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WO1992002556A1 - Facteur d'elongation de fibroblastes derive des cellules cancereuses du colon humain - Google Patents

Facteur d'elongation de fibroblastes derive des cellules cancereuses du colon humain Download PDF

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Publication number
WO1992002556A1
WO1992002556A1 PCT/AU1991/000337 AU9100337W WO9202556A1 WO 1992002556 A1 WO1992002556 A1 WO 1992002556A1 AU 9100337 W AU9100337 W AU 9100337W WO 9202556 A1 WO9202556 A1 WO 9202556A1
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Prior art keywords
fef
antibody
factor
collagen
fibroblasts
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PCT/AU1991/000337
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English (en)
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Michael Valentine Agrez
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Michael Valentine Agrez
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Publication of WO1992002556A1 publication Critical patent/WO1992002556A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is directed to a human colon cancer cell-derived fibroblast elongation factor capable of promoting or inducing elongation of fibroblasts.
  • a characteristic of fibroblasts is their ability to bind strongly to collagen and induce collapse of collagen matrices in vitro. This process is known as collagen lattice contraction and has been considered analogous to wound contraction in vivo (Bell et al, 1979; Steinberg et al, 1980).
  • the present invention arose, in part, following an investigation into the possibility that colon cancer cells influence binding between host fibroblasts and the surrounding collagen matrix.
  • colon cancer cells produce a factor which enhances the ability of colon fibroblasts to contract a collagen matrix by promoting or inducing elongation of fibroblasts.
  • the identification and isolation of this novel factor will inter alia enable the screening for early malignant transformations of the colon by assaying for the presence of the factor and the generation of antagonists to same.
  • the present invention provides a biologically pure factor comprising a protein capable of promoting or inducing elongation of fibroblasts.
  • the factor is derivable from human colon cancer cells.
  • the factor is human colon cancer cell-derived fibroblast elongation factor (hereinafter referred to as "FEF").
  • biologically pure is meant a homogeneous or near homogeneous preparation of FEF comprising at least 70% by weight, preferably at least 80% by weight and more preferably at least 90% by weight of FEF relative to other molecules.
  • the present invention extends to FEF, once purified to homogeneity or near homogeneity, being mixed with other molecules to facilitate storage, stability or administration to an animal to, for example, prepare antibodies.
  • a preparation of FEF may be used in which other molecules are present but which do not affect the activity of FEF. Accordingly, the present invention extends to preparations having substantial FEF activity whether or not such preparations comprise homogeneous or near homogeneous FEF.
  • FEF is meant a proteinaceous molecule with a molecular weight in the range of 20-70 kDa having the ability to promote or induce or otherwise facilitate the elongation of fibroblasts (see Example 3). It should be noted, however, that the molecular weight determined may vary depending on the procedure employed and on the multi eric nature of the factor. Accordingly, FEF molecules having molecular weight outside the range given above but which still have the ability to promote or induce or otherwise facilitate the elongation of fibroblasts are within the scope of the present invention. In association with this activity, is the ability of FEF to enhance, promote or otherwise facilitate the contraction of a collagen matrix by the elongation of fibroblasts.
  • FEF activity is readily determined by measuring fibroblast stretching, collagen lattice contraction, or DNA synthesis as herein described in the Examples. However, other forms of measuring FEF activity may be used without departing from the scope of the instant invention.
  • the present invention extends to naturally occurring, biologically pure, FEF or to derivatives and homologues thereof.
  • Derivatives and homologues of FEF include, but are not limited to, single or multiple amino acid substitutions, deletions or additions and where glycosylation of the FEF molecule is concerned, single or multiple substitutions, deletions or additions to the carbohydrate moiety.
  • Derivatives and/or homologues of FEF also extend to FEF in chemical and/or biological association, combination or fusion with another molecule whether such association, combination or fusion is naturally occurring or artificial, i.e. synthetic.
  • the present invention is also directed to recombinant or synthetic FEF and to any derivatives and/or homologues thereof.
  • FEF contemplated herein includes the naturally occurring, recombinant or synthetic molecule, or any derivatives or homologues thereof, in a homogenous or near homogenous form.
  • the present invention is described using FEF isolated from human colon cancer cell line SW480. This is done, however, with the understanding that other cell lines could also be used and the present invention is in no way limited to a FEF from a particular cell line.
