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WO2000076456A2 - Modulateurs de fibrose - Google Patents

Modulateurs de fibrose Download PDF

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Publication number
WO2000076456A2
WO2000076456A2 PCT/US2000/016366 US0016366W WO0076456A2 WO 2000076456 A2 WO2000076456 A2 WO 2000076456A2 US 0016366 W US0016366 W US 0016366W WO 0076456 A2 WO0076456 A2 WO 0076456A2
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Prior art keywords
eiiia
polypeptide
compound
sequence
domain
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WO2000076456A3 (fr
Inventor
Livingston Van De Water
Kenneth G. Wieder
Yung-Feng Liao
Jeanne M. Classen
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General Hospital Corp
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General Hospital Corp
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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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to promotion or inhibition of fibrosis.
  • FNs are a group of extracellular matrix (ECM) proteins that mediate cell adhesion, migration, proliferation, and differentiation (Hynes, Fibronectins, Springer- Verlag, New York, 1990). FNs play significant roles in embryonic development and are prominent components of the provisional matrix following tissue injury in adults
  • --$6Jje FNs are disulfide-linked, dimeric glycoproteins with structural domains that bind to cells, collagen, proteoglycans, and fibrin.
  • Each FN consists of homologous repeats designated as either type I, II, or III. Individual type III repeats within FN exhibit high sequence similarity between species (greater than 90% identity; Schwarzbauer et al, EMBO J. 6:2573-25S0, 19S7).
  • each type III repeat consists of two beta sheets, which are made up of four strands (G, F, C, C) and three strands (A, B, E), respectively, folded into a beta sandwich (Leahy et al., supra).
  • compositions and methods that can be used to either promote or inhibit fibrosis or scarring in various biological tissues. These compositions and methods are based on the discovery that the sequence or conformation of a particular FN domain, i.e., the EIIIA domain, must be maintained if FN is to participate in fibroblast activation. The importance of the EIIIA amino acid sequence and the conformation of this region were discovered by mapping the epitope to which two EIIIA- specific antibodies, IST-9 and DH1, bind. These mapping studies revealed that the IST-9 and DH1 "function-blocking" antibodies bind to the Ile 43 and residues within the EIIIA domain of FN in a conformation-dependent fashion. Thus, the conformation of the EIIIA domain, including a loop region encompassing both the Ile 43 and His 44 residues, is critical for mediating EIIIA function.
  • mutant EIIIA-containing FN polypeptides which are, accordingly, within the invention.
  • These mutants include FN polypeptides in which the sequence and/or conformation of the EIIIA domain has been altered so that the mutant polypeptides are unable to induce cellular activation (e.g., macrophage, endothelial cell, or fibroblast activation) to the same extent as wild type FN polypeptides.
  • cellular activation e.g., macrophage, endothelial cell, or fibroblast activation
  • TGF- ⁇ transforming growth factor ⁇
  • SMC smooth muscle cell
  • TGF- ⁇ e.g. 10 ng/ml
  • SMC ⁇ -actin expression e.g., by standard techniques such as Northern blot analysis, RNAse protection assays, or quantitative PCR.
  • the level of expression would be designated as 100% for purposes of comparison with cultures in which mutant FN polypeptides are included.
  • Mutant FN polypeptides in which the sequence and/or conformation of the EIIIA domain has been altered are within the scope of the invention if they inhibit TGF- ⁇ -induced SMC ⁇ -actin expression by at least 50%, for example, by at least 65%, 80%, 95% or more.
  • Assays of cellular activation can also be used to assess the ability of a therapeutic agent (e.g., a synthetic peptide, small molecule, or antibody) to inhibit cellular activation.
  • a therapeutic agent e.g., a synthetic peptide, small molecule, or antibody
  • TGF- ⁇ e.g. 10 ng/ml
  • SMC ⁇ -actin expression e.g., by standard techniques such as Northern blot analysis, RNAse protection assays, or quantitative PCR.
  • Other cell types e.g., macrophages and endothelial cells could also be tested.
  • the level of expression would be designated as 100% for purposes of comparison with cultures in which the cells were also exposed to a putative or candidate therapeutic agent.
  • Putative therapeutic agents positively identified by the assay would be those that inhibit TGF- ⁇ - induced SMC ⁇ -actin expression by at least 50%, for example, by 65%, 80%, 95% or more.
  • ⁇ -actin is only one of the markers of cellular activation. Those of ordinary skill in the art will recognize and be able to assay other markers of cellular activation.
  • collagen e.g., type I or type III collagen
  • metalloproteinases e.g., metalloproteinases
  • PAI-1 plasminogen activator inhibitor
  • Mutant FN polypeptides e.g., polypeptides that include the C-C region and those in which the sequence and/or conformation of the EIIIA domain has been altered
  • small molecules, and antibodies are within the scope of the invention if they inhibit expression of a marker of cellular activation (e.g., type I collagen) by at least 50%, for example, by 65%, 80%, 95% or more.
  • a marker of cellular activation e.g., type I collagen
  • These peptides, small molecules, and antibodies are useful in inhibiting cellular activation.
  • Mutant FN polypeptides, small molecules, and antibodies that, to the contrary, enhance expression of a marker of cellular activation are also within the scope of the invention and are useful in promoting cellular activation.
  • Therapeutic agents that induce cellular activation e.g., macrophage, endothelial cell, or fibroblast activation
  • therapeutic agents that inhibit cellular activation e.g., macrophage, endothelial cell, or fibroblast activation
  • inhibit cellular activation e.g., macrophage, endothelial cell, or fibroblast activation
  • the invention features mutant FN polypeptides in which the sequence of the EIIIA domain is not altered (i.e., the mutation (e.g., a deletion or substitution of one or more amino acid residues) lies within the FN polypeptide, but not within the EIIIA domain).
  • the invention features mutant FN polypeptides in which the conformation of the EIIIA domain is not substantially different from the conformation of the EIIIA domain in wild type FN (i.e., there is a mutation within the FN polypeptide, which may be within the EIIIA domain or elsewhere, but the mutation is one that does not substantially disrupt the conformation of the EIIIA domain).
  • a mutant FN polypeptide in which the conformation of the EIIIA domain is substantially disrupted is a polypeptide that One of ordinary skill in the art can assess these types of mutant FN polypeptides in a variety of routine assays, for example, in the SMC ⁇ -actin assay described above. That is, one can culture a cell (e.g., a fibroblast) on wild type FN and, in a parallel culture, on a mutant FN polypeptide in which the sequence or conformation of the EIIIA domain is not altered, and assess expression of SMC ⁇ -actin.
  • a cell e.g., a fibroblast
  • the mutant FN has increased cellular activation (e.g., macrophage, endothelial cell, or fibroblast activation) and would, in vivo, increase fibrosis and/or scarring.
  • cellular activation e.g., macrophage, endothelial cell, or fibroblast activation
  • Mutant FN polypeptides in which the sequence or conformation of the EIIIA domain has not been altered are within the scope of the invention if they increase SMC ⁇ -actin expression by at least 10% or more, e.g., about 25%, 40%, or 50% higher than 100%.
  • the mutant FN has decreased fibroblast activation and would, in vivo, decrease fibrosis and/or scarring.
  • mutant FN polypeptides are within the scope of the invention if they inhibit TGF- ⁇ -induced SMC ⁇ -actin expression by 50% or more, e.g., by about 65%, 80%o, or even 95% or more.
  • cell types other than fibroblasts e.g., macrophages and endothelial cells could also be tested.
  • the invention features nucleic acid molecules having a sequence that is the reverse and complement of (i.e., is antisense to) the sequence of the EIIIA domain of FN, and methods of administering these molecules to inhibit fibrosis or scarring in a patient.
  • antisense fibronectin molecules can be delivered to a patient using any of the known techniques for delivering gene constructs to cells.
  • retroviral vectors can be used to deliver antisense molecules that reduce the presence of the EIIIA domain in fibrotic tissue (as seen at the site of a wound, a bum, an implanted device, or within a fibrotic organ, such as the liver, lung, or kidney).
  • the invention features methods of screening for compounds that promote or inhibit fibrosis.
  • the invention features methods of screening for compounds that inhibit cellular activation by associating with (e.g., binding to or altering the conformation of) the EIIIA domain of FN.
  • the invention also features methods of screening for compounds that inhibit the interaction between cells and the EIIIA domain.
  • the invention features methods of modulating cellular activation (e.g., macrophage, endothelial cell, or fibroblast activation) by contacting the EIIIA region of FN with a compound that associates with the EIIIA region (e.g., that binds to the EIIIA region of the FN polypeptide in a manner that inhibits the ability of FN to function normally at the site of a wound (i.e., that inhibits the interaction between cellular receptors (e.g., ⁇ 9 ⁇ l) and FN or between TGF- ⁇ and FN)) or with any portion of the FN molecule in a way that alters the conformation of the EIIIA region.
  • a compound that associates with the EIIIA region e.g., that binds to the EIIIA region of the FN polypeptide in a manner that inhibits the ability of FN to function normally at the site of a wound (i.e., that inhibits the interaction between cellular receptors (e.g.,
  • the invention features a method of treating a patient who has, or who is at risk of developing, an unwanted growth of fibrous tissue or a scar by administering to the patient a compound that inhibits cellular activation by, for example, binding to the EIIIA domain of FN (e.g., to the C-C'-E region or to the C-C loop of EIIIA (residues 39-45 in any of SEQ ID NOs 1-5)) or by altering the conformation of the EIIIA domain of FN (e.g., the conformation of the C-C'-E region or the C-C loop of EIIIA) so that the EIIIA-containing FN polypeptide is unable to function as it otherwise would at the site of a wound.
  • a compound that inhibits cellular activation by, for example, binding to the EIIIA domain of FN (e.g., to the C-C'-E region or to the C-C loop of EIIIA (residues 39-45 in any of SEQ
  • compositions of the present invention include substantially pure polypeptides that include a FN polypeptide having a deletion or substitution mutation within the EIIIA domain and substantially pure polypeptides that include a FN polypeptide in which the conformation of the C-C loop is not substantially altered. Additional polypeptides that fall within the invention are described below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
  • Fig. 1A is a comparison of the amino acid sequence of the EIIIA segment of FN from human (SEQ ID NO:l), mouse (SEQ ID NO:2), rat (SEQ ID NO:3), chicken (SEQ ID NO:4), and Xenopus (SEQ ID NO:5), and a consensus sequence (SEQ ID NO:6).
