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WO1990008833A1 - Expression de la fibronectine de recombinaison dans des cellules mises au point par genie genetique - Google Patents

Expression de la fibronectine de recombinaison dans des cellules mises au point par genie genetique Download PDF

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Publication number
WO1990008833A1
WO1990008833A1 PCT/US1990/000650 US9000650W WO9008833A1 WO 1990008833 A1 WO1990008833 A1 WO 1990008833A1 US 9000650 W US9000650 W US 9000650W WO 9008833 A1 WO9008833 A1 WO 9008833A1
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fibronectin
recombinant
cells
cellular fibronectin
cell
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PCT/US1990/000650
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English (en)
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Jun-Lin Guan
Richard O. Hynes
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Massachusetts Institute Of Technology
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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

Definitions

  • Fibronectins are high molecular weight glycoproteins involved in cell adhesion, morphology and migration.
  • Fibronectin has been shown to consist of a dimer of two subunits, each about 250 kilodaltons in size.
  • the two subunits which are similar, but not necessarily identical, each fold into an elongated and flexible arm. They are joined by disulfide bonds very near their
  • Each subunit is made up of a series of tightly-folded globular domains, each of which is specialized for binding to other molecules or to cells.
  • Type I and II homologies which are disulfide-bonded loops each 45-50 amino acids long
  • Type III homologies which are 90 amino acids long and lack disulfide bonds.
  • fibronectins from different sources, e.g., fibroblasts
  • plasma fibronectin contains subunits of two different mobilities on SDS - polyacrylamide gels and fibronectin from fibroblasts (cellular fibronectin) shows a different subunit pattern.
  • EIIIA and EIIIB Type III repeats
  • Both EIIIA and EIIIB are always omitted by liver cells, although both can be included by other cell types and all possible combinations occur. Because both EIIIA and EIIIB are omitted by hepatocytes, neither repeat occurs in plasma fibronectin.
  • a third region, designated V is also alternatively alternatively
  • the V region can be present in both plasma fibronectin and fibroblast or cellular fibronectin.
  • fibronectin For cellular fibronectin, therefore, there are eight possible combinations or variants of these three alternatively spliced regions:
  • Fibronectins play an important role in many biological systems. They have been shown to be involved in cell adhesion and migration, cell morphology, hemostasis, thrombosis and oncogenic transformation. Hynes, R.O. and K.M. Yamada, J Cell. Biol., 95:369-377 (1982) and Hynes, R.O. Scientific American, 254:42-51 (1986).
  • fibronectin is thought to play an important role in the cell processes involved in tissue repair, particularly wound healing. The ability to repair damaged tissue, wound healing, represents an important response to injury that is common to all complex organisms. Just as in embryonic development, this process involves cell proliferation, migration and differentiation of a number of different cell types.
  • Fibronectin promotes cell migration in culture and is present in the embryo associated with many different cell migrations.
  • antibodies to fibronectin or to cell surface receptors of the integrin glycoprotein family can block migration when injected into the intact embryo.
  • Fibronectin is expressed at high levels In healing wounds. It is derived from two sources: plasma fibronectin, which is present in the exudate from damaged blood vessels, and cellular fibronectin, which is synthesized locally in the wound tissue. Fibronectin appears to be involved in the migration in vitro of four major cell types that migrate into the area of the wound. Fibroblasts and epithelial cells are stimulated to migrate by fibronectin.
  • the present invention relates to a method of producing cellular fibronectin of mammalian origin through the use of genetic engineering techniques, as well as to cellular fibronectins produced by the method.
  • the fibronectins of the present invention are homogeneous cellular fibronectins which include, as desired, region B, region A or both regions, as well as the V region, if desired. Recombinant fibronectins having portions of these regions can also be produced.
  • the fibronectins of the present invention differ from presently-available fibronectins in that the subject fibronectins are recombinant homogeneous cellular fibronectins which include region B and/or region A, alone or in combination.
  • a recombinant full length cDNA encoding cellular fibronectin is introduced into an appropriate host cell by means of a recombinant retrovirus (or other suitable vector).
  • the full length cDNA is expressed in the host cell, resulting in production of full length cellular fibronectin.
  • the eight possible variants of full length rat cellular fibronectin i.e., those in-cluding some, none or all of the three alternative splice domains designated A, B and V
  • A, B and V three alternative splice domains designated A, B and V
  • Figure 1 is a schematic representation of
  • Figure 2 is a schematic representation of the method of the present invention, by which recombinant full length fibronectin is produced.
  • Figure 3 shows the entire nucleic acid sequence encoding recombinant full-length rat fibronectin.
  • Figure 4 demonstrates expression of different variants of fibronectin in WEHI231 cells, which are lymphocytes which do not themselves produce fibronectin. Immunoprecipitation, using a polyclonal antibody (R61.1) which recognizes total fibronectin, was carried out, followed by reduced and nonreduced gel electrophoresis. WEHI231 cells infected with a recombinant vector containing one of the following were analyzed: pLJ, control
  • Figure 5 is a graphic representation of spreading or adhesion of melanoma cells (B16F10) on various variants and concentrations of recombinant fibronectin produced in WEHI231 cells.
  • 0, B, A and V indicate the fibronectin variants used (see Figure 4 for variant type).
  • Figure 6 is a photograph which demonstrates the ability of recombinant fibronectin variant A (B-A + V-) produced in WEHI231 cells to promote reversion of
  • Nil.8-HSV morphology to a normal morphology.
  • Figure 7 is a photograph which demonstrates the results of assessment of extracellular matrices of NIH 3T3 cells and incorporation of recombinant fibronectin variants B (B + A-V-) and V (B-A-V + ) produced in WEHI231 cells into the matrices at three concentrations: -, no added fibronectin; 30 ug/ml.; 90 ug/ml.
