes an non-naturally occurring glycosylation site, h one embodiment, the glycosylation site is glycosylated.
  • the protein can include one or more of the following features: glycosylation of the site alters the affinity of the protein for a cognate ligand (e.g., ICAM; e.g., at least 2, 5, 10, or 50 fold; typically the cognate ligand is other than an antibody); glycosylation of the site alters the preference of the protein for a conformational state; glycosylation of the site alters the angle between the I-like and the hybrid domains of the integrin subunits (e.g., at least 5, 10, 20 or more degrees); glycosylation of the site alters the distance between the I- like and the hybrid domains of the integrin subunits (e.g., the distance between centroid of each domain, by at least 5, 10, 15, 20, or 30 Angstroms).
  • a cognate ligand e.g., ICAM; e.g., at least 2, 5, 10, or 50 fold; typically the cognate ligand is other than an antibody
  • glycosylation of the site alters the preference of the
  • the beta subunit (e.g., beta-1, beta-2, or beta-3) includes the site.
  • the protein includes a single non-naturally occurring glycosylation site and the site is in the beta subunit.
  • the protein further includes a non-naturally occurring disulfide bond.
  • the beta subunit is human.
  • the site is at an amino acid position corresponding to at least a partially solvent accessible position in a structural model of a bent integrin, e.g., PDB 1 JV2.
  • the site is at an amino acid position located in the cleft between the I-like and the hybrid domain.
  • the site is located at an amino acid position in or between residues that form a structure corresponding to alpha helix 4 and beta sheet 6.
  • the site can be: at an amino acid position of the beta subunit whose wild-type beta-carbon of the wild-type residue is within 10 Angstroms of residues 305, 328, and 422 of the human ⁇ 3 subunit in the bent conformation; two amino acids c-terminal to a mutation that alters a naturally occurring amino acid residue to asparagine (Asn); at a mutation that alters a naturally occurring amino acid residue to serine (Ser) or threonine (Thr); and can be generated by a first mutation that alters a naturally occurring amino acid residue to asparagine (Asn) and a second mutation, two residues C-terminal, that alters a naturally occurring amino acid residue to serine (Ser) or threonine (Thr).
  • the beta subunit is a ⁇ 3 subunit and the site is at an amino acid residue corresponding to human ⁇ 3 residue 305.
  • the beta subunit is a ⁇ l subunit and the site is at an amino acid residue corresponding to mature human ⁇ l residue 335 or 431.
  • the beta subunit is a ⁇ 2 subunit and the site is at an amino acid residue corresponding to mature human ⁇ 2 residue 293.
  • the beta subunit is ⁇ 3 and the alpha subunit is ⁇ llb.
  • the beta subunit is ⁇ 3 and the alpha subunit is ⁇ V.
  • the protein can bind to a cognate ligand in the absence of an activating metal ion, an activating RGD-containing ligand or functional substitute thereof, e.g., an activating antibody.
  • the invention features an isolated or recombinant protein including extracellular domains of integrin ⁇ and ⁇ subunits, wherein at least one of the subunits includes an internal deletion relative to a corresponding wild-type subunit and the protein has an altered conformational preference relative to a corresponding wild-type protein.
  • the internal deletion is in the ⁇ subunit.
  • the internal deletion can include one or more of the following features: removes a turn of an ⁇ helix; deletes at least three or four amino acids; and deletes three or four amino acids.
  • the ⁇ helix is in the I-like domain, hi one embodiment, the internal deletion alters the length of an ⁇ helix corresponding to amino acids 340 to 352 of human ⁇ 3.
  • the invention features an isolated or recombinant protein including extracellular domains of integrin ⁇ and ⁇ subunits, wherein the subunits are linked by an engineered disulfide bond.
  • the invention features an isolated or recombinant protein including an extracellular domain of an integrin beta subunit that includes a mutation that alters a non-cysteine residue to cysteine.
  • the protein can include one or more of the following features: the protein includes two cysteine mutations that form a non- naturally occurring intra-molecular disulfide bond; the protein further includes an extracellular domain of an integrin alpha subunit; the alpha subunit includes a mutation that alters a non-cysteine residue to cysteine, and the cysteine in the alpha subunit forms an intermolecular disulfide with the cysteine in the beta subunit; the cysteine the beta subunit is in a solvent exposed loop; the cysteine of the beta subunit is at an amino acid corresponding to an amino acid in the amino acid sequence EDSSGKSI (residues 671-678 of SEQ ID NO:15); and the cysteine of the beta subunit is unpaired and coupled to a chromophore or fluorophore.
  • the proteins described herein can be soluble or may include a transmembrane domain.
  • they can be attached to a cell, an insoluble support, a label, or a cytotoxin.
  • the invention also includes cells that can express such proteins and that include the proteins in physical association with the cell surface.
  • the invention includes a virus or virus-like particle that includes a protein described herein and a cell (e.g., a host cell, culture cell, or stem cell) that includes one or more heterologous nucleic acids that encode one or more subunits of a protein described herein.
  • the cell can express the protein.
  • the cell is prokaryotic or eukaryotic (e.g., mammalian).
  • the invention also features a pharmaceutical composition that includes a protein described herein and a pharmacologically acceptable carrier.
  • the invention features a nucleic acid including a sequence that encodes at least a subunit of a protein described herein, e.g., a subunit altered relative to a wild- type integrin.
  • the invention features a kit including one or more nucleic acids that encode a protein described herein.
  • the kit includes a single nucleic acid that mcludes a first region encoding the alpha subunit and a second region that encodes the beta subunit.
  • the kit includes a first nucleic acid that mcludes a region encoding the alpha subunit and a second nucleic acid that includes a region that encodes the beta subunit.
  • the invention features an article of machine-readable or machine-accessible media that includes encoded information that provides a structural model of a protein described herein.
  • the model can be generated, e.g., by crystallography, modeling, NMR, or combinations thereof.
  • the invention features a method that includes providing a structural model of the extracellular domains of an integrin; selecting one or more partially-solvent accessible amino acid residues that are positioned with 20 Angstroms of a contact between non-contiguous amino acids in the model; and producing a protein in which the one or more selected amino acid residues are altered.
  • the method includes engineering a disulfide bond (e.g., an intra- or inter- subunit bond) between domains of the model.
  • the method includes engineering a glycosylation site.
  • the method can further include evaluating a binding property of the protein., e.g., evaluating binding in the absence of an activating metal ion, an activating RGD-containing ligand or functional substitute thereof.
  • the method can further include generating a structural model of the produced protein.
  • the invention features an antibody or artificial ligand that binds preferentially binds to a particular conformation of an integrin (e.g., a conformation schematized in FIG. 19 relative to another conformation schematized in FIG. 19).
  • the preference can be binds at least 4, 10, 100, 10 3 or 10 4 fold as measured by Kd.
  • Exemplary conformations include the bent confomer, an RGD or Mn2+ activated intermediate, an extended conformer with an open headpiece, an extended conformer with a closed headpiece, and an integrin activated by inside-out signalling.
  • the invention provides an antibody or artificial ligand that binds preferentially binds to an integrin in the bent state, relative to the extended state.
  • the ligand binds at least 4, 10, 100, 10 3 or 10 4 fold better to the bent state.
  • the invention provides an antibody or artificial ligand that binds preferentially binds to an integrin in the extended state, relative to the bent state.
  • the ligand binds at least a 2, 4, 10, 100, 10 3 or 10 4 fold better to the bent state.
  • the invention provides an antibody or artificial ligand that binds preferentially stabilizes an integrin in the bent state, relative to the extended state.
  • the invention provides an antibody or artificial ligand that binds preferentially stabilizes an integrin in the extended state, relative to the bent state.
  • interaction with the integrin causes at least 2, 4, 10, 100, 10 3 or 10 4 fold change in the percentage of integrin molecules in a population that adopt the stabilized state.
  • the invention provides an antibody or artificial ligand made by using a protein described herein as an immunogen, by using the protein in a selection, or by a combination thereof.
  • the invention features an antibody or artificial ligand that binds preferentially binds to or competes with another antibody for binding to an epitope whose solvent accessible surface area is altered between the integrin bent and extended conformations.
  • the invention features an antibody or artificial ligand that binds preferentially binds to or competes with another antibody for binding to an epitope whose population-averaged solvent accessible surface area is altered in a modified protein described herein relative to a corresponding wild-type protein.
  • the epitope is other than a ligand induced binding site (LIBS); the epitope is not recognized by an integrin-activating antibody; and/or the epitope is not recognized by a monoclonal antibody that binds to integrins on inactivated cells.
  • LIBS ligand induced binding site
  • the invention features an antibody or artificial ligand that binds preferentially binds to an epitope of a mutant integrin with a conformational preference for the bent state, relative to the extended state, wherein the epitope is not accessible in a wild-type integrin in an inactive state on a cell, or a wild-type integrin in an activated state on a cell.
  • the invention features an antibody or artificial ligand that binds in the cleft between the I-like and the hybrid domain of an integrin heterodimer and a small molecule compound that binds in the cleft between the I-like and the hybrid domain of an integrin heterodimer and alters the conformational preference of the heterodimer.
  • the invention features a hetero- or homo-multimeric cell surface protein that includes an engineered protein surface feature on the surface of at least one of the subunits of the protein, wherein the engineered surface feature alters the conformational preference of the protein relative to a corresponding wild-type protein that does not include the engineered surface feature.
  • the engineered protein surface feature is an inter-subunit disulfide bond or a glycosylation site, hi one embodiment, the glycosylation site is glycosylated.
  • the cell surface protein is a receptor, e.g., a protein that includes a cytoplasmic signalling domain. In one embodiment, the signalling domain has kinase activity.
  • the protein is dimeric (e.g., an integrin or a receptor tyrosine kinase). In another embodiment, the protein is trimeric (e.g., HIV gpl20).
  • the invention features proteins, e.g., integrins that are modified by introduction of a non-naturally occurring disulfide bond (or disulfide bonds) between amino acid residues that are in close proximity in a particular or desired protein conformation.
  • Introduction of the disulfide bond biases the conformation of the protein, e.g., integrin, in the desired conformation.
  • Proteins can be locked or fixed in a particular or desired global conformation or in a particular or desired local conformation.
  • the disulfide bond in introduced between two domains or between two chains of a protein resulting in a global conformational stabilization.
  • the disulfide bond is introduced within a domain in the protein resulting in a local conformational stabilization. Proteins can further be locked or fixed in a low-affinity conformation or a high-affinity conformation.
  • the method comprises introducing at least one disulfide bond into the protein such that the protein is stabilized in a desired conformation.
  • the disulfide bond is formed by the introduction of at least one cysteine substitution into the amino acid sequence of the protein.
  • the disulfide bond is formed by the introduction of a pair of cysteine substitutions in the amino acid sequence of the protein, e.g., within a domain or between two domains of a protein, or by the introduction of a pair of cysteine substitutions in a heterodimer, e.g., one cysteine substitution in each subunit of the heterodimer.
  • Proteins, e.g., integrin, locked in a desired conformation are particularly useful in screening assays designed to identify compounds that bind to and/or modulate the activity of the protein without eliciting further structural changes in the protein.
  • integrins locked in a low or high affinity conformation can be used in screening assays designed to identify compounds that are effective at modulating integrin activity without otherwise altering the conformation of the integrin.
  • Compounds so-identified are improved over prior art integrin modulators which can induce a shape change in integrins upon binding and elicit an autoimmune response to the changed or "non-self molecule.
  • the modified integrins of the invention can also be used in screening assays to identify compounds which stabilize a particular or desired global or local conformation.
  • compounds which stabilize low- affinity global and/or local integrin conformations are particularly useful as integrin inhibitors.
  • the strategy of inhibiting integrin activity by stabilizing low- affinity conformers, as compared to competitive inhibition of cognate ligand binding eliminates the unwanted "half-agonist" side effect (wherein competitive inhibitors shift the equilibrium of integrin conformers towards the high affinity state as inhibitory compound binds at or near the integrin active site).
  • Compounds identified according to the above-identified screening assays are particularly useful as therapeutic reagents for the treatment of integrin-related disorders. Accordingly, methods of preventing or treating integrin-related disorders are featured, hi particular, methods for preventing or treating inflammatory disorders, autoimmune disorders, thrombosis, cancer, osteoporosis, sickle cell anemia, psoriasis, and/or multiple sclerosis are provided.
  • the method of the invention is widely applicable to a variety of biologically and pharmaceutically important proteins that exist in two different three-dimensional conformations, including integrins, small G proteins, heterotrimeric G protein alpha subunits, tyrosine kinases, G protein-coupled receptors, enzymes under allosteric control, zymogens, complement C3, complement C4, and fibrinogen.
  • the protein is an integrin, e.g., an I-domain-containing integrin or non-I- domain containing integrin.
  • Computational design and other methods can be used to introduce a glycan wedge into a protein or polypeptide, e.g., an integrin, e.g., a ⁇ subunit of an integrin, such that the molecule is stabilized in a desired conformation, e.g., an extended global conformation.
  • Computational design can also be used to introduce an internal deletion into a protein or polypeptide, e.g., an integrin, e.g., a ⁇ subunit of an integrin, such that the molecule is stabilized in a desired conformation, e.g., an extended global conformation.
  • conformationally stabilized proteins e.g., integrins, antibodies, e.g., anti-LFA-1 antibodies, anti- ⁇ lib ⁇ 3 antibodies, anti- ⁇ V ⁇ 3 antibodies, or small molecule therapeutics that are specific for a desired protein conformation, e.g., an "extended” or active conformation or a "bent” or inactive conformation can be identified.
  • the invention pertains to methods for stabilizing a protein or polypeptide, e.g., a protein comprising a functional domain, a multi-domain polypeptide, or heterodimer, i.e., protein, in a desired conformation, e.g., a global, extended conformation.
  • the method of the invention is widely applicable to a variety of biologically and pharmaceutically important proteins that exist in two different three-dimensional conformations, including integrins, small G proteins, heterotrimeric G protein alpha subunits, tyrosine kinases, G protein-coupled receptors, enzymes under allosteric control, zymogens, complement C3, complement C4, and fibrinogen.
  • the protein is an integrin, e.g., an integrin ⁇ or ⁇ subunit or heterodimer including said subunits.
  • the invention provides a modified integrin which preferentially conforms to a high-affinity state, the integrin comprising an ⁇ and a ⁇ subunit and having an unnatural glycosylation site introduced into said ⁇ subunit such that the high-affinity state of said integrin is preferred, h one embodiment, a glycan wedge is introduced into the glycosylation site. In another embodiment, the unnatural glycosylation site is introduced between a domain-domain interface of said ⁇ subunit. h yet another embodiment, the domain-domain interface is the interface between the I-like domain and the hybrid domain of said ⁇ subunit and located at the inner side of the domain interface, e.g., the inner side of the bend in the subunit.
  • the ⁇ subunit contains at least one amino acid mutation that creates an unnatural glycosylation site. Since the consensus motif for N-glycosylation is Asn- Xaa-Ser/Thr, the unnatural glycosylation site can be created by mutating the first residue to Asn and/or the third residue to either Ser or Thr.
  • the modified integrin is ⁇ llb ⁇ 3, ⁇ V ⁇ 3, or ⁇ L ⁇ 2.
  • the ⁇ subunit is ⁇ 3, and the unnatural glycosylation is at amino acid residue 303 of the ⁇ 3 subunit. In another embodiment, the ⁇ subunit is ⁇ 2, and the unnatural glycosylation is at amino acid residue 293 of the ⁇ 2 subunit. hi a further embodiment, the ⁇ subunit is ⁇ l, and the unnatural glycosylation is at amino acid residue 333 of said ⁇ l subunit.
  • Another aspect of the invention provides methods for stabilizing an integrin comprising an ⁇ and a ⁇ subunit in a desired conformation, said method comprising introducing an unnatural glycosylation site into at least one subunit, such that the integrin is stabilized in a desired global conformation, e.g., an extended conformation, hi one embodiment, the unnatural glycosylation site is introduced into a ⁇ subunit. hi another embodiment, a glycan wedge is introduced into said glycosylation site. In one embodiment, the unnatural glycosylation site is introduced between a domain-domain interface of said ⁇ subunit, e.g., the interface between the I-like domain and the hybrid domain.
  • the ⁇ subunit contains at least one mutation that creates unnatural N-glycosylation site.
  • the ⁇ subunit is ⁇ 3, and wherein the unnatural glycosylation is at amino acid residue 303 of the ⁇ 3 subunit.
  • the ⁇ subunit is ⁇ 2, and the unnatural glycosylation is at amino acid residue 293 of the ⁇ 2 subunit.
  • the ⁇ subunit is ⁇ l, and wherein said unnatural glycosylation is at amino acid residue 333 of said ⁇ l subunit.
  • the invention provides isolated nucleic acid sequences encoding modified integrins which are fixed in a high-affinity state, the integrin comprising an ⁇ and a ⁇ subunit and having an unnatural glycosylation site introduced into said ⁇ subunit.
  • the invention provides a modified integrin which is fixed in a high-affinity state, said integrin comprising an ⁇ and a ⁇ subunit and having an internal deletion in said ⁇ subunit such that the high-affinity state of said integrin is fixed, h one embodiment, the integrin is ⁇ b ⁇ 3, ⁇ V ⁇ 3, or ⁇ L ⁇ 2. In a further embodiment, the internal deletion is within the C-terminal helix of said ⁇ subunit. In another embodiment, the internal deletion is of at least three amino acid residues. In yet another embodiment, the internal deletion is of at least four amino acid residues.
  • the ⁇ subunit is ⁇ 2, and the internal deletion is a deletion of Lys 340 -Leu-Ser-Ser.
  • the ⁇ subunit is ⁇ 2, and the internal deletion is a deletion of Asn 336 -Ala-Tyr-Asn.
  • the invention provides a method for stabilizing an integrin comprising an ⁇ and a ⁇ subunit in a desired conformation, said method comprising introducing an internal deletion in said ⁇ subunit, such that the integrin is stabilized in a desired global conformation.
  • the invention provides methods for the use of an integrin which is fixed in a high-affinity state in a screening assay.
  • the invention provides methods for the use of an integrin which is fixed in a high-affinity state in drug development.
  • the invention provides for the method for identifying a compound that selectively binds to an integrin, e.g., ⁇ JJb ⁇ 3, ⁇ V ⁇ 3, or ⁇ L ⁇ 2 (LFA-1), in a high-affinity state comprising determining the ability of said compound to bind to a modified integrin which is fixed in a high-affinity state as compared to a modified integrin which is fixed in a low-affinity state and selecting said compound based on its ability to selectively bind to the integrin which is fixed in a high-affinity state.
  • the compound stabilizes the high-affinity state.
  • the compound stabilizes the low-affinity state.
  • the invention provides a method for identifying a modulator of integrin activity comprising providing a modified integrin, contacting the modified integrin with a test compound; and assaying the ability of the test compound to bind to the modified integrin.
  • the compound is selected from the group consisting of a peptide, a peptidomimetic, and a small molecule or an antibody.
  • the invention provides for a compound identified by the methods described herein.
  • the invention provides isolated nucleic acid sequences encoding modified integrins which are fixed in a high-affinity state, said integrin comprising an ⁇ and a ⁇ subunit and having an internal deletion in said ⁇ subunit.
  • the invention provides methods for treating or preventmg an integrin-mediated disorder in a subject comprising administering to said subject a therapeutically effective amount of an integrin modulator compound which selectively binds to an integrin in the open conformation, thereby treating or preventing an integrin-mediated disorder in a subject.
  • the invention also features a reduction sensitive conformational switch, e.g., a molecule whose binding properties and conformation can be regulated by the redox potential of the immediate environment.
  • the switch can include a protein with a disulfide bond with a property described herein.
  • the invention also features a multimeric (e.g., heterodimeric or homodimeric) extracellular protein with a regulatable or "breakable" clasp.
  • the clasp can include a leucine zipper and a protease cleavage site, linked to at least one of the leucine zippers.
  • the protein can include other features described herein
  • integrins in addition to integrins, many pharmaceutically important proteins exist in two alternative three-dimensional structures, referred to as conformations or conformers. Often these proteins have important signaling functions, such as small G proteins, trimeric G protein subunits, tyrosine kinases (e.g., receptor tyrosine kinases), and G protein-coupled receptors. Typically, one of these conformations and not the other is enzymatically active or has effector functions. The methods described herein can be applied to such proteins.
  • a protein can be altered at one or more positions to cause the protein to preferentially adopt one conformation relative to another. Therefore, antibody or small molecule therapeutics that are specific for a protein in a particular conformation, for example, the active or inactive conformation, would have great advantages over non-selective alternatives. 5 This application incorporates by reference US Published Application 2002-
  • Figure 1 is a stereodiagram of the high affinity model of the LFA-1 1 domain, with mutations to introduce a disulfide bond.
  • the model was prepared using segments of the putative high affinity Mac-1 1 domain structure and a putative low affinity LFA-1 1 domain structure as templates.
  • the K287C and K294C mutations were included in the model.
  • the sidechains and disulfide bond of C287 and C294 are
  • Figure 2 depicts predicted disulfide bonds that are selective for high affinity
  • Figure 3 depicts the cell surface expression of LFA-1 cysteine substitution mutants on 293T transient transfectants (Panel A), and K562 stable transfectants (Panel B) as determined by flow cytometric analysis using monoclonal antibody TS2/4 (shaded histogram) to ⁇ L in oL//32 complex, or the nonbinding antibody X63 (open histogram). Numbers in the parentheses are clone numbers of the K562 stable transfectants.
  • Figure 4 depicts the binding of LFA-1 transfectants to immobilized ICAM-1.
  • Panel A 293T transient transfectants, and Panels B and C, K562 stable transfectants.
  • Panels A and B binding of the transfectants to immobilized ICAM-1 was determined in LI 5 medium containing Ca 2+ and Mg 2+ in the presence or absence (control) of the activating antibody CBRLFA-1/2 at 10 ⁇ g/ml.
  • the binding assay was performed in TBS, pH7.5 supplemented with divalent cations or EDTA as indicated. Numbers in the parentheses are clone numbers of the K562 stable transfectants. Results are mean + SD of triplicate samples and representative of at least three experiments.
  • Figure 5 depicts the binding of soluble ICAM-1 -IgA fusion protein to K562 transfectants that express wild-type LFA-1, the predicted liigh-affinity mutant K287C/K294C, or mutant L289C/K294C as assessed by flow cytometric analysis.
  • Mean fluorescent intensity of ICAM-1 -IgA binding is indicated on the upper right corner of the histogram plot. Numbers in the parentheses are clone numbers of the K562 stable transfectants. Results are representative of three experiments.
  • Figure 6 depicts the inhibitory activity of lovastatin on ligand binding by cells expressing activated wild-type and high affinity (K287C/K294C) LFA-1.
  • Figure 7 depicts the cell surface expression of the isolated LFA-1 1-domains.
  • the wild-type oL I-domain and the mutant K287C/K294C and L289C/K294C I- domains were expressed on the surface of the K562 transfectants by the PDGFR transmembrane domain.
  • the level of cell surface I-domain was determined by flow cytometry using monoclonal antibody TS1/22 to the I-domain (shaded histogram). Binding of the control mAb X63 is shown as open histograms. Mean fluorescent intensity of TS1/22 binding was indicated on the upper right corner of the histogram plot. Results of two individual clones (#1 and #2) from each I-domain transfectants are shown.