  • Fig. IB is a dendrogram of the polypeptide sequences aligned in Fig. 1 A. The numbers represent the percent identity of the corresponding sequence relative to the human EIIIA sequence.
  • Fig. 2 is a photograph of a Western blot of bacterial EIIIA fusion proteins bound by IST-9. Lane 1 contains wild type rat EIIIA protein, and Lane 2 contains a mutant rat EIIIA protein (rat EIIIA-H44R) in which the conserved His residue was replaced with arginine (Arg).
  • Lane 1 contains wild type rat EIIIA protein
  • Lane 2 contains a mutant rat EIIIA protein (rat EIIIA-H44R) in which the conserved His residue was replaced with arginine (Arg).
  • Fig. 3 is a map of various N-terminal, C-terminal, or internal peptides derived from wild type rat EIIIA (rEIIIA-WT, 90 amino acids). Deletion constructs were generated by PCR and subcloned into the pGEX-2T vector. Arrows indicate the length of individual deletion constructs relative to the wild type sequence shown at the top of the figure. A solid circle above the peptide sequence of rEIIIA-WT highlights the conserved His- M .
  • Figs. 4A-4C are graphs generated following ELISAs of deletion mutants with monoclonal antibodies.
  • the sigmoid plot in each graph represents the reactivity of wild type rat EIIIA with IST-9 (Fig. 4A), 3E2 (Fig. 4B), and DH1 (Fig. 4C). Dilution folds are expressed in log scale. Bar graph insets in each panel illustrate the titer (in percentage) of each deletion protein relative to rEIIIA-WT.
  • the points of OD 42 o at various dilution folds represent the average of quadruples; standard deviations greater than 0.1 OD are shown as bars.
  • Fig. 5 is a bar graph illustrating the reaction of monoclonal antibodies with rat EIIIA double mutants.
  • Recombinant EIIIA protein was mutagenized simultaneously at two amino acid residues, which were replaced with alanines.
  • the specificity of mAbs for mutated rat EIIIA (rEIIIA) fusion proteins was determined by ELIS A, and the titer of each mAb to mutant rat EIIIA determined relative to wild type rat EIIIA (rEIIIA-WT). Solid, gray, and striped bars represent the relative titer of IST-9, 3E2, and DH1, respectively.
  • Fig. 6 is a bar graph illustrating alanine scanning mutagenesis of rat EIIIA and reactivity to mAbs.
  • Mutagenized rat EIIIA (rEIIIA) fusion proteins were generated and purified. The amino acid replaced in each mutant is indicated (as described in the legend of Fig. 5). The titer values are given in percentage (%) using results obtained with wild type rat EIIIA (rEIIIA-WT) as the 100% reference value. Solid, gray, and striped bars indicate the relative titer of IST-9, 3E2, and DH1, respectively.
  • Fig. 7A is a comparison of wild type rat EIIIA (rEIIIA-WT; SEQ ID NO:3) and frog EIIIA (SEQ ID NO:5) sequences, with mutated residues highlighted.
  • Wild type frog EIIIA (fEIIIA-WT) fusion protein, with the sequence V 43 K 4 was mutated at these two residues to resemble rat EIIIA.
  • fEIIIA-WT was mutated only at one position (fEIIIA-K44H).
  • Capitalized letters denote the conserved peptide sequences among human, mouse, rat, chicken, and Xenopus EIIIA proteins.
  • Fig. 7B is a bar graph illustrating the titer of mAbs to rEIIIA-WT, fEIIIA-WT and mutated fEIIIA fusion proteins. Solid, gray, and striped bars indicate the relative titer of IST-9, 3E2, and DH1, respectively. Titer values are determined by using the result of rEIII-WT as 100% reference value.
  • Fig. 8 is a representation of the epitopes for IST-9 and DH1 within EIIIA.
  • the amino acid sequence of wild type rat EIIIA (rEIIIA-WT; SEQ ID NO:3) is shown, and the corresponding anti-parallel ⁇ -strands are indicated by short heavy lines denoted A, B, C, C, E, F, G, respectively, based on the X-ray crystallography of human fibronectin.
  • the predicted epitopes of the EIIIA segment that react with two mAbs, IST-9 and DH1, are highlighted by the shaded box which encompasses the two critical residues (Ile 43 and His 44 ) denoted by an arrow).
  • Figs. 9 A and 9B are line graphs demonstrating that TGF- ⁇ 1 binds the loop region of EIIIA.
  • Fig. 9A shows the OD 42 o following addition of various concentrations of TGF- ⁇ 1
  • Fig. 9B shows the effect of including soluble His-EIIIA in the same experimental paradigm. The results are expressed as relative binding (% of control, with the control being a reaction conducted in the absence of soluble EIIIA). The greater the concentration of soluble His-EIIIA (Competitor ( ⁇ g/ml)), the greater the inhibition of binding between TGF- ⁇ 1 and culture-bound His-EIIIA (% control).
  • Fig. 10 is a bar graph demonstrating that TGF- ⁇ 1 binds the loop region of EIIIA.
  • the graph shows the normalized band intensity (in arbitrary units/nmole) that was observed following exposure of peptide-coated agarose beads to TGF- ⁇ 1. Beneath the graph is the sequence of a portion of wild-type FN.
  • the mutant EIIIA peptides tested (E45A, H44R, H44A, 143 A and G42A) contain mutations within that sequence.
  • Fig. 11 is a bar graph demonstrating that a 22-amino acid peptide (pep #3) inhibits binding between TGF- ⁇ 1 and EIIIA.
  • the sequence of pep #3 is underlined in the sequence of EIIIA that appears beneath the bar graph.
  • Fig. 12 is a bar graph demonstrating that EIIIA binds to integrin ⁇ 9 ⁇ l .
  • the binding is assessed by determining the optical density (OD 5 o) following exposure of SW490 cells that have either been transfected with ⁇ 9 ⁇ l integrin (solid bars) or mock-transfected (striped bars) to peptide-coated (EIIIA-coated or 1114-coated) tissue culture plates. Some cells were preincubated with an anti- ⁇ 9 ⁇ l integrin antibody (EIIIA + Y9A2).
  • Fig. 13 is a bar graph demonstrating that EIIIA-specific antibodies inhibit the interaction between integrin ⁇ 9 ⁇ l and EIIIA.
  • SW490 cells were preincubated with various concentrations (Dilution) of either 3E2 (solid bars) or IST-9 (striped bars) before they were cultured on EIIIA-coated tissue culture plates.
  • Fig. 14 is a bar graph demonstrating that synthetic peptides containing Ile43 and His44 block integrin ⁇ 9 ⁇ l's adhesion to EIIIA.
  • Peptide #1 consists of residues 39-48 of the EIIIA domain with a cysteine residue flanking each of the 5' and 3' ends;
  • peptide #2 consists of residues 24-52 of the EIIIA domain;
  • peptide #3 consists of residues 31-52 of the EIIIA domain;
  • peptide #4 consists of residues 31-34 of the EIIIA domain, linked to a tyrosine residue, linked to residues 35-42 of the EIIIA domain (i.e., 31-32-33-34-Tyr-35-36-37-38- 39).
  • Fig. 15 is a summary of an epitope mapping study of three rat EIIIA-specific monoclonal antibodies (IST-9, 3E2, and DH1) using deleted and otherwise mutagenic rat EIIIA proteins.
  • Fig. 16 is a table of selected synthetic oligonucleotide sequences.
  • Compounds that inhibit cellular activation can be administered before, during, or after a surgical procedure (e.g., an abdominal surgery or a balloon angioplasty) to inhibit, and preferably prevent, adhesions (e.g., intraperitoneal fibrous adhesions) or vascular restenosis (e.g., that induced by injury from a balloon catheter, after which smooth muscle cells proliferate and express EIILA-containing fibronectin).
  • a surgical procedure e.g., an abdominal surgery or a balloon angioplasty
  • adhesions e.g., intraperitoneal fibrous adhesions
  • vascular restenosis e.g., that induced by injury from a balloon catheter, after which smooth muscle cells proliferate and express EIILA-containing fibronectin.
  • Compounds that inhibit cellular activation can also be administered to a patient who has, or is suspected of having, fibroplasia (i.e., an unwanted growth of fibrous tissue, as is known to occur in pulmonary fibrosis, hepatic fibrosis, hypertrophic scars, keloids, asthma, anterior capsular cataracts, kidney fibrosis, fibroid cysts, and fibromatosis (e.g., Dupuytren's disease or scleroderma).
  • fibroplasia i.e., an unwanted growth of fibrous tissue, as is known to occur in pulmonary fibrosis, hepatic fibrosis, hypertrophic scars, keloids, asthma, anterior capsular cataracts, kidney fibrosis, fibroid cysts, and fibromatosis (e.g., Dupuytren's disease or scleroderma).
  • compounds that inhibit cellular activation can be administered to treat epidermolysis bullosa (EB), a group of genodermatoses characterized by fragility and easy blistering of the skin and the mucous membranes (Fine et al., J. Am. Acad. Dermatol. 24:119-135, 1991). Although the initial blistering is in response to mechanical trauma, healing of erosions is compromised by continued fragility of the skin, and in some cases, by excessive granulomatous or fibrotic tissue response.
  • EB epidermolysis bullosa
  • EB can be divided into three major categories on the basis of clinical observations and the level of blister formation, as determined by transmission electron microscopy (Uitto et al., in The Molecular and Cellular Biology of Wound Repair (Second Edition), RA.F. Clark (Ed.), Plenum Press, New York, NY, pp. 547-548, 1996).
  • tissue separation occurs below the basement membrane within the papillary dermis at the level of anchoring fibrils, and healing of the lesion results in extensive scarring (Uitto et al., supra).
  • compounds that inhibit fibroblast activation can be administered to patients who have suffered burns or other traumatic injuries.
  • compounds that promote fibroblast activation can be administered to patients with an impaired wound healing ability (e.g., diabetic patients suffering from, for example, fibrotic retinopathy, or patients with chronic ulcers).