  • Figure 8 is a schematic representation of the rat fibronectin structure which is composed of three
  • Type I, II and III repeating peptide units termed Type I, II and III, which are shown by boxes.
  • Two alternatively spliced type III repeats, EIIIB and EIIIA are represented by the filled squares and are indicated above the diagram.
  • the V region is marked by the shaded box and the V25 segment is indicated with stripes, and its amino acid sequence is given below. Domains that interact with fibrin,
  • collagen, and heparin are illustrated above.
  • the sites that interact with cell surfaces are also shown.
  • Figure 9 shows the peptide inhibition of WEHI231 cell spreading on the V form of FN.
  • the average scores of three independent experiments for each peptide are shown with their standard deviations (A).
  • the peptide sequences are illustrated in (B).
  • 0 represents the score in the absence of any peptide competitor.
  • RGD and RGE represent peptides GRGDSP and GRGESP, respectively.
  • the present invention provides a method of producing recombinant cellular fibronectin which is essentially full length (recombinant cellular fibronectin) and of producing modified, essentially full-length recombinant cellular fibronectin.
  • cDNA encoding recombinant cellular fibronectin has been expressed in NIH 3T3 and WEHI231 cells and tested on several cell types (CHO, Rat-1, BHK, B16 melanoma) which are standard cell types used to assay fibronectins. It has been shown, using art-recognized methods, to be produced in the form of essentially pure homogeneous homodimers and to be biologically active.
  • fibronectin includes regions which bind to various proteins or other substances (e.g., fibrin, heparin, collagen, gelatin), as well as three regions, described above, designated EIIIB, EIIIA and V. Regions EIIIB and EIIIA can be present in cellular fibronectin, but not in plasma fibronectin.
  • Region V can be present in variants of both cellular and plasma fibronectin. As described above, there are eight possible combinations of these three regions (eight variants) in the case of full length cellular fibronectin. cDNA encoding each of these eight variants has been produced by combining or joining cDNA fragments, each of which encodes a portion or segment of cellular fibronectin. See, Patel, R.S. et al. , The EMBO J.,
  • the full length cDNA was introduced into appropriate cells, in which it was expressed and subsequently
  • Figure 3 shows the entire nucleic acid sequence encoding recombinant full-length rat fibronectin.
  • Recombinant full length cDNA encoding fibronectin was introduced into the pLJ vector, using known techniques. See, Schwarzbauer, J.E. et al., Proc. of the Natl. Acad. of Sci. , USA , 84:754-758 (1987).
  • Schwarzbauer has also modified the vector by removal of a splice site.
  • This modifed vector is called pLJ.
  • the characteristics of pLJ have been described in Korman, A . J . et al., Proc. of the Natl. Acad. o f Sci. , USA,
  • This vector is capable of expressing both the gene of interest and a dominant selectable marker, such as the neo gene.
  • the gene of interest is cloned in direct orientation into a BamHI/Smal/Sall cloning site just distal to the 5' LTR, while the Neo gene is placed distal to an internal promoter (from S-V40) which is located 3' of the cloning site. Transcription from pLJ is initiated at two sites: 1) the 5' LTR, which is responsible for expression of the gene of interest and 2) the internal SV40 promoter, which is responsible for expression of the neo gene.
  • the diagram at the top of Figure 2 is a representation of pLJ and the additional sequences inserted into it (e.g., full length cDNA encoding cellular fibronectin and a neomycin resistance-encoding gene, NEO-R), resulting in production of a recombinant retrovirus designated pLJ-FN.
  • the NEO-R gene product confers resistance to the antibiotic, G418, in mammalian cells and can be used to select cells containing the recombinant vector.
  • the resulting plasmid (pLJ-FN) was introduced into a packaging cell (e.g., Psi 2 cells), In which the cDNA was transcribed and the resulting fibronectin mRNA and NEO-R mRNA incorporated or packaged into viral particles which subsequently bud out of the cells.
  • a packaging cell e.g., Psi 2 cells
  • the Psi 2 cell line described by Mulligan and co-workers was created by transfecting NIH 3T3 fibroblasts with pMOV-Psi-, which is an ecotropic Moloney murine leukemia virus (Mo-MuLV) clone.
  • pMOV-Psi an ecotropic Moloney murine leukemia virus (Mo-MuLV) clone.
  • pMOV-Psi expresses all the viral gene products but lacks the Psi sequence, which is necessary for encapsidation of the viral genome.
  • pMOV-Psi expresses an ecotropic viral envelope glycoprotein which recognizes a receptor present only on mouse (and closely related rodent) cells.
  • Another cell line is the Psi am line, which are
  • Psi-2-like packaging cell lines contain a modified pMOV-Psi genome, in which the ecotropic envelope glycoprotein has been replaced with envelope sequences derived from the amphotropic virus 4070A. Hartley, J.W. and W.P. Rowe, Journal of Virology, 19:19-25 (1976). As a result, they are useful for production of recombinant virus with a broad mammalian host range, amphotropic host range.
  • the retrovirus used to make the Psi-am cell line has an amphotropic host range and can be used to infect human cells. If the recombinant genome has the Psi packaging sequence, the Psi-am cell line is capable of packaging recombinant retroviral genomes into infectious retroviral particles.
  • the retroviral genome has been modified by Cone and Mulligan for use as a vector capable of introducing new genes into cells.
  • the gag, the pol and the env genes have all been removed and a DNA segment encoding the neo gene has been inserted in their place.
  • the neo gene serves as a dominant selectable marker.
  • the retroviral sequence which remains part of the recombinant genome includes the LTRs, the tRNA binding site and the Psi packaging site. Cepko, C. et al., Cell, 37:1053-1062 ( 1984 ) . In this ins tance , full - length f ibronectin cDNA was constructed as described and inserted into the vector, as indicated in Figure 2.