  • Examples of other cell lines from which FEFs may be derivable include but are not limited to fibrosarcoma cell lines, and other epithelial cell lines derived from breast cancer, stomach cancer and melanomas.
  • the FEF contemplated herein will be useful in generating antibodies thereto, either monoclonal or polycl ⁇ nal, for use in screening for FEF in a biological sample.
  • biological sample is meant to include, but is not limited to, a biopsy specimen, blood sample, tissue fluid or extract or tissue culture or culture supernatant.
  • the i ⁇ ununological detection of FEF may provide a rapid and convenient screening technique for early malignant transformations of the colon.
  • Such an assay may be conducted by contacting a biological sample, or a purified or partially purified and/or concentrated form thereof, with a FEF-binding effective amount of an antibody for a time and under conditions sufficient for said antibody to bind to the FEF molecule and then detecting said FEF-antibody complex.
  • the antibody may be first labelled with a reporter molecule such as a radioactive isotope, chemiluminescent, bioluminescent or fluorescent molecule, enzyme or other signal producing or potentially producing molecule.
  • a reporter molecule such as a radioactive isotope, chemiluminescent, bioluminescent or fluorescent molecule, enzyme or other signal producing or potentially producing molecule.
  • the FEF- antibody complex is detected by use of a second antibody, labelled as described above, and which is capable of binding to, for example, the first antibody of the complex.
  • a reporter molecule such as a radioactive isotope, chemiluminescent, bioluminescent or fluorescent molecule, enzyme or other signal producing or potentially producing molecule.
  • the FEF- antibody complex is detected by use of a second antibody, labelled as described above, and which is capable of binding to, for example, the first antibody of the complex.
  • Antibodies to FEF or its derivatives and/or homologues may also be useful in purifying or substantially purifying same from biological samples.
  • the present invention also extends to antagonists of FEF capable of inhibiting, reducing or otherwise interfering with FEF-induced or enhanced elongation of fibroblasts and may, therefore, be useful in inhibiting tumour cell proliferation and ameliorating large bowel obstruction.
  • the antagonist may, for example, be an antibody to FEF. Alternatively, it may be a structural homologue of FEF and compete with FEF binding to target or effector sites.
  • this aspect of the present invention contemplates a method for inhibiting tumour cell proliferation and/or ameliorating large bowel obstruction in a human comprising administering to said human an antagonist to FEF for a time and under conditions sufficient to inhibit, reduce or otherwise interfere with FEF-induced or enhanced elongation of fibroblasts.
  • the present invention is also directed to a pharmaceutical composition
  • a pharmaceutical composition comprising FEF or an antagonist thereto and one or more pharmaceutically acceptable carriers and/or diluents.
  • the composition contemplated herein may be useful in stimulating antibodies specific to naturally occurring FEF, to compete against binding of naturally occurring FEF to receptor sites or to inhibit the activity of FEF. All such compositions will be useful in inhibiting, reducing or otherwise interfering with FEF-induced or enhanced elongation of fibroblasts.
  • the composition may also comprise other active molecules in synergistic combination with an FEF antagonist.
  • Figure 1 is a graphical representation depicting (a) Fibroblast-mediated contraction of collagen discs expressed as residual [ 3 H] 2 0 within gels in the absence and presence of SW480 tumour cells co- cultured within the gel (fibroblast and tumour cell density of 40 x 10 3 and 150 x 10 3 cells per gel, respectively).
  • Figure 2 is a pictorial representation showing the fibroblast-mediated contracton occurring in floating collagen rings (magnification x 2.5).
  • a and B Acellular control gels in the absence and presence, respectively, of the tumour cell monolayer (white arrow indicates indentation of gel due to transfer of rings at initiation of experiments);
  • C and D Fibroblast-containing gels (50 x 10 3 cells) in the absence and presence, respectively, of the tumour cell monolayer (black arrow).
  • Figure 3 is a graphical representation of Fibroblast- mediated contraction of floating collagen rings expressed as residual [ 3 H] 2 0 with gels (a) and assessed by indirect estimation of the area of the internal ring for the same experiment (b) in the absence ( - ⁇ - ) and presence ( - ⁇ - ) of the tumour cell monolayer.
  • Figure 4 is a graphical representation of the DNA synthesis by CCD-18 fibroblasts cultured in collagen rings in the absence ( - ⁇ - ) and presence ( - ⁇ - ) of the tumour cell monolayer for the same experiment as shown in Figure 3.