  • Impaired wound healing often occurs as a complication of a basal metabolic derangement, such as diabetes or poor vascular circulation, or as a consequence of treatment protocols such as those involving administration of steroids, chemotherapy, or radiation.
  • Compounds that promote fibroblast activation can also be usefully administered to patients who have received (or who will receive or who are in the process of receiving) a graft or an implant of a biological or non-biological material or device (e.g., a bioengineered machine, such as a valve, shunt, stent, lens, or pump; an ocular, auditory, or olfactory aid; an artificial organ or tissue (e.g., an islet-cell bearing device); or any other implantable device.
  • a biological or non-biological material or device e.g., a bioengineered machine, such as a valve, shunt, stent, lens, or pump; an ocular, auditory, or olfactory aid; an artificial organ or tissue (e.g., an islet-cell bearing device); or any other implantable device.
  • a biological or non-biological material or device e.g., a bioengineered machine, such as a valve, shunt, s
  • the FN at the wound site is derived from two sources: plasma FN in the exudate from damaged blood vessels, which is present in the ⁇ -granules of platelets, and cellular FN, which is synthesized locally at the wound site (Clark et al., J. Invest. Dermatol. 80:26-30S, 1983).
  • FN is involved in the migration of all the major cell types into the wound site (see, Yamada and Clark, in The Molecular and Cellular Biology of Wound Repair (Second Edition), R.A.F. Clark (Ed.), Plenum Press, N.Y., pp. 51-94, 1996) (the structure of FN is reviewed further, below).
  • PDGF platelet-derived growth factor
  • EGF epidermal growth factor
  • TGF- ⁇ growth factor- ⁇
  • neutrophils are ingested by the invading macrophages.
  • TGF- ⁇ directly stimulates the influx of both neutrophils and macrophages (Whal et al., Proc. Natl. Acad. Sci. USA 84:5788-5792, 1987).
  • fibroblast activation encompasses the differentiation of wound fibroblasts into myo fibroblasts.
  • compounds that modulate fibroblast activation may modulate the appearance (or disappearance) of myofibroblasts.
  • myo fibroblasts coexpress: (1) vimentin (V-type), (2) vimentin and desmin (VD-type), (3) vimentin and ⁇ -SM actin (also referred to as SMC ⁇ -actin), and (4) vimentin, desmin, and ⁇ -SM actin (VAD-type).
  • V-type vimentin
  • VD-type vimentin and desmin
  • SMC ⁇ -actin vimentin and ⁇ -SM actin
  • VAD-type vimentin, desmin, and ⁇ -SM actin
  • SMC ⁇ -actin is the actin isoform typical of contractile vascular SM cells and is also expressed by practically all myofibroblastic populations in vivo (Desmouliere and Gabbiani, supra).
  • MHC myosin heavy chains
  • any of the markers for myofibroblastic differentiation i.e., the cellular activation of fibroblasts listed above, as well as others known in the art, can be employed in assays to determine whether a given compound (e.g., a mutant FN polypeptide, a putative therapeutic agent that interacts with the EIIIA region of wild type FN, or a putative therapeutic agent that inhibits the interaction of EIIIA with either a cellular receptor for FN or with TGF- ⁇ ), inhibits or enhances cellular activation.
  • a given compound e.g., a mutant FN polypeptide, a putative therapeutic agent that interacts with the EIIIA region of wild type FN, or a putative therapeutic agent that inhibits the interaction of EIIIA with either a cellular receptor for FN or with TGF- ⁇
  • Compounds that inhibit expression of one or more of the gene products that serve as markers for myofibroblastic differentiation would be inhibitors of cellular activation (and therefore inhibitors of fibrosis or scar formation), and compounds that enhance expression of one or more of the gene products that serve as markers for myofibroblastic differentiation would be enhancers (or promoters) of cellular activation (and therefore enhancers of fibrosis or scar formation).
  • FN plays a key role in mediating cellular adhesion, promoting cell migration and monocyte chemotaxis, and in regulating cell growth and gene expression. FN functions via a series of functional domains and cell- binding sites that permit it to interact with a remarkably wide range of cell types, extracellular matrix molecules, and even cytokines.
  • FNs Although encoded by a single gene, FNs exist in a number of variant forms that differ in sequence at three general regions of alternative splicing of a precursor rnRNA.
  • the promoter region of the fibronectin gene has been characterized (see, e.g., Dean et al., J. Biol. Chem. 265:3522-3527, 1990; Srebrow et al. FEBSLett. 327:25-28, 1993).
  • tissue-specific (e.g., fibroblast-specific) promoters can be used to express the mutant nucleic acid molecules of the invention in the course of performing the screening methods described below (in culture or in vivo), as well as in the course of stimulating or inhibiting fibroblast activation in vivo (e.g., in a patient).
  • FN's wide ranging physiological functions have been mapped to specific segments of the highly defined FN polypeptide.
  • the reacting sequences are localized to short stretches of amino acids.
  • synthetic peptides that include the Arg-Gly-Asp (RGD) sequence from the FN-IIIio block interactions between FN and integrins (Pierschbacher and Ruoslahti, Nature 309:30-33, 1984; Yamada and Kennedy, J. Cell Biol. 99:29-36, 1984).
  • RGD Arg-Gly-Asp
  • FN-III 9 a short stretch of amino acid residues (Pro-His-Ser-Arg-Asn, or PHSRN (SEQ ID NO:48) in the ninth type III domain (FN-III 9 ), termed the synergy site, of FN has been found to enhance cell adhesion to the RGD sequence in FN (Aota et al., J. Biol. Chem. 266: 15938-15943, 1991 ; Aota et al., J. Biol. Chem. 269:24756-24761, 1994; Bowditch et al., J. Biol. Chem. 266:23323-23328, 1991).
  • the function of the EIIIA and EIIIB FN sequences is largely unknown.
  • DH1 (Nartio et al, J. Cell Sci. 88:419-430, 1987), are known to inhibit the function of F ⁇ s that include an EIIIA domain.
  • novel FN mutants described below (see, e.g., Fig. 3) were used to establish, for the first time, that residues within the C-C loop of the EIIIA domain mediate recognition between FN and these two "function-blocking" mAbs.
  • EIIIA-containing FN polypeptides the intracellular signaling mediated by EIIIA-containing FN polypeptides is likely to converge on the signaling pathway mediated by TGF- ⁇ , and thereby modulate the stimulatory activity of TGF- ⁇ .
  • the detailed characterization of the EIIIA structure provides a way to screen for and develop therapeutic agents that effectively modulate fibroblast activation which, in turn, can inhibit or promote fibrosis and scarring.
  • polypeptides of the invention are mutant FN polypeptides.
  • the polypeptides of the invention can include all or part of wild type FN so long as they have a sequence that differs from wild type FN and include the C-C loop of the EIIIA domain that has substantially the same conformation as the C-C loop in wild type FN.
  • polypeptides of the invention include EIIIA-containing FN polypeptides having a mutation in the EIIIA domain that does not substantially change the conformation of the C-C loop, as well as EIIIA-containing FN polypeptides having a mutation in domains other than the EIIIA region that, again, does not substantially change the conformation of the C-C loop.
  • a domain within a mutant FN polypeptide e.g., a truncation mutant that includes the EIIIA domain or the C-C loop, which consists of the seven amino acid residues between the boxes marked "C” and "C” in Fig. 1 A
  • the mutant FN polypeptide is not substantially altered if the mutant FN polypeptide is able to bind to one or more of the three EIIIA-specific antibodies disclosed herein (IST-9, 3E2, or DH1) with affinities comparable to that observed when these antibodies bind to a wild type FN polypeptide.
  • a mutation of as little as two amino acid residues can result in loss of antibody binding and, thereby, substantially alter the conformation of a portion of a mutant FN polypeptide.
  • the conformation of a domain within a mutant FN polypeptide e.g., the C-C ⁇ loop or a mutant FN polypeptide, such as peptide #1 described in Fig.
  • mutant FN polypeptide is able to bind TGF- ⁇ 1, ⁇ 9 ⁇ l integrin, or cells bearing ⁇ 9 ⁇ l integrin to an extent comparable to that observed when a wild type FN polypeptide binds TGF- ⁇ 1, ⁇ 9 ⁇ l integrin, or cells bearing ⁇ 9 ⁇ l integrin.
  • polypeptides consisting of 10 to 50 amino acid residues of FN (e.g., 10, 25, 35, or 45 residues) and including a C-C loop, the conformation of which is not substantially altered.
  • Such polypeptides can alter the ability of a cell grown in the presence of the polypeptide to express a marker of myofibroblast differentiation (e.g., SMC ⁇ -actin or collagen).
  • the marker of myofibroblast differentiation can be increased or decreased.
  • the polypeptide can also be limited to the wild type C-C loop or to mutants of the C-C loop in which the isoleucine and/or histidine residues are retained.
  • the invention does not encompass mutant FNs that consist of the 90 amino acid wild type EIIIA domain, but does encompass deletion and substitution mutants of the EIIIA domain (e.g., the polypeptides shown in Fig. 3 and the shorter synthetic peptides described in Fig.14).
  • the invention encompasses mutant FN polypeptides that are no longer than seven amino acid residues (e.g., the seven-amino acid peptide of the C-C loop) as well as polypeptides that are up to 10, 15, 20, 30, 40, 50, 60, 70, 80, or 90 amino acids long (e.g., the polypeptide can include the seven-amino acid C-C loop and one, five, ten, or twenty additional amino acid residues selected from the residues that normally flank one or both sides of the C-C loop within the EIIIA domain).
  • the peptide can include the C-C' loop and the C region of FN (see the boxed domain in Fig.
  • the peptide can consist of any of the foregoing peptides that also include substitution mutations within either the C region, the C region, or both.
  • the substituted amino acid residues can be, but are not necessarily, conservative amino acid substitutions.
  • the mutant FN polypeptide can consist of 10 amino acid residues that correspond to residues 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, or 80-90 of the EIIIA domain (e.g., the EIIIA domain of human FN; see SEQ ID NO: 1).
