WO1995030439A2 - Prevention de la metastase des tumeurs - Google Patents

Prevention de la metastase des tumeurs Download PDF

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Publication number
WO1995030439A2
WO1995030439A2 PCT/US1995/005819 US9505819W WO9530439A2 WO 1995030439 A2 WO1995030439 A2 WO 1995030439A2 US 9505819 W US9505819 W US 9505819W WO 9530439 A2 WO9530439 A2 WO 9530439A2
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Prior art keywords
antibody
cells
fragment
agent
invasion
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PCT/US1995/005819
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WO1995030439A3 (fr
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David Naor
Muayad Zahalka
Elimelech Okon
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Yissum Research Development Company Of The Hebrew University Of Jerusalem
Hadasit, Medical Research Services & Development Company Ltd.
Mcinnis, Patricia, G.
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Application filed by Yissum Research Development Company Of The Hebrew University Of Jerusalem, Hadasit, Medical Research Services & Development Company Ltd., Mcinnis, Patricia, G. filed Critical Yissum Research Development Company Of The Hebrew University Of Jerusalem
Priority to AU24796/95A priority Critical patent/AU2479695A/en
Publication of WO1995030439A2 publication Critical patent/WO1995030439A2/fr
Publication of WO1995030439A3 publication Critical patent/WO1995030439A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2842—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta1-subunit-containing molecules, e.g. CD29, CD49
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2884—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD44
    • 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 in the field of tumor metastasis and concerns compositions for preventing invasion of metastatic cells into specific organs.
  • CD44 Another integral membrane glycoprotein CD44, known to be a homing receptor of lymphocytes, was found to be involved in the metastatic potential of various tumor cell lines (6) .
  • CD44 Several variants of CD44 (vCD44) having additional extracellular domains have been detected in a variety of tissues and tumor cells.
  • a CD44 splice variant originally discovered on metastatic cells of a rat pancreatic adenocarcinoma (BSp73ASML), has been shown by transfection assays to confer metastatic behavior to non metastatic tumor cells whereas the standard type CD44 (sCD44) did not (7,S) .
  • sCD44 standard type CD44
  • CD44 has been proposed to be the principle cell surface receptor for hyaluronic acid (HA) and CD44 molecular species were found to be bound to HA (although sometimes only after activation with phorbol ester) (lt l >.
  • Primary tumor a tumor originating in an organ or tissue as a result of a cancerous transformation process of cells in this organ or tissue;
  • Secondary tumor a tumor which arose as a result of dissemina ⁇ tion of malignant cells from the primary tumor, their migration and infiltration into another organ, the term generally also known as
  • Primary organ and “Secondary organ” - the organ in which the primary tumor developed and the organ in which the secondary tumor developed, respectively;
  • Inner - the combined process of migration of malignant cells from a primary tumor and their infiltration into a secondary organ to develop the secondary tumor;
  • an anti-CD 18 antibody is capable of blocking invasion of tumor cells into the spleen. It was also found in accordance with the invention that anti-CD 18 antibody
  • CD44 antibodies particularly such directed against the 80-90 kilo Dalton (kDa) isoform of CD44 ("standard CD44 - sCD44") blocked the invasion of tumor cells into the lymph node.
  • anti-VLA-4 mAb in combination with an anti-VACM-1 mAb blocked invasion of tumor cells into the liver, while each one of them by themselves showed very little efficacy in such blocking, if at all.
  • Another finding of the present invention concerns blocking of invasion of CD44 expressing cells into lymph nodes by prior treatment with hyaluronidase.
  • the findings in accordance with the present invention open the way for new therapies for the treatment of cancer in general and for the treatment of cancer patients to avoid metastasis in particular.
  • the above antibodies with the anti-invasion activity can be used as is for blocking tumor invasion into secondary organs.
  • fragments thereof such as, for example, F(ab) fragments, F(ab') 2 fragments, single chain antibodies, as well as various proteins or peptides derived therefrom which have the same binding activity to an antigen as these antibodies may be used for the anti-invasion therapy of the invention.
  • Such a derivative may for example be a molecule derived from an antibody or fragment which comprise an antigen binding site having the same binding specificity as said antibody or fragment and in which one or more amino acid has been replaced, added or deleted.
