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WO1994005328A1 - Inhibitions des metastases tumorales par neutralisation de la fonction de la thromboplastine tissulaire - Google Patents

Inhibitions des metastases tumorales par neutralisation de la fonction de la thromboplastine tissulaire Download PDF

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Publication number
WO1994005328A1
WO1994005328A1 PCT/US1993/008047 US9308047W WO9405328A1 WO 1994005328 A1 WO1994005328 A1 WO 1994005328A1 US 9308047 W US9308047 W US 9308047W WO 9405328 A1 WO9405328 A1 WO 9405328A1
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Prior art keywords
antibodies
molecules
hutf
cells
cell
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Wolfram Ruf
Barbara M. Mueller
Ralph A. Reisfeld
Thomas S. Edgington
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Scripps Research Institute
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Scripps Research Institute
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • 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
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • 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 relates to methods and compositions for preventing or treating metastasis. More particularly, the present invention relates to molecules that bind tissue factor (TF) and disrupt the TF'VIIa complex, thereby inhibiting the onset and progression of metastasis. In addition, the present invention relates to molecules capable of disrupting tissue factor expression or function, or the function of TF-VIIa complexes. The present invention also discloses therapeutic compositions and methods for inhibiting or disrupting tumor-host interactions necessary for initiation and maintenance of the metastatic process.
  • TF tissue factor
  • the present invention also discloses therapeutic compositions and methods for inhibiting or disrupting tumor-host interactions necessary for initiation and maintenance of the metastatic process.
  • Metastasis is a multi-step process which requires highly adapted interactions of tumor cells with host target organs.
  • the metastatic process has specific requirements which must be met by both tumor cells and host factors, in order for the process to be completed successfully.
  • Hematogenous metastasis leads to disseminated tumor growth in distant sites and is the final stage in the progression of many neoplasias.
  • the metastatic process has been divided into several steps: (1) vascularization of the primary tumor; (2) local invasion and intravasation of tumor cells into the vasculature; (3) survival of tumor cells or tumor cell aggregates in the circulating blood; (4) arrest in the capillary bed of the target organ; (5) extravasation; and (6) proliferation into the target organ (Fidler, I. j. , Cancer Res. 50: 6130-6138 (1990)).
  • Tumor cells can only complete this multi-step process successfully if they have evolved to perform specific functions at each different step and if their specific abilities are complemented by host factors.
  • Clinically and experimentally, different tumors produce metastasis to specific organs, which requires specific contributions from tumor cells and cells in the host organ.
  • Malignant cells are characterized by their ability to invade locally and their ability to metastasize to distant sites. Tumor cells engage in various highly- adapted interactions with the host to accomplish the multi-step process of metastasis. This involves access of tumor cells to the vasculature by local invasion and entry into the vascular compartment, followed by arrest and implantation of tumor cells in the capillary bed of target organs an subsequent proliferation into metastases (Fidler, Cancer Res. 50: 6130-6138 (1990)). Melanoma metastasis is a paradigmatic model which is useful in the analysis of the molecular pathways of hematogenous metastasis (Clark, Br. J. Cancer 64: 631- 644 (1991)).
  • melanoma metastasizes from the skin to a variety of organs, including the lungs (Akslen, et al, Invasion Metastasis 8: 193-204 (1988)). This path of metastasis is recapitulated with many human melanoma cell lines when introduced intravenously into immune deficient mice. (See, eg, Cornil, et al, J. National Cancer Institute 81: 938-944 (1989) ; Mueller, et al, Cancer Met. Rev.
  • factor VII at the site of implantation may bind to tissue factor, its receptor and catalytic cofactor, which can be expressed on host endothelial cells in response to induction by the tumor secreted cytokine vascular permeability factor (Keck, et al., Science 246: 1309-1312 (1989); Clauss, et al., J. EX ⁇ . Med. 172: 1535-1545 (1990)).
  • cytokine vascular permeability factor Kerat, et al., Science 246: 1309-1312 (1989); Clauss, et al., J. EX ⁇ . Med. 172: 1535-1545 (1990)
  • the formation of an initiating TF*VIIa protease complex on host cells may locally trigger coagulation as an important step for tumor cell implantation and proliferation.