  • the mutant FN polypeptide can consist of 10 amino acid residues that correspond to residues 5-15, 15-25, 25-35, 35-45, 45-55, 55- 65, 65-75, or 75-85 of the EIIIA domain (e.g., the EIIIA domain of human FN; see SEQ ID NO:l).
  • the foregoing peptides can further include a C-C loop, the conformation of which is not substantially altered.
  • mutant FN polypeptides of the invention include substantially pure polypeptides that include a mutant FN polypeptide in which the conformation of the C-C loop is not substantially altered.
  • mutant FN polypeptides can include a mutant FN polypeptide in which the conformation of the C-C'-E region is not substantially altered.
  • Such a polypeptide can, when placed in a tissue culture vessel, alter the ability of a cell grown therein to express a marker of myofibroblast differentiation (e.g., SMC ⁇ -actin). Expression of the marker can be increased or decreased (relative to control) depending on the peptide used.
  • mutant FN polypeptides of the invention include those having a mutation at other than positions 39-45 of SEQ ID NO:l (e.g., the He residue at position 43, the His residue at position 44, or both (of SEQ ID NO:l) are not mutated).
  • the invention features a mutant fibronectin polypeptide that has a deletion mutation within the EIIIA domain.
  • the deletion can be such that the polypeptide includes 10, 20, 30, 40, 50, 60, 70, or 80 amino acid residues within the EIIIA domain.
  • the deletion can be a deletion of one or more amino acid residues at positions 67-90 of SEQ ID NO:l; a further deletion of one or more of amino acid residues 1-16 of SEQ ID NO: l ; and a further deletion of one or more of amino acid residues 17-23 of SEQ ID NO: l.
  • the deletion can be a deletion of one or more of amino acid residues 1-16 of SEQ ID NO:l; a further deletion of one or more of amino acid residues 18-29 of SEQ ID NO:l; a further deletion of one or more of amino acid residues 1-29 or 58-90 of SEQ ID NO:l.
  • the invention features a mutant fibronectin polypeptide having a substitution within the EIIIA domain.
  • mutant fibronectin polypeptides encoded in part by the sequences disclosed in Fig. 16 are within the invention.
  • the term “polypeptide” is used herein to describe a chain of two or more amino acid residues, regardless of post-translational modification.
  • the term “polypeptide” is used interchangeably with “peptide” and "protein,” all of which can be synthetic.
  • FN polypeptides of the invention can be full length FN polypeptides (e.g., polypeptides having the same number of amino acid residues as the corresponding wild type EIIIA-containing FN polypeptide, in which case the mutation would constitute a substitution of one or more of the amino acid residues in the wild type FN polypeptide); truncated FN polypeptides (e.g., polypeptides having fewer, and in some cases many fewer, amino acid residues than the corresponding wild type EIIIA-containing FN polypeptide, in which case the mutation would constitute a deletion of one or more of the amino acid residues in the wild type FN polypeptide); or expanded FN polypeptides (e.g., polypeptides have a greater number of amino acid residues than the corresponding wild type EIIIA-containing FN polypeptide or a region, portion, or domain thereof, in which case the mutation would constitute an addition of one or more amino acid residues to the wild type FN polypeptide).
  • polypeptides of the invention are not limited to those that can be wholly described as encoding substitution, deletion, or addition mutants, i.e., mutant EIIIA-containing FN molecules that have, for example, both a substitution and a deletion of one or more amino acid residues are encompassed by the invention (as are similar mutants, e.g., mutant polypeptides having both a substitution and an addition of one or more amino acid residues (e.g., a substitution within the C-C-E region of the EIIIA domain and a deletion at either the N-terminus, the C-terminus, or both)).
  • FN activity refers to an observable activity exerted by a FN polypeptide or FN-encoding nucleic acid molecule on a FN responsive cell.
  • the activity can be observed in culture or in vivo using the assays described herein.
  • FN activity can be exerted on a cell directly, as when an EIIIA-containing FN polypeptide directly binds to a second protein (e.g., a cell surface receptor).
  • the mutant EIIIA-containing FN polypeptides described herein can include conservative or non-conservative amino acid substitutions. However, it is expected that conservative amino acid substitutions will be more usually employed in the event that the mutant FN polypeptide is one in which the conformation of the EIIIA domain has not been substantially altered.
  • 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
  • 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
  • the polypeptides of the invention are useful in screening assays, in therapeutic regimes, and as immunogens to raise antibodies against mutant EIIIA-containing FN polypeptides.
  • antibodies e.g., humanized antibodies
  • mutant EIIIA-containing polypeptides in which the sequence of the EIIIA domain is not altered from wild type or in which the conformation of the C-C loop is not substantially disturbed can be administered to a patient to inhibit the manner in which the EIIIA domain functions at the site of a wound.
  • These antibodies may bind the EIIIA domain in such a way that the FN molecule cannot interact with TGF- ⁇ or a cellular receptor (e.g., integrin ⁇ 9 ⁇ l).
  • an “isolated,” “purified,” or “substantially pure” polypeptide, or a biologically active portion thereof, is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which it is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • preparations that are “isolated,” “purified,” or “substantially pure” include preparations of mutant EIIIA-containing FN polypeptides having less than about 30%, 20%, 10%, or 5% (by dry weight) of non-FN-like protein (also referred to herein as a "contaminating protein").
  • mutant EIIIA-containing FN polypeptide When the mutant EIIIA-containing FN polypeptide, 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%, 10%, or 5% of the volume of the protein preparation.
  • culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • a mutant EIIIA-containing FN polypeptide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals that are involved in the synthesis of the polypeptide. Accordingly, such preparations of mutant EIIIA-containing FN polypeptides have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or non-EIIIA-containing FN polypeptides.
  • the invention also encompasses fusion proteins containing mutant
  • a mutant EIIIA-containing FN polypeptide includes a mutant EIIIA-containing FN polypeptide operatively linked to at least one other polypeptide, none of which are mutant EIIIA-containing FN polypeptides.
  • One useful fusion protein is a GST-mutant EIIIA-containing FN fusion protein in which mutant EIIIA-containing FN sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant mutant EIIIA-containing FN polypeptides.
  • a chimeric or 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 that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Current Protocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons, 1992).
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a nucleic acid encoding a mutant EIIIA-containing FN can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the mutant EIIIA-containing FN polypeptide.
  • the mutant EIIIA-containing FN polypeptides described herein can function as either FN agonists (mimetics) or as FN antagonists.
  • variants of EIIIA-containing FN polypeptides can be generated by mutagenesis, e.g., discrete point mutation or truncation of the EIIIA-containing FN polypeptide.
  • An agonist of an EIIIA-containing FN polypeptide can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the EIIIA-containing FN polypeptide.
  • An antagonist of the EIIIA-containing FN polypeptide can inhibit one or more of the activities of the naturally occurring form of the EIIIA-containing FN polypeptide by, for example, competitively binding to a cellular receptor and, thereby, influencing the activity of a downstream member of a cellular signaling cascade that is involved in the activation of a cell (e.g., a fibroblast or smooth muscle cell; lipocytes in the liver can also be activated, differentiating into myofibroblasts).
  • a cell e.g., a fibroblast or smooth muscle cell; lipocytes in the liver can also be activated, differentiating into myofibroblasts.
  • compositions of the present invention encompass isolated nucleic acid molecules that encode a mutant EIIIA-containing FN polypeptide (including those described above).
  • the mutation can be one of several types.
  • the mutation can be a mutation in FN that alters the sequence of FN but does not substantially change the conformation of the EIIIA domain.
  • the mutation can alter the sequence within the EIIIA domain or in other regions of the FN polypeptide.
  • the mutation can also be a deletion mutation, in which case the FN molecule would have fewer amino acid residues than wild type FN (e.g., amino acids could be deleted from the C-terminal, the N-terminal, or both the C- and N-termini of the polypeptide; the amino acid(s) deleted are not necessarily those at either end of the polypeptide, i.e., one or more amino acid residues can be deleted from within the FN polypeptide).
  • the polypeptides of the invention can be used to inhibit or stimulate fibroblast activation, which, in turn, will inhibit or stimulate the development of fibrosis and/or scarring. It is well within the ability of one of ordinary skill in the art to determine whether any given FN polypeptide will inhibit or stimulate fibroblast activation. Assays suitable for this purpose are described below. For example, one can add mutant FN polypeptides to cultures of human subcutaneous fibroblasts that have been exposed to TGF ⁇ l. Cells can be seeded on simple gelatin or on gelatin containing 300 ⁇ g/ml of a mutant FN polypeptide and then stimulated (and, in parallel "control" cultures, not stimulated) with TGF ⁇ l (10 ng/ml) for three days.
  • a marker of fibroblast activation (e.g., SMC ⁇ -actin) can then be analyzed, for example, by Western blot analysis of equal amount of protein extracted from the two cultures (i.e., the TGF- ⁇ 1 -treated and untreated cultures). If the mutant FN polypeptide inhibits induction of the marker's expression (e.g., inhibits the expression of SMC ⁇ -actin or collagen), the mutant FN polypeptide is an inhibitor of cellular activation (e.g., macrophage, endothelial cell, or fibroblast activation).
  • cellular activation e.g., macrophage, endothelial cell, or fibroblast activation
  • mutant FN polypeptides have various uses.
  • a nucleic acid molecule that encodes a FN polypeptide that has a mutation that does not alter the conformation of the EIIIA domain can be delivered to a biological cell (e.g., a cultured cell or a cell in a patient) and, when expressed, can inhibit the ability of that cell or cells in the vicinity to participate in a wound healing process that culminates in excessive scarring.
  • this type of mutant peptide (which can be shorter than a full-length FN polypeptide) will inhibit fibroblast activation (also termed differentiation to myofibroblasts), which will in turn inhibit fibrosis or excessive scarring, by binding the molecules (e.g., cell surface receptors (e.g., integrin ⁇ 9 ⁇ l) or growth factors (e.g., TGF- ⁇ )) that normally interact with EIIIA-containing FN at the site of a wound.