  • the resulting recombinant retrovirus (pLJ-FN) was used to infect appropriate recipient/host cells (e.g., NIH 3T3, WEHI231). In these cells, reverse transcription of the viral RNA results in generation of a DNA copy, which integrated into the host cell genome. Infected cells, which were G418 resistant, synthesized the encoded recombinant fibronectin variant, which was subsequently secreted into the culture medium.
  • Each of the eight fibronectin variants in which EIIIB, EIIIA and/or V can be present was expressed in at least one of the following two types of mammalian cell: NIH 3T3 and WEHI231. Methods for making the variants are described in detail in the Exemplification. WEHI231 cells expressing the V fibronectin variant (B-A-V + ) have been deposited, according to the terms of the Budapest Treaty, at the American Type Culture Collection
  • the resulting recombinant fibronectin was tested on several cell types (e.g., CHO , Rat-1, BHK, B16 melanoma cells) which are, as mentioned previously, standard cell types used to assay fibronectins.
  • the resulting recombinant fibronectins were characterized and shown to be homodimers, rather than the heterodimers produced naturally. They have been shown, as described below, to be biologically functional.
  • Naturally-occurring fibronectin is known to bind anti fibronectin antibodies, gelatin, and heparin; to promote adhesion of several different cell types, cytoskeletal assembly and cell migration; and to participate in reversion of tumor cells (transformed cells) to normal morphology.
  • recombinant fibronectin produced as described herein has been shown to have these same capabilities.
  • Expression of recombinant fibronectin (BAV) in NIH 3T3 cells was clearly demonstrated and the results confirmed by Northern blot analyses. Two transcripts were detected from three individual clones infected with retrovirus containing the BAV form FN gene when the neo gene was used as probe.
  • the 11.6- and 3.1-kb messages which were observed corresponded with the sizes expected for the full-length genomic transcript from the viral LTR and subgenomic RNA from the SV-40 promoter. No band was detected in RNA isolated from parental 3T3 cells. RNA isolated from cells containing pLJ vector alone included the 3.1-kb subgenomic RNA and a minor band migrating at 3.9 kb, which corresponds with a transcript derived from the 5' viral LTR. The identity of the 11.6-kb transcript was confirmed by hybridizing the same blot with a probe derived from rat FN cDNA.
  • This probe also detects the endogenous murine FN message (8.1 kb), which is present in rat FN expressor clones as well as in the control cell lines.
  • the rat FN mRNA signal was -10% of the signal for endogenous murine FN mRNA. Therefore, cDNA clones for rat FNs are readily transcribed in murine 3T3 cells under the control of the MLV-LTR promoter.
  • the chimeric rat FN mRNAs are efficiently translated and the rat fibronectins expressed in stable infected NIH 3T3 cell clones are efficiently processed, assembled Into dimers and secreted into the medium. These cells also assemble the recombinant rat FNs into extracellular matrix.
  • the single cell clones secreting the corresponding rat FNs were isolated by limiting dilution from the G418 - resistant pools. Secretion of various forms of rat FNs was determined by Immunoprecipitation using the polyclonal antiserum R61 followed by SDS-PAGE analysis in the presence or absence of reducing agents.
  • a major protein product migrating at 220-250 kD was immunoprecipitated from the media of [ 35 S] methionine- labeled cells clones expressing the O, B, A or V forms of rat FNs.
  • the apparent molecular weights of the proteins are as expected for the various rat FN forms and also correspond with those of rat FNs expressed in NIH 3T3 cells as described above. This suggests that no major different pos ttranslational modifications occurred in recombinant FN synthesized in lymphoid WEHI231 cells which normally do not produce any endogenous FNs.
  • WEHI231 cells are lymphocytes and, thus, do not produce fibronectin. Ralph, P. Immunol. Rev., 48:107-121 (1979). Subsequent analyses demonstrated that all variants produced by WEHI231 cells bind to gelatin and heparin. For example, variants O (B-A-V-), B (B + A-V-),
  • a (B-A + V-) and V (B-A-V + ) bind both to gelatin and to heparin (as well as to a polyclonal antibody, R61.1, which recognizes total fibronectin).
  • fibronectin variants produced were also shown to promote cell adhesion or spreading, in a variety of cell types.
  • One representative experiment was performed with the mouse melanoma cell line B16F10. On control
  • Recombinant fibronectin variants were also shown to promote cytoskeletal organization, as assessed using antibodies against actin or vinculin. Through their transmembrane integrin receptors, extracellular FNs can induce cytoskeletal organization including organized actin bundles and focal contact formation.
  • B16F10 melanoma cells were cultured on substrata coated with various recombinant FNs. 2 hours later, cells were fixed and stained for F actin and vinculin distribution using double-label immunofluorescence. When low concentrations of the various FNs were used, actin bundles were visualized in only a small percentage of cells while diffuse and unorganized patterns were evident for most cells.
  • NIL8.HSV cells are rounded and detached from the substrate.
  • the cells assumed a more flattened and aligned morphology upon addition of the recombinant FN A form. Similar effects were also observed for O, B, V forms of FNs.
  • the differences in dose response among the different forms were, at most, two to threefold in different experiments. Therefore, in agreement with results obtained from basic adhesion and spreading assays using adherent cells, the ability of FN to revert the morphology of these transformed cells does not appear to reside in the EIIIB, EIIIA, or V regions.
  • fibronectin into cell matrices was also carried out and showed that all variants were incorporated and that variants B + (B + A-V-) and A + (B-A + V-) were incorporated somewhat more effectively than the other variants.
  • recombinant rat FNs formed fibrillar networks characteristic of the usual extracellular matrix distribution of FN.