  • TF is a single-chain, 263-amino acid membrane glycoprotein, and its primary sequence indicates structural similarity with the cytokine receptor family (Edgington, et al., Thromb. Hae ost. 66: 67-79 (1991)).
  • TF is a transmembrane cell surface receptor, and functions as the receptor and cofactor for factor Vila (Vila) to form a proteolytically active TF'VIIa complex on cell surfaces (Ruf, et al., J. Biol. Che . 267: 6375-6381 (1992) ; Ruf and Edgington, Thromb. Haemos. 66: 529-533 (1991)).
  • the TF extracellular domain binds factor Vila and serves as the catalytic cofactor for this serine protease, thereby initiating the plasma coagulation protease cascades (Edgington, et al. (1991) , supra) .
  • alternative pathways exist to activate coagulation including a tumor cell protease designated cancer procoagulant (Gordon, et al., Thromb. Res. 6: 127-137 (1975)
  • the in vivo activation of this protease system predominantly proceeds via the TF pathway (Davie, et al., Biochemistry 30: 10363-10370 (1991)).
  • TF is typically not expressed to any significant degree on cells of the blood or the endothelium, but is constitutively expressed on certain cell types with a differentiation-dependent regulation (Drake, et al.. Am. J. Pathol. 134: 1087-1097 (1989).
  • Promyelocytic leukemias Bauer, et al., Thromb. Res. 56: 425-430 (1989)
  • pancreatic carcinoma cells are examples of TF expression by non-differentiated precursors of cell types that normally do not express TF in their fully differentiated state.
  • TF tissue factor
  • TF tissue factor
  • TF expression and function represents not merely a correlate of the metastatic phenotype, but a direct causal role in facilitating metastatic implantation and growth of tumors.
  • a specific inhibitor of TF function may be used to block metastasis in vivo.
  • Only human melanoma cell lines with the metastatic phenotype were found to also express moderate to high levels of TF.
  • Blocking of tumor cell TF by binding of an inhibitory monoclonal antibody (mAb) prevented metastasis, but the binding of an equally avid and specific non-inhibitory mAb to TF did not have the same effect.
  • Inhibitory monoclonal antibodies disclosed herein recognize functional epitopes on TF, tending to inhibit TF-initiated coagulation of human plasma by more than 90% and inhibiting specific binding of factor VII to cultured cells expressing TF by more than 80%.
  • the non- inhibitory mAb apparently recognizes a distinct, nonfunctional epitope.
  • TF a function of TF is required for successful implantation of metastatic tumor cells, particularly in view of the presently- disclosed finding that specific interference with TF function, or specific interference with cell-surface expression of TF, profoundly inhibits metastasis.
  • other specific inhibitors of TF function and the TF*Vila complex also are disclosed to be effective as therapeutic agents useful in the prevention of metastatic spreading of tumors.
  • Targeting tumor cell TF function for therapeutic intervention in the metastatic process is further aided by the lack of TF expression in the vasculature. This would seem to allow efficient and specific intervention without side effects as expected from conventional anticoagulant therapy.
  • the present invention contemplates a method of preventing or inhibiting metastasis, comprising the administration of a therapeutically effective amount of molecules capable of preventing or inhibiting the expression of human tissue factor (huTF) on the surface of tumor cells.
  • the molecules are immunoglobulin molecules capable of binding to huTF.
  • the invention contemplates a method of preventing or inhibiting metastasis, comprising the administration of a therapeutically effective amount of molecules capable of inhibiting or disrupting huTF function.
  • the molecules are immunoglobulin molecules.
  • the immunoglobulin molecules are antibodies; in other variations, the antibodies are monoclonal or polyclonal.
  • monoclonal antibodies may be selected from the group consisting of TF8-5C4, TF8- 5G9, TF8-11D12, TF9-1F1, TF9-1D5, TF9-1E7, TF9-1B8,
  • Particularly preferred monoclonal antibodies include TF8-5G9, TF9- 6B4, and TF9-6G4.
  • methods using monoclonal antibodies capable of immunoreacting with huTF locus I or locus II epitopes, or both, are contemplated.
  • the invention also contemplates methods utilizing molecules which comprise immunologically active fragments of immunoglobulin molecules.
  • the fragments are selected from the group consisting of Fab, Fab 1 , F(ab) 2 , and F(v) fragments, and CD-grafted humanized mAbs or fragments thereof.