  • Figure 5 is a photomicrographic representation of colon fibroblasts cultured on collagen in the absence (left) and presence (right) of colon cancer cells embedded within the gel. Cells stained with coomassie blue after 72 hours in culture.
  • Figure 6 is a photomicrographic representation of colon fibroblasts cultured in collagen for 48 hours in the absence (left) and presence (right) of tumour-conditioned medium.
  • Figure 7 is a graphical representation showing dose- response relationship between increasing concentration of FEF and the percentage of cells stretched beyond the mean length of unstimulated cells plus 3 standard deviations.
  • Figure 8 is a graphical representation showing dose- response relationships between increasing concentration of FEF activity in tumour-conditioned medium and the number of cells stretched >110 ⁇ m (o——o) and [ 3 H]- thymidine uptake.
  • Figure 9 is a graphical representation depicting the profile of tumour-conditioned medium passed over a
  • FIG. 10 is a graphical representation showing estimation of FEF activity by comparing [ 3 H]-thymidine uptake for tumour-conditioned medium >50kDa versus tumour-conditioned medium 30 - 50 kDa.
  • Figure 11 is a graphical representation depicting the profile of FEF activity for bound and unbound fractions derived from the passage of tumour-conditioned medium across a heparin sepharose CL-6B column. Inset above shows [ 3 H]-thymidine uptake for bound and unbound fractions.
  • Figure 12 is a graphical representation showing an elution profile following purification of FEF by HPLC.
  • Figure 13 is a graphical representation showing an elution profile following purification of FEF by FPLC.
  • Figure 14 is a graphical representation showing an elution profile following purification of FEF by HPLC after FPLC.
  • Figure 15 is a photographic representation of SDS-PAGE analysis of fractions from an HPLC column.
  • Human colon cancer cells and fibroblasts obtained from normal colon were adapted to monolayer growth in tissue culture flasks using standard culture medium consisting of Dulbecco's Modified Eagle's Medium (DMEM, flow Laboratories, Virginia, USA) supplemented with glutamine, antibiotics (penicillin and streptomycin) and 10% (v/v) foetal calf serum.
  • DMEM Dulbecco's Modified Eagle's Medium
  • glutamine penicillin and streptomycin
  • 10% (v/v) foetal calf serum v/v) foetal calf serum.
  • Chemically-defined serum-free medium was used in all matrix experiments and consisted of DMEM supplemented with glutamine, hydrocortisone, non-essential amino acids, mercaptoethanol, insulin, transferrin and selenium.
  • both cell lines growing in standard culture medium were harvested by exposure to 0.05% (w/v) trypsin/0.02% (w/v) ethylenediaminotetracetic acid (Flow Laboratories) and washed once in standard medium. The cell preparations were then washed three times in serum- free medium before resuspension in serum-free medium and estimation of cell viability with 0.4% (w/v) trypan blue solution.
  • Collagen Gels Native type 1 collagen was prepared by acetic acid extraction from rat tail tendons according to the method reported by van Bockxmeer and Martin (1982), and protein concentration estimated (Bio-RAD protein microassay, Bio- RAD Laboratories, California, USA), according to a modification of the Lowry method (Lowry et al, 1951). Collagen gels were prepared in identical manner to that described previously except for the use of larger culture wells instead of microtitration plates (Agrez, 1989b). In brief, equal volumes of collagen and x 2 concentrate serum-free medium containing [ 3 H] 2 0 (Amersham,
  • Colon fibroblasts and colon cancer cells were seeded into each of triplicate wells at a cell density of 40 x 10 3 and 150 x 10 3 viable cells, respectively, and cultures were incubated at 37°C in 5% C0 2 and 100% relative humidity.
  • the retrieved gels were dissolved in 10 ml of scintillant overnight (Beckman Instruments, California, USA) and liquid scintillation counts provided estimates of gel volumes which were compared with uncontracted cell-free gels.
  • the extent of matrix contraction in test cultures was expressed as a percentage relative to the uncontracted 100% control cell-free gels.
  • the degree of collagen lattice contraction induced by tumour cells and fibroblasts when co-cultured together within the matrix was compared with that observed for either cell type when cultured alone in the gel. 2. Fibroblast-Mediated Contraction of Collagen Rings.