  • the finding of the invention that hyaluronidase has the ability to block invasion of CD44 expressing cells into lymph nodes may open the way of using hyaluronidase or enzymes having a similar activity in cancer therapy for blocking the occurrence of metastasis in lymph nodes.
  • the present invention thus provides an anti- invasion agent for blocking invasion of tumor cells into the spleen, comprising an effective amount of an active agent being one or more of the group consisting of: (a) anti-CD 18 antibody; (b) a fragment of the antibody of (a) comprising an antigen binding site thereof; and (c) a modified molecule derived from (a) and (b) comprising an antigen binding site having the same binding specificity as said antibody or fragment.
  • an anti-invasion agent for blocking invasion of tumor cells into lymph nodes, comprising an effective amount of an active agent selected from the group consisting of: (a) anti-sCD44 antibody; (b) a fragment of the antibody of (a) comprising an antigen binding site thereof; and (c) a modified molecule derived from (a) and (b) comprising an antigen binding site having the same binding specificity as said antibody or fragment.
  • an anti-invasion agent for blocking invasion of tumor cells into lymph nodes comprising an effective amount of an active agent being one or more of the group consisting of: (a) hyaluronidase;
  • the present invention provides an anti-invasion agent for blocking invasion of tumor cells into the liver, comprising a synergistic combination of a first and a second agent; said first agent being one or more of the group consisting of: (a) anti-VLA-4 antibody;
  • the amount of active agent to be used in each of the above embodiments should be such so as to achieve the desired clinical effect. - 8 -
  • Said active agent can be used in the therapy of various types of cancers so as to avoid the development of metastasis in secondary organs.
  • Various tumors have a tendency of developing a secondary tumor in a well defined organ or tissue and in such cases it may at times be advisable to administer the active agent directly into the suspected organ or tissue or in proximity thereof.
  • the active agent of the invention may be administered to an individual in need during treatment of his primary tumor, e.g. in combination with the administration of chemo-therapeutic agents intended to kill the primary tumor cells.
  • the administration of the active agent of the invention will be typically performed periodically, at least throughout the period of treatment of the primary tumor.
  • the present invention further provides use of any of the above agents in the preparation of pharmaceutical compositions for use in anti- cancer therapy.
  • the present invention still further provides a method of cancer treatment for the purpose of avoiding invasion of tumor cells from a primary organ to a secondary organ, comprising use of any of the above anti- invasion agents.
  • Fig. 1 shows FACS analysis of LB T-cell lymphoma cells.
  • the type of stain cells and the specificity of the first layer mAb used for staining are indicated in each panel.
  • the left histogram in each panel shows the non specific binding of the indicator secondary FITC-labeled antibody to various cell types.
  • the right histogram in each panel shows the specific binding of the first monoclonal antibody to the same cells.
  • Fig. 2 shows an electrophoretogram of [ 3S S] methionine-labeled LB lymphoma cells which were immunoprecipitated either with a control rat IgG mAb (lane 1 in the figure) or with an anti-CD 18 mAb (lane 2 in the figure) and the immunoprecipitate was subjected to SDS-PAGE under reducing conditions.
  • Fig. 3 shows histology of spleens (a-c in the figure) and lymph nodes (d-f in the figure) of BALB/c mice from the following groups: (i) non treated mice (a and d);
  • Fig. 4 shows the incorporation of [ 3 H]-Thymidine in cell suspensions derived from spleens and lymph nodes of BALB/c mice receiving the various treatments indicated in the figure. The results are expressed as cpm (mean ⁇ SD) and each experiment is representative of at least two experi ⁇ ments.
  • FIG. 5A shows the FACS analysis of non treated (Al in the figure) or PMA treated (A2 in the figure) LB lymphoma cells stained with a first anti-CD44 antibody and a secondary FIT C-conjugated antibody.
  • Fig. 5B shows a FACS analysis of direct double staining of LB cells with both an FITC-conjugated anti-CD 18 mAb and phycoerythrin-conjugated anti-CD44 mAb.
  • Fig. 6 shows an electrophoretogram of an LB cell extract which was immunoprecipitated using either the rat IgG antibody (lane 1 in the figure) or the anti-CD44 antibody (lane 2 in the figure) wherein the immunoprecip- itates were subjected to SDS-PAGE under reducing conditions.