  • Figure 8 depicts the ligand binding activity of the cell surface expressed LFA- 1 1-domains.
  • Panel A Binding of K562 transfectants to immobilized ICAM-1 in the presence or absence of DTT. Binding was performed in the presence (white bar) or absence (black bar) of DTT.
  • Panel B Effect of divalent cations on binding of K562 transfectants to ICAM-1. Binding was performed in the presence of Mn 2+ (black bar), Mg 2+ (shaded bar) or EDTA (white bar). In Panels A and B, two clones (#1 and #2) of the transfectants expressing the wild-type I-domain or mutant I-domain were tested.
  • Panel C Effect of LFA-1 blocking antibodies on binding of the
  • Figure 9 depicts the surface plasmon resonance sensograms by BIAcoreTM recording the interaction of the open (K287C/K294C) or wild-type I-domain with ligands, ICAM- 1 (Panels A and B), ICAM-2 (Panels C and D), and ICAM-3 (Panels E andF).
  • Figure 10 depicts the inhibition of LFA-1 -dependent adhesion in vitro by the open oiL I-domain.
  • Panel A depicts the adhesion of K562 stable transfectants expressing wild-type LFA-1 to immobilized ICAM-1 in the presence of soluble wild- type (closed circles) or open (K287C/K294C) I-domain (open circles);
  • Panel B depicts the homotypic aggregation of the murine EL-4 T lymphoma cell line in the presence of soluble wild-type (closed circles) or open (K287C/K294C) I-domain (open circles).
  • Figure 11 depicts the expression and ligand binding activity of the Mac-1 cysteine substitution mutants in transiently transfected 293T cells.
  • Panel A binding of monoclonal antibodies CBRMl/32 (open bars) and CBRMl/5 (black bars) to intact Mac-1 I-domain mutants.
  • Panel B adhesion of 293T transient transfectants expressing intact Mac-1 cysteine substitution mutants to iC3b coated on plastic.
  • Panel C adhesion of 293T transient transfectants expressing isolated Mac-lmutant I- domains to iC3b ligand in the presence (black bars) or absence (open bars) of antibody CBRM 1/5.
  • Figure 12 depicts the expression and ligand binding activity of the Mac-1 cysteine substitution mutants in K562 stable transfectants.
  • Panel A representative histogram showing binding of monoclonal antibodies CBRMl/32 and CBRMl/5 to intact Mac-1 I-domain mutants as assessed by flow cytometry. Mean fluorescent intensity is indicated in the upper right hand corner of the histogram plot.
  • Panel B adhesion of K562 stable transfectants expressing intact Mac-1 cysteine substitution mutants to iC3b coated on plastic.
  • Panel C adhesion of K562 stable transfectants expressing isolated Mac-1 1-mutant I-domains to iC3b ligand.
  • FIG. 13 depicts the domain organization in the bent conformation of the ⁇ V ⁇ 3 integrin crystal structure.
  • Hy hybrid domain
  • E3 and E4 integrin EGF domains 3 and 4, respectively; ⁇ T; ⁇ -tail domain; N, N-terminus; C, C-terminus.
  • Asterisks mark the position of ligand binding sites at a ⁇ - propeller - I-like domain interface in integrins that lack I domains, and on the "top" of the I domain in integrins that contain I domains.
  • the PSI domain at the N- terminus and the integrin EGF domains 1 and 2 following the hybrid domain are missing from the electron density, as is a portion of domain 3; these are symbolized with dashed black lines.
  • Polypeptide chain connections are shown as black lines. Note the two polypeptide chain connections between the I-like and hybrid domains in the ⁇ subunit, and between the I and ⁇ -propeller domains in the ⁇ subunit. Based on the crystal structure ((Xiong et al. (2001) Science 295:151-155).
  • Figure 14 are electron micrographs and representative projection averages of negatively stained ⁇ V ⁇ 3 integrin.
  • Clasped ⁇ V ⁇ 3 (A,C,E,G) and unclasped ⁇ V ⁇ 3 (B,D,F,H) were incubated in the presence of 5 mM Ca 2+ (A,B), ImM Mn 2+ (C,D), 1 mM Ca 2+ and 60 ⁇ M cyclo-RGDfV peptide (E,F), or 1 mM Mn 2+ and 60 ⁇ M cyclo- RGDfV (G,H) and subjected to gel filtration, negative staining, and electron microscopy.
  • Each panel shows a micrograph area as well as four representative projection averages. The number of individual particles represented by each projection average is shown beneath each average. The side length of the average images is 28.4 nm.
  • Figure 15 depicts a comparison of negatively stained ⁇ V ⁇ 3 projection averages to the ⁇ V ⁇ 3 crystal structure.
  • A Best correlating projected view calculated from the ⁇ V ⁇ 3 crystal structure (Xiong et al. (2001) Science 295:151-155).
  • B Projection average of clasped ⁇ V ⁇ 3 particles in the presence of Ca 2+ used for cross- correlation in A.
  • C Best correlating projected view calculated from the headpiece of the ⁇ V ⁇ 3 crystal structure (1 JV2); the corresponding headpiece from the ligand- complexed structure (1L5G) gave an identical cross-correlation rotation function and projected view.
  • D Representative projection average of an extended integrin with a closed headpiece (unclasped in Mn 2+ from Fig.
  • E Representative projection average of an extended integrin with an open headpiece (unclasped in Ca 2+ + cyclo-RGDfV, from Fig. 14F, average 1) used in cross correlation.
  • F Best correlating projected view calculated from the model of the open headpiece crystal structure. The model was made from the ⁇ V ⁇ 3 crystal structure (1 V2) by superimposing ⁇ 3 residues D109, A347 and Y348 on residues D 109, 1351, and R352 to simulate a 4 residue downward shift of the I-like domain C- terminal ⁇ -helix. Hybrid domain residues 55-109 and 348-434 from the superimposed structure were combined with the remaining headpiece residues from the 1 JV2 structure to make the open model.
  • the bar indicates 100 A.
  • the bent conformation observed in the crystal structure (1 JV2) with I-EGF domains 1 , 2, and a portion of 3 built in similar to the model described in (Beglova et al. (2002) Nat Struct Biol 9:282-287) is shown in G, and a corresponding model of the extended conformation is shown in H.
  • the model of the unbent molecule was created by breaking the bent form at the junction between the thigh and calf-1 domains in ⁇ and I-EGF 1 and 2 in ⁇ (dashed line) into the headpiece and tailpiece, and moving the headpiece relative to the tailpiece.
  • the approximate position of the cell membrane is indicated by a gray line.
  • Black bar indicates 100 A.
  • Figure 16 depicts a change in hydrodynamics of ⁇ V ⁇ 3 induced by Mn 2+ or ligand binding.
  • Unclasped ⁇ V ⁇ 3 (1.8 ⁇ g in 10 ⁇ l) was analyzed on a Superdex 200 column in a buffer containing ImM Ca 2+ (thin line), ImM Mn 2+ (dotted line), or ImM Mn 2+ + 1 ⁇ M cyclo-RGDfV (thick line).
  • Arrows indicate excluded volume (V 0 ) and total volume (V t ) of the column.
  • a rescaled view of the peak position is shown in inset.
  • Figure 17 show results from (A) a soluble ligand binding assay in which a wedge mutant integrin is evaluated and (B) a cell adhesion assay in which a wedge mutant integrin is evaluated. These results illustrate that the wedge mutant is constitutively active relative to a wild-type integrin.
  • Figure 18 shows that the cell surface ⁇ V ⁇ 3 kept in the bent conformation is locked in an inactive state.
  • a and B Disulfide bond formation between the headpiece of the ⁇ subunit and tailpiece of the ⁇ subunit.
  • 293T cells were co- transfected with wild type ( ⁇ and ⁇ ) or mutant ( ⁇ C and ⁇ C) integrin subunits to give the indicated ⁇ / ⁇ pairs.
  • ⁇ C denotes ⁇ V G307C (A) or ⁇ llb 3200 (B)
  • ⁇ C denotes ⁇ 3 .
  • Integrin heterodimers were immunoprecipitated from the [ S]-labeled cell extract with AP3 mAb to ⁇ 3 and subjected to non-reducing SDS-PAGE and fluorography.
  • C,D Binding of fibrinogen to ⁇ 3 integrins. The conversion to the high affinity state was assessed by binding of FITC-fibrinogen to ⁇ V ⁇ 3 (C) or ⁇ llb ⁇ 3 (D) using fluorescence flow cytometry.
  • the three confomiers defined by electron microscopy (A, D, E) and the hypothetical intermediates when the headpiece-tailpiece and ⁇ tailpiece - ⁇ tailpiece interfaces are destabilized in outside-in (B) or inside-out (C) signaling are schematized.
  • the following domains are shown schematically: ⁇ headpiece domains ( ⁇ -propeller and thigh), ⁇ tailpiece domains (calf-1 and calf-2), ⁇ headpiece domains (I-like, hybrid, PSI, and I-EGF1), and ⁇ tailpiece domains (I-EGF2, and the I-EGF3, I-EGF4, and ⁇ -tail which are merged together).
  • Black squiggly lines symbolize the transmembrane and cytoplasmic domains.
  • the conformers are shown in the same orientation as the cross-correlated headpiece in Figure 15C, except for rotation in the plane of the figure.
  • the rotation in the plane of the figure is such as to maximize comparison between the conformers, and is not uniform with regard to expected orientation relative to the cell membrane, especially in (D).
  • Figure 20 depicts structural models for exemplary mutations that introduce disulfide bonds that preferentially maintain an integrin in a bent conformation.
  • Figure 21 A depicts a structural model of the angle between the I-like and hybrid domains of an integrin in the bent conformation.
  • Figure 21B depicts a corresponding model for an angled-out conformation.
  • Figure 21 C depicts a corresponding model for an exemplary unnatural N-glycan attached at an amino acid position corresponding to residue Asn 303 of the ⁇ 3 integrin. The integrin is shown in an angled-out conformation.
  • Figure 22 is an amino acid sequence alignment for a region of different ⁇ subunits in the region that can form the ⁇ 4 helix to ⁇ 6 sheet secondary structures of the I-like domain.
  • a glycosylation site is introduced so that an N- glycan is attached at the amino acid position corresponding to amino acid 305 of the ⁇ 3 subunit (numbering in this figure is based on the ⁇ 3 subunit), for example, by mutating the residue corresponding to amino acid 305 to serine or threonine, and, if necessary, the residue corresponding to amino acid 303 to asparagine.
  • a glycosylation site is introduced so that an N-glycan is attached at the amino acid position corresponding to amino acid 328 of the ⁇ 3 subunit, for example, by mutating the residue corresponding to amino acid 326 to asparagine, and, if necessary, the residue corresponding to amino acid 328 to serine or threonine.
  • a cysteine is introduced at an amino acid position corresponding to amino acid 332 of the ⁇ 3 subunit.
  • the listed sequences correspond to SEQ ID NOs: 16-32
  • Figure 23 is an amino acid sequence alignment for a region of different ⁇ subunits.
  • a glycosylation site is introduced so that an N-glycan is attached at the amino acid position corresponding to amino acid 422 of the ⁇ 3 subunit (numbering in this figure is based on the ⁇ 3 subunit), for example, by mutating the residue corresponding to amino acid 420 to asparagine, and, if necessary, the residue corresponding to amino acid 422 to serine or threonine.
  • N-glycan at amino acid 431 of the mature ⁇ l subunit using a G429N mutation in the mature ⁇ 1 subunit.
  • the listed sequences correspond to SEQ ID NOs:33-49.
  • Figure 24 is an amino acid sequence alignment for a region of different ⁇ subunits.
  • a cysteine is introduced so that it is at the amino acid position corresponding to amino acid 307 of the ⁇ V subunit or at an amino acid position within two amino acids on either side of the gap indicated for the final eight sequences.
  • the listed sequences correspond to SEQ ID NOs:50-64.
  • the present invention is based, at least in part, on the principle that computational design can be used to introduce a disulfide bond into a protein or polypeptide, e.g., an integrin, such that the molecule is stabilized in a desired conformation.
  • computational design is used to introduce a disulfide bond that locks the protein in a particular conformation.
  • the invention features proteins, e.g., integrins that are modified by introduction of a non-naturally occurring disulfide bond (or disulfide bonds) between amino acid residues that are in close proximity in a particular or desired protein conformation.
  • Introduction of the disulfide bond essentially locks or fixes the protein, e.g., integrin, in the desired conformation.
  • Proteins can be locked or fixed in a particular or desired global conformation and/or in a particular or desired local conformation.
  • the disulfide bond in introduced between two domains or between two chains of a protein resulting in a global conformational stabilization.
  • the disulfide bond is introduced within a domain in the protein resulting in a local conformational stabilization. Proteins can further be locked or fixed in a low-affinity conformation or a high-affinity conformation.
  • the invention provides a modified integrin which is stabilized in a desired global conformation, the integrin having an ⁇ and a ⁇ subunit and having a disulfide bond introduced between the ⁇ and ⁇ subunits, such that the global conformation of the integrin is stabilized.
  • modified integrin includes any modified integrin, e.g., a modified I-domain-containing integrin or a modified non-I-domain-containing integrin, which comprises at least one subunit which has been altered with respect to the wild-type sequence or the native state such that at least one disulfide bond has been introduced into the integrin thereby stabilizing the integrin in a desired conformation.
  • the disulfide bond may be introduced within a single domain of a single subunit, between two domains of a single subunit, or between two subunits, e.g., the ⁇ subunit and the ⁇ subunit.
  • a "modified I-domain integrin” comprises a disulfide bond within the I-domain.
  • a "modified I-like domain integrin” comprises a disulfide bond within the I-like domain.
  • a modified integrin may or may not include a cytoplasmic region, a transmembrane domain, or unstructured residues immediately external to the transmembrane domain.
  • polypeptide and “protein” are used interchangeably throughout.
  • the terms “protein” or “polypeptide” include polypeptide chains, e.g., subunits, e.g., a and ⁇ subunits, and fragments thereof, proteins comprising a functional domain, e.g., an I domain or an I-like domain, multi- domain proteins, and heterodimeric protein, e.g., integrins, and fragments thereof.
  • a “conformation” or “conformer” refers to a three dimensional structure of a protein.
  • a “desired” conformation includes a protein conformation that is conducive to a particular use of the protein, e.g., a conformation that supports a particular biological function and/or activity, a therapeutic effect, or use in a particular screening assay.
  • a “domain” refers to a structural fold that includes an at least partially hydrophobic core. Many domains can also interact to form the complex structure of a protein, e.g., an integrin extracellular domain.
  • global conformation refers to the overall conformation of a protein, e.g., of a heterodimeric and/or multi-domain protein.
  • a global conformation can be an extended conformation or a bent conformation, as depicted in Figure 15, G-H.
  • local conformation refers to a conformation within a domain, e.g., an I domain or an I-like domain, of a multi-domain protein or within a portion or segment of a protein that is less than the entire protein.
  • a local conformation within an I domain or within an I-like domain can be an open or a closed conformation.
  • Structural conformation routinely imparts functional activity on a multi- domain or heterodimeric protein, in particular, an integrin.
  • structural conformations are routinely referred to interchangeably as functional conformations, e.g., "active” or “high affinity” or “inactive” or “low-activity” conformations.
  • active conformation and “high affinity conformation” are used interchangeably to refer to a conformation of a protein which favors, allows, facilitates, promotes or activates a biological function and/or activity.
  • An exemplary active, local conformation is an open conformer.
  • An exemplary active global conformation is an extended conformer.
  • inactive conformation and “low affinity conformation” are used interchangeably to refer a conformation of a protein which disfavors, prohibits, inhibits or suppresses a biological function and/or activity.
  • An exemplary inactive local conformation is a closed conformer.
  • An exemplary inactive global conformation is a bent conformer.
  • the invention provides a modified integrin which is stabilized in a desired global conformation, the integrin comprising an ⁇ and a ⁇ subunit and having a disulfide bond introduced between a first and second domain of the ⁇ or ⁇ subunit, such that the global conformation of the integrin is stabilized.
  • the distance between the C ⁇ carbons of the disulfide bond is loA or less. In another embodiment, the distance between the C ⁇ carbons in the disulfide bond is in the range of 3.00-8.09A. h still another embodiment, the distance between the C ⁇ carbons in the disulfide bond is in the range of 3.41-7.08A.
  • the integrin is ⁇ V ⁇ 3. In one embodiment, the disulfide bond is between the /3-propeller domain of the subunit and within the I-EGF4 domain of the /33 subunit.
  • the disulfide bond is introduced between a cysteine residue substituted for Gly307 of the oV subunit and a cysteine residue substituted for Arg563 of the ⁇ 3 subunit; or between a cysteine residue substituted for Gly307 of the oV subunit and a cysteine residue substituted for Asp565 of the /33 subunit.
  • the integrin is ⁇ llb ⁇ 3.
  • the disulfide bond is selected from the group consisting of a disulfide bond introduced between a cysteine residue substituted for Ala318 of the odlb subunit and a cysteine residue substituted for Asp552 of the 83 subunit; a disulfide bond introduced between a cysteine residue substituted for Arg320 of the ⁇ llb subunit and a cysteine residue substituted for Arg563 of the /33 subunit; and a disulfide bond introduced between a cysteine residue substituted for Arg320 of the cdlb subunit and a cysteine residue substituted for Asp565 of the /33 subunit.
  • the modified integrin is ⁇ V ⁇ 3.
  • the disulfide bond is introduced between an I-like domain and a /3-tail domain of the ⁇ subunit of the ⁇ subunit.
  • the disulfide bond is introduced between a cysteine residue substituted for Val332 and a cysteine residue substituted for residue Ser674 of the ⁇ 3 subunit.
  • the disulfide bond is introduced between a cysteine residue substituted for Val332 and a cysteine residue substituted for residue Ser674 of the ⁇ 3 subunit of the integrin, e.g., a ⁇ llb ⁇ 3 integrin.
  • the invention provides a modified integrin which is stabilized in a desired local conformation, the integrin comprising an ⁇ and a ⁇ subunit and having a disulfide bond introduced within a domain of the ⁇ or ⁇ subunits, such that the local conformation of the integrin is stabilized.
  • the disulfide bond is introduced into an I-like domain of the ⁇ subunit of the integrin.
  • the integrin is stabilized in a low-affinity conformation.
  • the integrin is ⁇ llb ⁇ 3.
  • the disulfide bond is introduced between a cysteine residue substituted for Thr329 and a cysteine residue substituted for Ala347 of the ⁇ 3 subunit; or between a cysteme residue substituted for Gly331 and a cysteine residue substituted for Leu343 of the ⁇ 3 subunit.
  • the integrin is ⁇ L ⁇ 2. In one embodiment, the integrin is stabilized in a low-affinity conformation.
  • the disulfide bond is selected from the group consisting of a disulfide bond introduced between a cysteine residue substituted for Gly321 and a cysteine residue substituted for Ala337 of the ⁇ 2 subunit; a disulfide bond introduced between a cysteine residue substituted for Ala319 and a cysteine residue substituted for Ala337 of the ⁇ 2 subunit; and a disulfide bond introduced between a cysteine residue substituted for Ala319 and a cysteine residue substituted for Lys340 of the ⁇ 2 subunit.
  • the integrin is stabilized in a high-affinity conformation.
  • the disulfide bond is introduced between a cysteine residue substituted for Met335 and a cysteine residue substituted for Val332 of the ⁇ 3 subunit.
  • the integrin is ⁇ L ⁇ 2.
  • the integrin is stabilized in a high-affinity conformation.
  • the disulfide bond is introduced between a cysteine residue substituted for Gly321 and a cysteine residue substituted for Leu333 of the ⁇ 2 subunit.
  • the disulfide bond is introduced into an I-domain of the ⁇ subunit of the integrin.
  • the integrin is stabilized in a high affinity conformation.
  • Methods for locking or fixing proteins, e.g., integrins, in a desired conformation are also featured. It is appreciated that the degree of locking or fixing can vary and can be reflected in at statistically significant alteration in the population of different conformers adopted by the protein. For example, covalent bonds may cause almost complete or complete rigidity at the sites of connection, but other modifications, e.g., a glycosylation site may cause a less drastic shift in conformer population.
  • the method of the invention includes modeling a protein, a functional domain thereof, on a template of the desired three-dimensional structure of the protein and introducing cysteines which are able to form a disulfide bond only in the desired conformation of the protein, thus stabilizing the protein in that particular conformation.
  • the protein can be any protein, for which a three dimensional structure is known or can be generated, and is preferably a protein that exists in two different conformations. Computational algorithms for designing and/or modeling protein conformations are described, for example, in WO 98/47089 and in US 2003-0054440 (see, e.g., below).
  • the SSBOND program (Hazes, B and Dijkstra, BW (1988) Protein Engineering 2:119-125) can be used to identify positions where disulfide bonds can be introduced in a protein structure by mutating appropriately positioned pairs of residues to cysteine. It is also possible to using a scanning mutagenesis (see, e.g., below) to find one or more sites into which a cysteine can be positioned.
  • Disulfide bond formation occurs between two cysteine residues that are appropriately positioned within the three-dimensional structure of a protein
  • a protein is stabilized in a desired conformation by introducing at least one cysteine substitution into the amino acid sequence such that a disulfide bond is formed.
  • the introduction of a single cysteine substitution is performed in circumstances in which an additional cysteine residue is present in the native amino acid sequence of the protein at an appropriate position such that a disulfide bond is formed.
  • two cysteine substitutions are introduced into the amino acid sequence of the protein at positions that allow a disulfide bond to form, thereby stabilizing the protein in a desired conformation.
  • the method comprises introducing at least one disulfide bond into the protein such that the protein is stabilized in a desired conformation.
  • the disulfide bond is formed by the introduction of at least one cysteine substitution into the amino acid sequence of the protein.
  • the disulfide bond is formed by the introduction of a pair of cysteine substitutions in the amino acid sequence of the protein, e.g., within a domain or between two domains of a protein, or by the introduction of a pair of cysteine substitutions in a heterodimer, e.g., one cysteine substitution in each subunit of the heterodimer.
  • the invention provides a method for stabilizing an integrin in a desired global conformation, wherein the integrin comprises an ⁇ and ⁇ subunit and wherein the method comprises introducing a disulfide bond between a first and second domain of the ⁇ or ⁇ subunit, such that the integrin is stabilized in the desired global conformation.
  • one cysteine substitution is introduced into one domain of a multi-domain protein within a heterodimeric protein, e.g., an ⁇ or ⁇ subunit of an integrin, and one cysteine substitution is introduced into another domain of the same multi-domain polypeptide resulting in a global conformational change of the heterodimer.
  • the invention provides a method for stabilizing an integrin in a desired global conformation, wherein the integrin comprises an ⁇ and ⁇ subunit and wherein the method comprises introducing a disulfide bond between the ⁇ and ⁇ subunits, such that the integrin is stabilized in the desired global conformation.
  • the method comprises introducing a disulfide bond between the ⁇ and ⁇ subunits, such that the integrin is stabilized in the desired global conformation.
  • one cysteine substitution is introduced into one subunit of a heterodimer and another cysteine substitution is introduced into the other subunit of a heterodimer, resulting in a global conformational change of the heterodimer.
  • the invention provides a method for stabilizing an integrin in a desired local conformation, wherein the integrin comprises an ⁇ and a ⁇ subunit and wherein the method comprises introducing a disulfide bond within a domain of the ⁇ or ⁇ subunit, such that the local conformation of the integrin is stabilized.
