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WO1999061060A1 - Conjugues de ligand d'amf et d'une molecule cytotoxique pouvant etre utilises dans la therapie du cancer - Google Patents

Conjugues de ligand d'amf et d'une molecule cytotoxique pouvant etre utilises dans la therapie du cancer Download PDF

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Publication number
WO1999061060A1
WO1999061060A1 PCT/CA1999/000438 CA9900438W WO9961060A1 WO 1999061060 A1 WO1999061060 A1 WO 1999061060A1 CA 9900438 W CA9900438 W CA 9900438W WO 9961060 A1 WO9961060 A1 WO 9961060A1
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amf
cells
conjugate
bamf
toxin
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PCT/CA1999/000438
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Ivan R. Nabi
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Universite De Montreal
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Priority to CA002333021A priority patent/CA2333021A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the invention relates to the use of the endocy- tosis or internalization of autocrine motility factor receptor (AMF-R) as a means to target motile cells, such as metastatic tumor cells.
  • AMF-R autocrine motility factor receptor
  • the invention also relates to AMF-conjugates for therapeutical treatment.
  • AMF-R autocrine motility factor receptor
  • AMF-R is localized not only to the plasma membrane but also to an intracellular tubular organelle, the AMF-R tubule (Nabi, I.R. et al . (1992) Cancer Met. Rev. 11 , 5-20; Benlimame, N. et al . (1995) J.
  • AMF-R tubules are distinct from endosomes and lysosomes; by post -embedding imm noelectron microscopy AMF-R is present primarily in smooth tubules which extend from ribosome-studded cisternae however AMF-R tubules do not colocalize with ERGIC-53, a marker for the ER-Golgi intermediate compartment (Benlimame, N. et al. (1995) J. Cell Biol . 129, 459-471; Wang et al . , 1997) .
  • AMF-R tubules acquire a fenestrated morphology typical of smooth ER suggesting that the AMF-R tubule is a distinct smooth subdomain of the endoplasmic reticulum (Wang, H.-J. et al . (1997) J. Cell Sci . 110 , 3043- 3053) .
  • the intracellular distribution of this cell surface receptor to smooth ER implicates AMF-R recycling in its function in cell motility and tumor cell metastasis .
  • motile cells such as metastatic tumor cells.
  • One aim of the present invention is to provide a means to target motile cells, such as metastatic tumor cells .
  • Another aim of the present invention is to provide the use of the endocytosis or internalization of autocrine motility factor receptor (AMF-R) as a means to target motile cells, such as metastatic tumor cells.
  • AMF-R autocrine motility factor receptor
  • AMF-R is concentrated at the cell surface within smooth plasmalemmal vesicles or caveolae and that AMF is internalized via a non-clathrin pathway to intracellular smooth ER tubules.
  • the results of the present invention identify an AMR-R-mediated clathrin- independent internalization pathway to the endoplasmic reticulum which may be implicated in AMF-R function in tumor cell motility and metastasis.
  • a therapeutical conjugate to specifically kill motile cells which comprises a first molecule which binds to autocrine motility factor receptor (AMF- R) attached to a second toxic molecule to kill said motile cells, such as metastatic tumor cells.
  • the preferred first molecule is AMF.
  • the said second molecule includes, without limitation, plant toxins, bacterial toxins, fungal toxins, drugs, and enzymes for treating prodrugs .
  • the preferred plant toxin includes, without limitation, ricin, abrin, modeccin, viscumin, pokeweed antiviral protein (PAP) , saporin, gelonin, momoridin, trichosanthin, barley toxin, and bryodin.
  • PAP pokeweed antiviral protein
  • the preferred bacterial toxin includes, without limitation, pseudomonas exotoxin (PE) , and diphtheria toxin.
  • PE pseudomonas exotoxin
  • the preferred fungal toxin includes, without limitation, ⁇ -sarcin, and restrictocin.
  • the preferred drug includes, without limitation, doxorubicin, 2-pyrrolinodoxorubicin, daunarubicin, methotrexate, neocarzinostatin, mitomycin C, cali- cheamicin, and vinca alkaloids.
  • the preferred enzyme includes, without limitation, carboxypeptidase, and alkaline phosphatase.
  • AMF-R autocrine motility factor receptor