  • total extracellular FN staining (with polyclonal antiserum R61) increased significantly upon addition of exogenous recombinant FNs, indicating their contribution to matrix formation. All forms of FNs incorporated into the existing matrices.
  • Fibronectins having a portion of regions B, A, V or combinations of these can also be produced.
  • the first 25 amino acids of the V region (referred to as V25 segment) which are shown in Figure 8 can be
  • V25 segment is important in the selective adhesion of various cell types and is recognized by the integrin ⁇ 4 ⁇ 1 fibronectin receptor.
  • V + recombinant fibronectins having the V25 segment alternatively spliced out can be produced as homodimers or heterodimers.
  • WEHI231 lymphoid cells interact with an alternatively spliced region of FN, specifically with the
  • GPEILDVPST C-terminal 10 amino acids
  • integrin ⁇ 4 ⁇ 1 binds specifically to the V25 peptide coupled to Sepharose.
  • integrin ⁇ 4 ⁇ 1 is an FN receptor distinct from the ⁇ 5 ⁇ 1 integrin receptor, which recognizes the RGDS site in FN (Pytela et al., Cell 40:191-198 (1985); Pytela et al., Science 231:1559-1562 (1986); Argraves et al., J. Cell Bio. 105:1183-1190 (1987); and Wayner et al., J.
  • the V25 segment can be selectively spliced out independently of the rest of the V region in mammals (Schwarzbauer et al., Cell 35:421-431 (1983); Kornblihtt et al., Nucl._Acids_Res. 12:5853-5868 (1985); Sekiguchi et al., Biochemistry 25:4936-4941 (1986)) and a 44 amino acid segment (V44) that includes V25 can be similarly spliced out in chickens (Norton, P.A. and R.O. Hynes,
  • V25 segment which can be omitted independently of other parts of the V region in mammals.
  • V25 -negative forms of FN may be found in specific locations.
  • the absence of the V segment from 50% of plasma FN subunits is of potential relevance given the fact that many circulating blood cells express ⁇ 4 ⁇ 1 (Helmer et al., J. Bio. Chem. 262: 3300 - 3309 (1987);
  • each molecule contains only a single binding site for a 4 ⁇ 1 . This appears to be insufficient for high affinity binding to cell surfaces since FN is not found as a surface component of circulating blood cells. If, as appears to be the case, matrix FN is largely V + , it should have a higher avidity for binding of cells bearinga 4 ⁇ 1 integrin. This could play a role in the adhesion of various blood cells to exposed extracellular matrix, such as endothelial basement membrane.
  • ⁇ 4 ⁇ 1 has recently been reported to be involved in homing of lymphocytes to Peyer's patch high endothelial venules (Holzmann et al., Cell 56:37-46 (1989)).
  • integrin ⁇ 1 subfamily there are three FN receptors: ⁇ 5 ⁇ 1 , ⁇ 4 ⁇ 1 and ⁇ 3 ⁇ 1 specific, respectively, for the RGDS site, 10 amino acids in the V25 segment, and an unknown site.
  • Other integrins are also known to interact with FN, including a I Ib ⁇ 3 (GPIIb/IIa), which binds to the RGDS site (Gardner and Hynes, Cell 42 :
  • Fibronectin of the present invention can be produced, as described previously, using an appropriate vector containing cDNA encoding full-length recombinant cellular fibronectin, which is introduced into and expressed by an appropriate host cell.
  • the DNA encoding the cellular fibronectin can be cDNA or DNA, synthesized by known methods, which has the same nucleotide sequence as the cDNA or a functional equivalent thereof (i.e., one which encodes a product having the same characteristics and exhibiting the same functions as the recombinant cellular fibronectin described herein).
  • Appropriate host cells include, but are not limited to, NIH 3T3 and
  • WEHI231 cells other cells in which the complex cellular fibronectin can be produced and properly processed can also be used.
  • Fibronectin produced by the process of the present invention has several therapeutic and clinical uses, and can be used wherever fibronectin is naturally utilized in the body.
  • fibronectin plays an important role in the cell migration associated with wound healing and tissue repair in general. Immunolocalization studies have shown that abundant fibronectin is present in healing wounds. In situ hybridization studies have shown that cellular fibronectin synthesized locally in wound tissue contains both the B and the A segments or regions.
  • Fibronectin can also be used to promote nerve regeneration in some cases. For example, while not strictly cell migration, the outgrowth of neurites from neurons involves many of the same principles and similar mechanisms. There is considerable evidence that some neurons will respond to fibronectin by extending
  • Fibronectin has been shown to promote neurite growth in chick ganglion cells (Carbonetto, S.T. et al., J. Neurosci., 3:2324-2335 (1983) and in human ganglion cells. (Baron-Van Evercooren, A. et al., J.
  • Fibronectin also plays a role in hemostasis and thrombosis and can be used in the treatment of blood or clotting disorders. Thrombotic diseases. are major killers, and the ability to control thrombosis would be a valuable therapeutic tool in treating them. Monoclonal antibodies raised against fibronectin, or specific peptides, could be used to intervene in the interactions between the ligand and its receptors. For example, fibronectin interacts with fibrin, becomes incorporated into clots, and is crosslinked to the fibrin by factor
  • fibronectin becomes an integral part of it. Intervening in this reaction by preventing fibronectin from performing its usual role provides a method of preventing dangerous blood clots. It has been shown that soon after wounding, fibronectin and fibrin appear in the area of a wound. These proteins then serve as a substrate for adhesion and migration of the cells repairing the defect and, in most cases, subsequently disappear. Fibronectin also plays a role in removal of debris by various cell types.
  • the first detectable event is the formation of a fibrin-fibronectin clot in the area of the wound.
  • Fibronectin can be used to treat corneal lesions.