  • the FN polypeptide can be a full length FN polypeptide (e.g., a polypeptide having the same number of amino acid residues as the corresponding wild type
  • EIIIA-containing FN polypeptide in which case the mutation would constitute a substitution of one or more of the amino acid residues in the wild type FN polypeptide); a truncated FN polypeptide (e.g., a polypeptide having fewer amino acid residues than the corresponding wild type EIIIA-containing FN polypeptide, in which case the mutation would constitute a deletion of one or more of the amino acid residues in the wild type FN polypeptide); or an expanded FN polypeptide (e.g., a polypeptide have a greater number of amino acid residues than the corresponding wild type EIIIA-containing FN polypeptide, in which case the mutation would constitute an addition of one or more amino acid residues to the wild type FN polypeptide).
  • a truncated FN polypeptide e.g., a polypeptide having fewer amino acid residues than the corresponding wild type EIIIA-containing FN polypeptide, in which case the mutation would constitute a deletion of one or more of
  • nucleic acid molecules of the invention are not limited to those that can be wholly described as encoding substitution, deletion, or addition mutants, i.e., nucleic acid molecules of the invention can encode mutant EIIIA-containing FN molecules that have, for example, both a substitution and a deletion of one or more amino acid residues.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • An "isolated” nucleic acid molecule is one that is separated from other nucleic acid molecules that are present within a natural source of the nucleic acid.
  • nucleic acid is substantially free of other cellular material, including the protein encoding sequences that 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 sequence is obtained.
  • nucleic acid molecule of the invention is used in recombinant techniques (e.g., to produce a recombinant protein), it is “isolated” when it is substantially free of the cells and culture medium with which it was associated in order to perform those techniques.
  • nucleic acid molecule of the invention is chemically synthesized, it is “isolated” when it is substantially free of chemical precursors or other chemicals used to carry out the synthesis.
  • a nucleic acid molecule of the present invention can have, or can contain, any of the nucleic acid sequences shown in Fig. 16 (e.g., any of SEQ ID NOs.16-40), their complements, or degenerate variants thereof.
  • a nucleic acid of the invention can be amplified using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Those of skill in the art are well able to clone and characterize a DNA sequence. Should guidance be required, one can consult, for example, Sambrook et al, Eds., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • oligonucleotides corresponding to some or all of the nucleotide sequence encoding a mutant EIIIA-containing FN polypeptide can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an oligonucleotide useful in the present invention will include a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 17, and more preferably about 25 consecutive nucleotides of the sense or anti-sense sequence of a FN, preferably a human FN (e.g., an human EIIIA-containing FN).
  • Stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50-65°C.
  • the sequence with which the oligonucleotide hybridizes can be that of a mutant FN sequence specifically disclosed herein or of another sequence that encodes an EIIIA-containing FN polypeptide having a mutation in the sequence of the EIIIA region or a mutation that disrupts the conformation of the EIIIA region.
  • Oligonucleotides such as those described above can be detectably labeled, if desired, and used as a probe.
  • the detectable label can be, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue that express a mutant EIIIA-containing FN polypeptide.
  • EIIIA-containing FN polypeptides can lead to amino acid substitutions at either "non-essential” or “essential” amino acid residues (depending on the intended use of the mutant polypeptide).
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of EIIIA-containing FN (e.g., the sequence of human E ⁇ iA-containing FN published by Komblihtt et al, EMBO J. 4:1755-1759, 1985) without altering the ability of FN to activate fibroblasts, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that reside in the C-C loop of the EIIIA domain are predicted to be essential for binding a cell surface receptor and, in the context of a functional FN polypeptide, to activate the cell that is bound (e.g., to activate fibroblasts or smooth muscle cells).
  • amino acid residues that reside in the EIIIA domain e.g., amino acid residues in the C-C-E region or in the C-C loop
  • that contribute to the conformational stability of the FN polypeptide in the region of the C- loop will, in the context of a non- functional FN polypeptide, bind, but not activate, a cell (in which case, cellular activation (e.g., macrophage, endothelial cell, or fibroblast activation) will be inhibited).
  • Mutations can, of course, be introduced by standard techniques, such as site- directed mutagenesis and PCR-mediated mutagenesis.
  • the oligonucleotide can include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. Proc. Natl. Acad. Sci. USA 86:6553- 6556, 1989; Lemaitre et al. Proc. Natl. Acad. Sci. USA 84:648-652, 1987; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al. Proc. Natl. Acad. Sci. USA 86:6553- 6556, 1989; Lemaitre et al. Proc. Natl. Acad. Sci.
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al Bio/Techniques 6:958-976, 1988) or intercalating agents (see, e.g., Zon Pharm. Res. 5:539-549, 1988).
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, or a hybridization-triggered cleavage agent.
  • Antisense Constructs and Therapies Treatment regimes based on an "antisense” approach involve the design of oligonucleotides (either DNA or RNA) that bind to complementary mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required.
  • a sequence "complementary" to a portion of an RNA, as referred to herein, is a sequence sufficiently complementary to be able to hybridize with the RNA, forming a stable duplex, within the environment of a cell; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be).
  • One of ordinary skill in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • Antisense oligonucleotides complementary to mRNA coding regions can be used in accordance with the invention, e.g., to treat patients who have or who may develop any of the fibrotic conditions described herein.
  • Antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 250 nucleotides in length. In specific aspects, the oligonucleotide is at least 10 nucleotides in length and can be quite lengthy (e.g., 15, 25, 50, 100, or 200 or more nucleotides long).
  • in vitro studies are usually performed first to assess the ability of an antisense oligonucleotide to inhibit gene expression. In general, these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. In these studies levels of the target RNA or protein are usually compared with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide.
  • control oligonucleotide is of approximately the same length as the test oligonucleotide, and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule or hybridization.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (as described, e.g., in Letsinger et al, Proc. Natl. Acad. Sci. USA 86:6553, 1989; Lemaitre et al, Proc. Natl Acad. Sci.
  • the oligonucleotide can be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization- triggered cleavage agent.
  • the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethyl-aminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosyl
  • the antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
  • the antisense oligonucleotide may also include at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal, or an analog of any of these backbones.
  • the antisense oligonucleotide can include an ⁇ -anomeric oligonucleotide.
  • oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al, Nucl Acids. Res. 15_:6625, 1987).
  • the oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al, Nucl. Acids Res. 1_5:6131, 1987), or a chimeric RNA-DNA analog (Inoue et al, EERS Lett. 215:327, 1987).
  • Antisense oligonucleotides of the invention can be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (Nucl. Acids Res. 15:3209, 1988), and methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al, Proc. Nat'l Acad. Sci. USA 85:7448, 1988).
  • the antisense molecules should be delivered to cells that express ⁇ fflA-containing FN in vivo.
  • a number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.
  • a recombinant DNA construct comprises an antisense oligonucleotide placed under the control of a strong pol III or pol II promoter.
  • the use of such a construct to transfect target cells in a patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous nonsense-mediated mRNA decay pathway transcript and thereby prevent translation of that mRNA.
  • a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Vectors can be constructed by recombinant DNA technology methods standard in the art.
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.
  • Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive.
  • Suitable promoters may include, but are not limited to: the SV40 early promoter region (Bemoist et al, Nature 290:304, 1981); the promoter contained in the 3' long terminal repeat of Rous sarcoma vims (Yamamoto et al, Cell 22:787-797, 1988); the herpes thymidine kinase promoter (Wagner et al, Proc. Natl. Acad. Sci. USA 78:1441, 1981); or the regulatory sequences of the metallothionein gene (Brinster et al, Nature 296:39, 1988). Constructs may also be contained on an artificial chromosome (Huxley, Trends. Genet.
  • Vectors to be used as described above include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or other viral vectors with the appropriate tropism for FN- expressing cells (e.g., cells that express EIIIA-containing FN) with activated nonsense- mediated mRNA decay pathways) can be used as a gene transfer delivery system for a therapeutic antisense nucleic acid constmct or other nucleic acid constmct that inhibits expression of an EIIIA-containing FN polypeptide.
  • Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al, N. Engl J. Med 323:370, 1990; Anderson et al, U.S. Pat. No. 5,399,346).
  • Non-viral approaches can also be employed for the introduction of therapeutic DNA into malignant cells.
  • an antisense EIIIA nucleic acid can be introduced into a cell by the techniques of lipofection (Feigner et al, Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al, Neurosci. Lett. 1JJ:259, 1990; Brigham et al, Am. J. Med. Sci. 298:278, 1989; Staubinger and Papahadjopoulos, Meth. Enz. 101:512, 1983); polylysine conjugation methods (Wu and Wu, J. Biol. Chem. 263: 14621, 1988; Wu et al, J. Biol. Chem. 264:16985, 1989); or, by micro injection under surgical conditions (Wolff et al, Science 247:1465, 1990).
  • Modulators of cellular activation can be identified through cell culture or in vivo assays. Such assays can be generally carried out by examining the effect of a test compound or molecule on the interaction between FN (e.g., a human EIIIA-containing FN polypeptide) and an anti-FN antibody, a molecule that binds FN (e.g., an integrin ⁇ 9 ⁇ l or TGF- ⁇ ), or a biological cell (e.g., a human fibroblast) that responds to (i.e., is activated by) FN.
  • FN e.g., a human EIIIA-containing FN polypeptide
  • an anti-FN antibody e.g., a molecule that binds FN (e.g., an integrin ⁇ 9 ⁇ l or TGF- ⁇ )
  • a biological cell e.g., a human fibroblast
  • parallel assays are performed in the presence and absence of the potential modulator, with the later assay
  • the invention features methods for screening for such compounds in vitro, in cell culture, or in vivo.
  • the data presented in the Examples below also demonstrate that TGF- ⁇ 1 binds to the loop region of EIIIA.
  • some of the compounds identified by way of the assays described herein, and by way of others known to and routinely practiced by those of ordinary skill in the art will inhibit fibroblast activation, and thereby inhibit fibrosis and/or scarring, by inhibiting the interaction between FN and TGF- ⁇ .
  • the invention is not limited to compounds, or to methods of treatment with compounds that inhibit fibrosis by any particular mechanism, the compounds that effectively inhibit fibrosis may be those that bind to the loop region of EIIIA and thereby prevent that region from binding to TGF- ⁇ in vivo.
  • Procedures for carrying out cell culture assays are well known to those of ordinary skill in the art and can include the following.