  • target motile cells which comprises at least one molecule which binds to autocrine motility factor receptor (AMF-R) and is internalized by motile cells, such as metastatic tumor cells.
  • a method to specifically kill cancer cells in vi tro and/or in vivo which comprises administering an effective amount of the conjugate of the pre- sent invention.
  • the cells in vi tro may be leukemias purging cells whereas the cells in vivo may be metastatic tumor cells .
  • Fig. 1 illustrates the electron microscopic localization of AMF-R in NIH-3T3 fibroblasts and He a cells ;
  • Fig. 2 illustrates the colocalization of AMF-R and caveolin by confocal microscopy
  • Fig. 3 illustrates bAMF and anti-AMF-R mAb colo- calize on the cell surface
  • Fig. 4 illustrates the internalization of bAMF to AMF-R tubules
  • Fig. 5 illustrates the localization of internalized bAMF to AMF-R tubules by confocal microscopy
  • Fig. 6 illustrates the electron microscopy of the internalization pathway of bAMF
  • Fig. 7 illustrates G25 SephadexTM chromatography of AMF-doxorubicin mixture following conjugation with 0.02% glutaraldehyde;
  • Fig. 8 illustrates the cell toxicity of a AMF- doxorubicin conjugate in accordance with one embodiment of the present invention.
  • Autocrine motility factor receptor is a cell surface receptor which is also localized to a smooth subdomain of the endoplasmic reticulum (ER) , the AMF-R tubule.
  • ER endoplasmic reticulum
  • AMF-R concentrates within smooth plasmalemmal vesicles or caveolae in both NIH-3T3 fibroblasts and HeLa cells.
  • confocal microscopy cell surface AMF-R labeled by the addition of anti-AMF-R antibody to viable cells at 4°C exhibits partial colocalization with caveolin confirming the localization of cell sur- face AMF-R to caveolae.
  • bAMF By confocal microscopy, the tubular structures labeled by internalized bAMF show complete colocalization with AMF-R tubules.
  • By electron microscopy bAMF internalized for 10 minutes is located to cell surface caveolae and after 30 minutes is present within smooth and rough ER tubules.
  • AMF is therefore internalized via a receptor- mediated clathrin- independent pathway to smooth endoplasmic reticulum.
  • the steady state localization of AMF-R to caveolae implicates these cell surface imaginations in AMF-R endocytosis.
  • NIH-3T3 fibroblasts obtained from the ATCC were cloned and a highly spread clone was used for these studies.
  • HeLa and NIH-3T3 cells were grown in an air-5% CO2 incubator at constant humidity in Dulbecco's minimum essential medium (DMEM) containing non-essential amino acids, vitamins, glutamine and a penicillin- streptomycin antibiotic mixture (Gibco, Burlington, Ontario) supplemented with 5% fetal calf serum (Immuno- corp, Montreal, Quebec) for HeLa or 10% calf serum (Gibco, Burlington, Ontario) for NIH-3T3 cells.
  • DMEM Dulbecco's minimum essential medium
  • fetal calf serum Immuno- corp, Montreal, Quebec
  • 10% calf serum Gibco, Burlington, Ontario
  • Monoclonal antibody against AMF-R was used m the form of concentrated hybridoma supernatant (Nabi, I.R. et al. (1992) Cancer Met. Rev. 11 , 5-20).
  • Rabbit anti-caveolm polyclonal antibody was purchased from Transduction Laboratories (Lexington, KY) , rabbit anti- biotin antibody from Sigma (St. Louis, Missouri), and rat antl-LAMP-1 from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City) .
  • Secondary antibodies conjugated to either fluorescem, Texas Red or 12 nm gold particles and streptavidm conjugated to fluorescem or Texas Red were purchased from Jackson Immunoresearch Laboratories (West Grove, PA) .
  • Texas Red conjugated human difer ⁇ c transferrm was kindly provided by Dr. Tim McGraw (Columbia University, New York, NY) . Streptavidm conjugated to 10 nm gold particles was purchased from Sigma. The secondary antibodies were designed for use m multiple labeling studies and no mterspecies cross-reactivity was detected. To detect antibodies to AMF-R, secondary antibodies specific for the ⁇ chain of rat IgM were used.
  • Rabbit phosphohexose isomerase was purchased from Sigma and biotmylated with NHS-LC-biot (Pierce, Rockford, Illinois) according to the manufacturer's instructions. To assess its purity, biotmylated phos- phohexose isomerase was separated by SDS-PAGE, transferred to nitrocellulose, probed with horseradish per- oxidase conjugated streptavidm (Jackson Immunoresearch Laboratories) and revealed by chemilummescence . Immunofluorescence