  • Fig. 7 shows the histology of the spleen and lymph node of BALB/c mice of the following groups: (i) lymph nodes (A in the figure) and spleen (E in the figure) of non treated BALB/c mice; (ii) lymph node (B) and spleen (F) of BALB/c mice inoculated s.c. with
  • Fig. 8 shows the incorporation of 3 H-Thymidine expressed as mean cpm ⁇ SD of cell suspensions prepared from lymph nodes and spleens of BALB/c mice receiving various treatments (as indicated in the graph).
  • Fig. 9 is a photograph of a cell suspension of spleen cells (A in the figure) and lymph node cells (B in the figure) which were , incubated with LB cells at a ratio of 8:1, respectively. The photograph was taken under an inverted microscope wherein the original magnification was x400. Aggregates are seen in the spleen cell suspension (A). The arrows in Fig. A show the LB cells participating in the aggregate (which are discriminated from the spleen cells participating in the same aggregates by their size).
  • Fig. 10 shows the histology of the liver of BALB/c mice of the following groups: (i) BALB/c mice inoculated s.c. with H-10 lymphosarcoma cells (A in the figure). H-10 cells are seen infiltrated into the liver parenchima via the blood vessels (the dark area in all the pictures);
  • Fig. 11 shows the [ 3 H]-Thymidine incorporation expressed as mean cpm ⁇ SD of cell-suspensions from livers taken from BALB/c mice receiving various treatments as indicated in the figure.
  • Fig. 13 shows the Thymidine incorporation expressed in mean cpm ⁇
  • Fig. 14 shows the histology of spleens and lymph nodes of BALB/c mice from the following groups:
  • mice inoculated s.c. with LB cells only A in the figure
  • mice inoculated s.c. with LB cells and injected s.c. with hyaluronidase near the lymph node B in the figure.
  • LFA-1 adhesion molecule on T-cell lymphoma LB cells was measured by FACS analysis. LB cells were removed from the ascitic fluid of 6 to 8 week old female mice. Lymph node cell suspensions were prepared by pressing the lymph nodes of
  • mice BALB/c mice through a stainless steel mesh.
  • the mAbs (0.1 ml) were added at the appropriate dilution to 3xl0 ⁇ prewashed and sedimented cells. The cells were kept for 30 min. on ice and then washed in RPMI 1640 containing 0.1% BSA. Then, 0.1 ml of species-specific anti-IgG (H and L chain) antibodies conjugated to FITC, diluted 1/20, was added to the sedimented cells. The cells were again kept for 30 mins. on ice, washed in RPMI 1640 containing 0.1% BSA, and resuspended in 0.5 ml of the same medium.
  • the cell suspensions were analyzed in a cell sorter (FACS 440; Becton-Dickenson, Mountain View, CA) tuned to 488 nm. Each cell suspension sample was also analyzed for non-specific binding of FITC-antibody. Specific and non-specific binding of fluorescent antibody was measured at wavelengths above 520 nm. Cell debris was gated out. As seen in Fig. 1, the LB cells express both the ⁇ -chain (detected by using the CDlla antibody) and /. -chain (detected by using the CD 18 antibody) of the LFA-1 molecule.
  • FACS 440 Becton-Dickenson, Mountain View, CA
  • LFA-1 molecules on LB cells were confirmed by the [ 32 S]-methionine immunoprecipitation assay.
  • LB lymphoma cells (10 7 /ml) were metabolically labeled with 0.5 mCi/ml [ 35 S] methionine (specific activity, 1000 Ci/mmol; Amersham, Buckinghamshire. UK) for 2 hours at 37°C.
  • Cells were lysed on ice at 5xl0 7 /ml, in lysis buffer containing 0.5% Triton X-100, 300 mM NaCl, 50 nM Tris-HCl, pH 7.6, 10 mM iodoacetaminde, 1 mM PMSF, 10 ⁇ g/ml Ieupeptin, and 10 ⁇ g/ml aprotinin.
  • Anti-CD 18 n AB or rat IgG (control) was bound to protein A-Sepharose 4B (Sigma, St. Louis, MO) at a ratio of 3 mg of antibody to 1 ml of packed beads, by using a polyvalent goat antiserum against rat Ig.