  • test compounds e.g., synthetic peptides
  • the test compounds can be added to fibroblasts cultivated under conditions in which the expression of SMC ⁇ -actin can be assessed (e.g., SMC ⁇ -actin is not expressed by cells cultured on plasma FN or plastic, but is induced when cells are cultured on cellular FN; SMC ⁇ -actin expression can also be influenced by growth factors, e.g., SMC ⁇ -actin expression is increased in cells exposed to TGF- ⁇ ; see, e.g., Serini et al, J. Cell Biol. 142:873-881, 1998).
  • fibroblasts can be cultured on dishes precoated with plasma FN, cellular FN, or albumin in the presence of a monoclonal antibody (mAb) to EIIIA and the test compound.
  • mAb monoclonal antibody
  • key synthetic peptides can be conjugated to albumin and absorbed to tissue culture dishes on which fibroblasts are cultivated.
  • SMC ⁇ -actin can be determined by Western blot analysis.
  • Assays of collagen gel contraction Analyzing collagen gel contraction in the presence of specific peptides and anti-EIIIA antibodies (e.g., antibodies that specifically bind mutant EIIIA-containing FN polypeptides) provides another way to test candidate modulators of fibroblast activation.
  • cells e.g., fibroblasts
  • collagen gels e.g., 2.5 mg/ml collagen which has been allowed to polymerize and rimmed to free its adhesion to the sides and bottom of the well
  • FN activity e.g., compounds identified in vitro by virtue of their ability to inhibit TGF- ⁇ -mediated fibroblast activation (as evidenced, e.g., by suppression of TGF- ⁇ -induced SMC ⁇ -actin expression), including those that bind to the EIIIA domain (e.g., the loop between the regions known as C and C; see Fig. 8) or that change the conformation of the EIIIA domain and, thereby, prevent the EIIIA-containing FN from interacting with cells in the vicinity of the wound.
  • compounds that modulate FN activity e.g., compounds identified in vitro by virtue of their ability to inhibit TGF- ⁇ -mediated fibroblast activation (as evidenced, e.g., by suppression of TGF- ⁇ -induced SMC ⁇ -actin expression), including those that bind to the EIIIA domain (e.g., the loop between the regions known as C and C; see Fig. 8) or that change the conformation of the EIIIA domain and, thereby
  • the SCID Mouse Model The potential consequences of modulating EIIIA function during wound healing can be assessed in numerous in vivo models of wounding, including a rat skin-SCID mouse model (Juhasz et al, Am. J. Pathol. 143: 1458-1469, 1993), which takes advantage of the species-specific nature of the mouse Mabs that react with, and block, the rat EIIIA domain, but that do not react with mouse FN. This eliminates the possibility that effects observed in the transplant model are due to an effect on host mouse FN, particularly mouse plasma FN, which is abundant. The delivery of Mabs to SCID/human wounds will not likely be a significant problem.
  • lupus immunoglobulin (Ro, SSA) are known to react with the human epidermis in unwounded human/nude and human/SCID mouse models (Lee et al, J. Clin. Invest. 83:1556-1562, 1989). Moreover, it has been shown that anti-integrins infused into a SCID mouse immunolabel human keratinocytes (Kaurmann et al, Arch. Dermatol. Res. 286:6-11, 1994). To perform the assay, a 1.5 cm circle of skin is excised from SCID mice. Full thickness skin grafts of the same size, obtained from adult rats, are then grafted onto the resulting fasciomuscular bed.
  • mice can be infused intravenously with graded concentrations of IgG (0.1 mg/kg), either a blocking anti-EIIIA segment or isotope- matched control IgG, or non-blocking anti-EIIIA Mab. These are doses that have been found suitable in independent studies performed on integrin function in rodents.
  • the mAbs available for these assays react with both human and rat EIIIA segments.
  • a test compound e.g., a synthetic peptide, anibody, or small molecule
  • Animals can receive the test compound just prior to wounding and at daily intervals thereafter to maximize the chance of observing modulation of fibroblast activation.
  • the graft can be bored with a sterile biopsy punch (e.g., having a 3 mm diameter) to create a partial thickness wound.
  • the transplanted rat skin can be harvested from euthanized SCID mice at intervals after wounding.
  • wound contraction which can be quantitated by marking the skin at the wound periphery on day 0 with carbon tattoos and measuring the relative positions of these tattoos on treated and untreated transplants at 7 to 14 days following wounding. Further data can be obtained by analysis of hematoxylin and eosin (H+E) stained paraffin sections taken at the center of the wound to quantitate two parameters: wound contraction and angiogenesis. Although occurring at intervals that precede the activation of fibroblasts, the study of H+E-stained sections allows one to determine whether the test compound alters the extent of epidermal migration or inflammatory cell infiltration.
  • H+E hematoxylin and eosin
  • the extent of angiogenesis that occurs in granulation tissue 4-7 days after a wound is inflicted can be quantitated by counting the vessels that positively stain for von Willebrand factor, or by staining for PECAM-1.
  • fibroblasts may have an indirect role in regulating angiogenesis, it may be important to assess the effect of any potential therapeutic agent on angiogenesis.
  • the vessels in these grafts are derived from both rat and mouse; if desired, similar assays can be carried out with human foreskin on SCID mice.
  • Antibodies reacting to human von Willebrand factor and PECAM-1, both of which give bright staining are commercially available.
  • Human foreskin is typically available through neonatology units and can be obtained by those of skill in the art pending receipt of approval for its use from the relevant medical center oversight committees.
  • An alternative rodent model for assaying the therapeutic agents described herein or identified by way of the screening methods described herein follows.
  • CD rats weighing 100-125 grams are acclimatized for one week prior to treatment.
  • animals are randomly divided into two experimental groups. Animals in one group are wounded and treated with a control substance (e.g., IgG), and animals in the other group are wounded and treated with test compounds (e.g., anti-EIIIA monoclonal antibodies or synthetic EIIIA peptides such as those containing the C-C loop,).
  • the rats are anesthetized before receiving full thickness excisional wounds using a sterile biopsy punch (4 mm diameter).
  • the wounds are inflicted at four separate sites on the dorsum of each of rat to control for cranial-caudal differences in wound healing.
  • the location of wounds are encoded in a blinded fashion so that subsequent comparisons will control for anatomic differences in healing.
  • a concentric circle of dye is placed 2 mm outside of each wound, against which wound contraction will be measured.
  • the study includes enough animals that those in the experimental groups can be assessed at various time points between zero and 28 days after treatment is begun. Additional injections or applications of the test compound can be made as needed.
  • Tissue samples are harvested and coded by investigators at the outset of the blinded study. Ample skin around, and including, each wound is excised and pinned out flat in fixative for a short period of time and a digital brightfield/fluorescent photograph of the wound site is taken. The wounds are then bisected and the cross-sectional (internal) faces are marked with a dot of India ink for proper orientation (carbon facing out) during the embedding process so that sections can be prepared from the center of the wound. The bisected specimen is then fixed overnight in cold 4% paraformaldehyde in PBS.
  • fixation of one half of the bisected tissue is quenched with glycine.
  • the piece is then infiltrated with 30% cold sucrose for 18 hours and snap frozen in OCT compound on dry ice.
  • the other half of the bisected wound is used for standard paraffin histology.
  • any compound (be., any d g, biological agent, small molecule, peptide, or chemical substance) that associates with the EIIIA domain of FN or alters the conformation of that domain, and thereby modulates the ability of the EIIIA-containing FN molecule to function as it otherwise would at the site of a wound can be developed as a therapeutic agent.
  • agents also referred to herein as "active compounds”
  • Such compositions typically include a pharmaceutically acceptable carrier, which is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, so long as they are compatible with pharmaceutical administration.
  • 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, or subcutaneous administration), oral, transmucosal (e.g., nasal or rectal administration, and transdermal (e.g., topical) administration.
  • Solutions or suspensions used for parenteral or oral 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 ethylenediamine-tetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity, such as sodium chloride or dextrose.
  • the pH of the formulation can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials, which are typically made of glass or plastic.
  • 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).
  • the composition injected must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • 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 mannitol, sorbitol, sodium chloride in the composition.
  • Absorption of the injectable compositions can be prolonged by including an agent that delays absorption, such as aluminum monostearate or gelatin, in the composition.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that 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 com 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 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.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Patent 5,328,470) or by stereotactic injection (see, e.g., Chen et al, Proc. Natl. Acad. Sci. USA 9J_:3054-3057, 1994).
  • 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 embedded.
  • the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • Bioproducts Indianapolis, IN), Locus Genex (Helsinki, Finland) and Sigma Chemical Co. (St. Louis, MO), respectively.
  • BCA Protein Assay Kit, Gel Code Blue, SuperBlock, Super Signal and HRP-conjugated goat anti-mouse IgG were from Pierce (Rockford, IL).
  • GST gene fusion vectors and U.S.E. Mutagenesis kit were obtained from Amersham Pharmacia Biotech (Piscataway, NJ).
  • QIAGEN Plasmid Maxi kit, QIAprep 8 Miniprep kit, QIAprep 8 Ml 3 kit, and QIAquick Gel Extraction kit were from QIAGEN Inc. (Valencia, CA).
  • Glutathione (reduced form), glutathione-immobilized agarose were obtained from Sigma Chemical Co.
  • AEBSF Hydrochloride was from CalBiochem (San Diego, CA).
  • Synthesized oligonucleotides were purchased from Gibco/BRL Life Technologies (Gaithersburg, MD). All other reagents were at least reagent grade and obtained from standard suppliers.
  • PCR Polymerase chain reaction
  • Full-length wild type rat EIIIA cDNA was amplified using PCR in a 50 ⁇ l reaction mixture containing 1 ng of a rat cDNA (clone 74T, kindly provided by R.O. Hynes, Massachusetts Institute of Technology) (Schwarzbauer et al, EMBO J. 6:2573-2580 1987) (peptide sequences of EIIIA proteins are available through GenBankTM under the following
  • the PCR contained 50 mM KC1, 10 rnM Tris-HCI, pH 8.3, 1.5 mM MgCl 2 and 0.001% (w/v) gelatin, 200 ⁇ M each dNTP, 0.5 ⁇ M primers (EIIIA-sense and EIIIA-antisense, Fig. 16) and 2.5 units of Taq DNA polymerase (Perkin-Elmer, Norwalk, CT).