  • Cells were plated on glass cover slips 2 days prior to each experiment at a concentration of 30,000 cells/35 mm dish.
  • AMF-R surface labeling the cells were incubated m DMEM minus bicarbonate supplemented with 25 mM Hepes pH 7.2 and 2.5% serum for 15 mm at 4°C prior to labeling with anti-AMF-R primary antibody or biotmylated AMF at 4°C for 30 mm.
  • the cells were washed at 4°C and then fixed with 3% paraformaldehyde phosphate buffered saline (pH 7.4) supplemented with
  • Biotmylated AMF was revealed with rabbit anti-biotm antibody and fluorescent anti- rabbit secondary antibody.
  • AMF internalization studies NIH-3T3 cells were pulsed with biotmylated AMF (-250-500 ⁇ g/ml) and chased at 37°C for the indicated periods of time prior to fixation by the addition of precooled (- 80°C) methanol/acetone directly to the cells. After fixation, internalized bAMF was revealed with Texas Red streptavidm and lysosomes and AMF-R tubules by anti- LAMP-1 and anti-AMF-R antibodies, respectively, followed by the corresponding FITC-conjugated secondary antibodies.
  • NIH-3T3 cells were pre-treated with acidification medium (DMEM containing 5% calf serum and 50mM MES pH 5.5) for 15 minutes at 37°C prior to addition of bAMF m acidification medium for one hour at 37°C.
  • acidification medium DMEM containing 5% calf serum and 50mM MES pH 5.5
  • Texas Red trans- fer ⁇ n 50 ⁇ g/ml was added to cells m regular or acidification medium for 30 minutes at 37°C after which the cells were fixed with 3% paraformaldehyde . After labeling the coverslips were mounted m
  • Airvol Air Products and Chemicals Inc., Allentown, PA
  • Confocal microscopy was performed with the 6OX Nikon Plan Apochromat objective of a dual channel BioRad 600 laser scanning confocal microscope equipped with a krypton/argon laser and the corresponding dichroic reflectors to distinguish fluorescem and Texas Red labeling.
  • Confocal images were printed using a Polaroid TX 1500 video printer.
  • Post -embedding immunolabeling for AMF-R was performed as previously described (Benlimame, N. et al . (1995) J. Cell Biol . 129 , 459-471) .
  • Cells grown on petri dishes were rmsed and incubated at 37°C m Ringer's solution for 15 minutes before fixing m Ringer's solution containing 2% paraformaldehyde and 0.2% glutaraldehyde for 30 minutes at 37°C.
  • the fixed cells were rinsed in PBS/CM, scraped from the petri dish and collected by centrifugation.
  • the cell pellet was post -fixed for 30 minutes with 1% osmium tetroxide in PBS/CM containing 1.5% potassium ferrocyanide (reduced osmium) , dehydrated and embedded in LR-White resin.
  • Ultra-thin sections 80 nm were blocked with 2% BSA, 0.2% gelatin in PBS/CM for 1 hour, and then incubated at room temperature with anti-AMF-R antibody for 1 hour followed by 12 nm gold conjugated goat anti -rat antibodies for 1 hour. The sections were then stained with 5% uranyl acetate and examined in a Philips 300 electron microscope.
  • the numerical density of gold particles associated with plasma membrane, caveolae, clathrin coated pits and vesicles, smooth tubules and vesicles, and rough ER was determined.
  • the length of the limiting membrane of the indicated organelles was measured using a Sigma-Scan measurement system and the gold particles associated with these organelles counted.
  • Rough ER was defined by the presence of a linear array of membrane-associated ribosomes . Smooth vesicles attached to the plasma membrane or within 100 nm of the plasma membrane were considered to be caveolae. Control labeling with non-immune rat IgM antibod- ies was analyzed similarly.
  • biotinylated AMF was internalized as described for the fluorescence studies and detected by poste bedding labeling with streptavidin conjugated to 10 nm gold as described above. No labeling was observed in the absence of biotinylated AMF.
  • the AMF/PHI polypeptide has been shown to be expressed m various tissues m the mouse including muscle, salivary gland, brain, liver and kidney.
  • PHI mRNA exhibits particularly high expression m the muscle, brain and kidney of adult mouse, rat and chicken.
  • PHI is identical to neuroleukm, which increases the survival of cultured sensory neurons and which is secreted by lectm-stimulated T cells and induces matu- ration of B-cells into antibody secreting cells, and to maturation factor, which induces the differentiation of human myeloid cells into monocytes .