  • Immunoabsorbents were incubated with cell lysates for 2 hours in the cold and washed with immunoprecipitation buffer, which was identical to the lysis buffer except that 0.15% Triton was used instead of 0.5%. After thorough washing the bound material was eluted from the beads with 50 mM Tris-HCl, pH 6.8, 1% SDS, 10% glycerol. The sample was analyzed by reducing SDS-PAGE (7% polyacrylamide; exposure time, 8 days). As seen in Fig. 2, the anti-CD 18 mAb, but not the control reagent (rat IgG), precipitated the a- and /3-chains of the LB cell LFA-1 integrin molecule in LB cells.
  • the control reagent rat IgG
  • mice were injected subcutaneously (s.c.) in the left flank with 3xl0 6 LB T cell lymphoma cells. Two hours after the s.c. inoculation of LB cells, and then every other day until the end of the experiment, some of the mice were injected intravenously (i.v.) with the 50% ammonium sulfate fraction (0.5 mg of protein) of anti-CD18 mAb (M18/2) or anti- CDlla mAb (M7/14) or with the same amount of one of the following control mAbs: anti-CD lib (Ml/70), anti-CD3 (145-2C11) or anti-D d (35- 5-8C).
  • mice On day 12 the mice were killed and their spleens and axillary and brachial lymph nodes were removed. Half of the spleens and lymph nodes derived from each treatment group were examined histologically (see Fig. 3), whereas cell suspensions from the other half were simultaneously tested using the proliferation assay (see Fig. 4).
  • Fig. 3 shows the histological examination of spleen and lymph nodes of the treated mice. Portions of the spleens and axillary and brachial lymph nodes were fixed in 10% buffered formalin and imbedded in paraffin and 5-/. m section were stained with hematoxylin and eosine. As seen, in spleens of mice injected with LB cells (Fig. 3b), the normal structure of the spleen was completely effaced: the compartmentalized red and white pulps of the normal spleen were replaced by the infiltrating nuclear vesiculated LB cells, with many of them presenting mitotic figures. In contrast, in mice inoculated with LB cells and injected i.v.
  • lymph nodes' architecture of mice inoculated with LB cells was also completely effaced showing enlargement and heavy infiltration by the lymphoma cells (Fig. 3e).
  • i.v. injection of anti- CD18 mAb did not block the invasion of nuclear vesiculated LB cells into the lymph nodes which were totally effaced by the invading cells (Fig. 4f).
  • local s.c. injection of anti-CDl8 mAb near the axillary or brachial lymph nodes of the mice injected with LB cells at a different and remote site did however, partially block the proliferation of infiltrating LB cells into these organs (not shown).
  • mice inoculated with LB cells were confirmed by a corresponding [ 3 H]-Thymidine incorporation assay.
  • Spleens and axillary and brachial lymph nodes were removed from the LB cell-inoculated mice and cell suspensions were prepared in RPMI 1640. The cells were incubated (5% C0 2 atmosphere, 37°C) for 15 hours in U-shaped 96-well microplates (Nunc, Roskilde, Denmark) at 2xl0 5 cells/well).
  • the cells were pulsed with 1 of [ 3 H]Thymidine (specific activity, 5 Ci/mmol; Nuclear Research Center, Negev, Israel) and were harvested with a Titertek cell harvester (Flow Laboratories, Irvine, Scotland, UK) 20 hours later.
  • the incorporation o ' t [ '3 H]Thymidine was determined with a beta counter (Betamatic; Kontron, Montigny le Bertonneux, France). Results are expressed as cpm (mean ⁇ SD). Each experiment is representa- tive of at least two experiments, showing similar results. Control experi ⁇ ments demonstrated that in these mice, the only cells proliferating in the spleen and lymph nodes are the inoculated LB cells and not local cells (results not shown).
  • a strong cell proliferation (indicated by 3 H-Thymidine uptake) was revealed in spleen and lymph node cell suspensions of the mice injected with LB cells, whereas the cell proliferation of the corresponding lymph node organs derived from normal mice was very low.