  • the cornea of the eye is a relatively simple, nonvas- cularized system.
  • the cornea consists of a layer of epithelial cells on a basement membrane, beneath which lies a thick stromal layer composed largely of collagen with a few keratocytes. Beneath the stroma is a specialized basement membrane (Descemet's membrane) with a layer of endothelial cells attached to it.
  • Fibronectin is believed to be involved in the migration of the endothelial cells and stromal cells during development, but in the mature cornea the only significant concentration of fibronectin is in Descemet's membrane; the basement membrane of the corneal epithelium has little fibronectin. Kurkinen, M. et al., Dev._Biol., 69:589-600 (1979) and Cintron, C. et al., Curr .___Eye R es ., 3:4
  • fibronectin and fibrin appear on the surface of the cornea. Between one and two days after wounding, the corneal epithelium grows back over this fibronectin and fibrin-coated surface and the fibronectin and fibrin disappear over the next several days. Fibronectin also appears in significant amounts within the stroma which normally contains only small amounts.
  • Nishida and co-workers have cultured blocks of cornea in vitro and shown that the epithelial layer migrates over the cut stromal surface. Nishida, T. et al., Jpn. J. Ophthalmol . , 26:410-415 (1982) and Nishida, T. et al., Jpn._J. Ophthalmol_., 26:416-424 (1982).
  • the migrating epithelium is underlain by a layer of fibronectin. Addition of autologous serum or, more significantly, purified plasma fibronectin to the cultures accelerated the epithelial migration and anti- fibronectin antisera inhibited it.
  • fibronectin can be used in the treatment of corneal epithelial wounds.
  • Fibronectin has therapeutic value in promoting corneal epithelial wound healing and in leading to curing of persistent corneal ulcers.
  • Fibronectin can be used to prepare eye drops, for example. Treatment with these eye drops leads to accelerated healing of corneal ulcers and other defects.
  • the protease inhibitor, aprotinin has dramatic therapeutic effects on the healing of corneal lesions.
  • combinations of fibronectin and protease inhibitors may prove particularly efficacious.
  • Past efforts in improving wound healing have made use of plasma fibronectin, which, as mentioned, is a mixture of types. Homogeneous cellular fibronectin might be more effective in promoting wound healing (e.g., because of the presence of B and/or A regions).
  • Fibronectin treatment may be useful in treatment of periodontal disease, which is characterized by failure of attachment of gingival tissue to the tooth roots.
  • fibronectin promotes attachment of gingival tissue to tooth roots in vivo and this appears to be the case.
  • connective tissue attachment during healing is significantly promoted by treatments with citric acid and fibronectin.
  • the fibronectin, or even conjugated peptides can be used to promote cell adhesion and migration on the wound bed.
  • the accumulation of fibronectin and other matrix molecules, and the cells that produce them must be controlled, for example, by intervention in the stimulatory events between cells br in the biosynthesis of fibronectin. It is also possible to interfere with fibronectin function by the use of antibodies or peptides.
  • the quantity of the present fibronectin to be administered will be determined on an individual basis, and will be based at least in part on consideration of the severity of the symptoms to be treated and the result sought.
  • the agent or drug containing fibronectin can be administered by subcutaneous or other form of injection, intravenously, parenterally, transdermally or topically.
  • the form in which it will be administered will depend upon the route by which it is administered.
  • a fibronectin composition of the present invention can optionally include other components.
  • the components included in a particular composition are determined primarily by the manner in which the composition is to be administered.
  • a composition to be applied topically can include, in addition to fibronectin, or a derivative thereof, a binder (e.g., carboxymethyl cellulose, gelatin) , a protease inhibitor, (e.g., aprotinin), a filler (e.g., lactase) or an emulsifier.
  • a composition to be administered dropwise may contain a liquid carrier (e.g., saline).
  • a liquid carrier e.g., saline
  • composition of the present invention to be applied to a skin wound would be applied directly to the wound for a period of time necessary to Induce healing. More than one application may be necessary.
  • the dosage, or concentration of fibronectin, in the composition will also vary on an Individual basis and be determined by the type and severity of the symptoms to be treated.
  • NIH 3T3 and Psi 2 cells were grown in Dulbecco's modified Eagle's medium (DME) plus 10% calf serum (CS, Gibco Laboratories, Grand Island, NY).
  • DME Dulbecco's modified Eagle's medium
  • CS calf serum
  • Murine melanoma B16F10 cells were generous gifts of I.J. Fidler (M.D. Anderson Hospital, Houston) and cultured as described (Fidler, I.J., (1974), Cancer Res. 34:1074-1078). Plasmid Construction
  • Retroviral vectors containing the 3' third of rat FN cDNA including or excluding EIIIA or V have been described previously (Schwarzbauer, J.E. et al., (1987), Proc. Natl. Acad. Sci. USA 84:754-758). Genomic clones were isolated from a rat genomic library in EMBL3B
  • Genomic fragments were subcloned into a murine retroviral vector, pLJ, which is a derivative of DOL (Korman et al., (1987) Proc. Natl. Acad. Sci. , USA
  • Clones ⁇ rFN2 to ⁇ rFN5 cover the central part of the gene which includes all the type-Ill repeats ( Figure 3).
  • cDNA clones isolated from a rat liver ⁇ gtll library cover type-Ill repeats 9-15 (Schwarzbauer et al., (1983) Cell 35: 421-431).
  • cDNA clones covering type-Ill repeats 1-9 and the two alternatively spliced EIII repeats were obtained by passage of genomic clones through retroviral vectors.