  • AMF activity has been detected rheumatoid synovial fluid. Therefore, m addition to its enzymatic activity, the secreted PHI polypeptide functions as a neuro- trophic factor, a lymphokme and a motility factor.
  • AMF-R was expressed not at all or at significantly reduced levels m adjacent normal tissue. Studies performed m the Nabi laboratory have shown that while AMF-R is expressed m brain, liver and lung but not kidney and muscle of young (postnatal day 5 and 12) rats, AMF-R expression is significantly reduced or absent m these tissues m the adult. In the cerebellum, AMF-R was localized to cere- bellar neurons, including Purkmje cells, and may play a role m neuronal plasticity and establishment of neu- ronal contacts during development.
  • AMF and AMF-R may regulate their motile activity during development; deregulation of the expression of AMF and its receptor during tumo ⁇ genesis may contribute to the acquisition of motile and invasive properties by tumor cells.
  • AMF-R is primarily localized to smooth intracellular membranous tubules (Figs. 1A,D), similar m morphology to those previously described m
  • MDCK cells (Benlimame, N. et al . (1995) J. Cell Biol.
  • FIG. 1D,E,F cells were post-embedding immunolabeled with anti-AMF-R and 12 -nm gold-conjugated anti-rat IgM secondary antibodies.
  • Typical AMF-R labeling of smooth tubules (Figs. 1A,D, arrows) and cell surface caveolae (Figs. 1B,C,E,F, arrowheads) is shown.
  • AMF-R label localizes to smooth mvagmations of the plasma membrane morphologically equivalent to caveolae (Figs. 1B,C,E,F). Quantification of the labeling revealed that the predominant AMF-R label is localized to smooth tubules and vesi- cles, flat regions of the plasma membrane and caveolae (Table 1) .
  • Gold particles associated with the indicated membrane organelles were counted and the density per ⁇ m membrane length determined.
  • Control labeling was determined using a nonimmune rat IgM antibody (Benlimame, N. et al. (1995) J. Cell Biol. 129 , 459-471). While specific label was previously detected in the rough ER of MDCK cells (Benlimame, N. et al . (1995) J. Cell Biol. 129 , 459-471), the density of labeling of rough ER tubules in NIH-3T3 and HeLa cells is reduced and at control levels.
  • the density of AMF-R labeling of caveolae is equal to that of intracellular smooth tubules and vesicles m NIH-3T3 cells and greater than that of intracellular smooth tubules and vesicles m HeLa cells and essentially no AMF-R label is found withm clathrm coated pits and vesicles.
  • the density of AMF-R labeling m caveolae is increased relative to flat regions of the plasma membrane. However, based on the total number of gold particles at the plasma membrane, only 13% of cell surface AMF-R m NIH-3T3 and 26% m HeLa cells is found withm caveolae.
  • NIH-3T3 cells were surface labeled for AMF-R by the addition of anti-AMF-R antibodies to viable cells at 4°C (Nabi, I.R. et al . (1992) Cancer Met. Rev. 11 , 5-20) and then double lmmunofluo- rescently labeled after fixation and permeabilization with antibodies to caveolin (Fig. 2) .
  • Viable NIH-3T3 cells were labeled for cell surface AMF-R at 4°C (Fig. 2A) and for caveolin after fixation and permeabilization (Fig. 2B) .
  • Fig. 2C AMF-R m green and caveolin m red
  • colocalization appears m yellow. Bar 20 ⁇ m.
  • AMF phosphohexose isomerase
  • NIH-3T3 cells by the addition of both biotinylated AMF (bAMF) (Fig. 3B) and anti-AMF-R at 4°C (Fig. 3C) revealed a high degree of colocalization (Fig. 3D, yellow) demonstrating that AMF and antibodies to AMF-R recognize the same receptor.
  • the presence of spots labeled exclusively with either bAMF or anti-AMF-R may be due to the fact that the two were added together and may compete for the same site.
  • Pulse labeling of NIH-3T3 cells with bAMF for one or two hours resulted in the ability to detect both punctate structures as well as fainter tubular structures which colocalized with AMF-R tubules (Figs.
  • NIH-3T3 cells were pulse labeled with bAMF at 37°C for one hour (Figs. 4A, B) , for two hours and chased for 4 hours (Figs. 4C, D) or for one hour in medium acidified to pH 5.5 to disrupt clathrin-mediated endocytosis (Figs. 4E, F) .
  • Figs. 4A, C, E After fixation with metha- nol/acetone, cells were double labeled with Texas Red- streptavidin to reveal bAMF
  • Figs. 4A, C, E anti- AMF-R mAb and FITC-conjugated anti-rat secondary anti- body to reveal AMF-R labeling