  • a disulfide bond within a domain of the ⁇ or ⁇ subunit, such that the local conformation of the integrin is stabilized.
  • two cysteine substitutions are introduced into a single domain of a protein within a heterodimeric protein, e.g., an I domain or an I-like domain, resulting in a local conformational change of the heterodimeric protein.
  • the distance between the substituted cysteines is less than 10 A in a particular model, e.g., a model of a bent or extended conformation, e.g., PDB 1 JV2.
  • the distance between the C/3 carbons of the residues that are substituted for cysteine is 3.00-8.09A.
  • the distance between the C/3 carbons in the disulfide bond is in the range of 3.41- 7.08A.
  • cysteine substitutions are introduced such that the formation of a disulfide bond is favored only in one protein, e.g., integrin, conformation, such that the protein is stabilized in that particular conformation.
  • Preparation of a modified protein of the invention by introducing cysteine substitutions is preferably achieved by mutagenesis of DNA encoding the protein of interest (e.g., an integrin).
  • the protein of interest e.g., an integrin
  • an isolated nucleic acid molecule encoding a modified integrin can be created by introducing one or more nucleotide substitutions into the nucleotide sequence of an integrin gene, e.g., within the nucleotide sequence of the ⁇ or ⁇ subunit such that one or more amino acid substitutions, e.g., cysteine substitutions, are introduced into the encoded protein.
  • Mutations can be introduced into a nucleic acid sequence by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Proteins, e.g., integrins, locked in a desired conformation are particularly useful in screening assays designed to identify compounds that bind to and/or modulate the activity of the protein without eliciting further structural changes in the protein.
  • integrins locked in a low or high affinity conformation can be used in screening assays designed to identify compounds that are effective at modulating integrin activity without otherwise altering the conformation of the integrin.
  • Compounds so-identified are improved over prior art integrin modulators which can induce a shape change in integrins upon binding and elicit an autoimmune response to the changed or "non-self molecule.
  • the modified integrins of the invention can also be used in screening assays to identify compounds which stabilize a particular or desired conformation, e.g., a global or local conformation.
  • compounds which stabilize low-affinity global and/or local integrin conformations are particularly useful as integrin inhibitors.
  • the strategy of inhibiting integrin activity by stabilizing low-affinity conformers, as compared to competitive inhibition of cognate ligand binding eliminates the unwanted "half-agonist" side effect (wherein competitive inhibitors shift the equilibrium of integrin conformers towards the high affinity state as inhibitory compound binds at or near the integrin active site).
  • the invention provides a method for identifying integrin ligands (e.g., inhibitors) for pharmaceutical use.
  • the ligands can be inhibitors that are capable of inhibiting integrin activity without eliciting an autoimmune response.
  • the method can include: contacting the modified integrin, or a cell expressing the integrin with a test compound; evaluating whether the compound stabilizes the low-affinity state of the integrin; and selecting the compound as an inhibitor capable of inhibiting integrin activity without eliciting an autoimmune response.
  • the invention provides a method for identifying a compound that selectively binds to an integrin in a low-affinity state comprising determining the ability of the compound to bind to a modified integrin which is fixed in a low-affinity state as compared to a modified integrin which is fixed in a high-affinity state and selecting the compound based on its ability to selectively bind to the integrin which is fixed in a low-affinity state, one embodiment, the compound stabilizes the low- affinity state.
  • the compound is selected from the group consisting of a peptide, a peptidomimetic, and a small molecule or an antibody, or antigen binding fragment thereof,
  • the integrin is selected from the group consisting of ⁇ V ⁇ 3, ⁇ llb ⁇ 3, ⁇ L ⁇ 2 and ⁇ M ⁇ 2.
  • the invention also provides compounds identified by the above-identified methods.
  • Compounds identified according to the above-identified screening assays are particularly useful as therapeutic reagents for the treatment of integrin-related disorders. Accordingly, methods of preventing or treating integrin-related disorders are featured. In particular, methods for preventing or treating inflammatory disorders, autoimmune disorders, thrombosis, cancer, osteoporosis, sickle cell anemia, psoriasis, and/or multiple sclerosis are provided.
  • the invention provides a method for preventing or treating an integrin-mediated disorder in a subject comprising administering to the subject a therapeutically effective amount of a compound identified by the methods described herein, thereby treating or preventing an integrin-associated disorder in a subject.
  • the compound is an antibody, or antigen-binding fragment thereof, or small molecule.
  • the invention provides a method of modulating platelet adhesion in a patient having thrombosis, the method comprising administering a compound identified by the methods described herein.
  • a method of modulating angiogenesis in a patient having cancer the method comprising administering a compound identified by the methods described herein.
  • a method of modulating integrin activity osteoporosis, or sickle cell anemia comprising administering a compound identified by the methods described herein.
  • a method of modulating leukocyte adhesion in a patient having Crohn's disease, psoriasis, or multiple sclerosis comprising administering a compound identified by the methods described herein.
  • Additional proteins suitable for use in the modification methodologies of the invention include, but are not limited to, industrially and therapeutically important proteins such as: 1) signaling molecules, such as small G proteins, trimeric G protein alpha subunits, tyrosine kinases, and G protein-coupled receptors; 2) enzymes under allosteric control, 3) zymogens that undergo conformational change after activation by proteolytic cleavage, such as the proteases (convertases and factors) of the complement and clotting cascades, and 4) proteolytically activated effector molecules such as complement components C3 and C4, and fibrinogen.
  • signaling molecules such as small G proteins, trimeric G protein alpha subunits, tyrosine kinases, and G protein-coupled receptors
  • enzymes under allosteric control such as the proteases (convertases and factors) of the complement and clotting cascades
  • proteolytically activated effector molecules such as complement components C3 and C4, and fibrinogen.
  • the method of the invention can be used to stabilize a protein in a biologically active conformation, e.g., a conformation that is enzymatically active or has ligand binding capacity and/or effector functions.
  • the method of the invention can be used to stabilize a protein in a biologically inactive conformation, e.g., a conformation that is enzymatically inactive or does not have ligand binding capacity and/or effector functions.
  • Proteins that are stabilized in a particular conformation may find further use in, for example, in proteomic screening technologies.
  • proteomic screens of tissues and disease states antibodies, polypeptide, and/or small molecules that are specific for, e.g., an active protein conformer or an inactive protein conformer, can be used to assess the activity of different cellular signaling, metabolic, and adhesive pathways.
  • an active protein conformer or an inactive protein conformer can be used to assess the activity of different cellular signaling, metabolic, and adhesive pathways.
  • associations can be made between specific diseases and the activation of specific biochemical and signaling pathways.
  • the invention relates to the polypeptides, antibodies, and small molecules identified using the methods described herein and uses for same, e.g., to treat integrin-related disorders, for example, inflammatory or immune system disorders, cellular proliferative disorders, thrombotic disorders, or endothelial cell disorders.
  • Conformer-specific reagents can also be placed on chips and used to screen tissue extracts, or used to stain tissue sections.
  • drugs or antibodies which specifically recognize a modified integrin , e.g., an anti-LFA-1 antibody which specifically recognizes a modified LFA-1 protein, an anti- ⁇ V ⁇ 3 antibody which specifically recognizes a modified ⁇ V ⁇ 3 protein, or an anti- ⁇ llb ⁇ 3 antibody which specifically recognizes a modified ⁇ - ⁇ llb ⁇ 3 protein, that are selective for a particular conformer, or act to stabilize a particular conformer, e.g., an extended, active conformer or a bent, inactive conformer, may provide differential therapeutic effects.
  • anti-integrin antibodies which specifically recognize a modified integrin e.g., an anti-LFA-1 antibody which specifically recognizes a modified LFA-1 protein, an anti- ⁇ V ⁇ 3 antibody which specifically recognizes a modified ⁇ V ⁇ 3 protein, or an anti- ⁇ llb ⁇ 3 antibody which specifically recognizes a modified ⁇ - ⁇ llb ⁇ 3 protein, that are selective for a particular conformer, or act to stabilize a particular conformer, e.g., an extended
  • integrins which are fixed in a particular state, e.g., an inactive state, can be used in screening and designing drugs and antibodies that stabilize the receptor molecule in a desired state and do not induce shape changes in the receptor molecule, thus preventing inducement of auto-antibody production against exposed portions of the receptor. Therefore, selective screening assays using a protein stabilized in a particular conformer can be used to rationally obtain compounds with a desired activity and with reduced adverse effects.
  • the "glycan wedge,” or “glycan chain” which is introduced into the integrin is, in one embodiment, an N-glycosylation.
  • the amino acid sequence of the subunit e.g., the ⁇ subunit is mutated.
  • the consensus motif for N-glycosylation is Asn-Xaa-Ser/Thr. Accordingly, either one or two amino acid substitutions may be made to the amino acid sequence.
  • An unnatural glycosylation site can be created by mutating the first residue of the consensus motif to Asn and/or the third residue to either Ser or Thr.
  • the invention provides a modified integrin which is biased for a high-affinity state, said integrin comprising an and a ⁇ subunit and having an unnatural glycosylation site introduced into said ⁇ subunit such that the high-affinity state of said integrin is more populated.
  • the integrin is ⁇ llb ⁇ 3 or ⁇ V ⁇ 3.
  • a "modified integrin,” as used herein includes any modified integrin, e.g., a modified I-domain-containing integrin or a modified non-I-domain- containing integrin, which comprises at least one subunit which has been altered (i.e., which has an amino acid sequence which has been altered by the hand of man) with respect to the wild-type sequence or the native state.
  • the ⁇ subunit is ⁇ 3, the unnatural glycosylation produced by a mutation at amino acid residue 303 of the ⁇ 3 subunit.
  • the ⁇ subunit is ⁇ 2, and wherein said unnatural glycosylation is produced by a mutation at amino acid residue 293 of the ⁇ 2 subunit.
  • the ⁇ subunit is ⁇ l, and the unnatural glycosylation is produced by a mutation at amino acid residue 333 of said ⁇ l subunit.
  • the ⁇ subunit is ⁇ l, and the unnatural glycosylation is at amino acid residue 431 of said ⁇ l subunit, this glycosylation site is produced by mutation of amino acid residue 429 in the mature sequence.
  • the integrin is ⁇ llb ⁇ 3 or ⁇ V ⁇ 3.
  • the invention features proteins, e.g., integrins that are modified by an internal deletion of amino acids of the ⁇ subunit, e.g., the ⁇ l, ⁇ 2, or ⁇ 3 subunit of an integrin, thereby biasing the protein to an extended conformation.
  • Deletion of at least three internal amino acids of the terminal C-helix of the ⁇ subunit alters the conformational preference of the protein, e.g., integrin, in an extended conformation.
  • the internal deletion is a deletion of Lys 340 -Leu- Ser-Ser in the ⁇ 2 subunit.
  • the internal deletion is a deletion of Asn 336 -Ala-Tyr-Asn in the ⁇ 2 subunit.
  • the method comprises introducing at least one unnatural glycosylation site into the protein.
  • the protein is stabilized in a desired conformation by the introduction of a glycan wedge.
  • the glycosylation site is formed by the mutation of at least one amino acid of the protein, e.g. the ⁇ subunit of an integrin.
  • the glycosylation site is introduced at the inner side of the hybrid domain I-like domain interface of the ⁇ subunit to alter the conformational preference.
  • Sites to modify can be at least partially solvent accessible, e.g., at least 40, 50, 70, 80, or 90% solvent accessible. Sites can be found, e.g., by visual inspection, computation, distance measurements, and by scanning mutagenesis (e.g., by making a set of mutations and evaluating proteins encoded by each - such strategies have been used in other contexts such as alanine scanning). Accordingly, in one aspect of the invention, a method is provided for stabilizing an integrin comprising an ⁇ and a ⁇ subunit in a desired conformation, said method comprising introducing an unnatural glycosylation site into one subunit, such that the integrin is stabilized in a desired global extended conformation.
  • Proteins e.g., integrins, locked in a desired conformation are particularly useful in screening assays designed to identify compounds that bind to and/or modulate the activity of the protein without eliciting further structural changes in the protein.
  • Integrins that have an altered conformation e.g., that favor a low affinity (inactive) or high affinity (active) conformation can be used in differential screening assays designed to identify compounds, that are specific to the low or high affinity conformation.
  • the modified integrins of the invention can also be used in screening assays to identify compounds which stabilize or preferentially bind to an active conformation.
  • the modified, constitutively active integrins of the invention can also be used as a standard or control or as an assay reagent in an assay designed to identify compounds that preferentially bind to or stabilize an inactive conformation.
  • the compound may be selected from the group consisting of a peptide, a peptidomimetic, a small molecule, or an anti-integrin antibody.
  • the invention provides methods for identifying a compound that selectively binds to an integrin in a high-affinity state comprising determining the ability of said compound to bind to a modified integrin which is fixed in a high- affinity state as compared to a modified integrin which is fixed in a low-affinity state and selecting said compound based on its ability to selectively bind to the integrin which is fixed in a high-affinity state.
  • the compound stabilizes the high-affinity state.
  • the invention provides a method for identifying a modulator of integrin activity comprising providing a modified integrin; contacting the modified integrin with a test compound; and assaying the ability of the test compound to bind to the modified integrin to thereby identify a modulator of integrin activity, hi one embodiment, the integrin is ⁇ llb ⁇ 3, ⁇ V ⁇ 3, or ⁇ L ⁇ 2 (LFA-1).
  • the invention also provides compounds identified by the above-identified methods. Integrins
  • Integrins exist on cell surfaces in an inactive conformation that does not bind ligand. Upon cell activation, integrins change shape (conformation) and can bind ligand. Over 20 different integrin heterodimers (different ⁇ and ⁇ subunit combinations) exist that are expressed in a selective fashion on all cells in the body. After activation, integrins bind in a specific manner to protein ligands on the surface of other cells, in the extracellular matrix, or that are assembled in the clotting or complement cascades. Integrins on leukocytes are of central importance in leukocyte emigration and in inflammatory and immune responses.
  • Ligands for the leukocyte integrin Mac-1 include the inflammation-associated cell surface molecule ICAM-1, the complement component iC3b, and the clotting component fibrinogen.
  • Ligands for the leukocyte integrin LFA-1 include ICAM-1, ICAM-2, and ICAM-3.
  • Antibodies to leukocyte integrins can block many types of inflammatory and auto-immune diseases, by, e.g., modulating, e.g., inhibiting, for example, cell to cell interactions or cell to extracellular matrix interactions and inflammatory or immune system disorders.
  • Integrins on platelets are important in thrombotic disorders and in heart disease; approved drugs include the antibody abciximab (ReoproTM) and the peptide-like antagonist eptifibatide (IntegrilinTM).
  • Integrins on connective tissue cells, epithelium, and endothelium, e.g., ⁇ V ⁇ 3, are important in disease states affecting these cells. They regulate cell growth, differentiation, wound healing, fibrosis, apoptosis, and angiogenesis and therefore modulate endothelial cell disorders, cellular proliferation disorders, and tumorigenic disorders. Integrins on cancerous cells regulate invasion and metastasis.
  • drugs are needed that bind to a particular conformation.
  • Most antibodies bind to both the active and inactive conformations, since only a small portion of the surface of the integrin molecule changes shape.
  • Drugs currently used as anti-integrins have limitations based on the development of autoimmune response induced by integrin conformational changes. Therefore, antibodies or other compounds which selectively bind to and stabilize the bent or inactive conformation are superior because they prevent autoimmune responses and thus reduce harmful side effects.
  • the methods described herein have been successfully used to introduce disulfide bonds into the I domains of the integrins, e.g., LFA-1 and Mac-1.
  • the methods described herein have also been successfully used to introduce disulfide bonds into the I-like domains of integrins which may or may not contain an I domain.
  • ⁇ V ⁇ 3 and ⁇ llb ⁇ 3 do not contain I domains.
  • the methods described herein have been used to introduce disulfide bonds into I-like domains contained within ⁇ subunits.
  • the methods described herein have also been used to introduce disulfide bonds into between two domains of multi-domain proteins, e.g., ⁇ subunits, and to introduce disulfide bonds between the ⁇ and ⁇ subunits of integrins.
  • the invention provides a modified integrin containing at least one disulfide bond, such that said modified integrin is stabilized in a desired conformation, e.g., a global or local conformation.
  • a modified integrin of the invention may be derived from a modification to an integrin subunit including ⁇ l, ⁇ 2, ⁇ lO, ⁇ l 1, oD, oE, ⁇ L (CDl la), ⁇ M (CDl lb) and ⁇ X (CDl lc), ⁇ V ⁇ 3 and ⁇ llb ⁇ 3.
  • a modified integrin of the invention may be also be derived from a modification to an integrin ⁇ subunit including ⁇ 1 , ⁇ 2, and ⁇ 3. The integrin may or may not contain an I domain.
  • derived from or “derivative”, as used interchangeably herein, are intended to mean that a sequence is identical to or modified from another sequence, e.g., a naturally occurring sequence.
  • Derivatives within the scope of the invention include polynucleotide and polypeptide derivatives.
  • Polypeptide or protein derivatives include polypeptide or protein sequences that differ from the sequences described or known in amino acid sequence, or in ways that do not involve sequence, or both, and still preserve the activity of the polypeptide or protein.
  • Derivatives in amino acid sequence are produced when one or more amino acid is substituted with a different natural amino acid, an amino acid derivative or non-native amino acid.
  • protein derivatives include naturally occurring polypeptides or proteins, or biologically active fragments thereof, whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions, which typically have minimal influence on the secondary structure and hydrophobic nature of the protein or peptide.
  • Derivatives may also have sequences which differ by one or more non-conservative amino acid substitutions, deletions or insertions which do not abolish the biological activity of the polypeptide or protein.
  • Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics (e.g., charge, size, shape, and other biological properties) such as substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • the non- polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • derivatives with amino acid substitutions which are less conservative may also result in desired derivatives, e.g., by causing changes in charge, conformation and other biological properties.
  • substitutions would include, for example, substitution of hydrophilic residue for a hydrophobic residue, substitution of a cysteine or proline for another residue, substitution of a residue having a small side chain for a residue having a bulky side chain or substitution of a residue having a net positive charge for a residue having a net negative charge.
  • the derivatives may be readily assayed according to the methods disclosed herein to determine the presence or absence of the desired characteristics.
  • polypeptides and proteins of this invention may also be modified by various changes such as insertions, deletions and substitutions, either conservative or nonconservative where such changes might provide for certain advantages in their use.
  • a modified integrin is stabilized in the open conformation, and binds ligand with high affinity.
  • a modified integrin is stabilized in the bent or closed, e.g., inactive conformation, and binds ligand with low affinity.
  • a modified integrin e.g., ⁇ llb ⁇ 3, ⁇ V ⁇ 3, or ⁇ L ⁇ 2 (LFA-1), or a modified integrin subunit, e.g., an ⁇ or ⁇ subunit, may be modified such that it is stabilized in a desired local or global conformation.
  • a disulfide bond can be introduced within a single domain of a protein, e.g., in an I domain of an ⁇ subunit or in an I-like domain of a ⁇ subunit of an integrin.
  • a disulfide bond can be introduced between two domains of a multi- domain protein, e.g., an ⁇ or ⁇ subunit of an integrin, or between two chains or subunits of a heterodimer, e.g., an integrin heterodimer.
  • Exemplary locally-stabilized I domain integrins include, but are not limited to, the ⁇ L K287C/K294C, E284C/E301C, L161C/F299C, K160C/F299C, L161C/T300C, and L289C/K294C mutants, and the ⁇ M Q163C/Q309C and D294C/Q311C mutants which are stabilized in the "open" conformation that bind ligand with high or intermediate affinity; and the ⁇ L L289C/K294C mutant and the ⁇ M Q163C/R313C mutants which are stabilized in an inactive or "closed" conformation that does not bind ligand.
  • the affinity of E284C/E301C is nearly comparable to that of K287C/K294C, e.g., high-affinity.
  • the affinity of L161C/F299C, K160C/F299C, and L161C/T300C are significantly higher than wild-type, but 20-30 times lower than high-affinity ⁇ L I-domain, K287C/K294C.
  • LI 61 C/F299C, Kl 60C/F299C, and L161C/T300C are referred to herein as intermediate affinity ⁇ L I-domains.
  • the invention provides a modified integrin I-like domain protein containing at least one disulfide bond, such that said modified I-like domain protein is stabilized in a desired conformation.
  • a modified integrin I-like domain protein is stabilized in the open conformation, and binds ligand with high affinity. In another embodiment, a modified integrin I-like domain protein is stabilized in the closed conformation, and binds ligand with low affinity. In one embodiment, a modified integrin I-like domain proteins of the invention is encoded by an amino acid sequence containing at least one cysteine substitution, and preferably two cysteine substitutions, as compared to the wild-type sequence. In another embodiment, the invention provides a modified integrin I-like domain which is comprised within an integrin ⁇ subunit, and which may be further associated with an integrin ⁇ subunit. In another embodiment, a modified integrin I- like domain protein of the invention is a soluble protein. Furthermore, the invention provides a modified integrin I-like domain protein which is operatively linked to a heterologous protein.
  • an inactive, locally-stabilized conformer for ⁇ llb ⁇ 3 comprises a wild type ⁇ subunit and a ⁇ 3 subunit wherein cysteine substitutions have been introduced at Thr329 and Ala347 or at Val332 and Ala347, or corresponding residues in other subunits.
  • An example of an inactive, locally-stabilized conformer for ⁇ llb ⁇ 3 comprises a wild-type ⁇ subunit and a ⁇ 3 subunit wherein cysteine substitutions have been introduced Gly331 and Leu343 or Val332 and Leu332 or corresponding residues in other subunits.
  • an inactive, locally-stabilized conformer for ⁇ L ⁇ 2 (LFA-1) comprises a wild-type ⁇ subunit and a ⁇ 2 subunit wherein cysteine substitutions have been introduced at Gly321 and Ala337.
  • Another inactive, locally-stabilized conformer for ⁇ L ⁇ 2 comprises a wild-type ⁇ subunit and a ⁇ 2 subunit wherein cysteine substitutions have been introduced at Ala319 and Ala337 or Lys340.
  • an active, locally-stabilized conformer for ⁇ 3 integrins comprises an integrin wherein cysteine substitutions have been introduced into the ⁇ 3 subunit at Met335 and Val332 and/or Gly321 and Leu333, and/or corresponding amino acids in other subunits.
  • An example of the design of an active conformer for ⁇ 2 integrins comprises an integrin wherein cysteine substitutions have been introduced into the ⁇ 2 subunit at Gly321 and Leu333.
  • An inactive, globally-stabilized conformation for ⁇ V ⁇ 3 or ⁇ llb ⁇ 3 comprises, for example, a wild-type ⁇ subunit and a ⁇ 3 subunit wherein cysteine substitutions have been introduced between two domains, e.g., the I-like domain and the /3-tail domain, at Val332 and Ser674, respectively.
  • an inactive, globally-stabilized conformation for ⁇ V ⁇ 3 comprises an ⁇ subunit wherein a cysteine substitution has been introduced within the /3-propeller domain of the ⁇ subunit at, e.g., Gly307, and a ⁇ 3 subunit wherein cysteine substitutions have been introduced within the I-EGF4 domain at, e.g.,
  • an inactive, globally-stabilized conformation for ⁇ llb ⁇ 3 comprises an ⁇ subunit wherein a cysteine substitution has been introduced within the /3-propeller domain of the ⁇ subunit at, e.g., Ala318 and a ⁇ 3 subunit wherein cysteine substitutions have been introduced within the I-EGF4 domain at, e.g., Asp552.