  • the samples were placed in a DNA thermal cycler 480 (Perkin-Elmer) programmed for a temperature-step cycle of 94°C (45 sec), 55°C (1 min), and 72°C (1 min) for 30 cycles. After the final cycle, the reaction was maintained at 72°C for additional 7 minutes. The final reaction products were resolved on a 1 % agarose gel containing ethidium bromide (0.5 ⁇ g/ml). Amplified DNA fragments were purified from the gel, digested with Eco RI and subcloned into the pGEX-2T vector. The recombinant plasmid (EIIIA-pGEX-2T) was isolated from liquid bacterial cultures using QIAfilterTM Plasmid Maxi kit or QIAprepTM Spin Miniprep kit and subjected to DNA sequencing.
  • a DNA thermal cycler 480 Perkin-Elmer
  • Various deletion constructs of rat EIIIA cDNA were generated by using PCR. The amplification was performed in a 25 ⁇ l reaction volume containing 1 ng of EIIIA- pGEX-2T plasmid DNA, 10 mM Tris-HCI, pH 9.0, 50 mM KC1, 0.1% Triton X-100, 1.5 mM MgCl , 200 ⁇ M each dNTP, 0.5 ⁇ M sense strand and antisense strand primer, and 2.5 units of Taq DNA polymerase (Promega, Madison, WI). The sequences of 5' and 3' primers are listed in Fig. 16. Constmcts were then subcloned and propagated as described above. Deletion of EIIIA cDNA was confirmed by DNA sequencing analysis.
  • Mutagenesis mixtures consisted of 0.025 pmol of plasmid DNA, 1.25 pmol of U.S.E. selection primer (Fig. 16), 1.25 pmol of target mutagenic primer and One-Phor-All Buffer PLUS in a total volume of 20 ⁇ l. Following the incubation at 100°C for 5 minutes, the reaction mixtures were cooled in ice for 5 minutes and then incubated at room temperature for 30 minutes.
  • the mutagenesis reaction mixture was digested with 10 units of Rstl in a final volume of 50 ⁇ l, and transformation was carried out as described by the manufacturer. Transformed cells were incubated in 4 ml L-broth with 100 ⁇ g/ml of ampicillin at 37°C overnight with shaking at 250 rpm. Plasmid DNAs were prepared from the overnight cultures using QIAprep 8TM Miniprep kit (Qiagen) and were subjected to a second restriction digestion by Pstl. One microgram of digested DNA from the second round of restriction enzyme selection was transformed into E. coli competent cells, followed by plating of the transformed cells onto LB plates containing 100 ⁇ g/ml ampicillin. The plates were incubated at 37°C overnight, and individual transformant colonies were selected to prepare plasmids for sequencing to verify the presence of the desired point mutations.
  • DNA sequencing was performed on subcloned fragments in multicopy plasmids, PCR amplimer fragments using Taq polymerase in a dideoxy dye-terminator reaction (Sanger et al, Proc Natl Acad Sci USA 74:5463-5467 1977). The sequencing reactions were resolved on an Applied Biosystems ABI 377 Sequencer (DNA Sequencing Core Facility, Department of Molecular Biology, Massachusetts General Hospital). The sequencing results were assembled and analyzed using the GCG Software Package (version 9.0, the University of Wisconsin and Genetics Computer Group), which includes "Pileup,” “Bestfit,” and "Pretty.”
  • cell pellets were washed with PBS and used for protein purification or stored at -80°C until ready to use.
  • cell pellets were resuspended in 10 ml of PBS with 1 mM AEBSF (PBS-AEBSF) and sonicated on ice using a Sonifier 450 (Branson Ultrasonics Corp., Danbury, CT) with microtip at full power for 1 minute.
  • Sonifier 450 Branson Ultrasonics Corp., Danbury, CT
  • 100 ⁇ l of Triton X- 100 was then added into the sonicated suspension, and an incubation at 4°C was carried out for 30 minutes. Cell debris was removed by a centrifugation at 14,000 x g for 30 minutes.
  • the clarified supernatant was collected and mixed with 1 ml of glutathione-agarose (50% slurry pre-equilibrated with PBS-AEBSF) at 4°C for 2 hours.
  • Protein-bound agarose beads were collected by a centrifugation at 1,000 x g for 1 minute and washed with 10 ml of PBS- AEBSF 5 times. Washed beads were mixed with 1 ml of elution buffer (25 mM glutathione, 120 mM NaCl and 100 mM Tris-HCI, pH 8.0) at 4°C for 10 minutes to elute the GST- fusion proteins, followed by a centrifugation at 1,000 x g for 1 minute. The elution step was repeated twice, and the eluted fractions were pooled and dialyzed against PBS. Purified proteins were quantitated using the BCA protein assay reagent and stored at -80°C
  • the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Piere) (1 :5000 in SuperBlock), followed by three washes in PBST.
  • the immunoblot was then incubated with Supersignal chemiluminescence substrate for 10 minutes and exposed to a phosphor cassette. Images of the blots and gels were processed with the Molecular Image System GS-525 and the Fluor-S Multilmager, respectively, using Multi-Analysis software version 1.1 (Bio-Rad, Hercules, CA).
  • Enzyme-linked i munosorbent assays The reactivity of mutant EIIIA-GST fusion protein was tested by enzyme-linked immunosorbent assays (ELISA). Microtiter plates (96-well) were coated with 100 ⁇ l/well of 10 ⁇ g/ml purified EIIIA-GST proteins in coating buffer (100 mM NaHCO , pH 8.6) at 4°C in a humidified chamber overnight. The plates were briefly rinsed in washing buffer (0.1% Tween-20 in PBS), blocked with
  • Protein sequence comparison and antibody reactivities implicate the C-C'-E segment of EIIIA encompassing the His residue as epitopes.
  • Fig. 1A protein sequences derived from mRNAs for human, mouse, rat, chicken and frog EIIIA segments show extensive sequence similarity. See Fig. 1 A, where amino acid residues conserved in all five species are shown in capital letters, and dashes indicate the positions where the aligned amino acids are only partially conserved. Rectangular boxes and letters above each box delineate the stmctural domains of anti-parallel ⁇ -sheets. The arrow denotes His 44 , which is conserved in human, rat, and chicken EIIIA, but not in mouse or Xenopus. Note that the underlined amino acids, Asp 53 and Glu 5 , of mouse EIIIA sequence obtained by the present inventors differ from those reported by others
  • the EIIIA domains in human, mouse, rat, chicken, and Xenopus are all 90 amino acids in length, and the consensus sequence for these five species is 70% conserved.
  • the EIIIA protein sequences for mouse, rat, chicken and frog display 96.7%, 94.4%, 85.6% and 80%) identity, respectively, to the human EIIIA protein (Fig. IB). All sequences conform to a domain structure in which seven ⁇ -strands (denoted by A, B, C, C, E, F, G) are conserved in the type III repeat crystal stmcture (Leahy et al, Cell 84:155-164, 1996).
  • the mAb IST-9 raised against human cellular fibronectin (cFN), specifically recognizes the EIIIA segment in rat and human cFN (Carnemolla et al, FEBS Lett. 215:269- 273, 1987). This mAb exhibits function-blocking activities in these species (Jamagin et al, J. Cell Biol. 127:2037-2048, 1994; Serini et al., J. Cell Biol. 142:873-881, 1998). The reactivities of IST-9 and 3E2 were tested against the EIIIA segment in chicken, frog and mouse FN by either immunofluorescence, immunob lotting or ELISA.
  • cFN human cellular fibronectin
  • IST-9 reacted with chicken cFN, but not appreciably with either mouse or frog EIIIA (see below).
  • DH1 which binds chicken FN (Gehris et al, Dev. Biol. 190:191-205, 1997), also does not react with mouse or frog EIIIA (see below).
  • 3E2 reacts with chicken and mouse (Peters et al, Cell Adhes. Commun. 4:103-125, 1996) but not frog EIIIA (see below). Comparisons of these reactivities with the protein sequences (Fig.
  • rat EIIIA The "native" conformation of full-length rat EIII protein is required for its IST-9 reactivity.
  • the wild-type sequence of rat EIIIA was cloned into a bacterial expression vector, pGEX-2T, and then used to generate various deletion constructs of rat EIIIA by PCR (Fig. 3). All of these deletion mutants retain the hypothesized epitope sequence of domain C-C'-E. Wild type and deletion mutant constructs were expressed as GST fusion proteins in E. Coli and purified by glutathione-affinity chromatography.
  • Antibody reactivities of wild type rat EIIIA and the derived deletion mutants were tested by ELISA, and the dilution yielding 50% binding (titer) for each mAb was determined. Three EIIIA-specific mAbs, IST-9, 3E2, and DH1, were included in these analyses. When reacted with wild type rat EIIIA, the titers of IST-9, 3E2 and DH1 were 5 x 10 4 , 4 x 10 3 and 1 x 10 4 , respectively (Fig. 4). However, unlike the strong reactivity exhibited by rat EIIIA toward these mAbs, none of the six deletion mutants displayed any detectable antibody reactivity (insets, Fig. 4).
  • rat EIIIA Thr 35 ⁇ Tyr 6 , Ser 7j GI1140 and Asp 4 j residues of rat EIIIA are important for maintaining an optimal conformation for antibody binding.
  • C-C'-E domain of EIIIA protein is crucial for antibody reactivity
  • a series of rat EIIIA double mutants was generated in which two adjacent amino acids were simultaneously replaced by alanines and then tested for reactivity to mAbs by ELISA. In some preliminary screenings, antibody reactivities were determined by Western blotting. Most of the double mutants retain some or all of the antibody reactivity of the wild-type rat EIIIA protein (Fig. 15).
  • rat EIIIA- D53A+E54A representing a potential polymorphism is mouse EIIIA (Figs. 1 and 15).
  • some double mutants sharply reduced antibody reactivity (Fig. 5).
  • One of these mutants, rat EIIIA-V34A+T35A completely lost its ability to react with all three mAbs tested.