  • injection of anti-CDIS mAb blocked the proliferation of LB cells invading the spleen, but not those invading the lymph nodes.
  • the proliferation in the spleens of the mice injected with LB cells and also with an anti-CDlla mAb or a different control mAb (anti-CDUb, anti-CD3, anti-D d and anti-ICAM-1) was not blocked.
  • anti-CD 18 mAb purified with protein G or its F(ab') 2 fragments also blocked the proliferation of s.c. inoculated LB cells in the spleens of these mice, indicating that the elimination of LB cells by the anti-CD18 mAb is not by C-dependent lysis or antibody-dependent cellular clearance.
  • LB cells were removed from the ascitic fluid of 6 to 8 week old female BALB/c mice. Some of these cells (10 ⁇ /ml) were incubated (5% C0 2 atmosphere, 37°C) with PMA (20 ng/ml) for 16 hours. Ammonium sulfate precipitated anti-CD 18 mAb (Ml 8/2, rat IgG2a) was added (140 ⁇ g) to lxlO 6 prewashed PMA activated or nonactivated LB cells. The bound antibody was detected with anti-rat IgG antibody [F(ab') 2 , Sigma, St. Louis, MO] conjugated to fluorescein isothiocyanate (FITC), following analysis by FACS 440 (Bacton-Dickenson, Mountain View, CA) tuned to 488 nm.
  • FACS 440 Bos-Dickenson, Mountain View, CA
  • LB cells expfess the CD44 (PgP-1) adhesion molecule on their cell surface.
  • Double staining of the LB cells with anti- CD18 and an t ⁇ -CD44 antibodies was performed by adding a mixture of FITC-conjugated rat anti-mouse CD 18 mAb (C71/16, IgG2a, PherMingen, San Diego, CA) and phycoerythrin-conjugated rat anti-mouse CD44 mAb (IM7, IgG2b, FarMingen) to the cell suspension.
  • the cells were kept 30 mins. on ice, washed and analyzed by FACS, as indicated above.
  • the vast majority of the LB cells simultaneously expressed both the CD18 and the CD44 adhesion molecules on their cell surface.
  • mice Female BALB/c were inoculated s.c. with 3xlO ⁇ LB cells and two hours later some of the mice were injected s.c. near the draining lymph nodes with a 50% ammonium sulfate fraction of an anti-CD44 mAb. The antibody injection was repeated every other day until termination of the experiment on day 12. The spleens and lymph nodes of the treated mice were removed and subjected to histological examination and cell suspensions of these organs were prepared and used for proliferation assays, as described in Example 3 above. Other mice were also inoculated s.c. with LB cells and then received i.v. injections of anti-CD18 mAb as described in Example 3 above. As seen in Fig.
  • LB cells ability of LB cells to form in vitro aggregates with cells derived from normal spleens or lymph nodes was examined by incubation of the LB cells with spleen or lymph node cells in a 24-well microplate
  • the percentage of cell aggregation was calculated by using the following formula: 100 - cell concentration (cells/ml) after filtration/cell concentration before filtration.
  • the LB cells formed aggregates with spleen cells (A in the figure) at various spleen cell to LB cell ratios but did not form aggregates with lymph node cells (B in the figure) at any lymph node cell to LB cell ratio.
  • Addition of the anti-CD18 mAb to the spleen cell-LB cell suspension blocked the aggregate formation between LB cells and spleen cells by 82% (not shown).
  • anti-CDl8 mAb injected i.v. blocked ex vivo aggregates formed spontaneously in the spleen after s.c. inoculation, of LB cells whereas the other control antibodies did not.
  • mice were inoculated with RAW 117-H-10 lymphosar ⁇ coma cells s.c. and then divided into four groups: (i) mice receiving no further treatment (control); (ii) mice receiving a subsequent i.v. injections with anti-VACM-1 mAb (MW2.7); '
  • Mice from the various groups were killed on day 14, their livers were removed and analyzed histologically.
  • the H-10 cells infiltrated the liver parenchima via the blood vessels (the dark area in the pictures) of livers from the control mice (Fig. 10A) in the mice injected with anti-VACM-1 mAb (Fig. 10B) and in the livers of mice injected with the anti-VLA-4 mAb (Fig. IOC).