  • Overlapping cDNA clones covering the 5' regions of the gene were generated from the respective genomic clones, ⁇ rFN-3, ⁇ rFN-5, ⁇ rFN-8, and ⁇ rFN-9 (Patel, R.S. et al., (1987), EMBO J. 6:2565-2572; Schwarzbauer, J.E. et al., (1987), EMBO J. 6: 2673 - 2580), using a fusion rescue method as outlined before (Schwarzbauer, J.E. et al., (1987), EMBO J. 6:2673-2580; Schwarzbauer, J.E. et al., (1987), Proc. Natl. Acad. Sci. USA 84:754-758).
  • the expression vector, pDOP was constructed from pMSV-gpt
  • pDOP contains a unique BamHI cloning site followed by a fragment of pBR322, the simian virus 40 (SV40) origin and early promoter, and the neo gene (Cepko, C.L. et al., (1984) Cell 37:1053-1062; Korman, A.J. et al., (1987) Proc.
  • the neo gene product confers resistance to the antibiotic, G418, In mammalian cells
  • the polyoma virus early region Increases the plasmid copy number after transfection into
  • FN cDNAs were isolated from a rat liver ⁇ gtll library (Schwarzbauer, J.E. et al., (1983) Cell 35:421- 431). BamHI and Bel I linkers were added to the 5' and 3' ends, respectively, of an EcoRI partial-Sac II fragment of ⁇ rlf 3 containing the 3'-terminal 2400 bases of coding sequence including the 360-base variable segment (V120) plus 169 bases of the 3'-untranslated region.
  • V120 360-base variable segment
  • prepro coding sequences of parathyroid hormone was then ligated to the 5' end of the linkered FN cDNA, The hybrid cDNA was inserted into the BamHI site of pDOP.
  • FN cDNA containing the EIII segment was obtained by passage through ⁇ 2 cells of a retroviral vector, DOL (Korman, A.J. et al. (1987) Proc. Natl. Acad Sci.,
  • constructs include the natural signal and propeptide segments of rat FN to allow normal secretion and processing.
  • the full length clones were inserted into the retroviral expression vector pLJ to generate pLJ-FN plasmids.
  • pLJ is a derivative of pDOL (Korman, A.J. et al., (1987), Proc.
  • neomycin resistance (neo r ) gene driven by the SV-40 early promoter and a pBR322 origin of replication.
  • G418 r ⁇ 2 clones were tested for virus production. Viral titers for these clones ranged from 10 3 to 10 5 G418 r colony-forming units/ml of supernatant. The following modifications were performed. After infection, NIH 3T3 cells were selected for neo expression in G418 (Gibco Laboratories) at a concentration of 0.5 mg/ml. A subset of G418 - resistant clones was then isolated and clones were expanded for further analysis. Infected WEHI231 cells were selected with G418 at a concentration of 3 mg/ml. The selected pool of cells was then cloned by limiting dilution. Single cell clones that produced the highest amounts of recombinant FNs, as determined by immunoprecipitations, were expanded for further analysis. Northern Blot Analysis
  • RNA samples were used for hybridizations with probes corresponding to rat FN cDNA or the neo gene. The probes were labeled
  • Conditioned media were immunoprecipitated using either a rabbit anti-rat FN serum R61 and goat anti-rabbit IgG or a mouse monoclonal anti-rat FN M9 (a generous gift of M. Chiquet) and goat anti-mouse IgG, as described (Choi, M. and R.O. Hynes, (1979), J. Biol Chem. 254:12050-12055). Immunoprecipitates were analyzed either with or without reduction by electrophoresis through SDS-PAGE followed by fluorography. Direct binding of FNs in the conditioned media to gelatin-coupled Sepharose (Pharmacia Fine).
  • Recombinant FNs produced from expressor WEHI231 cell clones were purified by affinity-chromatography using a gelatin-coupled Sepharose column as described by Engvall, E. and E. Ruoslahti, (1977), Int. J. Cancer 20:201-205. Briefly, the expressing clones were grown to saturation in 3 liters of growth medium. The cells were then washed with PBS and resuspended in 10 liters of RPMI 1640 plus 5% FCS that had been passed through gelatin-Sepharose 4B to deplete FN in the serum. The cells were incubated further for 3 d and the conditioned media were
  • FNs were added in 100 ⁇ l PBS to give the desired concentration. Photographs were taken on the Nikon inverted phase-contrast microscope 24 h later.
  • recombinant FNs were used to coat coverslips at various concentrations.
  • Murine melanoma B16F10 cells or fibroblastic BHK cells were then plated onto the coated coverslips in the absence of serum. 2 h later, the distribution of actin bundles and vinculin was visualized by immunofluorescence. Briefly, cells were rinsed twice in PBS and fixed for 15 min in a freshly prepared 4% solution of paraformaldehyde (Fluka Chemical Co., Bern, Switzerland) in PBS, rinsed and permeabilized with 0.5% NP-40 in PBS for 15 min.
  • paraformaldehyde Feluka Chemical Co., Bern, Switzerland
  • antiserum R61 were used as the primary antibody, and fluorescein-conjugated goat anti-mouse IgG and
  • a rat monoclonal antibody R1-2 against mouse integrin ⁇ 4 was kindly provided by Drs. Holzmann and Weissman (Stanford University). This antibody was raised against the ⁇ 4 subunit as a part of the murine Peyer's patch-specific lymphocyte homing receptor (Holzmann, B. et al. (1989), Cell 56 : 37-46).
  • V25, V14, V10a, V10b and V10 peptides are derived from the sequences in the alternatively spliced V segment of rat FN (see Figures 8 and 9).
  • the V10/1 and V10/2 peptides are two scrambled versions of the V10 peptide containing the same amino acids but in a
  • tissue culture dishes were coated with 60 ⁇ g/ml V form of FN. Purified
  • Iodinated cells were extracted with 0.5% NP-40, and immunoprecipitation was performed as described previously (Marcantonio, E.E. and R.O. Hynes, (1988), J. Cell. BIol. 106 : 1765-1772).