  • FIGs. 4B, D, F AMF-R labeling
  • bAMF internalized for one hour is localized to intracellular AMF-R tubules (Figs. 4E,F).
  • Figs. 4E,F intracellular AMF-R tubules
  • Figs. 4G,H acidified medium
  • bAMF is therefore internalized via a clathrm- independent endocytic pathway to the smooth ER.
  • bAMF labeled tubules was confirmed by confocal microscopy (Fig. 5) .
  • NIH-3T3 cells were pulse labeled with bAMF at 37°C for one hour m regular medium (Figs. 5A-F) , for one hour in medium acidified to pH 5.5 to disrupt clathrm-mediated endocytosis (Figs. 5G-I), or m regular medium m the presence of 10 -fold excess unlabeled AMF (Figs. 5J-L) prior to fixation with metha- nol/acetone.
  • bAMF was revealed with Texas Red-strepta- vidm (Figs.
  • bAMF is localized to tubular structures which colocalize with AMF-R tubules (Figs. 5A-C) as well as to punctate structures which exhibit partial colocalization with LAMP-1 positive lysosomes (Figs. 5D-F) .
  • the intense punctate labeling can hide the fainter tubular labeling of bAMF m some cells (Figs. 4A, 5D) .
  • acidification medium the vast majority of bAMF labeling, aside from cell surface fibrils, is localized to tubules which colocalize with AMF-R tubules (Figs. 5G-I).
  • bAMF internalized for 1 hour m the presence of 10 -fold excess unlabeled AMF is localized only to punctate structures and no labeling of AMF-R tubules can be detected (Figs. 5J-L) . While the extent of tubular labeling of bAMF varies between cells under control conditions (Figs. 5A,D) , m the presence of excess unlabeled AMF the localization of bAMF to AMF-R tubules is never observed (Figs. 5J-L) . bAMF internalization to intracellular AMF-R tubules therefore occurs via a receptor-mediated process .
  • NIH-3T3 cells were pulsed with bAMF at 37°C for 10 (Figs. 6A,B,H) or 30 minutes (Figs. 6C,D,E, F,G, I) .
  • R is localized to morphologically identifiable caveolae as well as to smooth ER tubules (Fig. 1; Table 1) .
  • Fig. 1 smooth ER tubules
  • labeling of rough ER tubules was not above background in either
  • AMF-R is a specific marker for smooth ER in these two cell types.
  • the localization of AMF-R to caveolae was confirmed by the colocalization of cell surface AMF-R, labeled by the addition of anti-AMF-R to viable cells at 4°C, with caveolin by confocal fluorescence microscopy (Fig. 2) .
  • Fig. 2 confocal fluorescence microscopy
  • heterotrimeric G pro- teins as well as tyrosine kinase and protein kinase C activities in transduction of the AMF motility signal is consistent with the localization of AMF-R to cell surface caveolae.
  • AMF exhibits sequence identity to phosphohexose isomerase (Watanabe, H. et al . (1996) Cancer Res. 56, 2960-2963) .
  • Biotinylated phosphohexose isomerase or bAMF colocalizes with cell surface AMF-R labeled with antibodies to AMF-R at 4°C and is endocytosed by cells at 37°C to tubules which colocalize with smooth ER AMF- R tubules by confocal microscopy and to morphologically identifiable ER tubules by electron microscopy.
  • bAMF internalization to smooth ER tubules is a receptor- mediated process as it can be blocked by the presence of excess unlabeled AMF.
  • SV40 virus associates with caveolae and is internalized via smooth plasmalemmal vesicles to smooth tubules which are extensions of the ER (Schwn- beck, J. et al . (1989) J. Cell Biol. 109 , 2721-2729).
  • the internalization pathway of SV40 to the ER (Schwn- beck, J. et al . (1989) J. Cell Biol.
  • AMF activation of AMF-R may stimulate both transduction of the AMF motility stimulating signal and internalization of AMF-R, perhaps within the same cell surface caveolar domain.
  • AMF recycling and cell motility The established role of AMF-R in cell motility and metastasis implicates AMF-R internalization, and subsequent recycling to the cell surface, in the motile process (Nabi, I.R. et al . (1992) Cancer Met. Rev. 11 , 5-20; Silletti, S. et al . (1996) Am. J. Pathol. 148 , 1649-1660) .
  • This recycling pathway stimulated by the cytokine, AMF may represent a motility specific membrane targeting pathway.
  • EXAMPLE I AMF-R as a target for tumor therapy The internalization of cancer specific ligands as a means to target metastatic tumor cells has previ- ously been demonstrated for the BR96 antigen.
  • the monoclonal antibody BR96 is specific for the Le ⁇ polylactosamine carbohydrate antigen expressed abundantly on numerous carcinomas (Hellstr ⁇ m, I. et al . (1990) Cancer Res. 50:2183-2190) .