  • an inactive, globally-stabilized conformation for ⁇ llb ⁇ 3 comprises an ⁇ subunit wherein a cysteine substitution has been introduced within the
  • ⁇ subunit at, e.g., Arg320 and a ⁇ 3 subunit wherein cysteine substitutions have been introduced within the I-EGF4 domain at, e.g.,
  • Integrins are key targets in many diseases. Accordingly, isolated modified integrins of the invention, as well as integrin modulating compounds of the invention, e.g., integrin antibodies, or small molecules selective for integrins in the bent conformation can be used to modulate, e.g., inhibit or prevent, integrin-mediated disorders. Integrins locked in a low affinity (inactive) conformation can be used in differential screening assays designed to identify compounds that are specific to the low affinity conformation. Furthermore, co-crystals of integrins bound to natural ligands and/or small molecule antagonists can readily be made, which will enable computational drug design, and advance modification and improvement of drug development candidates.
  • the invention provides a method for identifying a modulator of integrin activity comprising assaying the ability of a test compound to preferentially bind to and/or stabilize a modified integrin which is locked in the bent (inactive) conformation.
  • the modified, inactive integrins of the invention can also be used as a standard or control or as an assay reagent in an assay designed to identify compounds that preferentially bind to or stabilize an active conformation.
  • the invention also provides a composition comprising a modified integrin or an anti-mtegrin antibody (or an antigen binding fragment thereof) which selectively binds to a modified integrin e.g., an integrin in the bent conformation, and a pharmaceutically acceptable carrier.
  • a modified integrin e.g., an integrin in the bent conformation
  • a pharmaceutically acceptable carrier e.g., a pharmaceutically acceptable carrier.
  • the compositions of the invention are used in therapeutic methods of the invention.
  • the invention provides methods for treating or preventing an integrin-mediated disorder (e.g., an inflammatory disorder, an autoimmune disorder, a thrombotic disorder, an endothelial cell disorder, or a cellular proliferative disorder) in a subject.
  • the modified integrin for therapeutic use is a soluble polypeptide, e.g., a fusion protein.
  • an integrin mediated disorder includes, for example, an inflammatory or immune system disorder, a cellular proliferative disorder, a thrombotic disorder, or an endothelial cell disorder.
  • An inflammatory or immune system disorder includes, but is not limited to adult respiratory distress syndrome (ARDS), multiple organ injury syndromes secondary to septicemia or trauma, viral infection, inflammatory bowel disease, ulcerative colitis, Crohn's disease, multiple sclerosis, leukocyte adhesion deficiency II syndrome, thermal injury, hemodialysis, leukapheresis, peritonitis, chronic obstructive pulmonary disease, lung inflammation, asthma, acute appendicitis, dermatoses with acute inflammatory components, wound healing, septic shock, acute glomerulonephritis, nephritis, amyloidosis, reactive arthritis, rheumatoid arthritis, chronic bronchitis, Sjorgen's syndrome, sarcoidosis, scleroderma,
  • a “cellular proliferative disorder” includes those disorders that affect cell proliferation, activation, adhesion, growth, differentiation, or migration processes.
  • a “cellular proliferation, activation, adhesion, growth, differentiation, or migration process” is a process by which a cell increases in number, size, activation state, or content, by which a cell develops a specialized set of characteristics which differ from that of other cells, or by which a cell moves closer to or further from a particular location or stimulus.
  • Such disorders include cancer, e.g., carcinoma, sarcoma, lymphoma or leukemia, examples of which include, but are not limited to, breast, endometrial, ovarian, uterine, hepatic, gastrointestinal, prostate, colorectal, and lung cancer, melanoma, neurofibromatosis, adenomatous polyposis of the colon, Wilms' rumor, nephroblastoma, teratoma, rhabdomyosarcoma; tumor invasion, angiogenesis and metastasis; skeletal dysplasia; hematopoietic and/or myeloproliferative disorders.
  • cancer e.g., carcinoma, sarcoma, lymphoma or leukemia
  • thrombootic disorder includes any disorder or condition characterized by excessive or unwanted coagulation or hemostatic activity, or a hypercoagulable state.
  • Thrombotic disorders include disorders diseases involving platelet adhesion and thrombus formation, and may manifest as an increased propensity to form thromboses, e.g., an increased number of thromboses, thrombosis at an early age, a familial tendency towards thrombosis, and thrombosis at unusual sites.
  • thrombotic disorders include, but are not limited to, thromboembolism, deep vein thrombosis, pulmonary embolism, stroke, myocardial infarction, miscarriage, thrombophilia associated with anti-thrombin HI deficiency, protein C deficiency, protein S deficiency, resistance to activated protein C, dysf ⁇ brinogenemia, fibrinolytic disorders, homocystinuria, pregnancy, inflammatory disorders, myeloproliferative disorders, arteriosclerosis, angina, e.g., unstable angina, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, cancer metastasis, sickle cell disease, and glomerular nephritis, or thrombotic events during or after therapeutic clot lysis or procedures such as angioplasty or surgery.
  • angina e.g., unstable angina, disseminated intravascular coagulation, thrombotic thrombocytopen
  • an "endothelial cell disorder” includes a disorder characterized by aberrant, unregulated, or unwanted endothelial cell activity, e.g., proliferation, migration, angiogenesis, or vascularization.
  • Endothelial cell disorders include tumorigenesis, tumor metastasis, psoriasis, diabetic retinopathy, endometriosis, Grave's disease, ischemic disease (e.g., atherosclerosis), and chronic inflammatory diseases (e.g., rheumatoid arthritis), and osteoporosis.
  • a "tumorigenic disease or disorder” includes a disease or disorder characterized by aberrantly regulated cell growth, proliferation, differentiation, adhesion, or migration, resulting in the production of or tendency to produce tumors.
  • a "tumor” includes a normal benign or malignant mass of tissue.
  • tumorigenic diseases include cancer, e.g., carcinoma, sarcoma, lymphoma or leukemia, examples of which include, but are not limited to, ovarian, lung, breast, endometrial, uterine, hepatic, gastrointestinal, prostate, colorectal, and brain cancer.
  • Modified integrins of the invention include modified soluble modified integrin I-domain and I-like domain proteins; and modified integrins that are operatively linked to a heterologous polypeptide, e.g., fusion proteins.
  • cDNAs for multiple human integrin and ⁇ subunit polypeptides have been cloned and sequenced, and the polypeptide sequences have been determined (see, for example, GenBank Accession Numbers: NM_002203 (d ⁇ ), AFl 12345 ( ⁇ lO), NM_012211 ( ⁇ l 1), NM_005353 (oD), NM_002208 ( ⁇ E), NM_000887 ( ⁇ X),
  • NM_000632 ( ⁇ M), NM_002209 ( ⁇ L), X68742 ( ⁇ l), NM_033669 ( ⁇ l), NM_000211 ( ⁇ l), NM_000212 (/33), NM_002214 ( ⁇ 8)).
  • polypeptide sequences encoding human ⁇ L and ⁇ M are set forth as SEQ ID NO:2 (GenBank Accession No. P20701) and SEQ ID NO:4 (GenBank Accession No. PI 1215), respectively.
  • the polypeptide sequence encoding human ⁇ llb is set forth as SEQ JD NO:6 (GenBank
  • the polypeptide sequence encoding human ⁇ V is set forth as SEQ ID NO:8 (GenBank Accession No. NP_00201.
  • the polypeptide sequences encoding human ⁇ l, 82, and ⁇ 3 axe set forth as SEQ ID NO.T0 (GenBank Accession No. NP_391989), SEQ ID NO:12 (GenBank Accession No. 000202), and SEQ ID NO:14 (GenBank Accession No. 000203), respectively.
  • References to amino acid positions are with respect to mature protein sequences, not the unprocessed, unless indicated. Typically, however, references to amino acid positions in human ⁇ 3 are according to the numbering from the PDB file 1 JV2 (chain B) and is set forth as follows:
  • references to amino acid positions in human ⁇ 1 are according to the numbering of the mature human ⁇ l protein which begins at amino acid 21 of SEQ ID NO.T0 (an offset of 20).
  • amino acids Asn310-Ile311-Gln312 of mature human ⁇ l protein correspond to residues 350-352 of SEQ ID NO: 10.
  • references to amino acid positions in human ⁇ 2 are according to the numbering of the mature human ⁇ 2 protein which begins at amino acid 23 of SEQ ID NO:12 (an offset of 22).
  • amino acids Asn336-Ala-Tyr-Asn of mature human ⁇ 2 protein correspond to residues 358-361 of SEQ ID NO:12.
  • FIG. 13 describes the three-dimensional organization of an integrin in a bent state.
  • the domains are organized as follows: PSI domain from about amino acids 1- 54;
  • a ⁇ integrin extracellular domain includes at least seven of these eight domains, or more commonly all eight domains.
  • Corresponding amino acids in other integrins can be easily identified by the methods described herein (e.g., alignment and threading). It is appreciated, that at domain boundaries, sequence dependent effects may determine whether a particular position is within one domain or an adjacent one.
  • Isolated modified integrins of the present invention preferably have an amino acid sequence that is sufficiently identical to the amino acid sequence of a native integrin, yet which comprise at least one, and preferably two cysteine substitutions, such that a disulfide bond is formed that stabilizes the protein in a desired conformation.
  • the term "sufficiently identical” refers to an amino acid (or nucleotide) sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue that has a similar side chain) amino acid residues (or nucleotides) to a integrin amino acid (or nucleotide) sequence such that the polypeptide shares common structural domains or motifs, and/or a common functional activity with a native integrin (e.g., ability to bind the cognate ligand of the integrin).
  • a sufficient or minimum number of identical or equivalent e.g., an amino acid residue that has a similar side chain amino acid residues (or nucleotides) to a integrin amino acid (or nucleotide) sequence such that the polypeptide shares common structural domains or motifs, and/or a common functional activity with a native integrin (e.g., ability to bind the cognate ligand of the integrin).
  • amino acid or nucleotide sequences which share at least 90%, 91%, 92%, 93%, 94%, 95%, 97, 98, 99% or greater identity and share a common functional activity (e.g., an activity of a modified integrin as described herein) are defined herein as sufficiently identical.
  • An integrin may differ in amino acid sequence from the integrins disclosed herein due to natural allelic variation or mutagenesis. The integrin may differ by less than 7, 5, 4, 3, or 2 alterations (e.g., substitutions, insertions, or deletions).
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Bioscl, 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • a "biologically active portion" of a modified protein e.g., a modified integrin or an ⁇ or ⁇ subunit includes a fragment of a modified integrin or integrin subunit which retains a modified integrin activity.
  • a biologically active portion of a modified integrin comprises at least one domain or motif with at least one activity of the modified integrin, e.g., ligand binding.
  • Biologically active portions of a modified integrin may comprise amino acid sequences sufficiently identical to or derived from the amino acid sequence of a modified integrin, which include less amino acids than the full length modified integrin, and exhibit at least one activity of a modified integrin.
  • Biologically active portions of a modified integrin can be used as targets for developing agents which modulate a integrin activity, e.g., ligand binding, adhesion, e.g., cell to cell adhesion or cell to extracellular matrix adhesion, and/or signaling activity.
  • a biologically active portion of a modified integrin comprises a protein which can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a modified integrin.
  • modified integrin are produced by recombinant DNA techniques.
  • a modified integrin can be isolated from a host cell transfected with a polynucleotide sequence encoding a modified integrin (e.g., an ⁇ or ⁇ subunit) using an appropriate purification scheme using standard protein purification techniques.
  • a modified integrin can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” polypeptide or protein, or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the source, e.g., the cellular source, from which the modified integrin is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of modified integrin in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of modified integrin having less than about 30% (by dry weight) of non- modified mtegrin (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-modified integrin, still more preferably less than about 10% of non-modified integrin, and most preferably less than about 5% non-modified integrin.
  • non-modified mtegrin also referred to herein as a "contaminating protein”
  • the modified integrin or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of modified integrin in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of modified integrins having less than about 30% (by dry weight) of chemical precursors or non-modified integrin chemicals, more preferably less than about 20% chemical precursors or non-modified integrin chemicals, still more preferably less than about 10% chemical precursors or non- modified integrin chemicals, and most preferably less than about 5% chemical precursors or non-modified integrin chemicals.
  • a modified integrin "chimeric protein" or “fusion protein” comprises a modified integrin operatively linked to a non-modified integrin, e.g., a heterologous integrin.
  • a modified integrin fusion protein comprises at least an I-domain or an I-like domain.
  • the term "operatively linked” is intended to indicate that the modified integrin and the heterologous polypeptide sequences are fused in-frame to each other.
  • the heterologous polypeptide can be fused to the N-terminus or C-terminus of the modified integrin.
  • the fusion protein is a modified integrin- fusion protein in which the Fc region, e.g., the hinge, CI and C2 sequences, of an immunoglobulin, (e.g., human IgGl) is fused to the C-terminus of the modified integrin sequences.
  • an immunoglobulin e.g., human IgGl
  • Integrin immunoglobulin chimeras can be constructed essentially as described in WO 91/08298. Such fusion proteins can facilitate the purification of recombinant modified integrins.
  • the fusion protein is a modified integrin fused to a heterologous transmembrane domain, such that the fusion protein is expressed on the cell surface
  • the fusion protein is designed so that each subunit is attached to amino acid sequences that can interact, e.g., a clasp, or a small-molecule dependent dimerization domain (e.g., FKBP which can be dimerized by FK1012).
  • modified integrins and fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo.
  • a soluble modified integrin stabilized in an open, ligand binding conformation, or fusion protein thereof may be used to modulate integrin activity (e.g., integrin binding to a cognate ligand) in a subject.
  • a soluble modified integrin or fusion protein may be used to treat an inflammatory disorder, an autoimmune disorder, a thrombotic disorder, an endothelial cell disorder, or a cellular proliferative disorder.
  • soluble modified integrins and fusion proteins can also be used to affect the bioavailability of a mtegrin ligand, e.g., ICAM-1, ICAM-2, fibrinogen, fibronectin, von Willebrand factor, thrombospondin, vitronectin, osteopontin, or collagen.
  • a mtegrin ligand e.g., ICAM-1, ICAM-2, fibrinogen, fibronectin, von Willebrand factor, thrombospondin, vitronectin, osteopontin, or collagen.
  • modified integrins and fusion proteins of the invention can be used as immunogens to produce anti-integrin antibodies in a subject, e.g., anti-LFA-1, anti- ⁇ IIb/33, or anti- ⁇ V/33 antibodies, and in screening assays to identify molecules which modulate integrin activity, and/or modulate the interaction of a integrin with a integrin ligand or receptor.
  • a modified integrin fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a modified integrin -encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the modified integrin.
  • the methods of the present invention may also include the use of modified integrins which function as either integrin agonists (mimetics) or as integrin antagonists.
  • An agonist of an integrin can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of an integrin.
  • An antagonist of an integrin can inhibit one or more of the activities of a native form of the integrin by, for example, competitively modulating an integrin activity.
  • specific biological effects can be elicited by treatment with a modified integrin stabilized in a desired conformation.
  • the invention features ligands that preferentially bind to an integrin in a particular conformation, e.g., relative to another conformation.
  • exemplary ligands include antibodies, modified scaffold proteins, peptides, and small molecules.
  • the ligands bind at least 0.5, 1, 2, 5, 10, 20, 25, 50, or 100 fold better to an integrin in a first conformation relative to an integrin in a second conformation, h one embodiment, the first conformation is a bent conformation and the second conformation is extended. In another embodiment, the first conformation is extended, and the second conformation is bent. In one embodiment, the first conformation is favored by a first integrin modification.
  • the second conformation may be, e.g., an inactive or active conformation adopted by the wild-type integrin or a conformation is favored by the second integrin modification.
  • the first integrin modification can create an artificial glycosylation site or a disulfide bond.
  • An integrin described herein e.g., a modified integrin, e.g., modified LFA-1, modified ⁇ llb ⁇ 3, modified ⁇ V ⁇ 3, or a portion or fragment thereof
  • a ligand that binds to a integrin e.g., a ligand that preferentially binds to a particular conformer.
  • a modified integrin e.g., modified LFA-1, modified ⁇ llb ⁇ 3, modified ⁇ V ⁇ 3, or a portion or fragment thereof
  • an immunogen can be used as an immunogen to generate antibodies that bind to a specific conformation of an mtegrin, e.g., a global bent or extended conformation, using standard techniques for polyclonal and monoclonal antibody preparation (see, generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, e.g., an integrin in an extended or bent conformation, or a modified integrin domain, such as an I-domain or an I-like domain, e.g., an open or closed I-domain or an open or closed I-like domain.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind a modified integrin, or a portion or fragment thereof.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular modified integrin, or a portion or fragment thereof with which it immunoreacts.
  • a monoclonal anti-integrin antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with a modified integrin, e.g., a modified integrin stabilized in the open or closed conformation, to thereby isolate immunoglobulin library members that bind to an conformation specific epitope on an integrin, e.g., an open or closed confo ⁇ nation.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No.
  • recombinant anti-integrin antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, can also be used in the methods of the present invention.
  • chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No.
  • the antibody is human or at least partially human.
  • variable domains of the antibody can include at least one, two, or three framework regions (e.g., FR1, FR2, FR3, or FR4) that are human.
  • all four FR regions can be human.
  • Some or all of the constant regions can be human, e.g., one of the human isoforms of such regions.
  • one or more CDRs of one or both of the immunoglobulin variable domains is human.
  • CDR1, CDR2, and/or CDR3 can be human.
  • Human CDRs can be obtained, e.g., by amplifying immunoglobulin coding mRNA from human spleen cells.
  • part of the antibody may be synthetic, e.g., one or more the CDRs of one or both the immunoglobulin variable domains is synthetic (e.g., non-human and non-mammalian in origin). See, e.g., de Wildt et al. (2000) Eur. J. Immunol. 30:254- 261.
  • an anti-integrin antibody of the invention binds selectively to an integrin in the open, high-affinity conformation, e.g., at an epitope that is unique to an activated integrin (also referred to herein as an activation specific epitope).
  • an anti-integrin antibody of the invention modulates (e.g., inhibits) the binding interaction between an activated integrin and its cognate ligand.
  • an anti-integrin antibody inhibits leukocyte adhesion and/or aggregation, cellular proliferation, migration, or growth, or thrombosis.
  • An integrin-binding ligand e.g., an anti-integrin antibody (e.g., a monoclonal antibody)
  • an integrin-binding ligand can be used in the methods of the invention to modulate the expression and/or activity of an integrin.
  • the ligand or anti-integrin antibody can also be used to isolate modified integrins or integrin polypeptides (e.g., in a particular conformation), or fusion proteins by standard techniques, such as affinity chromatography or immunoprecipitation.
  • an integrin-binding ligand an anti- integrin antibody
  • the ligand can be used to remove and/or kill cells expressing activated integrin.
  • an anti-integrin antibody can be used to detect integrins in a particular conformation (e.g., an activated integrin), for example, for the localization of stimulated and/or activated leukocytes.
  • an anti-integrin antibody can be used therapeutically as described herein. Accordingly anti-integrin antibodies can be used diagnostically to monitor protein levels in blood as part of a clinical testing procedure, e.g., to, for example, detect inflammation, cell adhesion, or cell proliferation. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin biotin;
  • suitable fluorescent materials include umbelliferone, fiuorescein, fiuorescein isothiocyanate, rhodamine, dichlorotriazinylamine fiuorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 1, 131 I, 35 S or 3 H.
  • the ligand or antibody binds to a unique and novel epitope.
  • the epitope can differ (e.g., can be non-overlapping with, or partially overlapping, but detectable distinct) from the epitope bound by a monoclonal or recombinant antibody publically available prior to July 17, 2003.
  • the epitope can differ from the epitope bound by BL5, F8.8, CBRLFA-1/9, TS2/6, May.035, TS1/11, TS1/12, TS1/22, TS2/14, 25-3-1, CBRLFA-1/1, S6F1, , TS1/18, YFC51, CLBLFA-1/1, and CBRLFA-1/7.
  • the ligand (e.g., the antibody) does not bind to an mtegrin molecule described in US 2003-0054440 or US 2002-0123614, or binds preferentially to an integrin described herein relative to an integrin described in US 2003-0054440 or US 2002-0123614 (e.g., an integrin with an intra-domain disulfide bond in the I-domain of the integrin ⁇ subunit.
  • the invention includes the use of isolated nucleic acid molecules that encode modified integrin proteins or polypeptides or biologically active portions thereof.
  • the nucleic acid can encode at least one modified domain of an integrin protein, e.g., a domain, which when assembled into a heterodimeric protein complex has an altered conformational preference to a corresponding wild-type heterodimeric protein.
  • the nucleic acid can be used, e.g., to express the domain (e.g., for a binding assay, structure determination, or therapeutic purpose) or to construct a nucleic acid which includes a full integrin ectodomain, or a full-length integrin subunit.
  • the isolated nucleic acid molecules of the present invention include the nucleotide sequences of SEQ ID NO:9, SEQ ID NO:ll, and SEQ ID NO:13, encoding the modified amino acid sequences of the ⁇ l, 82, and ⁇ 3 mutants described herein.
  • isolated nucleic acid molecule includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • isolated m cludes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • an isolated nucleic acid molecule encoding a modified integrin I- domain polypeptide can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • nucleotide sequence encoding a modified integrin thereby leading to changes in the amino acid sequence of the encoded modified integrin, without further altering the structural characteristics or functional ability of the modified integrin.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence encoding a modified integrin.
  • non-essential amino acid residue is a residue that can be altered from the sequence of a modified integrin without further altering the structure and/or biological activity, hi accordance with the methods of the invention, computational design and modeling are used to determine which amino acid residues are amenable to alteration in order to achieve the desired protein conformation.
  • the methods of the invention may include the use of nucleic acid molecules encoding modified integrins that contain changes in amino acid residues that are not essential for activity.
  • conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • a predicted nonessential amino acid residue in a modified integrin is preferably replaced with another amino acid residue from the same side chain family.
  • vectors for example, recombinant expression vectors, containing a nucleic acid encoding a modified integrin (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the methods of the invention may include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., modified integrins, fusion proteins, and the like).
  • the invention provides a method for producing a modified integrin, by culturing in a suitable medium, a host cell of the invention (e.g., a prokaryotic or eukaryotic host cell) containing a recombinant expression vector such that the protein is produced.
  • a host cell of the invention e.g., a prokaryotic or eukaryotic host cell
  • a recombinant expression vector such that the protein is produced.
  • modified integrins or fusion proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility and/or stability of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • integrin fusion proteins e.g., soluble I-domain-Ig
  • GST glutathione S-transferase
  • maltose E binding protein or protein A, respectively, to the target recombinant protein.
  • Purified modified integrin fusion proteins e.g., soluble I-domain-Ig
  • Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, (1988) Gene 69:301-315) and pET lid (Studier et al, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET lid vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, (1992) Nucleic Acids Res. 20:2111- 2118).
  • Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari, et al, (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (InVitrogen Corp, San Diego, CA).
  • modified integrins can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include ⁇ CDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBOJ. 6:187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalo virus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.