  • extracts were immunoprecipitated using Sepharose coupled with the rat monoclonal antibody R1-2, followed by direct recovery by boiling for 3 min in the sample buffer (2% SDS , 100 mM Tris-HCl [pH 6.8], 10 mM EDTA, 10% glycerol and bromophenol blue).
  • integrin complexes extracted from labeled WEHI231 cells were first dissociated by heating at 100°C for 2 min in 1% SDS. After cooling, a 5-fold excess of Triton X-100 was added, and the extracts were precipitated with antiserum against ⁇ 1 as described above. SDS-PAGE was performed by the method of Laemmli, U.K. (1970), Nature 227 680-6.85. Separation gels were 7% acrylamide with a 3% stacking gel. The V25 peptide was covalently coupled to CNBr- activated Sepharose 4B at 2 mg peptide per ml beads according to instructions provided by the manufacturer (Pharmacia Biochemicals Co., Piscataway, NJ).
  • iodinated WEHI231 cells were extracted using 200 mM n-octyl- ⁇ -D-glucopyranoside in 50 mM Tris[pH 7.5], 150 mM NaCl, 1 mM MnCl 2 , 1 mM MgCl 2 , 1 mM CaCl (TBMMC) for 30 min on ice. These extracts were incubated with V25-Sepharose beads for 3 hr at 4°C, followed by washing in 50 mM n- octyl - ⁇ -D- glucopyranos ide in TBMMC (washing buffer) four times. The bound material was then eluted with the sample buffer and subjected to SDS-PAGE as described above.
  • integrin ⁇ 5 ⁇ 1 is a major functional FN receptor on many cells including NIH 3T3 cells
  • Sepharose beads coupled with this antibody did not react with any proteins from NIH 3T3 cells but precipitated a major protein complex from WEHI231 cells, which comigrated with that precipitated by antibody against the Integrin ⁇ 1 subunit from the same cells.
  • heterogeneous smear ranging from M 120,000 to M 150,000 was indeed composed of two noncovalently associated subunits typical of integrins.
  • immunoprecipitations with antiserum against ⁇ 1 both before and after SDS denaturation were carried out. Only the lower part of the smear was precipitated by the antiserum after dissociation of the complex by SDS. This also suggested that the upper part was the ⁇ 4 subunit, in agreement with its molecular weight of 150,000 (Hemler, et al., Ibid (1987); Holzmann et al., Ibid).
  • V25 region is probably the active site in the V segment
  • this peptide was coupled to Sepharose beads and used to identify the WEHI231 cell receptor for the alternatively spliced V segment of rat FN .
  • V25- Sepharose beads were used in a direct binding assay.
  • WEHI231 cells were surface labeled with 125 I, extracted with n-octyl glucoside, and then incubated either with mock- activated Sepharose beads or with V25 - Sepharose beads in the absence or presence of various competing peptides. The bound materials were eluted with SDS and analyzed by SDS-PAGE. Cell surface proteins appearing on the gel as a heterogenous smear between 120-150 kd were shown to bind to V25-Sepharose beads specifically. The heterogenous smear in this region is reminiscent of the integrin ⁇ 4 ⁇ 1 immunoprecipitated from the surface of
  • V25 peptide is the active site for mediating WEHI231 cell spreading rather than inhibiting the cell spreading by interfering with a nearby site.
  • the parallelism between the ability of various peptides to inhibit WEHI231 cell spreading and their ability to interfere with the binding of the 120-150 kd proteins to V25- Sepharose strongly suggests that the 120-150 kd proteins are the cell surface receptor mediating the spreading.
  • V25 - Sepharose column was then used to purify the receptor by affinity chromatography.
  • WEHI231 cells were Iodinated and extracted with n-octyl glucoside in buffer containing divalent cations. The extracts were loaded onto the V25 -Sepharose column by incubating with the beads for 1 hour at 4°C and then washed extensively. The bound materials were eluted sequentially with buffers containing 1 mg/ml V14 peptide and 1 mg/ml V10 peptide. Aliquots of each fraction were analyzed on SDS-PAGE.
  • proteins migrating as a 120-150 kd smear on the gel were bound to V25 - Sepharose beads. About 40% of the bound material (as estimated by cpm) was eluted with the V14 peptide, and the remainder was eluted with the V10 peptide. Furthermore, the same bound material could be eluted completely with either the V25 peptide or the V10 peptide when applied first. Fractions from both V14 peptide eluates (peak I) and V10 peptide eluates (peak II) were then pooled and analyzed along with the starting material by immunoprecipitation with several antibodies specific for integrin subunits.
  • the antibodies against either the integrin ⁇ 1 or ⁇ 4 subunits precipitated the 120-150 kd proteins from both peak I and peak II, as well as from the starting material. These results suggested that the 120-150 kd proteins bound to V25 - Sepharose beads were indeed the integrin ⁇ 4 ⁇ 1 complex.
  • the results also suggest that the interaction between integrin ⁇ 4 ⁇ 1 and the V25 peptide occurs in the V10 peptide region but could be affected partially by the overlapping V14 peptide.
  • WEHI231 cells were preincubated with the rat monoclonal antibody R1-2 specific for integrin ⁇ 4 , anti-IgM or rat IgG antibodies for 30 min at 4°C The cells were then seeded in the dishes coated with saturating amounts (60 ⁇ g/ml) of the V form of FN . The percentage of cell spreading was then quantitated. At two different concentrations, R1-2 almost completely abolished the spreading of WEHI231 cells (>95% reduction).