  • Colon fibroblasts embedded in collagen rings were seeded at a cell density in the range of 20-65 x 10 3 cells per well and triplicate collagen rings were prepared at each fibroblast cell density tested.
  • Preliminary studies confirmed that a linear relationship existed between collagen ring contraction during the first 24 hours as estimated by residual tritium in the gel and increasing fibroblast cell density in the range of 20 - 65 x 10 3 cells per gel. These cell densities comprised only a tiny proportion of the total gel volume.
  • 50 x 10 3 fibroblasts constituted ⁇ 0.03% of the uncontracted microtitre gel volume.
  • total cell volumes were so small relative to the uncontracted gel volume that transfer of tritiated water in and out of the cell compartment was considered unlikely to impact significantly on estimates of residual tritium within contracted matrices.
  • the degree of gel contraction was then measured in two ways. Collagen rings cultured in the presence of a tumour cell monolayer were photographed 22-24 hours after "rimming". Gels were then retrieved with a hooked needle and gel size estimated by liquid scintillation counting as described for collagen discs. From photographic prints of each well (128 mm x 78 mm) the circumference of the central hole or "lumen” or each collagen ring was traced onto transparent plastic sheets. The tracings were cut out as discs and the weight of each disc provided an indirect measure of the degree of constriction of the "lumen”. 3. Fibroblast Proliferation Within Collagen Rings.
  • collagen discs were prepared within 35 mm diameter tissue culture wells (Linbro) in identical manner to that described for contraction experiments except for omission of [ 3 H] 2 0 and the "rimming" step at the initiation of experiments.
  • Colon fibroblasts at a cell density in the range of 15 - 45 x 10 3 cells per well, were cultured in and on collagen gels (1 ml gel per well) either alone or co-cultured with SW480 tumour cells embedded within the collagen at a cell density in the range of 250 - 400 x 10 3 tumour cells per gel. All gels were overlain with 2 mis of serum-free medium.
  • tumour-conditioned medium was obtained from monolayer cultures of SW480 cells grown in 150 cm 2 tissue culture flasks (Corning, New York, USA). The TCM, collected from flask cultures 4 days after seeding 5 x 10 ⁇ viable cells, was centrifuged at 25,000 G for one hour to remove all cell debris, adjusted to pH 7.2-7.4, and sterilised by filtration through a 0.2 urn filter before use (Minisart, Sartorius Instruments, Surrey, England).
  • fibroblast morphology was examined at low and high power magnification using a Leitz Labovert inverted microscope. Fibroblast cultures were photographed either unstained or after fixation and staining of gels with coomassie blue (0.1% (w/v) solution in 10% (v/v) acetic acid and 40%(v/v) methanol). Photomicrography of cultures was performed using a 35 mm camera attachment and technical pan film (Kodak) at 100 ASA setting.
  • the aim of this study was to determine if interactions between when fibroblasts, the extracellular matrix and colon cancer cells could be responsible for malignant large bowel obstruction and to determine whether a FEF was involved.
  • EXAMPLE 3 Establishment of the bioassays to detect the presence of FEF in tumou -conditioned medium.
  • tumour-conditioned medium Large volumes of tumour-conditioned medium were obtained from serum-free roller bottle cultures of SW480 colon cancer cells. After ultracentrifugation of the tumour- conditioned medium as described in Example 1 the medium was concentrated in an Amican ultrafiltration cell by passage over a YM30 membrane. The concentrated medium ( - x 1000 concentrate) was allowed to stand at 4°C for 72-96 hours to facilitate sedimentation of large molecular aggregates. Following cryo-sedimentation, bioactivity of the concentrated supernatant sample was tested for FEF activity at progressive dilutions in two culture systems. (i) CCD-18 cells seeded on collagen type III:
  • the concentrated tumour-conditioned supernatant was passed across a Sephadex G200 molecular sizing column and peak FEF activity identified between the molecular standards 66kDa and 44kDa as shown in Figure 9.
  • the bioactive fractions were pooled, reconcentrated over a XM50 ultrafiltration membrane and subsequent [ 3 H]- thymidine uptake studies confirmed that the molecular weight of FEF was >50kDa but less than 66kDa as shown in Figure 10.
  • other molecular weight determination procedures indicate at least a subunit size of 20-30 kDa.
  • the molecular weight of FEF is taken as from about 20 to about 70 kDa but may also be from 30 to 70 or 50 to 70 kDa.