  • T cell receptors Winoto and Baltimore (1989) EMBO J. 8:729-733
  • immunoglobulins Bonerji etal. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748
  • neuron-specific promoters e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477
  • endothelial cell-specific promoters e.g., KDR/flk promoter; U.S. Patent No. 5,888,765
  • pancreas-specific promoters Edlund et al.
  • mammary gland-specific promoters e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166.
  • Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Grass (1990)
  • Another aspect of the invention pertains to host cells into which a nucleic acid molecule encoding a modified integrin of the invention is introduced, e.g., a modified integrin nucleic acid molecule within a recombinant expression vector or a modified integrin nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome.
  • a nucleic acid molecule encoding a modified integrin of the invention is introduced, e.g., a modified integrin nucleic acid molecule within a recombinant expression vector or a modified integrin nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome.
  • the terms "host cell” and "recombinant host cell” are used interchangeably herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a modified integrin or fusion protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as hematopoietic cells, leukocytes, K562 cells, 293T cells, human umbilical vein endothelial cells (HUV ⁇ C), human microvascular endothelial cells (HMV ⁇ C), Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as hematopoietic cells, leukocytes, K562 cells, 293T cells, human umbilical vein endothelial cells (HUV ⁇ C), human microvascular endothelial cells (HMV ⁇ C), Chinese hamster ovary cells (CHO) or COS cells).
  • HMV ⁇ C human umbilical vein endothelial cells
  • HMV ⁇ C human microvascular endo
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, D ⁇ A ⁇ -dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a modified integrin or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a modified integrin for use in the methods of the invention
  • a host cell into which a recombinant expression vector encoding a modified integrin or fusion protein has been introduced
  • a suitable medium such that a modified integrin or fusion protein is produced
  • a modified integrin or fusion protein is isolated from the medium or the host cell.
  • a recombinant cell expressing a modified integrin or fusion protein can also be administered to a subject to modulate integrin activity.
  • the host cells of the invention can also be used to produce non-human transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a modified integrin-coding sequences have been introduced.
  • Such host cells can then be used to create non- human transgenic animals in which exogenous modified integrin sequences have been introduced into their genome or homologous recombinant animals in which endogenous integrin sequences have been altered.
  • Such animals are useful for studying the function and/or activity of a modified integrin molecule and for identifying and/or evaluating modulators of modified integrin activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • Other examples of transgenic animals include non- human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous integrin gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a transgenic animal of the invention can be created by introducing a modified integrin-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g. , by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to a modified integrin transgene to direct expression of a modified integrin to particular cells.
  • a vector is prepared which contains at least a portion of a modified integrin gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the modified integrin gene.
  • the modified integrin gene can be a human gene, but more preferably, is a non-human homologue of a human modified integrin gene.
  • a mouse modified integrin gene can be used to construct a homologous recombination nucleic acid molecule, e.g., a vector, suitable for altering an endogenous modified integrin gene in the mouse genome.
  • the homologous recombination nucleic acid molecule is designed such that, upon homologous recombination, the endogenous modified integrin gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the homologous recombination nucleic acid molecule can be designed such that, upon homologous recombination, the endogenous modified integrin gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous modified integrin protein).
  • the altered portion of the modified integrin gene is flanked at its 5' and 3' ends by additional nucleic acid sequence of the modified integrin gene to allow for homologous recombination to occur between the exogenous modified integrin gene carried by the homologous recombination nucleic acid molecule and an endogenous modified integrin gene in a cell, e.g., an embryonic stem cell.
  • the additional flanking modified integrin nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene.
  • homologous recombination nucleic acid molecule typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the homologous recombination nucleic acid molecule (see, e.g., Thomas, K.R. and Capecchi, M. R. (1987) Cell 51 :503 for a description of homologous recombination vectors).
  • the homologous recombination nucleic acid molecule is introduced into a cell, e.g., an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced modified integrin gene has homologously recombined with the endogenous modified integrin gene are selected (see e.g., Li, E. et al.
  • the selected cells can then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, EJ. Robertson, ed. (JJ L, Oxford, 1987) pp. 113-152).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • homologous recombination nucleic acid molecules e.g., vectors, or homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec et al; WO 91/01140 by Smithies et al; WO 92/0968 by Zijlsfra et al; and WO 93/04169 by Berns et al.
  • transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI.
  • cre/loxP recombinase system of bacteriophage PI.
  • FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355.
  • mice containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • the invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, antibodies, peptidomimetics, small molecules (organic or inorganic) or other drugs) which modulate integrin activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, antibodies, peptidomimetics, small molecules (organic or inorganic) or other drugs) which modulate integrin activity.
  • an integrin e.g., an integrin in an inactive conformation
  • binds to other proteins that interact with an integrin induce binding, and modulate the interaction of an integrin with other proteins, e.g., an integrin ligand, e.g., ICAM-1, ICAM-2, fibrinogen, fibronectin, von Willebrand factor, thrombospondin, vitronectin, osteopontin, or collagen, and thus modulate integrin activity.
  • an integrin ligand e.g., ICAM-1, ICAM-2, fibrinogen, fibronectin, von Willebrand factor, thrombospondin, vitronectin, osteopontin, or collagen, and thus modulate integrin activity.
  • modulator of integrin activity includes a compound or agent that is capable of modulating or regulating at least one integrin activity, as described herein.
  • Modulators of integrin activity may include, but are not limited to, small organic or inorganic molecules, nucleic acid molecules, peptides, antibodies, and the like.
  • a modulator of integrin activity can be an inducer or inhibitor of integrin activity, e.g., cell adhesion or ligand binding.
  • an "inducer of integrin activity” stimulates, enhances, and/or mimics an integrin activity.
  • an “inhibitor of integrin activity” reduces, blocks or antagonizes an integrin activity.
  • an "integrin activity”, or an “integrin- mediated activity” refers to an activity exerted by an integrin polypeptide or nucleic acid molecule on an integrin responsive cell, or on integrin ligand or receptor, as determined in vitro and in vivo, according to standard techniques.
  • an integrin activity is the ability to mediate cell adhesion events, e.g., cell to cell or cell to extracellular matrix adhesion.
  • an integrin activity is the ability to transduce cellular signaling events.
  • an integrin activity is the ability to bind a ligand, e.g., ICAM-1, ICAM- 2, fibrinogen, fibronectin, von Willebrand factor, thrombospondin, vitronectin, osteopontin, or collagen.
  • a ligand e.g., ICAM-1, ICAM- 2, fibrinogen, fibronectin, von Willebrand factor, thrombospondin, vitronectin, osteopontin, or collagen.
  • an assay is a cell-based assay comprising contacting a cell expressing a modified integrin on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., induce or inhibit) an integrin activity.
  • a cell expressing a modified integrin stabilized in an extended conformation on the cell surface is contacted with a test compound, and the ability of the test compound to modulate adhesion to an integrin ligand is determined, as described and exemplified herein.
  • the modified integrin e.g., the extracellular domains thereof
  • the modified integrin are attached to a insoluble support.
  • the modified integrin e.g., the extracellular domains thereof
  • Animal-based model systems such as an animal model of inflammation, cellular proliferation disorders, e.g., cancer, thrombotic disorders, or endothelial disorders may be used, for example, as part of screening strategies designed to identify compounds which are modulators of integrin activity.
  • the animal- based models may be used to identify drugs, pharmaceuticals, therapies and interventions which may be effective in modulating, for example, inflammation, thrombosis, cellular proliferation, growth and migration, or cell adhesion, and treating integrin-mediated disorders.
  • animal models may be exposed to a compound, suspected of exhibiting an ability to modulate integrin activity, and the response of the animals to the exposure may be monitored by assessing inflammatory activity, thrombosis, cellular proliferation, growth and migration, or cell adhesion before and after treatment.
  • Transgenic animals e.g., transgenic mice, which express modified integrins as described herein can also be used to identify drugs, pharmaceuticals, therapies and interventions which may be effective in modulating inflammation, thrombosis, cellular proliferation, growth and migration, or cell adhesion, and treating integrin-mediated disorders
  • the invention pertains to a combination of two or more of the assays described herein.
  • a modulator of integrin activity can be identified using a cell-based assay, and the ability of the agent to modulate integrin activity can be confirmed in vivo, e.g., in an animal model such as an animal model for inflammation cellular proliferation disorders, e.g., cancer, thrombotic disorders, or endothelial disorders.
  • an animal model such as an animal model for inflammation cellular proliferation disorders, e.g., cancer, thrombotic disorders, or endothelial disorders.
  • screening assays can be used to identify inducers of integrin activity, for example, that mimic the activity of an integrin, e.g., the binding of an integrin to a ligand or receptor, or the activity of an integrin towards an integrin responsive cell.
  • inducers of integrin activity for example, that mimic the activity of an integrin, e.g., the binding of an integrin to a ligand or receptor, or the activity of an integrin towards an integrin responsive cell.
  • Such compounds may include, but are not limited to, peptides, antibodies, or small organic or inorganic compounds.
  • test compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A.
  • Any sequence alignment or protein modeling program can be used to compare different integrin amino acid sequences. It is also possible to do such comparisons by hand or using a spreadsheet. Using such methods a "corresponding" amino acid position can be identified relative to a reference sequence, e.g., the mature human ⁇ 3 sequence or SEQ ID NO: 15. Correspondence can also be indicated by a similar three- dimensional position in a structural model, e.g., generated by threading, modeling, combination of alignment and modeling, energy-minimization, or structure solution (e.g., crystal structure solution by replacment). Typically, however, an alignment method is sufficient. Exemplary alignments are also provided in FIG. 22, 23, and 24.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987); the method is similar to that described by Higgins & Sharp CABIOS 5:151-153 (1989).
  • Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
  • Another example of a useful algorithm is the BLAST algorithm, described in:
  • a particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., Methods in Enzymology, 266:460-480 (1996); and at related on-line resources. Still another example, is PFAM and other hidden variables.
  • PF00362 in release 9 of PFAM provides an alignment of ⁇ integrins.
  • Structural alignment of structurally related proteins can be done to generate sequence alignments (Orengo et al., Structure 5(8):1093-108 (1997); Holm et al., Nucleic Acids Res. 26(l):316-9 (1998)). These sequence alignments can then be examined to determine the observed sequence variations.
  • Libraries can be generated by predicting secondary structure from sequence, and then selecting sequences that are compatible with the predicted secondary structure.
  • forcefield calculations that can be used to optimize the conformation of a sequence within a computational method, or to generate de novo optimized sequences as outlined herein include, but are not limited to, OPLS-AA [Jorgensen et al., J. Am.
  • a single cysteine can be positioned at a solvent accessible site and coupled to a chemical agent of varying size (e.g., between 100-10 000 Daltons.
  • the agent can include a labile bond (e.g., a light sensitive bond or pH sensitive bond) which can be used to remove at least part of the agent (e.g., by irradiation, acid, or base) from the protein and trigger a conformational change in the protein to which it is attached.
  • bifunctional crosslinkers and other molecules can be used to connect non-contiguous amino acids in the protein (e.g., on the same or different subunits).
  • the linkers can include labile bonds, e.g., photolabile, acid-labile, or pH-labile bonds.
  • Modified integrins, modified integrin proteins (e.g., modified proteins and fusion proteins), and active fragments thereof, anti-integrin antibodies, and integrin modulators can be incorporated into pharmaceutical compositions suitable for administration.
  • active compounds also referred to herein as "active compounds”, e.g., compounds that interact with a particular integrin conformation or which are identified by a method described herein
  • nucleic acid molecules encoding modified integrins, modified integrin proteins (e.g., modified proteins and fusion proteins), and active fragments thereof, anti-integrin antibodies, and integrin modulators (also referred to herein as "active compounds"), DNA vaccines, or DNA vectors can be incorporated into pharmaceutical compositions suitable for administration.
  • a "modulator" of integrin activity includes a compound that modulates an integrin activity, e.g., an integrin-mediated signaling event, an integrin-mediated adhesion event, or integrin binding to a cognate ligand.
  • Integrin modulators include modified integrins of the invention, anti-integrins, as well as compounds identified in a screening assay described herein.
  • Such compositions typically comprise the compound, nucleic acid molecule, vector, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, ophthalmic, and rectal administration, including direct installation into a disease site.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a soluble modified integrin I-domain fusion protein) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a soluble modified integrin I-domain fusion protein
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • a "therapeutically effective dose” refers to that amount of an active compound sufficient to result in a detectable change in the physiology of a recipient patient.
  • a therapeutically effective dose refers to an amount of an active compound sufficient to result in modulation of an inflammatory and/or immune response, thrombosis, cell adhesion, or cellular proliferation, growth, or migration.
  • a therapeutically effective dose refers to an amount of an active compound sufficient to result in the amelioration of symptoms of an inflammatory disorder, an autoimmune disorder, a thrombotic disorder, an endothelial cell disorder, or a cellular proliferative disorder.
  • a therapeutically effective dose refers to an amount of an active compound sufficient to prevent an inflammatory disorder, an autoimmune disorder, a thrombotic disorder, an endothelial cell disorder, or a cellular proliferative disorder.
  • a therapeutically effective dose refers to that amount of an active compound sufficient to modulate an integrin activity (e.g., a signaling activity, an adhesion activity or a ligand binding activity) as described herein.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred.
  • While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a therapeutically effective amount of an integrin- modulating compound ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. Ranges intermediate to the above recited values, also are intended to be part of this invention. For example, ranges of span values using a combination of any of the above recited values as upper and/or lower limits are intended to be included.
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with an integrin-modulating compound, e.g., an integrin antibody, in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • an integrin-modulating compound e.g., anti-integrin antibody used for treatment
  • an integrin-modulating compound e.g., anti-integrin antibody used for treatment
  • Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
  • a subject is treated with an initial dosing of a therapeutically effective amount of an integrin-modulating compound, e.g., anti- integrin antibody which reacts with, binds, or stabilizes a modified integrin, e.g., an integrin stabilized in the bent conformation followed by a subsequent intermittent dosing of a therapeutically effective amount of the integrin-modulating compound that is less than 100%, calculated on a daily basis, of the initial dosing of the integrin- modulating compound, e.g., anti-integrin antibody wherein the an integrin-modulating compound, e.g., anti-integrin antibody is administered not more than once per week during the subsequent dosing.
  • an integrin-modulating compound e.g., anti- integrin antibody which reacts with, binds, or stabilizes a modified integrin, e.g., an integrin stabilized in the bent conformation followed by a subsequent intermittent dosing of a therapeutically effective amount of
  • the subsequent dosing is two or more times per week, hi another embodiment, the subsequence dosing is one or more time every two weeks. In still another embodiment, the subsequence dosing is one or more times every three weeks. In yet another embodiment, the subsequence dosing is one or more times every four weeks. In one embodiment, the subsequent dosing is less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% ⁇ , 2%, or 1%, calculated on a daily basis, of the initial dosing of the antibody.
  • the initial dosage is between 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • the initial dosage is less than 0.3 mg/kg body weight, e.g., between 0.001 to 0.30, e.g., 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, and 0.275. Ranges intermediate to the above recited values, also are intended to be part of this invention.
  • a subject is treated with an initial dosing of a therapeutically effective amount of an integrin-modulating compound, e.g., anti- integrin antibody which reacts with, binds, or stabilizes a modified integrin, e.g., an integrin stabilized in a particular conformation (e.g., the bent conformation), followed by a subsequent intermittent dosing of a therapeutically effective amount of the antibody that is greater than 100%, calculated on a daily basis, of the initial dosing of the antibody wherein the antibody is administered to the mammal not more than once per week during the subsequent dosing.
  • the subsequence dosing is two or more times per week.
  • the subsequence dosing is one or more time every two weeks, h still another embodiment, the subsequence dosing is one or more times every three weeks. In yet another embodiment, the subsequence dosing is one or more times every four weeks.
  • the initial dosage is between 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • the initial dosage is less than 0.3 mg/kg body weight, e.g., between 0.001 to 0.3, e.g., 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, and 0.275. Ranges intermediate to the above recited values, also are intended to be part of this invention. In still another example, an initial dosage is followed by the same dosage, for example, not more than once per week during the subsequent dosing. In another embodiment, the subsequence dosing is two or more times per week. In another embodiment, the subsequence dosing is one or more time every two weeks, hi still another embodiment, the subsequence dosing is one or more times every three weeks. hi yet another embodiment, the subsequence dosing is one or more times every four weeks.
  • an effective amount of an anti-inflammatory or immunosuppressive agent to the mammal in combination with the antibody, either at the same time, or at different time points.
  • the present invention encompasses active agents which modulate an integrin activity.
  • An agent may, for example, be a small molecule.
  • such small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • organic or inorganic compounds i.e., including heteroorganic and organometallic compounds
  • doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.
  • Exemplary doses include milligram or micro gram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • a modulator of integrin activity is administered in combination with other agents (e.g., a small molecule), or in conjunction with another, complementary treatment regime.
  • an inhibitor of integrin activity is used to treat an integrin-mediated disorder such as an inflammatory disorder, an autoimmune disorder, a thrombotic disorder, an endothelial cell disorder, or a cellular proliferative disorder.
  • the subject may be treated with an inhibitor of integrin activity, and further treated with an anti-inflammatory or immunosuppressive agent, an anti- thrombotic or an anti-cancer agent, for example.
  • an antibody e.g., an anti-integrin antibody, (or fragment thereof) or a modified integrin may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • the conjugates of the invention can be used for modifying a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a coagulation factor such as tissue factor; a protein such as vascular endothelial growth factor ("VEGF”), platelet derived growth factor, and tissue plasminogen activator; biological response modifiers such as, for example, lymphokines, cytokines and growth factors; or a toxin.
  • a coagulation factor such as tissue factor
  • VEGF vascular endothelial growth factor
  • platelet derived growth factor vascular endothelial growth factor
  • tissue plasminogen activator vascular endothelial growth factor
  • biological response modifiers such as, for example, lymphokines, cytokines and growth factors
  • lymphokines cytokines and growth factors
  • toxin a toxin.
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
  • the nucleic acid molecules of the invention e.g., a nucleic acid molecule encoding, for example, a modified integrin, or active fragment thereof, can be used as a gene-based therapy alone, or, can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy is the insertion of a functioning gene into the cells of a patient (i) to correct an inborn error of metabolism, or (ii) to provide a new function in a cell (Kulver, K. W., "Gene Therapy", 1994, p.
  • Vectors e.g., viral vectors
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).
  • the gene therapy vector can include, for example, DNA encoding an antigen of interest to induce an immune response in the subject in vivo.
  • the modified integrin acts as an adjuvant to produce an increased antibody reaction to the antigen.
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • the nucleic acid molecules of the invention can also be used in DNA vaccine formulations for therapeutic or prophylactic treatment of integrin-mediated disorders, e.g., an inflammatory disorder, an autoimmune disorder, a thrombotic disorder, an endothelial cell disorder, or a cellular proliferative disorder, hi one embodiment, the DNA vaccine formulation comprises a nucleic acid molecule encoding a modified integrin, e.g., a modified integrin, or fragment thereof, coupled with an antigenic component, e.g., DNA encoding an antigenic component.
  • integrin-mediated disorders e.g., an inflammatory disorder, an autoimmune disorder, a thrombotic disorder, an endothelial cell disorder, or a cellular proliferative disorder
  • the DNA vaccine formulation comprises a nucleic acid molecule encoding a modified integrin, e.g., a modified integrin, or fragment thereof, coupled with an antigenic component, e.g., DNA encoding an
  • an antigenic component is a moiety that is capable of binding to a specific antibody with sufficiently high affinity to form a detectable antigen-antibody complex
  • the DNA vaccine further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of an integrin-mediated disorder or having an integrin- mediated disorder such as an inflammatory disorder, an autoimmune disorder, a thrombotic disorder, an endothelial cell disorder, or a cellular proliferative disorder.
  • an integrin-mediated disorder such as an inflammatory disorder, an autoimmune disorder, a thrombotic disorder, an endothelial cell disorder, or a cellular proliferative disorder.
  • Treatment is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder, with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving or affecting the disease or disorder, the symptoms of disease or disorder or the predisposition toward a disease or disorder.
  • a therapeutic agent includes, but is not limited to, nucleic acid molecules, DNA vaccines, gene-based therapies, small molecules, peptides, antibodies, e.g., anti-integrin antibodies, which react with or bind to modified integrins, ribozymes and antisense oligonucleotides.
  • “Pharmacogenomics” refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market.
  • the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or "drug response genotype”).
  • a drug response genotype e.g., a patient's "drug response phenotype", or "drug response genotype”
  • another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the integrins of the present invention or modulators thereof according to that individual's drug response genotype.
  • Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.
  • the invention provides a method for preventing in a subject a disease or condition associated with a integrin-mediated disorder by administering to the subject one or more integrin-modulating compounds, e.g., anti-integrin antibodies.
  • integrin-modulating compounds e.g., anti-integrin antibodies.
  • Subjects at risk for an integrin-mediated disorder can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the integrin-mediated disorders, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • integrin-modulating compounds e.g., anti-integrin antibodies of the present invention, or modulators thereof, can be used for treating the subject.
  • the appropriate agent can be determined based on screening assays described herein.
  • Another aspect of the invention pertains to methods of modulating expression of integrins or their activity for therapeutic purposes (e.g., treating a subject at risk of an integrin-mediated disorder or having an integrin-mediated disorder such as an inflammatory disorder, an autoimmune disorder, a thrombotic disorder, an endothelial cell disorder, or a cellular proliferative disorder).
  • the modulatory method of the invention involves contacting a cell with one or more integrin-modulating compounds of the present invention, e.g., an antibody which stabilizes, binds, or reacts to an integrin in a bent conformation, hi a preferred embodiment, the integrin-modulating compound stabilizes a modified integrin in a bent conformation.
  • An agent that modulates integrin expression or activity can be an agent as described herein, such as a nucleic acid or a protein, a target molecule of an integrin (e.g., a substrate), an antibody which reacts or binds to a modified integrin, an integrin agonist or antagonist, a peptidomimetic of an integrin agonist or antagonist, or other small molecule.
  • the agent stimulates integrin expression or one or more integrin activities. Examples of such stimulatory agents include active integrin protein and a nucleic acid molecule encoding integrin that has been introduced into the cell.
  • the agent inhibits stimulates integrin expression or one or more integrin activities.
  • inhibitory agents include antisense integrin nucleic acid molecules, gene therapy vectors, DNA vaccines, anti-integrin antibodies, and integrin inhibitors.
  • modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized associated with an integrin-mediated disorder.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) integrin expression or activity.
  • integrin molecules of the present invention as well as agents, or modulators which have a stimulatory or inhibitory effect on integrin expression or activity as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) an integrin-mediated disorder such as an inflammatory disorder, an autoimmune disorder, a thrombotic disorder, an endothelial cell disorder, or a cellular proliferative disorder.
  • an integrin-mediated disorder such as an inflammatory disorder, an autoimmune disorder, a thrombotic disorder, an endothelial cell disorder, or a cellular proliferative disorder.
  • pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer an integrin modulatory compound as well as tailoring the dosage and/or therapeutic regimen of treatment with such molecule.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp.Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254- 266.
  • two types of pharmaco genetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymo ⁇ hisms.
  • G6PD glucose-6- ⁇ hosphate aminopeptidase deficiency
  • oxidant drugs anti-malarials, sulfonamides, analgesics, nitrofurans
  • consumption of fava beans One pharmacogenomics approach to identifying genes that predict drag response, known as "a genome-wide association", relies primarily on a high- resolution map of the human genome consisting of already known gene-related markers (e.g., a "bi-allelic" gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants).
  • Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/III drag trial to identify markers associated with a particular observed drug response or side effect.
  • a high resolution map can be generated from a combination of some ten million known single nucleotide polymorphisms (SNPs) in the human genome.
  • SNPs single nucleotide polymorphisms
  • a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA.
  • a SNP may be involved in a disease process, however, the vast majority may not be disease-associated.
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • drug metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and the cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • the gene coding for CYP2D6 is highly polymo ⁇ hic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite mo ⁇ hine. The other extreme are the so called ultra- rapid metabolizers who do not respond to standard doses.
  • a method termed the "gene expression profiling" can be utilized to identify genes that predict drug response.
  • a drug e.g., an integrin molecule or integrin modulator
  • the gene expression of an animal dosed with a drug can give an indication whether gene pathways related to toxicity have been turned on.
  • Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment an individual. This knowledge, when applied to dosing or drag selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an integrin I-domain polypeptide molecule or modulator thereof, such as a modulator identified by one of the exemplary screening assays described herein.
  • LFA-1 is expressed on all leukocytes and is the receptor for three Ig superfamily members, intercellular adhesion molecule- 1, -2 and -3) (Marlin, SD et al. (1987) Cell 57:813-819; Staunton, DE et al. (1989) Nature 339:61 -64; de Fougerolles, et al. (1991) JExp Med 1 74: 253-267). Substantial data indicate that the I-domain of LFA-1 is critical for interaction with ligands.
  • I-domain deleted LFA-1 lacks ligand recognition and binding ability, further demonstrating the role of the I-domain in LFA-1 function (Leitinger, B et al (2000) Mol Biol Cell 11 , 677-690; Yalamanchih, P et al. (2000) J Biol Chem 275:21877-82).
  • the I-domains of other I-domain containing integrins have also been implicated in ligand binding (Diamond, MS (1993) J Cell Biol 120:545556; Michishita, M et al. (1993) Cell 72:857-867; Muchowski, PJ et al. (1994) JBiol Chem 269:26419-26423; Zhou, L et al.
  • LFA-1 activation LFA-1 can be activated by signals from the cytoplasm, called “inside-out” signaling (Diamond, MS et al. (1994) Current Biology 4:506-517). Divalent cations Mn 2+ , Mg 2+ and Ca + can directly modulate ligand-binding function of LFA-1 (Dransfield, I et al. (1989)
  • LFA-1 can be activated by certain mAbs that bind the extracellular domains of the ⁇ L or ⁇ 2 subunit (Keizer, GD et al. (1988) J Immunol 140:1393- 1400; Robinson, MK et al. (1992) J Immunol 148:1080-1085; Andrew, D et al. (1993) Eur J Immunol 23:2217-2222; Petruzzelli, L et al.
  • the lido and ljlm stractures were aligned by their sequence, and Ufa molecule A and lzon were aligned by structural similarity to ljlm.
  • the distances between all C ⁇ carbons at equivalent sequence positions were calculated using a Microsoft Excel spreadsheet. This was analogous to the comparison between ljlm and lido (Lee, J-O et al. (1995) Structure 3:1333-1340), except that LFA-1 I domain structures were included.
  • segments from Ufa molecule A were chosen where differences between all four I domains were small, or differences between Ufa and ljlm (low affinity, closed LFA-1 and Mac-1 1 domains) were greater than between lido and ljlm (open and closed Mac-1 1 domains).
  • Segments from lido were chosen when differences between lido and ljlm were greater than between Ufa and ljlm. These segments were spliced together in regions where the backbones were as similar as possible.
  • the template utilized segments G128 to F136, Ml 54 to L203, F209 to L234, T243 to 1255, and E272 to A282 of Ufa; and segments D140 to F156, G207 to T211, V238 to K245, R266 to R281, and R293 to K315 of lido.
  • No chain breaks were detected by LOOKTM (Molecular Application Group, Palo Alto, CA) in the spliced template, dubbed lfa- mac.
  • Models of a high affinity open form of LFA-1 were made with MODELLER 4TM using this template, the Mg 2+ and water molecules 403 and 404 of lido, with heteroatom, water, and hydrogen input turned on, and dynamic Coulomb turned on.
  • the SSBOND program (Hazes, B and Dijkstra, BW (1988) Protein Engineering 2:119-125) was used to identify positions where disulfide bonds could be introduced by mutating two appropriately positioned pairs of residues to cysteine. It was hypothesized that it might be possible to use disulfide bonds to trap the LFA-1 1 domain in either the open or closed conformations.
  • the high affinity open LFA-1 1 domain model (the lfa_hi.063 model) was examined and two low affinity closed LFA-1 I domain structures, Ufa and lzon, with SSBOND and found 14 to 19 pairs of such residues in each structure.
  • one pair of residues in the high affinity open model, and one pair of residues in the low affinity closed structures underwent large movements between the two conformers, such that disulfide bond formation could only occur in one conformer (Figure 1).
  • These disulfides bridge /3-strand 6 to the C-terminal ⁇ -helix, ⁇ 6.
  • the numbering of /3-strands and ⁇ -helices differs among I domains; a uniform nomenclature is used(Huang, C et al.
  • Helix ⁇ 6 moves 10 A along its axis down the body of the I domain in the high affinity open structure, and this movement is accompanied by a complete remodeling and downward shift of the loop between ⁇ 6 and ⁇ 6.
  • Cysteines introduced in place of K287 and K294 were predicted to form a disulfide only in the high affinity open conformer, and thus lock the I domain in the high affinity open state ( Figure 2).
  • the C/3 carbons of K287 and K294 are predicted to be 3.8 A apart in the high affinity open model (lfa_hi.063), within the range of 3.41 to 4.25A that is ideal for disulfide formation, and after checking for C/3-S7 and S -S ⁇ distances, were found to have four favorable sidechain-disulfide conformations.
  • the C/3 atoms of these residues are 8.9 to 9.2 A apart ( Figure 2).
  • Cysteines introduced in place of L289 and K294 were predicted to form a disulfide only in the low affinity closed conformer ( Figure 2), and thus lock the I domain in the low affinity closed state.
  • the C ⁇ carbons of L289 and K294 are 3.9 to 4.0 A apart in the low affinity closed Ufa and lzon conformers, within the favorable range, although favorable cysteine sidechain conformations were not found.
  • the region of the conformationally mobile C- terminal ⁇ -helix and the preceding loop were examined for positions in which one cysteine could be introduced, and structurally adjacent regions were searched for positions where a second cysteine could be introduced that would form a disulfide bond. Pairs of residues whose side-chains face towards one another were chosen. The distance between the C ⁇ and C ⁇ atoms of each of these pairs was measured by software LookTM both in the open and closed conformation. The ideal separation for cysteine C ⁇ carbons for formation of a disulfide bond is reported to be 3.41 to 4.25 A. However, the crystal structures or models from which these were measured represent average positions of snapshots, whereas proteins are dynamic and exhibit atomic mobility.
  • L161C/T300C was made by substituting cysteines for the L161 and T300.
  • the low affinity closed mutant L289C/K294C was made by substituting cysteines for the L289 and K294.
  • the distance between mutated residues for these six mutant is shown in Table 1, below.
  • single cysteine substitution mutants K287C, L289C and K294C were generated. Table 1.
  • the distance between wild-type residues was measured by LookTM software in open conformation (lfa_hi.063) or closed conformation (llfaA).
  • the human ⁇ L cDNA was contained in vector AprM8, a derivative of CDM8 (Seed, B and Araffo, A (1987) Proc Natl Acad Sci USA 84:3365-3369).
  • Overlap extension PCR was used to generate cysteine substitution mutations in the ⁇ L I- domain (Ho, SN et al. (1989) Gene 77:51-59; Horton, RM et al. (1990) BioTechniques 8:528).
  • the outer left primer for PCR extension was complementary to the vector sequence at 5' to the EcoRI site at position 1826, and the outer right primer was 3' to the EcoRI site in the ⁇ L cDNA.
  • the inner primers were designed for each individual mutation and contained overlapping sequences.
  • Wild-type ⁇ L CDNA in AprM8 was used as template for the first PCR reaction.
  • the second PCR product was digested with EcoRI and ligated into the same site in the wild-type ⁇ L cDNA in AprM ⁇ .
  • the correct orientation of the insert was confirmed by restriction enzyme digestion. All mutations were confirmed by DNA sequencing.
  • the Xbal fragment of ⁇ L wild-type and mutant cDNA was subcloned into the same site of the stable expression vector pEFpuro (Lu, C and Springer, TA. (1997) J Immunol 159:268-278).
  • the mutated ⁇ L subunit was transiently coexpressed with the ⁇ 2 subunit in 293T cells, and cell surface expression of the ⁇ L//32 complex was determined by flow cytometry with monoclonal antibody TS2/4 to the ⁇ L subunit in the ⁇ L//32 complex.
  • human embryonic kidney 293T cells (SV40 transformed) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine and 50 ⁇ g/ml gentamycin. 293T cells were transiently transfected using the calcium phosphate method (DuBridge, RB et al. (1987) Mol Cell Biol 7:379-387; Heinzel, SS et al. (1988) J Virol 62:3738-3746).
  • FBS fetal bovine serum
  • 293T cells were transiently transfected using the calcium phosphate method (DuBridge, RB et al. (1987) Mol Cell Biol 7:379-387; Heinzel, SS et al. (1988) J Virol 62:3738-3746).
  • 7.5 ⁇ g of wild-type or mutant ⁇ L cDNA in plasmid AprM8 and 7.5 ⁇ g of /32 cDNA in AprM8 were used to co-transfect one 6-cm plate of 70-80% confluent cells. Two days after transfection, cells were detached from the plate with Hanks' balanced salt solution (HBSS) containing 5 mM EDTA for LFA-1 expression and functional analyses.
  • HBSS Hanks' balanced salt solution
  • the monoclonal antibodies used in these studies are as follows: The mouse anti-human ⁇ L (CD 11a) monoclonal antibodies TS 1/11 , TS 1/12,
  • TS1/22, TS2/4, TS2/6 and TS2/14; anti-,32 (CD18) monoclonal antibodies TS1/18, CBRLFA-1/2, and CBRLFA-1/7; mAb YFC51; and the nonbinding mAb X63 have been described previously (Sanchez-Madrid, F et al. (1982) Proc Natl Acad Sci USA 79:7489-7493; Hale, LP et al. (1989) Arthritis Rheum 32:22-30; Petruzzelli, L et al. (1995) J Immunol 155:854-866).
  • Monoclonal antibodies BL5, F8.8, 25-3-1, May.035, CBRLFA-1/9, CBRLFA-1/1, S6F, and May.017 were described in Leukocyte Type V and were obtained from the Fifth International Leukocyte Workshops.
  • Monoclonal antibodies X63 and TS1/11 were used as hybridoma supernatants at a 1:20 dilution; monoclonal antibodies TS1/12, DBRLFA-1/2, CBRLFA-1/7 and YFC51 were used as purified IgG at lO ⁇ g/ml; monoclonal antibodies TS1/2, TS2/14, TS1/18 and TS2/4 used as ascites at a 1:200 dilution; and all monoclonal antibodies from the Fifth International Leukocyte Workshops were used at a 1 :200 dilution.
  • Wild-type LFA-1 and LFA-1 mutant K287C/K294C, L289C/K294C, K287C, L289C, and K294C were transiently expressed on the surface of 293T cells or stably expressed on K562 transfectants. Reactivity of antibodies with the transfectants was determined by flow cytometry. Mean fluorescence of each antibody binding was normalized to the mean fluorescence of mAb TS2/4 binding, except for CBRLFA-1/9 that was normalized to mAb TS1/22 binding. TS2/4 bound to wild-type LFA- 1 and the mutants equally well. The results are expressed as percent of wild-type binding. Data are mean ⁇ SD of at least two independent FCAS experiments. For some antibodies, only one experiment was done. ND: not determined.
  • LFA-1 cysteine substitution mutants The ability of the LFA-1 cysteine substitution mutants to bind to the LFA-1 ligand ICAM-1 was determined. 293T cell transfectants that express wild-type LFA- 1 and the predicted high-affinity open I-domain mutant K287C/K294C showed constitutively strong binding to immobilized ICAM-1 ( Figure 4A). By contrast, the low-affinity closed mutant L289C/K294C did not bind to ICAM-1. Whereas the single cysteine substitution mutants K287C and L289C exhibited reduced binding to ICAM-1, binding of mutant K294C was comparable to that of the wild-type.
  • the human erythroleukemia cell line K562 was cultured in RPMI 1640, 10% FBS and 50 ⁇ g/ml gentamycin.
  • RPMI 1640 10% FBS
  • 50 ⁇ g/ml gentamycin 50 ⁇ g/ml gentamycin.
  • 2 ⁇ g of Pvul-linearized pEFpuro containing ⁇ L subunit cDNA was cotransfected with 40 ⁇ g of Sfil-linearized AprM8 containing the /32 subunit cDNA by electroporation at 250V and 960 ⁇ F.
  • Transfectants were selected for resistance to 4 ⁇ g/ml puromycin (Sigma), and subcloned by limiting dilution. All stable cell lines were maintained in RPMI 1640, 10% FBS supplemented with 4 ⁇ g/ml puromycin.
  • Cells were labeled with a florescence dye 2',7'-bis-(carboxyethyl)-5(and-6)- carboxyfluorescein, acetoxymethyl ester (BCECF-AM), and resuspended to 1 x 10 6 /ml in L15/FBS.
  • 50 ⁇ l cell suspension was mixed in ICAM-1 coated wells with an equal volume of L15/FBS in the absence or presence of monoclonal antibody (CBRLFA-1/2, 10 ⁇ g/ml).
  • Monoclonal antibodies were used at final concentration of 1 :20 hybridoma supernatant, 1 :200 ascites, or 10 ⁇ g/ml purified IgG.
  • BCECF-AM- labeled cells were washed 2 x with TS buffer, pH7.5 (20 mM Tris, pH 7.5, 150 mM NaCl) containing 5 mM EDTA, followed by 2 washes with TS buffer, pH7.5. Cells were then resuspended to 5 x 10 5 /ml in the TS buffer, pH7.5 supplemented with 1 mM MgCl 2 /CaCl 2 , MgCl 2 , MnCl 2 or 5 mM EDTA, and 100 ⁇ l cell suspension was added to ICAM-1 coated wells.
  • K562 transfectants that express wild-type LFA-1 showed low basal binding to ICAM-1, and binding was greatly increased by the activating monoclonal antibody CBRLFA-1/2 ( Figure 4B).
  • cells expressing the predicted high-affinity open mutant K287C/K294C strongly bound to ICAM-1, and monoclonal antibody CBRLFA-1/2 did not further enhance binding of this mutant, whereas the predicted low-affinity closed mutant L289C/K294C did not binding to ICAM-1 even in the presence of the activating antibody.
  • LFA-1, mutant K287C/K294C, or mutant L289C/K294C was also assessed. Briefly, a soluble ICAM-1-IgA chimera containing the 5 Ig domains of human ICAM-1 was purified from the culture supernatant of stable CHO transfectants by monoclonal antibody R6.5 affinity chromatography as previously described (Martin, S et al. (1993) J Virol 67:3561-3568). K562 transfectants were washed once with L15/FBS, and resuspended in the same buffer to 1 x 10 7 /ml.
  • 25 ⁇ l cell suspension was mixed with 25 ⁇ l L15/FBS containing ICAM-1-IgA fusion protein at final concentration 100 ⁇ g/ml in the presence or absence of antibody CBRLFA-1/2 (10 ⁇ g/ml), and incubated at 37°C for 30 minutes. After incubation, cells were washed once in L15/FBS, and incubated with FITC-conjugated anti-human IgA (Sigma) at room temperature for 20 minutes. After 2 washes, cells were resuspended in PBS, and analyzed on a FACScan (Becton Dickinson, San Joe, CA).
  • FACScan Becton Dickinson, San Joe, CA
  • the soluble ICAM-1 -IgA fusion protein bound to cells expressing the high-affinity open mutant K287C/K294C, and binding was further increased in the presence of the activating monoclonal antibody CBRLFA-1/2.
  • the ICAM-1 fusion protein did not bind to the transfectants that expressed wild-type LFA-1 or the low affinity closed mutant L289C/K294C in the absence or presence of monoclonal antibody CBRLFA-1/2, and binding was not detected at a higher ICAM-1 fusion protein concentration (300 ⁇ g/ml).
  • the I-domain antibodies differentially inhibited binding of wild-type LFA-1 and the high affinity open mutant K287C/K294C to ICAM-1.
  • Antibodies to the /3-propeller domain and to the C-terminal region of ⁇ 2 did not inhibit binding of wild-type LFA-1, or mutant K287C/K294C.
  • Antibodies to the I-like domain of the ⁇ subunit blocked binding of wild-type LFA-1 to ICAM-1, but did not block mutant K287C/K294C.
  • Wild-type LFA-1 and LFA-1 mutant K287C/K294C were transiently expressed on the surface of 293T cells or stably expressed in K562 transfectants. Binding of the transfectants to immobilized ICAM-1 was determined in the presence of the indicated antibodies. For binding of K562 transfectants that express wild-type LFA-1, the cells were preincubated with the activating mAb CBRLFA-1/2 at 10 ⁇ g/ml for 30 min. Data shown are % inhibition + SD of at least two independent experiments. % inhibition is defined as % bound cells in the presence of the indicated mAb/% bound cells in the presence of the nonbinding mAb X63 x 100. For some antibodies, only one experiment was done.
  • the high affinity open I-domains of the invention can be used to discriminate between direct/competitive and indirect/non-competitive modes of inhibition of LFA- 1.
  • the LFA-1 inhibitor lovastatin binds to the I-domain in a hydrophobic pocket formed by the ⁇ sheet and the C-terminal ⁇ -helix (Kallen, J et al. (1999) JMol Biol 292: 1-9) and thus inhibits LFA-1 by an indirect mechanism.
  • the ability of lovastatin to inhibit ligand binding of the high-affinity I- domain K287C/K294C was assessed.
  • Lovastatin dissolved in DMSO at 50 mM was diluted in assay buffer.
  • Cells (10 6 /ml) labeled with BCECF-AM were preincubated with lovastatin (0-50 ⁇ M) at 37°C for 15 minutes, then transferred to a 96 well plate coated with ICAM-1 and further incubated at 37°C for 30 minutes in the presence or absence of activating monoclonal antibody (CBR LFA1/2) or MnCl .
  • LI 5 medium supplemented with fetal bovine serum (L15/FBS) which contains Ca2+ and Mg2+ was used for wild-type ⁇ L/32 activated by antibody CBR LFA1/2.
  • 20 mM HEPES pH7.4, 140 mM NaCl, ImM MnC12, 2 mg/ml glucose, 1% BSA was used for activation by Mn2+.
  • lovastatin inhibits ICAM-1 binding by cells expressing wild-type LFA-1 and stimulated with Mn 2+ or antibody (CBRLFAl/2), but does not interfere with ligand binding by the high affinity open K287C/K294C mutant (HA aLb2).
  • EXAMPLE 4 EXPRESSION AND FUNCTION OF ISOLATED WILD-TYPE AND MUTANT LFA-1 I-DOMAINS
  • the wild-type I-domain and the I-domains of mutant K287C/K294C and L289C/K294C from residues V130 to A338 were expressed on the surface of K562 cells by the transmembrane domain of the PDGF receptor.
  • mutant K287C/K294C I-domain showed strong binding to ICAM-1. If the constitutive ligand binding activity of mutant K287C/K294C is due to the formation of a disulfide bond between the introduced C287 and C294, disruption of the disulfide bond with a reducing agent would abolish ligand binding ability of the mutant. Accordingly, the transfectants were treated with the reducing agent DTT (10 mM) in LI 5/FBS containing Mg 2+ and Ca 2+ , and the ability of transfectants to bind to ICAM-1 was assessed.
  • DTT 10 mM
  • ICAM-1 was divalent cation dependent, as EDTA treatment abolished the binding.
  • Mn 2+ or Mg 2+ did not activate ligand binding of the isolated wild-type I-domain or the mutant L289C/K294C I-domain.
  • Transfectants expressing intact LFA-1 were pre-incubated with the activating antibody CBRLFA-1/2, and binding of the cells to ICAM-1 was performed in the presence of the I-domain antibodies TS1/22, TS2/6, TS1/11, TS1/12, CBRLFA-1/9, CBRLFA-1/1, 25.3.1, TS2/14, or the nonbinding antibody X63, as indicated.
  • a soluble ⁇ L I-domain mutant stabilized in the open conformation by a disulfide bond (K287C/K294C) was made in E. coli.
  • recombinant mutant ⁇ L I-domain stabilized in the open conformation (K287C/K294C), or recombinant wild-type ⁇ L I-domain from amino acid residue G128 to Y307 were cloned into pETl lb (Novagen) and expressed in E. coli induced with 1 mM IPTG for 4 hours.
  • the recombinant proteins were purified from inclusion bodies by solubilization of inclusion bodies in 6M guanidine HC1 and were refolded by dilution in the presence of 0.1 mM Cu 2+ /phenanthrolin to enhance formation of disulfide bonds.
  • Protein was concentrated by ammonium sulfate precipitation, dialyzed, and purified over a monoQ ion-exchange column. To remove any material in which the disulfide bond did not form, free sulfhydryls were reacted with activated biotin and passed over a streptavidin column. The recombinant proteins were then purified by gel filtration and concentrated by Centriprep. For BIAcoreTM analysis, recombinant ICAM-1, ICAM-2 and ICAM-3 Fc chimeras were immobilized on the BIAcoreTM sensor chip by an amine-coupling method.
  • Recombinant ⁇ L I-domains were flowed in, and BIAcoreTM assays were performed with Tris-buffered saline supplemented with 1 mM MgCl 2 or 2 mM EDTA, at a flow rate of 10 ⁇ l/minute at 25°C.
  • the purified open I-domain showed high affinity to its ligands, ICAM-1, -2, and -3, in the presence of 1 mM MgCl as assessed by BIAcoreTM analysis, whereas binding of a soluble wild-type I domain was not detectable ( Figure 9, Panels A, C and E; Table 4).
  • the interaction of the open I-domain with ligands was divalent cation- dependent, and was abolished in the presence of 2 mM EDTA, suggesting that the interaction depends on MIDAS. Since the wild-type I-domain showed no interaction with ligands, the open I-domain allowed for the detailed analysis of the binding kinetics of LFA-1 with its ligands.
  • ICAM-3 0.19 x lO 5 0.0749 3942 k on , k off , and K D were calculated based on 1 : 1 interaction model using BIAevaluationTM software.
  • K562 cells stably expressing wild-type LFA-1 were fluorescently labeled by BCECF and LFA-1 on the cell surface was activated by the activating monoclonal antibody, CBRLFA-1/2 in L15 media supplemented with FCS.
  • the cells were subsequently incubated in ICAM-1 coated 96-well plastic plates in the presence or absence of I-domains. After incubation for 40 minutes at 37°C, unbound cells were washed off on a Microplate Autowasher. The fluorescence content of total input cells and the bound cells in each well was quantitated on a
  • murine T lymphoma cell line EL-4 which expresses both murine LFA-1 and its ligands, including murine ICAM-1, and which exhibits LFA-1 - dependent homotypic aggregation upon activation by PMA was used.