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Abstract

L'invention concerne un procédé de production de la fibronectine cellulaire homogène d'origine mammifère, qui est un homodimère, ainsi que de la fibronectine cellulaire de recombinaison. Les fibronectines ainsi produites peuvent présenter toutes les régions B, A, V ou une partie de ces dernières, ou des combinaisons de celles-ci. Spécifiquement, on peut produire des homodimères et des hétérodimères dont les 25 acides aminés à terminaison carboxy de la région V sont supprimés. La fibronectine cellulaire de recombinaison est utile dans toutes les applications où on peut utiliser la fibronectine naturelle.
PCT/US1990/000650 1989-02-02 1990-02-02 Expression de la fibronectine de recombinaison dans des cellules mises au point par genie genetique WO1990008833A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0489837A4 (en) * 1989-09-01 1993-06-16 Hutchinson Cancer Research Center Fred Inhibition of lymphocyte adherence to vascular endothelium utilizing a novel extracellular matrix receptor-ligand interaction
US5610148A (en) * 1991-01-18 1997-03-11 University College London Macroscopically oriented cell adhesion protein for wound treatment
US5629287A (en) * 1991-01-18 1997-05-13 University College London Depot formulations
US5641483A (en) * 1995-06-07 1997-06-24 Beaulieu; Andre Wound healing formulations containing human plasma fibronectin
US6251859B1 (en) 1995-06-07 2001-06-26 BEAULIEU ANDRé Deepithelialized skin diffusion cell system
WO2001096599A3 (fr) * 2000-06-15 2003-04-17 Philogen Srl Procedes de determination quantitative de b-fibronectine dans des fluides et des tissus biologiques
US7112320B1 (en) 1995-06-07 2006-09-26 Andre Beaulieu Solid wound healing formulations containing fibronectin
US7238668B1 (en) 1989-09-01 2007-07-03 Fred Hutchinson Cancer Research Center Inhibition of lymphocyte adherence with CS-1-peptides and fragments thereof
WO2020228390A1 (fr) * 2019-05-10 2020-11-19 美尔健（深圳）生物科技有限公司 Peptide de fibronectine recombinant à petites molécules pour fermentation de levure, son procédé de préparation et son application
CN115976031A (zh) * 2022-07-18 2023-04-18 烟台市华昕生物医药科技有限公司 一种重组纤连蛋白及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0207751B1 (fr) * 1985-06-28 1990-11-14 Delta Biotechnology Limited Fibronectines

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0207751B1 (fr) * 1985-06-28 1990-11-14 Delta Biotechnology Limited Fibronectines

Non-Patent Citations (4)

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Title
EMBO Journal, Volume 4, No. 7, 1985, IRL Press Limited, (Oxford, GB), A.R. KORNBLIHTT et al.: "Primary Structure of Human Fibronectin: Differential Splicing may Generate at least 10 Polypeptides from a Single Single Gene", see pages 1755-1759 *
EMBO Journal, Volume 6, No. 9, 1987, IRL Press Limited, (Oxford, GB), J.E. SCHWARZBAUER et al.: "Multiple Sites of Alternative Splicing of the Rat Fibronectin Gene Transcript", pages 2573-2580 *
Proc. Natl. Acad. Sci. USA, Volume 84, April 1987, (Washington, D.C., US), A.J. KORMAN et al.: "Expression of Human Class II Major Histocompatibility Complex Antigens using Retrovirus Vectors", see pages 2150-2154 *
The Journal of Cell Biology, Volume 109, No. 6, pt. 2, December 1989, the Rockefeller University Press, (New York, NY, US), J.E. SCHWARZBAUER et al.: "Selective Secretion of Alternatively Spliced Secretion of Alternatively Spliced Fibronectin Variants", see pages 3445-3453 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0489837A4 (en) * 1989-09-01 1993-06-16 Hutchinson Cancer Research Center Fred Inhibition of lymphocyte adherence to vascular endothelium utilizing a novel extracellular matrix receptor-ligand interaction
GR1001372B (el) * 1989-09-01 1993-10-29 Hutchinson Fred Cancer Res Αναστολή της προσκόλλησης λεμφοκυττάρων στο αγγειακό ενδοθήλιο με την χρησιμοποίηση μιάς αλληλεπίδρασης νέου υποδοχέα εξωκυτταρικής μήτρας-προσδέτη.
US7238668B1 (en) 1989-09-01 2007-07-03 Fred Hutchinson Cancer Research Center Inhibition of lymphocyte adherence with CS-1-peptides and fragments thereof
US5610148A (en) * 1991-01-18 1997-03-11 University College London Macroscopically oriented cell adhesion protein for wound treatment
US5629287A (en) * 1991-01-18 1997-05-13 University College London Depot formulations
US5641483A (en) * 1995-06-07 1997-06-24 Beaulieu; Andre Wound healing formulations containing human plasma fibronectin
US6251859B1 (en) 1995-06-07 2001-06-26 BEAULIEU ANDRé Deepithelialized skin diffusion cell system
US7112320B1 (en) 1995-06-07 2006-09-26 Andre Beaulieu Solid wound healing formulations containing fibronectin
WO2001096599A3 (fr) * 2000-06-15 2003-04-17 Philogen Srl Procedes de determination quantitative de b-fibronectine dans des fluides et des tissus biologiques
WO2020228390A1 (fr) * 2019-05-10 2020-11-19 美尔健（深圳）生物科技有限公司 Peptide de fibronectine recombinant à petites molécules pour fermentation de levure, son procédé de préparation et son application
US12221470B2 (en) 2019-05-10 2025-02-11 Mellgen (Shenzhen) Biotechnology Co., Ltd. Yeast-fermented recombinant fibronectin peptide in small molecule, and its preparation method and applications thereof
CN115976031A (zh) * 2022-07-18 2023-04-18 烟台市华昕生物医药科技有限公司 一种重组纤连蛋白及其应用

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