  • livers of H-10 cell i ⁇ noculated mice which were i.v. injected with a combination of the anti- VACM-1 and anti-VLA-4 mAbs was almost totally protected from invasion by the lymphoma (Fig. 10D).
  • Proliferation of the lymphosarcoma H-10 cells in the liver was also measured by 3 H-thymidine incorporation (as described in Example 3 above).
  • BALB/c mice were inoculated s.c. with either cells of the parent line RAW 117-P or with cells of the lymphosarcoma RAW 117-H-10.
  • the mice inoculated with the H-10 cells were divided into the same groups as described above and received either no further treatment, i.v.
  • LB cells were labeled with [ 3 H]-Thymidine and their in vitro binding ability to plastic coated hyaluronic acid (HA) was tested as follows: A flat bottom 96-well microplate (Nunc, Roskilde, Denmark) was coated with 0.5 mg/well HA (Sigma) or other glycosaminoglycans. After 16 hours incubation at room temperature, the microplate was washed three times with phosphate buffered saline. LB cells (10 6 /ml) were incubated (5% C0 2 , 37°C) with 5 Ci/ml [ 3 H]Thymidine (specific activity 5 Ci/mmole; Nuclear Research Center, Negev, Israel) in the presence of 20 ng ml PMA.
  • HA hyaluronic acid
  • radiolabeled LB cells were washed and resuspended in RPMI 1640 containing 10% fetal calf serum and 3x10 labeled cells were added to each HA coated well.
  • the HA coated microplate also contained tested mAbs (300 .g protein well), the enzyme hyaluronidase (100 // g/well) or other related enzymes at concentrations of equivalent specific activity.
  • the bound cells were washed after one hour of incubation (5% C0 2 , 37°C) and harvested with a Titertek cell harvester (Flor Laboratories, Irvine, Scotland).
  • Radiolabeled bound cells were counted with a beta counter (Betamatic, Kontron, Montigny le Bertonneux, France). Percent adhesion was calculated according to the following equation: bound cpm per well divided by total cpm added to each well x 100. Results of the binding assay showed that whereas non-activated LB cells did not bind to HA (results not shown), LB cells activated by PMA. (after incubation with 20 ng per/ml PMA) demonstrated extensive binding to HA (with a maximum level of about 40% binding at a concentration of 0.5 ml HA per well).
  • the binding of the activated LB cells to HA was also blocked by the enzyme hyaluronidase (at a concentration of 100 g/well) whereas the enzymes heparinase and chondroitinase AC, at equivalent specific activities did not block the binding of LB cells to HA (Fig. 12-4).
  • mice were inoculated s.c. with 3xl0 6 LB cells. Some of the mice were also locally s.c. injected with hyaluronidase (0.5 mg/mouse) near the lymph node or with other enzymes at doses adjusted to an equivalent specific activity. Enzyme injection was repeated every other day until termination of the experiment. The mice were killed on day 12, their lymph nodes and spleens were removed, cell suspensions were prepared and the level of their proliferation was measured by 3 H-thymidine incorporation assay, as described in Example 3 above. As seen in Fig.

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Abstract

On inhibe la métastase des tumeurs en utilisant des agents actifs qui bloquent l'infiltration des cellules tumorales dans des organes cibles secondaires. Ces agents comprennent un certain nombre d'anticorps et différents fragments et dérivés de ceux-ci, ainsi que la hyaluronidase, l'acide hyaluronique et leurs analogues.