  • the BR96 antibody has been shown to be internalized via coated pits to multivesicular bodies, endosomes and finally to lysosomes where it is degraded (Garrigues, J. et al . (1993) Am. J. Pathol.
  • Toxin conjugates of this internalizing monoclonal antibody such as BR96 doxoru- bicm immunoconjugates or Pseudo onas PE40 exotoxin fusion proteins, effected complete regression of xeno- grafted human carcinomas in athymic mice (Friedman, P.N. et al. (1993) J. Immunol. 150 : 3054-3061; Trail, P.A. et al. (1993) Science 261 : 212-215) .
  • AMF-R is an attractive target for tumor therapy because (1) its expression is specifically associated with an essential element of tumor cell metastasis, cell motility, and it exhibits minimal expression in normal cells, (2) its ligand is a common cellular protein, phosphohexose isomerase, which should not generate an immune response and which should exhibit a relatively long biological half-life, and (3) AMF is internalized by its receptor. This last point is critical: it distinguishes the use of AMF-R as a target from many cell surface tumor markers and permits the internalization of a toxic com ⁇ pound. Normal and tumor cells will therefore be discriminated at two levels: receptor expression and receptor internalization.
  • Glutaraldehyde is a non-specific crosslinker which links a ine groups and has previously been used to conjugate various proteins to DOX, including transferrm which is internalized by its receptor to early endocytic compartments.
  • DOX transferrm which is internalized by its receptor to early endocytic compartments.
  • transferrm which is internalized by its receptor to early endocytic compartments.
  • AMF-DOX will be prepared by glutaraldehyde crosslinking as previously described for transferrm.
  • the maximal glutaraldehyde concentration which does not generate high molecular weight oligomers of AMF will be determined by SDS-PAGE.
  • AMF Using this gluta- raldehyde concentration as well as moderately higher and lower concentrations, AMF will be reacted with an excess molar ratio (20:1) of DOX m order to favor DOX conjugation to AMF.
  • Reactive glutaraldehyde will be neutralized with lysme and the DOX-AMF conjugate separated from free DOX by either dialysis or Sephadex G25 chromatography.
  • the extent of DOX incorporation into the conjugate will be determined by spectrophotometry at 495 nm (absorbance of DOX) after establishing con- centration curves w th known concentrations of free DOX.
  • Protein concentration of the conjugate will be determined by spectrophotometry at 280 nm (for AMF protein) m comparison with known concentrations of AMF and taking into consideration potential interference of DOX. Alternatively, protein concentration can be determined by densitometry of coomassie stained SDS-PAGE gels m comparison with known amounts of uncon ugated AMF.
  • SPDP-conjugated ricm ⁇ -cham will be reduced with DTT to generate free SH sites which can attack the intact SH-reactive sites of SPDP-PHI.
  • Crosslinking will be assessed by SDS-PAGE under reducing and non-reducing conditions; successful crosslinking of the two proteins should result in the presence of a protein band of -100-110 kD only under non-reducing conditions.
  • the crosslinking will also be performed using long chain LC-SPDP which contains an internal spacer between the reactive groups thereby reducing inefficient cross-linking due to steric hindrance.
  • Ricin conjugated AMF can be purified from free AMF by affinity chromatography on Blue sepharose CL-6B.
  • doxorubicin also called adriamycin
  • 2-pyrrolinodoxorubicin a more toxic derivative of DOX
  • daunarubicin methotrexate neocarzinostatin mitomycin C calicheamicin vmca alkaloids (vmblastm, vmcristme etc)
  • Enzyme conjugates for subsequent treatment with pro- drugs carboxypeptidase alkaline phosphatase
  • the reaction mix was passed over a G25 Sephadex column to separate PHI from the smaller free doxorubicm (Fig. 7) .
  • One ml fractions were collected and the major protein peak absorbing at 280 nm (Peak 1) corresponding to PHI also absorbs at 495 nm, the absorbance maximum of doxorubicm, demon- stratmg that doxorubicm was successfully conjugated to PHI .
  • Free doxorubicm eluted more slowly and corresponds to the peak of 495 nm absorbance after fraction 25.
  • B16-F10 cells were plated at a density of 4,000