  • the concentrated factor was further purified by means of affinity chromatography using heparin-sepharose CL-6B. FEF was eluted at approximately 0.25M NaCl and bioactivity confirmed by estimating [ 3 H]-thymidine uptake as shown in Figure 11.
  • FEF induced stretching of colon fibroblasts was demonstrated on collagen types I and III matrices but not on wells coated with either collagen IV, laminin or fibronectin. FEF activity was not destroyed by either lyophilisation or boiling at 100°C for 5 minutes, but was destroyed by exposure of FEF to trypsin (lOO ⁇ g/ml) and dithiothreitol (lOm ). These data confirm that FEF is a polypeptide and that its molecular configuration is stabilised by disulphide bonds.
  • FEF fibroblast elongation factor
  • the elution profile is shown in Figure 12 and fractions were collected as 1 ml aliquots at minute intervals. Individual fractions were lyophilised, reconstituted with H 2 0, and filter-sterilised using a 0.2 ⁇ m Millipore filter.
  • Bioactivity within each fraction was determined by means of visual assessment of fibroblast stretching.
  • CCD-18 colon fibroblasts were seeded on lOO ⁇ l Collagen Type I gels within microtitre wells (2 x 10 3 cells per well) in the presence of 35 ⁇ l of each fraction and bioactive fractions identified by their ability to induce obvious stretching of colon fibroblasts after 48 hours in culture.
  • the cells were stained with Coomassie blue solution (0.1%) as previously described and the culture photographed.
  • the degree of cell stretch was scored in a semi-quantitative manner (0-4+) as shown in Table 1. Bioactivity was identified in fractions 28 - 36 inclusive and these samples pooled, lyophilised, and reconstituted in 0.5ml H 2 0.
  • the concentrated sample was then subjected to FPLC using a strong anion-exchange Mono Q column.
  • the column was equilibrated with 20mM TRIS buffer pH 7.4 and developed with a dual pump linear gradient of 0 - 1 M sodium chloride over 40 minutes.
  • the elution profile is shown in Figure 13 and fractions were collected as 1ml aliquots at minute intervals, filter-sterilised and tested for bioactivity by measureing fibrolast stretching on collagen semi-quantitatively as previously described and shown in Table II.
  • Eluate fractions numbered 26 - 33 obtained from FPLC were pooled, lyophilised, and reconstituted in 0.5ml of H 2 0.
  • the concentrated sample was further purified by means of HPLC in identical manner to that described above and the lution profile is shown in Figure 14(a).
  • Each eluted 1ml fraction was tested for bioactivity in the fibroblast- stretch bioassay and activity identified in fraction 29 corresponding to the twin protein peaks seen on the HPLC profile ( Figure 14(a), (b)).
  • Bioactivity in FPLC fractions obtained between 6 - 40 minutes and tested on colon fibroblasts cultured on collagen type I gels for 48 hours.

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Abstract

On a mis au point un facteur biologiquement pur désigné 'FEF' et comportant une protéine capable de promouvoir ou de provoquer l'élongation des fibroblastes. On peut dériver ce facteur des cellules cancéreuses et il présente un poids moléculaire apparent compris entre environ 20 kDa et environ 70 kDa. On a prévu un procédé d'inhibition de la prolifération des cellules tumorales et/ou d'amélioration de l'occlusion du gros intestin par une interférence dans l'élongation provoquée ou facilitée par FEF des fibroblastes.
PCT/AU1991/000337 1990-08-02 1991-07-31 Facteur d'elongation de fibroblastes derive des cellules cancereuses du colon humain WO1992002556A1 (fr)

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AUPK1526 1990-08-02
AUPK152690 1990-08-02

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU6660886A (en) * 1985-12-20 1987-06-25 Bristol-Myers Squibb Company Novel cell growth regulatory factor
AU6605690A (en) * 1989-10-27 1991-05-31 Du Pont Merck Pharmaceutical Company, The Monoclonal antibodies to basic fibroblast growth factor that inhibit its biological activity

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU6660886A (en) * 1985-12-20 1987-06-25 Bristol-Myers Squibb Company Novel cell growth regulatory factor
AU6605690A (en) * 1989-10-27 1991-05-31 Du Pont Merck Pharmaceutical Company, The Monoclonal antibodies to basic fibroblast growth factor that inhibit its biological activity

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