PCT/US1995/005819 1994-05-09 1995-05-09 Prevention de la metastase des tumeurs WO1995030439A2 (fr)

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AU24796/95A AU2479695A (en) 1994-05-09 1995-05-09 Prevention of tumor metastasis

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US24006494A 1994-05-09 1994-05-09
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19732323A1 (de) * 1996-08-06 1998-06-04 Dunzendorfer Udo Priv Doz Dr M Medikamentenkombination zur erhöhten Wirkstoffkonzentration in Geweben
WO2004092361A1 (fr) * 2003-04-15 2004-10-28 Ista Pharmaceuticals, Inc. Procede d'isolation et de purification d'hyaluronidase ovine
WO2007028196A1 (fr) * 2005-09-07 2007-03-15 Alchemia Oncology Pty Limited Compositions thérapeutiques comprenant de l’hyaluronane et des anticorps therapeutiques, et procédes thérapeutiques
DE102007054877A1 (de) * 2007-11-15 2009-05-20 Dieter Dr. med. Wetzel Verwendung von Hyaluronidase als Arzneimittel zur Behandlung einer Lebererkrankung mit profibrogener Stoffwechsellage
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WO2022162164A1 (fr) 2021-01-29 2022-08-04 INSERM (Institut National de la Santé et de la Recherche Médicale) Procedes d'évaluation du risque de développement d'une leuco-entéphalopathie multifocale progressive chez les patients traités par des antagonistes de la vla-4

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Cited By (18)

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DE19732323C2 (de) * 1996-08-06 1999-08-19 Dunzendorfer Medikamentenkombination zur erhöhten Wirkstoffkonzentration in Geweben
DE19732323A1 (de) * 1996-08-06 1998-06-04 Dunzendorfer Udo Priv Doz Dr M Medikamentenkombination zur erhöhten Wirkstoffkonzentration in Geweben
US8038998B2 (en) 1997-09-18 2011-10-18 Ivan Bergstein Methods of cancer therapy targeted against a cancer stemline
US8846325B2 (en) 1997-09-18 2014-09-30 Ivan Bergstein Methods of cancer diagnosis and therapy targeted against a cancer stem line
US8715945B2 (en) 1997-09-18 2014-05-06 Ivan Bergstein Methods of cancer diagnosis and therapy targeted against a cancer stem line
US8741970B2 (en) 1999-01-13 2014-06-03 Alchemia Oncology Pty Limited Composition and method for the enhancement of the efficacy of drugs
US8287894B2 (en) 2000-07-14 2012-10-16 Alchemia Oncology Pty Limited Hyaluronan as a drug pre-sensitizer and chemo-sensitizer in the treatment of disease
US8388993B2 (en) 2000-07-14 2013-03-05 Alchemia Oncology Pty Limited Hyaluronan-chemotherapeutic agent formulations for the treatment of colon cancer
US9066919B2 (en) 2000-07-14 2015-06-30 Alchemia Oncology Pty Limited Hyaluronan as a chemo-sensitizer in the treatment of cancer
WO2004092361A1 (fr) * 2003-04-15 2004-10-28 Ista Pharmaceuticals, Inc. Procede d'isolation et de purification d'hyaluronidase ovine
US8937052B2 (en) 2005-07-27 2015-01-20 Alchemia Oncology Pty Limited Therapeutic protocols using hyaluronan
EA013877B1 (ru) * 2005-09-07 2010-08-30 Алкемиа Онколоджи Пти Лимитед Терапевтические композиции, содержащие гиалуроновую кислоту и терапевтические антитела, а также способы лечения
US8623354B2 (en) 2005-09-07 2014-01-07 Alchemia Oncology Pty Limited Therapeutic compositions comprising hyaluronan and therapeutic antibodies as well as methods of treatment
WO2007028196A1 (fr) * 2005-09-07 2007-03-15 Alchemia Oncology Pty Limited Compositions thérapeutiques comprenant de l’hyaluronane et des anticorps therapeutiques, et procédes thérapeutiques
DE102007054877A1 (de) * 2007-11-15 2009-05-20 Dieter Dr. med. Wetzel Verwendung von Hyaluronidase als Arzneimittel zur Behandlung einer Lebererkrankung mit profibrogener Stoffwechsellage
WO2011020874A1 (fr) 2009-08-20 2011-02-24 Inserm (Institut National De La Sante Et De La Recherche Medicale) Vla-4 en tant que biomarqueur pour le pronostic et le ciblage pour thérapie dans la dystrophie musculaire de duchenne
US20150258194A1 (en) * 2012-08-31 2015-09-17 Vib Vzw Modulating transendothelial migration and recruitment of granulocytes by modulating c-met pathway
WO2022162164A1 (fr) 2021-01-29 2022-08-04 INSERM (Institut National de la Santé et de la Recherche Médicale) Procedes d'évaluation du risque de développement d'une leuco-entéphalopathie multifocale progressive chez les patients traités par des antagonistes de la vla-4

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