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Abstract

L'invention concerne un conjugué thérapeutique servant à détruire de façon spécifique des cellules mobiles et comprenant une première molécule se liant au récepteur du facteur autocrine de mobilité (AMF-R) fixé à une deuxième molécule toxique afin de détruire lesdites cellules mobiles.
PCT/CA1999/000438 1998-05-22 1999-05-13 Conjugues de ligand d'amf et d'une molecule cytotoxique pouvant etre utilises dans la therapie du cancer WO1999061060A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU38066/99A AU3806699A (en) 1998-05-22 1999-05-13 Conjugates of an amf ligand and a cytotoxic molecule for use in cancer therapy
CA002333021A CA2333021A1 (fr) 1998-05-22 1999-05-13 Conjugues de ligand d'amf et d'une molecule cytotoxique pouvant etre utilises dans la therapie du cancer

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CA2,238,257 1998-05-22
CA002238257A CA2238257A1 (fr) 1998-05-22 1998-05-22 Endocytose d'un recepteur de facteur autocrine de mobilite (amf-r) et utilisations dans le traitement des cancers

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US09700844 A-371-Of-International 2001-02-08
US10/366,319 Continuation-In-Part US20030223978A1 (en) 1998-05-22 2003-02-14 Conjugates of an AMF ligand and a cytotoxic molecule for use in cancer therapy

Publications (1)

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WO1999061060A1 true WO1999061060A1 (fr) 1999-12-02

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AU (1) AU3806699A (fr)
CA (2) CA2238257A1 (fr)
WO (1) WO1999061060A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001074897A3 (fr) * 2000-04-03 2002-06-20 Curagen Corp Proteines et acides nucleiques codant pour lesdites proteines

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988009797A1 (fr) * 1987-06-05 1988-12-15 The United States Of America, As Represented By Th Facteurs de motilite de l'autocrine dans le diagnostic et le traitement du cancer
WO1994001777A1 (fr) * 1992-07-14 1994-01-20 Michigan Cancer Foundation Procede pour determiner le potentiel metastatique de cellules tumorales
US5728383A (en) * 1987-10-05 1998-03-17 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Treatment of tumors of the central nervous system with immunotoxins
WO1998010795A2 (fr) * 1996-09-10 1998-03-19 The Burnham Institute Molecules se logeant dans une tumeur, conjugues tires de ces molecules et leurs procedes d'utilisation
WO1998018493A2 (fr) * 1996-10-30 1998-05-07 Merck & Co., Inc. Conjugues utilises dans le traitement du cancer de la prostate

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988009797A1 (fr) * 1987-06-05 1988-12-15 The United States Of America, As Represented By Th Facteurs de motilite de l'autocrine dans le diagnostic et le traitement du cancer
US5728383A (en) * 1987-10-05 1998-03-17 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Treatment of tumors of the central nervous system with immunotoxins
WO1994001777A1 (fr) * 1992-07-14 1994-01-20 Michigan Cancer Foundation Procede pour determiner le potentiel metastatique de cellules tumorales
WO1998010795A2 (fr) * 1996-09-10 1998-03-19 The Burnham Institute Molecules se logeant dans une tumeur, conjugues tires de ces molecules et leurs procedes d'utilisation
WO1998018493A2 (fr) * 1996-10-30 1998-05-07 Merck & Co., Inc. Conjugues utilises dans le traitement du cancer de la prostate

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001074897A3 (fr) * 2000-04-03 2002-06-20 Curagen Corp Proteines et acides nucleiques codant pour lesdites proteines

Also Published As

Publication number Publication date
CA2333021A1 (fr) 1999-12-02
AU3806699A (en) 1999-12-13
CA2238257A1 (fr) 1999-11-22

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