  • rat EIIIA-Y36A+T35A displayed a complete loss of reactivity to IST-9 and 3E2 but still retained low (about 1%>) reactivity to DH1, as compared to wild type EIIIA.
  • An additional double mutant, rat EIIIA-E40A+D41A showed a dramatic reduction in reactivity to IST-9 (3% of rat EIIIA- WT) had no significant effect on IST-9 reactivity and only minor effects on the reactivities of 3E2 and DHL
  • rat EIIIA-V34A+T35A and rat EIIIA- Y36A+S37A could result directly from dismption of the antibody-recognition epitope or indirectly by changing the tertiary stmcture of the EIIIA segment, which in turn alters the conformation of the epitope.
  • the double mutants (rat EIIIA- V34A+T35A, rat EIIIA-Y36A+S37A and rat EIIIA-E40A+D41 A) showed either a complete loss or a dramatic reduction in antibody reactivity
  • the single mutants (rat EIHA-T35 A, rat EIIIA- Y36A, rat EIIIA-S37A, and rat EIIIA-E40A+D41 A) exhibited antibody reactivity levels comparable to those of wild type rat EIIIA (Figs. 6 and 15).
  • the amino acid residues Thr 35 , Tyr 36 , Ghuo, and Asp 4 j are implicated in maintaining an optimal conformation of the epitope.
  • Ile 3 and His 44 segment are critical for IST-9 binding.
  • Site-directed mutagenesis of the rat EIIIA segment was conducted to generate a panel of single mutants. These mutants were tested for reactivity to IST-9, 3E2 and DHl (Figs. 6 and 15).
  • rat EIIIA-H44R an arginine residue was substituted for histidine to mimic the Arg-, 4 (H44R) in the mouse EIIIA segment.
  • a striking loss of reactivity for IST-9 and DHl was observed (solid and striped bars, Fig. 6).
  • reactivity with 3E2 was retained (gray bar, Fig. 6), consistent with the reported use of this mAb in immunohistochemistry of mouse tissues (Peters et al, Cell Adhes. Commun. 4: 103-125, 1996).
  • Ile 4 and His 44 are a necessary part of the epitope for a function-blocking monoclonal antibody (IST-9) that reacts with the E ⁇ iA segment of human, rat and chicken FN.
  • the Ile 4 and His 44 residues lie in a loop between two ⁇ strands (C and C).
  • the epitopes for DHl and 3E2 are also conformationally-dependent and appear to reside in the same loop.
  • the key residues for DHl and 3E2 binding overlap with, and yet are distinct from, those for IST-9.
  • the loop between the C and C ⁇ strands is critical to the mechanism by which the EIIIA segment regulates cell function.
  • FNs that include the EIIIA or EIIIB segments are present in rather low amounts in many adult tissues (Peters et al, Cell Adhes. Commun. 4: 127-148, 1996).
  • FNs that lack the EIIIA and EIIIB segments i.e., plasma FN
  • plasma FN plasma FN
  • EIIIA and EIIIB differ, suggesting distinct roles for each segment (Jamagin et al, J. Cell Biol. 127:2037-2048, 1994; Coito et al, Am. J. Pathol. 150:1757-1772, 1997).
  • Splicing in a non-homologous repeat, the V region also occurs following injury.
  • the V95 segment is included in most FNs, but variations occur in the inclusion of the cell adhesive portion, termed CS-1, during regeneration of peripheral nerves (Mathews et al, J. Neurobiol. 26:171- 188, 1995) and following cardiac transplantation (Coito et al. Am. J. Pathol. 150:1757-1772, 1997).
  • IST-9 blocks the conversion of lipocytes to myofibroblasts (Jamagin et al, J. Cell Biol. 127:2037-2048, 1994).
  • Another mAb, DHl has been shown to block chondrogenesis (Gehris et al, Dev. Biol. 190:191-205, 1997).
  • Recent studies demonstrate that IST-9 blocks the stimulatory activity of TGF- ⁇ on SMC ⁇ -actin expression during myofibroblast differentiation (Serini et al, J. Cell Biol.
  • TGF- ⁇ controls the expression of ECM molecules and certain integrins
  • TGF- ⁇ signaling pathway may result in tissue fibrosis (Border and Ruoslahti, J. Clin. Invest. 901-7 1992), Spom et al, J. Cell Biol. 119:1017-1021, 1992; Border et al, N.
  • the Ile 43 and His 4 residues of EIIIA are necessary for IST-9 binding, and partially constitute the reactive epitopes for DHl an 3E2. None of the three mAbs react with frog EIIIA unless the Lys 44 is replaced by a His residue found in human, rat and chicken FN (Fig. 7). Replacement of the His 4 with Arg by site-directed mutagenesis in rat EIIIA (Fig. 6) or Lys which occurs in frog FN (Fig. 7) does not suffice for either IST-9 or DHl binding. In contrast, mutation at His-w made too either Arg or Ala does not significant affect 3E2 binding (Fig. 6).
  • rat EIIIA deletion constmcts demonstrates that these epitopes are only active in an intact polypeptide (Fig. 4).
  • the GST moiety in the purified EIIIA fusion proteins does not appear to alter the conformation of rat EIIIA because comparable reactivities are obtained when the GST and EIIIA moieties are separated by thrombin cleavage.
  • the influence of molecular conformation on rat EIIIA reactivity is further demonstrated by the antibody reactivities of rat EIIIA double mutants.
  • TGF- ⁇ 1 binds to the loop region of EIIIA
  • TGF- ⁇ 1 was bound to His- EIIIA in a dose-dependent manner.
  • His-EIHA (10 ⁇ g/ml) was also coated onto a 96-well microtiter plate, allowed to dry overnight, and then exposed to TGF- ⁇ 1 in the presence of soluble His-EIIIA.
  • the soluble His-EIIIA served as an effective competitor; the ability of TGF- ⁇ 1 to bind to immobilized EIIIA was dramatically reduced in the presence of sufficient soluble His-EIIIA (i.e., in the presence of about 25 ⁇ g/ml of soluble His-EIIIA; Fig. 9B).
  • TGF- ⁇ 1 binds to the loop region of EIIIA.
  • Wild-type EIIIA peptides, five mutant EIIIA peptides containing single amino acid substitutions, and a control GST peptide, which contains no EIIIA sequence (10 ⁇ g) were applied to gutathione-agarose beads.
  • the mutant EIIIA peptides included a peptide in which A was substituted for E at position 45 of the EIIIA peptide shown beneath the graph in Fig.
  • EIIIA binds to integrin ⁇ 9 ⁇ l.
  • SW490 cells (5 x 10 5 ) were either transfected with a constmct encoding integrin ⁇ 9 ⁇ l or mock-transfected and then cultured in tissue culture wells that had been previously coated with EIITA or III4 peptides (the fourth type III repeat; 10 ⁇ g/ml). The cells were allowed to adhere to the coated wells by incubating the tissue culture plates at 37° C for one hour. A subset of the cells was preincubated with an antibody, Y9A2 (at 10 ⁇ g/ml) that specifically binds integrin ⁇ 9 ⁇ l. The preincubation was carried out at 4°C for 15 minutes. As shown in Fig.
  • EIIIA-specific antibodies also inhibit the interaction between integrin ⁇ 9 ⁇ l and EIIIA.
  • the experiment described in the preceding paragraph was repeated except that the EIIIA-coated plates were preincubated with two EIIIA-specific antibodies, IST-9 and 3E2 (diluted 1:100, 1:1000, and 1:10,000, as shown in Fig. 13).
  • the transfected SW490 cells were then added to the culture plates and incubated as described above. Both of these EIIIA- specific antibodies significantly inhibited adhesion between integrin ⁇ 9 ⁇ l and EIIIA (Fig. 13).
  • the experiment described in the preceding paragraph was also repeated except that the ⁇ 9 ⁇ 1 -transfected S W490 cells were preincubated with one of four synthetic peptides (100, 10, and 1 ⁇ M) as shown in Fig. 14.
  • the first peptide (#1) consisted of residues 39-48 of the EIIIA domain flanked by additional Cys residues at the 5' and 3' ends;
  • the second peptide (#2) consisted of residues 24-52 of the EIIIA domain;
  • the third peptide (#3) consisted of residues 31-52 of the EIIIA domain;
  • the fourth peptide consisted of residues 31-42 of the EIIIA domain, interrupted following residue 34 by an additional Tyr residue.

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Abstract

L'invention concerne des compositions et des méthodes permettant d'inhiber ou de stimuler une fibrose.
PCT/US2000/016366 1999-06-14 2000-06-14 Modulateurs de fibrose Ceased WO2000076456A2 (fr)

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AU54889/00A AU5488900A (en) 1999-06-14 2000-06-14 Modulators of fibrosis

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1913954A4 (fr) * 2005-06-13 2011-01-12 Proyecto Biomedicina Cima Sl Agents et methodes fondes sur l'utilisation du domaine eda de la fibronectine
US11597769B2 (en) * 2018-01-25 2023-03-07 Massachusetts Institute Of Technology Nanobody based imaging and targeting of ECM in disease and development

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIAO ET AL.: 'Identification of two amino acids within the EIIIA (ED-A) segment of fibronectin constituting the epitope for two function-blocking monoclonal antibodies' J. BIOL. CHEM. vol. 274, no. 25, 18 June 1999, pages 17876 - 17884, XP002934458 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1913954A4 (fr) * 2005-06-13 2011-01-12 Proyecto Biomedicina Cima Sl Agents et methodes fondes sur l'utilisation du domaine eda de la fibronectine
AU2006259041B2 (en) * 2005-06-13 2012-02-02 Proyecto De Biomedicina Cima, S.L. Agents and methods based on the use of the EDA domain of fibronectin
US9155783B2 (en) 2005-06-13 2015-10-13 Proyecto De Biomedicina Cima, S.L. Agents and methods based on the use of the EDA domain of fibronectin
US11597769B2 (en) * 2018-01-25 2023-03-07 Massachusetts Institute Of Technology Nanobody based imaging and targeting of ECM in disease and development
US12454576B2 (en) 2018-01-25 2025-10-28 Massachusetts Institute Of Technology Nanobody based imaging and targeting of ECM in disease and development

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