  • Cells were incubated in a 96 well plate in the presence of 50 ng/ml PMA and varying amounts of soluble I-domains.
  • the degree of aggregation was scored under the microscope as follows: 0 indicated that essentially no cells were clustered; 1 indicated that ⁇ 10% of cells were aggregated; 2 indicated clustering of ⁇ 50%; 3 indicated that up to 100% of cells were in small, loose aggregates; 4 indicated that nearly 100% of cells were in larger clusters; and 5 indicated that nearly 100% of cells were in very large, tight clusters.
  • the soluble open I-domain also inhibited PMA-induced LFA-1 dependent homotypic aggregation of the murine T-cell line EL-4.
  • the ability of the open I-domain mutants to inhibit LFA-1 function in vivo was tested by visualizing microcirculation in the peripheral lymph node (LN) with intravital microscopy. Briefly, a small bolus (20-50 ⁇ l) of LN cell suspensions from T-GFP mice were retrogradely injected through a femoral artery catheter and visualized in the subiliac LN by fluorescent epi-illumination from a video-triggered xenon arc stroboscope. After recording control T GFP cell behavior in the absence of I- domain, the mouse was preheated by intra-arterial injection of I-domain (10 ⁇ g/g of weight) 5 minutes before T GFP cell injection.
  • I-domain 10 ⁇ g/g of weight
  • the rolling fraction was calculated as percentage of rolling cells amount the total number of T GFP cells that entered a venule.
  • the sticking (firm adhesion) fraction was determined as the percentage of T GFP cells becoming firmly adherent for >20 seconds in the number of T GFP cells that rolled in a venule. Results were semi-quantitatively scored as follows: -: 0%, ⁇ : 0-5%, +: 5-20%, ++: 20- 40%, +++: 40-60%, ++++: 60-80%, +++++: 80-100%.
  • Recombinant soluble ⁇ L I-domains were expressed in E. coli, refolded and purified.
  • Kinetics of binding of the I-domains to ICAM-1 was measured by BIAcoreTM instruments.
  • Kinetics was analyzed BIAevaluationTM software.
  • KD was calculated by Scatchard plots using data at steady states.
  • Koff was obtained by curve fitting of the dissociation phase using 1 : 1 binding model.
  • Kon was calculated by Koff/KD.
  • the distance between wild-type residues was measured by LoobkTM software in open conformation (lido) or closed conformation (ljlm).
  • plasmids encoding the wild-type or mutant ⁇ M subunits and the 82 subunit were co-transfected into 293T and K562 cells.
  • a ⁇ heterodimer formation was confirmed using monoclonal antibody CBRMl/32 which recognizes an epitope in the putative /3-propeller domain of the ⁇ M subunit only after association with the /32 subunit, and antibody CBRMl/5 was used to detect integrin activation.
  • the Q163C/Q309C pair of mutations worked well ( Figure 1 IB, Figure 12B and C).
  • This mutant introduces a putative disulfide bond near the bottom front of the I-domain, between residues that are in the lower one-third of the last ⁇ -helix and the first ⁇ -helix, and have C/3 carbons that are 6.36A apart in the lido structure.
  • the C ⁇ carbons for the D294C/T307C and D294C/N311 C substitutions are 8.67A and 7.08A apart, respectively.
  • the C/3 carbons for the Q298C/N301C and F297C/A304C substitutions are within the ideal range, however these substitutions are closer to the loop between the last /3-strand and ⁇ -helix, and must have unfavorable effects such as distorting the ligand binding site.
  • the Q163C/Q309C mutant When expressed within an intact heterodimer in transiently transfected 293T cells, the Q163C/Q309C mutant is expressed half as well as wild-type as measured by CBRMl/32 antibody, but the ratio of the CBRMl/5 activation-dependent epitope to CBRMl/32 expression is markedly higher ( Figure 11 A).
  • the adhesion of 293T cells expressing the Mac-1 Q163C/Q309C mutant to iC3b coated on plastic as assayed in LI 5/FBS medium at room temperature, was higher than wild-type, despite its lower expression (Figure 1 IB).
  • isolated Mac-1 mutant I-domains were expressed on the cell surface in conjunction with an artificial signal sequence and transmembrane domain of the PDGF receptor. Adhesion was assayed in L15/FBS/MnCl 2 at 37°C. The isolated wild-type I-domain showed no binding to iC3b, whereas the previously described mutants with computationally redesigned hydrophobic cores, idolr and ido2r, were active ( Figure 1 IC) (Shimaoka, M et al. (2000) Nature Structural Biology 7:674-678). The Q163C/Q309C mutant I-domain exhibited strong specific ligand binding that was completely blocked by the inhibitory I-domain monoclonal antibody CBRMl/5 ( Figure 12C).
  • the open I-domain mutants Q163C/Q309C and D294C/Q31 IC were stably expressed in K562 cells, and clones expressing the same levels of receptors were selected.
  • Adhesion assays to immobilized iC3b were performed with LI 5/FBS at 37°C. hi contrast to 293T cells, wild-type Mac-1 has little basal activity for ligand binding in these cells ( Figure 12A and 12B).
  • Both Q163C/Q309C and D294C/Q31 IC showed increased CBRMl/5 activation-dependent epitope expression and increased ligand binding when expressed in an intact cM.82 heterodimer, as compared to wild-type ( Figure 12A and 12B).
  • K562 cells expressing isolated open I-domain mutants on the cell surface showed strong specific binding to iC3b as compared to wild-type ( Figure 12C).
  • the effect of the reducing agent DTT was tested. Binding of ⁇ M/32 transfectants containing mutant I- domains to immobilized iC3b on plastic was tested in the presence and absence of DTT.
  • locked open ⁇ M I-domains (Q163C/Q309C) and (D294C/Q31 lc), are active in the absence of activation and their activities are partly reduced by disulfide reduction by DTT.
  • locked closed ⁇ M I-domain Q163C/R313C is inactive and resistant to activation, but becomes activatable after disulfide reduction by DTT.
  • DTT disulfide reduction by DTT treatment.
  • a soluble extracellular fragment of ⁇ V ⁇ 3 was made that contained a releasable, C-terminal clasp.
  • the complete extracellular domains of the ⁇ V and ⁇ 3 chains were fused at the C-terminus to peptides that formed a disulfide-linked ⁇ - helical coiled-coil to mimic the closely positioned transmembrane helices on the cell surface (Takagi et al., 2001).
  • One peptide also contained a TEV protease site to enable release of the C-terminal intersubunit clasp by specific cleavage, and a His tag to enable purification under mild conditions.
  • Protein was purified in the presence of 1 mM Ca 2+ and Mg 2+ , and half was treated with TEV protease to cleave the clasp.
  • the clasped and unclasped ⁇ V ⁇ 3 preparations were then further purified by gel filtration in buffer containing either 5 mM Ca 2+ (a condition resembling the crystallization buffer used by Xiong et al), 1 mM Mg 2+ / 1 mM Ca 2+ , or 1 mM Mn 2+ .
  • the crystal structure projection appears slightly less dense than the EM projection average near the genu and between the genu and the headpiece. This is however the location of the PSI domain and integrin EGF domains 1 and 2 that are present in the protein preparations but missing in the crystal structure.
  • the angles between the legs of the V in the crystal structure and the predominant EM projections are the same, showing that the degree of bending is not a crystal structure artifact but occurs in individual unconstrained molecules as well.
  • the crystal structure thus represents the predominant native conformation of ⁇ V ⁇ 3 in the presence of Ca 2+ or Ca 2+ /Mg 2+ .
  • Mn 2+ Although it binds to the extracellular segment of integrins, Mn 2+ appears in many respects to mimic inside-out signaling, both by activating in the absence of a bound ligand, and in exposing a similar array of activation epitopes (Lu et al., 2001b). In the presence of Mn 2+ , a fraction of the ⁇ V ⁇ 3 molecules (-15 and -20% for clasped and unclasped ⁇ V ⁇ 3, respectively) remained in the compact conformation; however, the vast majority underwent a dramatic, global change in conformation and converted to an extended form (Fig. 14C and D).
  • the ⁇ V ⁇ 3- RGDfV complex adopted a fully extended conformation even in the presence of Ca 2+ , indicating that ligand binding results in the global conformational rearrangement of the integrin molecule. This is consistent with the observation that binding of small molecule ligands induces neoepitopes recognized by antibodies to LIBS epitopes (Frelinger et al., 1990; Kouns et al., 1992; Pelletier et al., 1996).
  • the side of the headpiece containing the ⁇ subunit is distinguished by the thicker coating of negative stain outlining the ⁇ subunit ⁇ - propeller domain (Fig. 14C-H), a consequence of this domain extending further above the carbon film than the other integrin domains because of its larger size.
  • Fig. 14C-H all averaged images are shown with the headpiece toward the top of the page and the ⁇ subunit on the left.
  • the ⁇ subunit leg appears in a relatively uniform orientation whether clasped or unclasped, and whether in Mn 2+ alone (Fig. 14C,D), Ca 2+ + cyclo-RGDfV (Fig.
  • the upper portion of the ⁇ 3 leg differs in the angle it makes with the headpiece among different classes of averaged projections, hi Mn 2+ and absence of cyclo-RGDfV, an acute angle is seen in some averages (Fig. 14C, averages 1 and 2 and Fig. 14D, average 2) and a more obtuse angle in others (Fig. 14C, averages 3 and 4 and Fig. 14D, averages 1, 3, and 4). These are referred to as the closed and open conformations of the headpiece, respectively. In Mn 2+ alone, a higher proportion of unclasped particles (-70%) had open headpieces than clasped particles (-30%).
  • Binding to cyclo-RGDfV in Ca 2+ or Mn 2+ resulted in uniform adoption of the open conformation of the headpiece, with all averages showing wide separation between the ⁇ and ⁇ legs near the headpiece (Fig. 14E-H).
  • the images of the extended conformation were compared to the ⁇ V ⁇ 3 crystal stractures.
  • the extreme bend occurs in ⁇ V at the genu between the thigh and calf-1 domains and in ⁇ 3 between the hybrid domain and I- EGF domain 3, which are connected by domains that are disordered and not defined (Fig. 13).
  • the crystal structure coordinates were truncated at the positions of these bends in ⁇ V and ⁇ 3 to obtain the fragment corresponding to the headpiece, and cross- correlated to representative EM projection averages in which the ⁇ leg was in the acute (closed) or obtuse (open) orientation to the headpiece (Fig. 15D, E).
  • Cross- correlation confirmed that in the orientation shown in Fig.
  • ⁇ and ⁇ subunits are to the left and right, respectively (Fig. 15C).
  • the ⁇ subunit ⁇ -propeller and ⁇ subunit I-like domains combine to form a globular head, while the ⁇ thigh and ⁇ hybrid domains project from the bottom of the globule on the left and right, respectively.
  • the fatter, C-terminal end of the thigh domain corresponds to a density in the EM projections located at a bend at the junction between the thigh and calf-1 domains (Fig. 15C).
  • the hybrid domain corresponds to the dense, upper part of the ⁇ leg that has two orientations relative to the head.
  • the hybrid domain Since the hybrid domain connects to the I-like domain in the head, the hybrid domain has two different orientations relative to the I- like domain.
  • the crystal structures (Fig. 15C) match the closed orientation found in averages in Mn 2+ (Fig. 15D), but clearly do not match the open orientation seen in averages in Mn 2+ and in all the averages in which cyclo-RGDfV is bound in Ca 2+ or Mn 2+ (Fig. 15E).
  • a conformational change occurs upon activation by Mn + or cyclo-RGDfV that changes the orientation of the I-like domain relative to the hybrid domain.
  • Both integrin I domains and I-like domains are inserted within other domains, i.e.
  • Integrin I domains are activated by a downward movement of the C-terminal ⁇ -helix (Shimaoka et al., 2002). Modeling the effect of a one turn (four residue) downward movement of the C-terminal ⁇ -helix of the I-like domain shows that it causes the hybrid domain to pivot about its connection to the N- terminus of the I-like domain, and swing away from the ⁇ thigh domain and make the headpiece more open. Projection averages were made of this model of an activated headpiece (Fig. 15F).
  • Cross-correlation showed an excellent match to the images of ⁇ V ⁇ 3 with the open conformation of the headpiece (Fig. 15E).
  • the cross-correlation coefficient of the open headpiece model (Fig. 15F) with the open EM images in cyclo-RGDfV (Fig. 15E) was 893, significantly better than the coefficients of 729 and 730 obtained with the headpieces from the uncomplexed and peptide-complexed crystal structures (Fig. 15C), respectively.
  • the extended but not the bent conformation has high affinity for biological ligands.
  • Binding of the biological ligands fibrinogen or vitronectin to ⁇ V ⁇ 3 was measured in solution phase using surface plasmon resonance. Binding was measured in the presence of Ca 2+ /Mg 2+ or Mn 2+ , with the same preparations as used in EM and gel filtration studies. The traces were made at increasing concentrations (50, 100, and 150 nM) of ⁇ V ⁇ 3 analyte. In the presence of physiological divalent cation concentrations of 1 mM Ca 2+ and 1 mM Mg 2+ , little or no binding of fibrinogen or vitronectin to clasped ⁇ V ⁇ 3 was observed, and release of the C-terminal clasp only slightly increased binding.
  • the k on values and extent of binding were similar for clasped and unclasped ⁇ V ⁇ 3 in Mn 2+ .
  • the k on values are 5.1 x IO 4 M “1 s “1 for fibrinogen and 7.9 x 10 3 M “1 s “1 for vitronectin. These are comparable to the k on values of 2.5 x IO 4 M “1 s “1 for fibrinogen and 2.3 x 10 3 M “1 s "1 for vitronectin reported for detergent-solubilized ⁇ V ⁇ 3 in a solid phase binding assay in the presence of Mn 2+ (Smith et al, 1994). Ligand dissociated more slowly from unclasped than clasped ⁇ V ⁇ 3.
  • the initial rate of dissociation from fibrinogen was 9.8 x IO "4 and 2.8 x IO "3 s "1 for unclasped and clasped ⁇ V ⁇ 3, respectively.
  • the rate of dissociation from vitronectin was 2.1 x IO "4 and 9.2 x IO "4 s "1 respectively. Therefore, release of the C-terminal intersubunit constraint, which mimics uncoupling of the transmembrane/cytoplasmic domain interaction between the ⁇ and ⁇ subunits, results in stronger association with ligand.
  • integrins exist in three conformational states: a bent conformer (Fig. 19 A), an extended conformer with a closed headpiece (Fig. 19D), and an extended conformer with an open headpiece (Fig. 19E).
  • the bent conformer is seen in Ca /Mg and Ca , is stabilized by Ca , strongly destabilized by cyclo-RGDfV, and less strongly destabilized by Mn 2+ .
  • a higher proportion of ⁇ V ⁇ 3 molecules is present in the V-shaped bent conformation in Ca 2+ .
  • the bent ⁇ V ⁇ 3 conformer does not bind the biological ligands fibrinogen or vitronectin as shown in solution phase binding assays in Ca 24 VMg 2+ .
  • ⁇ V ⁇ 3 and ⁇ flb ⁇ 3 cannot be stimulated to bind their biological ligand fibrinogen with high affinity when locked in the bent conformation with a disulfide bond.
  • the bent conformer does not detectably bind biological ligands, it clearly can bind high affinity ligand-mimetic peptides as demonstrated by a co-crystal structure (Xiong et al., 2002).
  • the bent conformer is therefore referred to as a low affinity rather than an inactive conformation.
  • Cyclo-RGDfV was used at a concentration (60 to 1 ⁇ M) much higher than its IC 50 for ⁇ V ⁇ 3 of 2.5 nM (Dechantsreiter et al., 1999). Generally, IC 50 values are >K D values. Ligand concentrations far above the Kp will drive ligand binding to completion, and drive the conformational equilibrium to the conformer with the highest affinity for ligand. This identifies the extended conformer with the open headpiece as the conformation with highest affinity for ligand (Fig. 19E).
  • the extended conformer with the closed headpiece represents an intermediate conformation, because it shares the closed headpiece with the bent conformer, and shares the lack of a tailpiece-headpiece interface with the extended conformer with the open headpiece.
  • the presence of all three species of conformers in Mn 2+ , and only the bent conformer in Ca 2+ alone and only the extended, open conformer in cyclo-RGDfV also suggests that the extended, closed conformer is an intermediate in the conformational equilibrium between the low and high affinity forms. Because the equilbria of conformational change and ligand binding are thermodynamically linked (Marvin and Hellinga, 2001), it is reasonable to assume that the extended, closed conformer has an intermediate affinity for ligand (Fig. 19D).
  • Integrins vary markedly in their susceptibility to activation, e.g. as demonstrated among different ⁇ l integrins in ability of Mn 2+ or soluble ligands to induce expression of ⁇ l subunit activation epitopes (Bazzoni et al., 1998). Electron microscopy of clasped ⁇ 5 ⁇ l and ⁇ llb ⁇ 3 integrins prepared similarly to clasped ⁇ V ⁇ 3 shows that in Ca 2+ they also adopt the bent conformation. However, a significantly higher proportion of particles adopted the extended conformation in Ca 2+ than seen with ⁇ V ⁇ 3. Therefore, the equilibrium point between the bent and extended conformations may differ depending on the integrin.
  • ⁇ V ⁇ 3 is particularly stable in the bent conformation compared to other integrins, favoring crystallographic studies that require homogenous preparations. It is not yet clear for a resting integrin, or for a fully activated integrin, whether all conformational states are kinetically accessible. However, it is useful to think of activators or inhibitors as influencing the equilibria that relate conformers, and changing the lifetimes of particular conformers, rather than locking in a particular conformation. Almost all factors with which integrins interact, such as cytoplasmic proteins (Liu et al., 2000) and the TM4 superfamily proteins (Hemler, 1998), may influence the conformational equilibria.
  • the C-terminal covalent clasp helped to stabilize ⁇ V ⁇ 3 in the bent, low affinity conformation as revealed by the higher percentage of bent particles in our electron micrographs, and the lower ligand binding activity in Ca 2+ and Mg 2+ .
  • Previous EM images of integrins with widely separated legs are consistent with integrins in a high affinity conformation in which inter-subunit restraints between membrane proximal segments have been broken.
  • Many anti-integrin mAbs have been reported that bind preferentially to the active and/or ligand-occupied form of integrins (Bazzoni and Hemler, 1998; Humphries, 2000).
  • ⁇ subunit residues predicted to restrain integrin activation by stabilizing an ⁇ subunit - ⁇ subunit interface are indeed present in an ⁇ - ⁇ interface in the bent conformation (Beglova et al., 2002).
  • the location of epitopes within a combined ⁇ V ⁇ 3/ ⁇ 2 model (Beglova et al., 2002) and the electron microscopic, ligand binding, and cysteine cross bridging studies presented here are consistent with the same switchblade model of integrin activation.
  • cyclo-RGDfV The structural rearrangements demonstrated here after binding of cyclo- RGDfV to ⁇ V ⁇ 3 define a pathway for communication from the ligand binding site in the headpiece to the membrane proximal segments of the ⁇ and ⁇ legs in integrin outside-in signaling.
  • the cyclo-RGDfV peptide bears the RGD sequence that is paradigmatic of many biological integrin ligands. This peptide differs only 4-fold in IC 50 and in one methyl group from its N-methyl-Val derivative, cyclo-RGDf-mV (Dechantsreiter et al., 1999), which was used in ⁇ V ⁇ 3 co-crystals (Xiong et al, 2002).
  • the N-methyl-Val residue does not interact with ⁇ V ⁇ 3, and instead helps stabilize the conformation of the cyclic peptide.
  • cyclo-RGDf-mV bound to the presumptive ligand binding site, with its Asp sidechain coordinating the Mn 2+ in the MIDAS of the ⁇ 3 I-like domain, and its Arg sidechain bound to the ⁇ V ⁇ -propeller domain.
  • the crystal stracture visualized the peptide bound to the bent conformation of ⁇ V ⁇ 3.
  • binding to ⁇ V ⁇ 3 in solution results in a dramatic quaternary rearrangement to an extended conformation with an open headpiece.
  • peptide binds to the low affinity, bent ⁇ V ⁇ 3 conformer, which has a closed headpiece; and in a second step, in the absence of crystal lattice constraints, binding causes a dramatic quaternary rearrangement to the high affinity, extended conformer with an open headpiece.
  • cyclo-RGDfV can augment binding of ⁇ V ⁇ 3 to fibrinogen, fibronectin, and vitronectin when used near its IC 5 o, but is inhibitory at high concentrations (Legler et al., 2001).
  • peptidomimetic and antibody antagonists of ⁇ llb ⁇ 3 are in wide use clinically for treatment of heart disease (Coller, 2001; Scarborough and Gretler, 2000).
  • the peptidomimetic antagonists induce conformational change in ⁇ llb ⁇ 3 on the surface of circulating platelets, as well documented with LIBS monoclonal antibodies (Kouns et al., 1992). This has important clinical implications, because a small subset of patients have pre-existing LIBS antibodies that result in thrombocytopenia (Billheimer et al., 2002; Scarborough and Gretler, 2000).
  • integrins Inside-out activation of integrins occurs as a result of breakage of interactions between the membrane proximal regions of the ⁇ and ⁇ subunits (Hughes et al., 1996; Lu et al, 2001c; Takagi et al, 2001; Weljie et al, 2002).
  • the talin head domain can directly activate integrins by binding to the ⁇ subunit cytoplasmic domain (Calderwood et al., 2002).
  • Destabilizing the headpiece-tailpiece interface, and thereby favoring the extended integrin conformation provides a mechanism for communicating conformational change from the membrane to the headpiece.
  • the headpiece - tailpiece and ⁇ tailpiece - ⁇ tailpiece interfaces in integrin activation The headpiece-tailpiece and ⁇ tailpiece - ⁇ tailpiece interfaces have key roles in regulating integrin activation and deactivation, and in relaying signals between the ligand binding site and the cytoplasmic domains. Initially, these interfaces either were not mentioned or were suggested to be small and discontinuous (Xiong et al., 2001). To the contrary, each of these buries over 2000 A 2 (Table 10).
  • the combined size of the two interfaces is 4,400 A 2 ; interestingly, with interfaces in the range of 2,000 to 4,000 A 2 , significant conformational changes are often seen when associated and unassociated structures are compared (Conte et al., 1999). Such changes at the genu and at the I-like domain - hybrid domain interface have already been resolved here by electron microscopy. Detailed characterization of the extended conformation shows that when the headpiece-tailpiece interface is broken, the ⁇ tailpiece - ⁇ tailpiece interface is also broken. The ⁇ subunit adopts a fairly uniform orientation in the extended conformation, showing that the genu snaps into a stable alternative conformation after unbending and that the other interdomain interfaces in the ⁇ leg are stable.
  • the flexibility of the lower ⁇ leg illustrates the difficulty of transmitting conformational signals through long, extended structures.
  • Other surface receptors that transmit signals have ligand binding sites that are much closer to the membrane (Falke and Hazelbauer, 2001; Remy et al., 1999). How signals could be transmitted through the long integrin legs that are seen in the extended conformation has been a mystery.
  • Transition between bent and extended conformations provides a solution to the problem of signal transmission over long distances. It enables an active ligand binding site to be displayed far above the surface of a cell, while equilibration with a bent, inactive conformation enables signals between the ligand binding site and the cytoplasmic domains to be relayed over relatively short distances to protein interfaces present in the bent conformation.
  • Our findings suggest that both inside-out and outside-in signals destabilize the bent conformation and are consistent with the observation that ligand binding to integrins and inside-out activation of integrins induce an overlapping or even identical set of epitopes.
  • Mn 2+ may activate by a similar mechanism (Fig. 19B). Only a proportion of extended molecules in Mn 2+ have the open headpiece; however, if swing-out of the hybrid domain occurred in a portion of the bent molecules, this would provide a mechanism for triggering extension. Open extended molecules (Fig. 19E) could then convert to closed extended molecules (Fig. 19D) and in turn to bent molecules, providing a mechanism for equilibration between all three conformers in Mn 2+ .
  • the obtuse orientation of the hybrid domain is unequivocally distinct from the acute orientation seen in the ⁇ V ⁇ 3 crystal stracture, and in a subset of EM averages in Mn 2+ . Therefore, a conformational change occurs at the I-like domain - hybrid domain interface upon conversion to the high affinity state.
  • the change in orientation of the hybrid domain relative to the I-like domain occurs as a result of a downward movement of the C-terminal ⁇ -helix of the I-like domain that is analogous to that seen in I domain activation, and is similarly linked to a change in affinity.
  • the I and I-like domains are each connected through both their N and C-termini to the domains in which they are inserted, the ⁇ -propeller and hybrid domains, respectively (Fig. 13).
  • the I-like domain's C-terminus is closer than its N-terminus to the ⁇ subunit; therefore, a downward movement of the C-terminal ⁇ -helix would pivot the hybrid domain away from the ⁇ subunit about a fulcrum at the N-terminus of the I-like domain.
  • a stracture in which this movement was modeled fit the projection averages with the open headpiece significantly better than the crystal structure, which exhibits a closed headpiece.
  • the EM projection averages demonstrate swing-out of the hybrid domain in the ligand-bound, open configuration of the headpiece. They do not define the underlying detailed structural rearrangements; for example, unwinding of a portion of the C-terminal ⁇ -helix from a rigid body movement of this helix cannot be distinguished. However, alternative movements such as unwinding do not provide a mechanism for coupling cyclo-RGDfV binding at the top of the I-like domain to hybrid domain swing-out at the bottom of the I-like domain, which the EM studies clearly establish.
  • Soluble, clasped ⁇ V ( ⁇ V-AHCys) and ⁇ 3 ( ⁇ 3-tev-BHCys) constructs were prepared from wild type human ⁇ V and ⁇ 3 cDNAs by overlap extension PCR using the same design as for soluble clasped ⁇ 5 ⁇ l (Takagi et al., 2001), except that a hexahistidine tag was fused to the C-terminus of ⁇ 3.
  • Soluble integrin was purified from culture supernatant of stably transfected CHO lee 3.2.8.1 cells (Stanley, 1989) using Ni-NTA agarose (QIAGEN) followed by anion-exchange (monoQ) and gel filtration (Superdex 200 HR) chromatographies. The final preparation was devoid of aggregated materials and eluted as a single peak around 300 kDa, and was stored at 0.5 - 1 mg/ml at 4°C in 50 mM Tris HC1 pH 7.5, 150 mM NaCl (TBS), containing ImM CaCl 2 and 1 mM MgCl 2 . Release of the C- terminal clasp of ⁇ V ⁇ 3 was achieved by incubation with 250 U/ml TEV protease (Invitrogen) at 25 °C for 16 h.
  • TEV protease Invitrogen
  • Grids were washed with two drops of deionized water and stained with two drops of freshly prepared 0.75% uranyl formate. Specimens were inspected with a Philips Tecnai 12 electron microscope operated at 120 kV and images were recorded at a nominal magnification of 52,000x using low-dose procedures. Images were digitized with a Zeiss SCAI scanner using a step size of 7 ⁇ m and 3 x 3 pixels were averaged to yield a final pixel size of 4 A at the specimen level. Particles were windowed into smaller images of 100 x 100 pixels, grouped into different classes using the multi-reference alignment procedure, and the particles in each class were averaged to produce class averages.
  • the X-ray models were resolution-filtered to 25 A and projections were calculated at an angular interval of 2 degrees. These re-projections were cross-correlated to selected class-averages and the re-projections with the highest correlation coefficient are shown in Figure 15. All image processing was carried out with the SPIDER image processing package (Frank et al., 1996).
  • Full-length human ⁇ 3 cDNA was transferred to pcDNA3.1/Myc (Invitrogen) so that the C- terminus was in-frame with the Myc and hexahistidine tags.
  • Full-length human ⁇ V and ⁇ llb cDNAs were transfened to pEFl/V5-His (Invitrogen) so that the C-terminus was in- frame with the V5 tag.
  • Cysteine mutations (G307C in D V, R320C in ⁇ llb, and R563C in ⁇ 3) were made by site-directed mutagenesis.
  • FITC- fibrinogen Binding of FITC- fibrinogen was determined as previously described (Pampori et al., 1999). Activating mAbs AP5 (5th International Leukocyte Workshop) and PT25-2 (gift from M. Handa, ) were used at 1:50 ascites and 7.5 ⁇ g/ml, respectively. After a final staining with Cy3 labeled AP3 (nonfunctional anti- ⁇ 3 mAb, American Type Culture Collection), the ⁇ 3-positive cell population was analyzed for the binding of FITC-fibrinogen.
  • Table 10 Solvent accessible surface area buried in tailpiece-headpiece and ⁇ tailpiece- ⁇ tailpiece interfaces in ⁇ V ⁇ 3 a .
  • the ⁇ V ⁇ 3 crystal structure (1 JV2) (Xiong et al., 2001) was extended by moving the headpiece ( ⁇ 1-594, ⁇ 55-434) relative to the tailpiece ( ⁇ 595-956, ⁇ 532-690) so that the covalent connection between residues 594 and 595 at the genu was maintained and the thigh - calf-1 interface was minimized.
  • the areaimol function of the CCP4 suite was used to compare solvent exposed surface areas of the extended and bent stractures (Bailey, 1994; Lee and Richards, 1971).
  • the ⁇ and ⁇ tailpieces were placed in separate PDB files and compared to the file containing both tailpieces. Due to a technical problem comparing residues with atoms in different orders in the PDB files, the size of the headpiece-tailpiece interface was previously underestimated (Beglova et al., 2002).
  • This Example describes the production of modified integrins that include an unnatural glycosylation site.
  • /33 integrins ⁇ V/33 and ⁇ TIb/33
  • the ⁇ 3 subunit was modified such that Asn305/3 was substituted with Threonine, resulting in an unnatural glycosylation at Asn303.
  • ⁇ l integrins the ⁇ l subunit was modified such that Pro333 was substituted with Asparagine, resulting in an unnatural glycosylation at Asn333.
  • 82 integrins ⁇ L/32, in one example, the 82 subunit was modified such that Gln295 was substituted with Threonine, resulting in an unnatural glycosylation at Asn293.
  • the glycan wedge high affinity ⁇ IIb/33 mutants are constitutively active in the presence of calcium without the addition of Mn/PT25, a monoclonal antibody which activates integrin heterodimers (see Figure 17, left panel).
  • Figure 17 (right panel) illustrates that the ⁇ lTb/33 glycan wedge mutant adheres to the fibrinogen ligand at a lower concentration of fibrinogen as compared to the wild type.
  • the ⁇ 4- ⁇ 5 loop includes amino acid residues at positions that corresponding to the amino acids between about 295 to 308 or about 302 to 306 of SEQ ID NO: 15.
  • This site is far from the ligand binding site on the "upper" face of the I-like domain containing the MIDAS, and thus should only enhance ligand binding allosterically. Furthermore, the site is distal from both the headpiece-tailpiece interface and the ⁇ -propeller-I-like domain interface, and thus should not directly affect either of these interfaces which have also been suggested to regulate ligand binding.
  • the ⁇ 3 mutation Asn-305 ⁇ Thr introduced the N303-I304- T305 N-glycosylation sequence, and is predicted to result in attachment of a bulky N- glycan "wedge" at N303 in the ⁇ 3 protein.
  • Wild type or mutant ⁇ 3 ( ⁇ 3 N305T ) was co-transfected with wild type ⁇ llb subunit into CHO-K1 cells. Stable clones expressing similar levels of mutant and wild type ⁇ llb ⁇ 3 were selected. Similar binding to wild type and mutant receptors by mAbs to multiple ⁇ llb and ⁇ 3 epitopes including AP3 (anti- ⁇ 3), 7E3 (anti- ⁇ 3), HA5 (anti- ⁇ llb) and AP2 (anti- ⁇ llb ⁇ 3 complex specific) suggested that the mutant receptor adopts a native fold. Soluble ligand binding was measured using two-color flow cytometry.
  • mutation of Asn-303 to Trp to introduce a bulky sidechain was not activating, consistent with ample space for the Trp sidechain in the closed interface.
  • the affinity state of ⁇ IIb ⁇ 3 N305T was further tested in cell adhesion assays. Although high affinity is required for soluble ligand binding, basally active, i.e., low affinity ⁇ llb ⁇ 3 can mediate cell adhesion to high density substrates. Wild type ⁇ llb ⁇ 3 transfectants adhered to fibrinogen coated substitutes at coating concentrations of ⁇ l ⁇ g/ml. Parent CHO-K1 cells showed no adhesion even at the highest concentration of fibrinogen. In contrast, cells expressing ⁇ llb ⁇ 3 adhered to fibrinogen surfaces at coating concentrations as low as 10 ng/ml. The overall shift in the dose-response curve showed that the mutant receptor had at least 10 times higher activity than wild type.
  • the wedge mutant was hyperactive in soluble fibrinogen and PAC-1 binding assays just as in CHO-K1 cells.
  • Transiently transfected 293T cells were metabolically labeled and ⁇ llb ⁇ 3 complex was immunoprecipitated with ⁇ 3 mAb AP3.
  • Non-reducing SDS-PAGE showed that the ⁇ llb subunits migrated similarly, whereas the mutant ⁇ 3 migrated slower than wild type ⁇ 3, with -3 kDa increase in molecular mass (Fig.3A, lanes 1, 2).
  • this difference between wild type and mutant ⁇ 3 disappeared upon deglycosylation by PNGaseF (Fig.3A, lanes 3 and 4).
  • the increased molecular mass of the mutant ⁇ 3 is therefore due to additional N-glycosylation, suggesting that the N305T mutation resulted in the attachment of a glycan chain at Asn303.
  • transient transfectants were treated prior to and after transfection with the N-glycosylation inhibitor tunicamycin.
  • Ligand-binding by wild-type ⁇ llb ⁇ 3 was unaffected by tunicamycin, since treated and untreated cells showed similar fibrinogen binding after stimulation with Mn and activating mAb (Fig. 3B).
  • tunicamycin treatment abolished the constitutive ligand binding activity by ⁇ llb ⁇ 3 , whereas like wild type it was still activatable by Mn and mAb (Fig. 3B). Therefore, carbohydrate attachment at residue ⁇ 3-N303 endowed the receptor with high affinity, rather than the mutation of Asn305 to Thr.
  • Wild type human ⁇ 1 transfectants bound a biotinylated fibronectin fragment containing domains 9 and 10 (FN 9-10 ) only when the integrin was activated with mAb TS2/16 to human ⁇ l.
  • ⁇ l P333N -transfectants bound Fn -10 in the absence of mAb TS2/16 activation. Binding was mediated by transfected mutant human ⁇ l, since it was inhibited by anti-human ⁇ l blocking mAb 13.
  • a glycan wedge in the cleft between the I-like and hybrid domains can activate both ⁇ l and ⁇ 3 integrins.
  • ligand induced binding sites Such epitopes are buried in the bent conformation in headpiece-tailpiece and ⁇ tail- ⁇ tail interfaces, and are exposed in the extended conformation 4 .
  • binding of a panel of anti-LIBS mAbs was determined.
  • the mAbs AP5 (anti- ⁇ 3, residues 1-6), LIBSl (anti- ⁇ 3), LQ3S6 (anti- ⁇ 3, residues 602-690), and PMI-1 (anti- ⁇ llb, residues 844-859) bound poorly to the cells stably expressing the wild-type ⁇ llb ⁇ 3 in Ca 2+ but bound maximally when incubated with Mn 2+ and RGD peptide.
  • all LIBS mAbs stained cells expressing the glycan wedge mutant ⁇ llb ⁇ 3 brightly even in Ca 2+ in the absence of ligand, and no further increase was observed upon addition of Mn 2+ and RGD peptide.
  • Another LIBS mAb D3 (anti- ⁇ 3, residues 422-490) also bound constitutively to the mutant ⁇ llb ⁇ 3.
  • the hybrid domain is located in the headpiece and has a larger contribution than any other domain to the headpiece-tailpiece interface of 2,370 A 2 .
  • the glycan wedge is predicted not to push the hybrid domain toward the tailpiece, but laterally out of the headpiece-tailpiece interface, and thus to indirectly destabilize it.
  • Ligand- induced swing out of the hybrid domain has been previously predicted to destabilize the bent conformation by the same mechanism, and the maximal exposure of LIBS epitopes in the wedge mutant accords with this model of integrin activation. The above studies did not define whether high ligand binding affinity was a proximate effect of straightening the bend between the I-like and hybrid domains or secondary to transition from the bent to extended integrin conformation.
  • the ⁇ V ⁇ 3 integrin can be locked in the bent conformation by an intersubunit disulfide bond between the ⁇ subunit ⁇ -propeller domain and ⁇ subunit I-EGF4 domain.
  • the disulfide-locked integrin is inactive and resistant to activation by Mn 2+ and antibodies unless a disulfide reducing agent is added.
  • the postulated lateral swing out of the hybrid domain would not be blocked by this disulfide bond. Therefore, the two types of mutations were introduced into the same ⁇ V ⁇ 3 integrin molecules. Immunoprecipitation and non-reducing SDS-PAGE confirmed complete formation of the disulfide bond between the ⁇ V head and ⁇ 3 tail, suggesting that the doubly mutant receptor was locked in the overall bent conformation.
  • This result demonstrates that activation of high affinity ligand binding by the wedge mutant is not secondary to adoption of the extended conformation of the integrin.
  • the ⁇ -subunit I-like domain has a similar fold to the I domain which is present in a subset of integrin ⁇ subunits and serves as the major ligand binding site in the integrins in which it is present.
  • the I and I-like domains are each connected through both their N and C termini to the domains in which they are inserted, the ⁇ -subunit ⁇ - propeller and ⁇ -subunit hybrid domains, respectively.
  • downward movement of the C-terminal ⁇ -helix causes rearrangement of residues that form the metal ion dependent adhesion site (MIDAS), thereby increasing ligand affinity.
  • MIDAS metal ion dependent adhesion site
  • the structural similarity between I and I-like domains suggests a similar regulatory mechanism, in which a downward movement of the C-terminal ⁇ -helix of the I-like domain would allosterically regulate the conformation of the MIDAS, and increase affinity for ligands such as RGD.
  • the glycan wedge-induced widening of the angle between the I-like and hybrid domains is predicted to be tightly linked to downward displacement of the C-terminal ⁇ -helix of the I-like domain, based on the overall topology of the interdomain connections.
  • three are internal ⁇ -sheet strands, and hence are fixed in position by hydrogen bond networks.
  • the hydrogen bonds in an ⁇ -helix are all local, within the secondary stracture element, and permit displacement relative to neighboring structural elements.
  • the I-like domain ⁇ -helix connection to the hybrid domain ⁇ -sheet is the innermost connection, and a piston-like downward ⁇ -helix movement at this connection would result in swiveling about a fulcrum at the other connection, opening the interface to accommodate the nearby glycan wedge.
  • a disulfide bond that stabilizes the bent conformation favors low affinity for ligand; interestingly, the wedge mutant is dominant over this mutant in its effect on affinity, consistent with a more downstream location of the I-like domain-hybrid domain interface in the affinity regulation pathway.
  • the rational design of mutations that allosterically stabilize high affinity or low affinity conformations of integrins demonstrates marked advances in our understanding of the molecular basis of affinity regulation. Further, drugs might be designed that stabilize the low affinity conformation of integrins, in contrast to the current generation of "ligand-mimetic" integrin antagonists that stabilize the high affinity conformation..
  • Plasmid construction, expression, and ligand binding activity of integrin on CHO- 1 cells Plasmids coding for full-length human ⁇ llb, ⁇ 3 and ⁇ l were subcloned into pcDNA3.1/Myc-His(+) or pEFl/V5-HisA as described previously, and were introduced into the CHO-K1 cells using calcium phosphate precipitation. After selecting the transfected cells in a medium containing 5 mg/ml geneticin G418 for one week, the surviving cells were assessed for integrin expression by flow cytometry using anti- ⁇ 3 7E3 (gift from B.S. Coller) or anti- ⁇ 1 TS2/16.
  • the cells were then sorted by fluorescence-activated cell sorting to obtain cell lines expressing the desired level of ⁇ llb ⁇ 3 or ⁇ 5 ⁇ l integrins. Binding of fluorescein-labeled human fibrinogen and ligand mimetic antibody PAC-1 (gift from S.J. Shattil) were performed as previously described. For the binding of fibronectin fragment to human ⁇ l- expressing cells, the cells were incubated with 50 ⁇ g/ml biotinylated Fn 9-10 in the presence or absence of activating mAb TS2/16 (lO ⁇ g/ml) at room temperature for 30 min, and stained with FITC-streptavidin (Zymed) followed by flow cytometry. Cell adhesion to human fibrinogen (Enzyme Research Laboratories) was determined by assaying cellular phosphatase.
  • Anti-LIBS mAb AP5 was from the Fifth International Leukocyte Workshop, and LIBS-1, LIBS-6 and PMI-1 were from M.H. Ginsberg.
  • CHO-K1 cells stably expressing wild type or mutant ⁇ llb ⁇ 3 were incubated with anti- LIBS mAbs at concentration of 10 ⁇ g/ml at room temperature for 30 min, followed by staining with FITC-conjugated secondary antibody and flow cytometry.
  • 293T cells were transiently transfected with wild type or mutant integrin cDNAs using calcium phosphate precipitation, and were metabolically labeled with [ 35 S]cysteine/methionine as described. Labeled cell lysates were first immunoprecipitated with anti- ⁇ 3 AP3 (ATCC), eluted with 0.5% SDS, and after addition of 1% NP-40 treated with or without 500 units of PNGase F (NEB) at 37°C for lh. Material was subjected to non- reducing 7% SDS-PAGE and fluorography.
  • Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels, Cell 79, 1157-1164.
  • Anti-GPIIb/IIIa drugs current strategies and future directions, Thromb Haemost 86, 427-43.
  • N-Methylated cyclic RGD peptides as highly active and selective alpha(V)beta(3) integrin antagonists J Med Chem 42, 3033-3040.
  • Integrin activation involves a conformational change in the alpha 1 helix of the ⁇ subunit A-domain, J Biol Chem in press.
  • Synthetic peptides derived from fibrinogen and fibronectin change the conformation of purified platelet glycoprotein Ilb-IIIa, J Biol Chem 262, 12597-12602.
  • the integrin ⁇ b ⁇ 3 platelet glycoprotein Ilb-IIIa, can form a functionally active heterodimer complex without the cysteine-rich repeats of the ⁇ subunit, J Biol Chem 269, 8754-8761.
  • Gly lie Ser Ala Asp Leu Ser Arg Gly His Ala Val Val Gly Ala Val 355 360 365 Gly Ala Lys Asp Trp Ala Gly Gly Phe Leu Asp Leu Lys Ala Asp Leu 370 375 380
  • Gly lie Gin Trp Phe Gly Arg Ser lie His Gly Val Lys Asp Leu Glu 580 585 590 Gly Asp Gly Leu Ala Asp Val Ala Val Gly Ala Glu Ser Gin Met He 595 600 605

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Abstract

L'invention concerne, entre autres, une protéine isolée ou recombinée qui comprend des domaines extracellulaires de sous-unités α et β d'intégrine. Au moins une des sous-unités comprend une ou plusieurs mutations qui provoquent une différence dans la préférence conformationnelle de la protéine, entre les états conformationnels plié et étendu ; ou au moins une des sous-unités comprend une caractéristique de surface modifiée de sorte que la protéine présente (1) une différence dans la préférence conformationnelle de la protéine entre les états plié et étendu, ou (2) une différence d'affinité pour un ligand apparenté dans une ou plusieurs conditions de réaction.
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WO2011020529A3 (fr) * 2009-08-19 2011-06-16 Merck Patent Gmbh Anticorps pour la détection de complexes d'intégrine dans une matière ffpe
WO2016022851A1 (fr) * 2014-08-06 2016-02-11 Children's Medical Center Corporation Polypeptides d'intégrine modifiés, dimères de polypeptides d'intégrine modifiés, et leurs utilisations
WO2017132620A1 (fr) * 2016-01-29 2017-08-03 La Jolla Institute For Allergy And Immunology Méthodes et compositions utilisant des produits thérapeutiques à base d'intégrine
CN113272317A (zh) * 2018-11-02 2021-08-17 华盛顿大学 正交蛋白异二聚体

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BAJT M.L. ET AL.: 'Characterization of a gain of function mutation of integrin alphaIIbBeta3 (Platelet Glycoprotein IIb-IIIa)' JOURNAL OF BIOLOGICAL CHEMISTRY vol. 267, no. 31, 05 November 1992, pages 22211 - 22216, XP002975728 *
SALAS A. ET AL.: 'Transition from rolling to firm adhesion is regulated by the conformation of the I domain of the integrin lymphocyte function-associated antigen-1' JOURNAL OF BIOLOGICAL CHEMISTRY vol. 277, no. 52, 27 December 2002, pages 50255 - 50262, XP002975729 *
TAKAGI J. ET AL.: 'Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling' CELL vol. 110, 06 September 2002, pages 599 - 611, XP002975730 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011020529A3 (fr) * 2009-08-19 2011-06-16 Merck Patent Gmbh Anticorps pour la détection de complexes d'intégrine dans une matière ffpe
JP2013502205A (ja) * 2009-08-19 2013-01-24 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング Ｆｆｐｅ材料におけるインテグリン複合体検出のための抗体
US8420348B2 (en) 2009-08-19 2013-04-16 Merck Patent Gesellschaft Mit Beschrankter Haftung Antibodies for the detection of integrin complexes in FFPE material
US8993261B2 (en) 2009-08-19 2015-03-31 Merck Patent Gmbh Antibodies for the detection of integrin complexes in FFPE material
EA032728B1 (ru) * 2009-08-19 2019-07-31 Мерк Патент Гмбх Антитела для выявления комплексов интегрина в ffpe материале
WO2016022851A1 (fr) * 2014-08-06 2016-02-11 Children's Medical Center Corporation Polypeptides d'intégrine modifiés, dimères de polypeptides d'intégrine modifiés, et leurs utilisations
US10273283B2 (en) 2014-08-06 2019-04-30 The Children's Medical Center Corporation Modified integrin polypeptides, modified integrin polypeptide dimers, and uses thereof
US11104713B2 (en) 2014-08-06 2021-08-31 The Children's Medical Center Corporation Modified integrin polypeptides, modified integrin polypeptide dimers, and uses thereof
WO2017132620A1 (fr) * 2016-01-29 2017-08-03 La Jolla Institute For Allergy And Immunology Méthodes et compositions utilisant des produits thérapeutiques à base d'intégrine
CN113272317A (zh) * 2018-11-02 2021-08-17 华盛顿大学 正交蛋白异二聚体

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