  • the present invention also contemplates methods and compositions used to inhibit the cell surface expression of TF.
  • molecules capable of inhibiting cell surface expression of TF are capable of modulating the expression of nucleotide sequences regulating the expression of TF, particularly huTF.
  • the molecules are capable of modulating cellular processing mechanisms or expression levels of TF.
  • Methods of identifying useful, cell- surface expression-inhibiting molecules are also disclosed.
  • the method may comprise the culturing of tumor cells or other TF-expressing cells; admixing the cells with a test compound — preferably, one that is non-cytotoxic; incubating the resulting admixture for a predetermined period of time; and then determining the amount of TF expressed per cell.
  • therapeutically-effective molecules and compositions of the present invention may be administered at various times and dosages, according to the needs of a particular individual receiving treatment.
  • administration of therapeutically effective amounts of compounds (e.g., immunoglobulin molecules) according to the present invention begins subsequent to diagnostic procedures indicating the presence of tumors, neoplasias, or other malignancies.
  • administration of an anti-huTF antibody or antibody-containing composition as described herein occurs within about 48 hours, preferably within about 12-36 hours, more preferably within about 2-8 hours and most preferably immediately prior to or substantially concurrently with invasive surgical procedures directed at diagnosis or removal of a tumor or malignancy.
  • the molecules are administered within about 1-8 hours of invasive surgical procedures directed at diagnosis or removal of a tumor or malignancy. In another embodiment, the molecules are administered substantially concurrently with invasive surgical procedures directed at diagnosis or removal of a tumor or malignancy. In yet another variation, the molecules are administered as a single bolus 30 minutes prior to surgery. In one embodiment, the bolus comprises a dose of about 500/zg/kg.
  • the invention further contemplates administration of therapeutically effective amounts of preferred molecules, wherein preferred amounts for administration are in the range of about 0.05 to 20 milligrams per kilogram body weight per day (mg/kg/day) . More preferably, the administered dose is in the range of 0.1 to lOmg/kg/day. In one variation, the molecules are administered intravenously. Other routes/modes of administration are also contemplated.
  • the therapeutically effective amount is an amount sufficient to produce plasma levels of the effective ingredient (e.g., antibody molecules) of about 0.05 ⁇ g/ml to about 25 ⁇ g/ml; in another embodiment, an effective amount is in the range of about O.l ⁇ g/ml to about lO ⁇ g/ml.
  • the therapeutically effective amount is an amount sufficient to produce plasma levels of the effective ingredient of about l ⁇ g/ml to about 5 ⁇ g/ml.
  • Therapeutically effective amounts of the presently-disclosed molecules and compositions administrable according to the within-disclosed methods also include amounts sufficient to inhibit, and preferably inhibit by at least about 10-20 percent, more preferably 30 percent, even more preferably by at least about 50 percent, or about 70 percent, and most preferably by at least about 90 percent, the cell surface expression of TF, or its function, on potentially metastatic cells.
  • a therapeutically effective amount is one sufficient to inhibit binding of huTF to factor VII or Vila by at least about 40%, more preferably by about 60%, and even more preferably by at least about 80%.
  • the present invention also contemplates the administration of therapeutically effective amounts of the presently- disclosed compounds sufficient to produce similar degrees or percentages of inhibition of a clinically significant increase in the surface expression of TF on potentially metastatic cells, e.g., tumor cells.
  • the present invention contemplates methods using molecules capable of inhibiting the binding of factor VII or Vila to huTF.
  • the molecules are capable of disrupting huTF'VIIa complex function.
  • the molecules are capable of preventing or inhibiting the activation of substrates and the coagulation cascade via binding to TF-VIIa complex.
  • the present invention also contemplates methods of identifying molecules capable of inhibiting metastasis.
  • cells derived from metastatic tissue — or from another TF-producing source — are isolated and suspended; the suspension is then incubated with a sample of the molecules for a predetermined period of time, to form an admixture; and the admixture is subsequently assayed for TF specific activity, thereby assessing the ability of the molecules to inhibit metastasis.
  • An assay according to the present invention may comprise (a) incubating the admixture with a TF-specific antibody-containing solution for a predetermined period of time; (b) incubating the second admixture of (a) with a labeled immunoglobulin or a labeled immunoglobulin fragment for a predetermined period of time; and (c) assaying for the presence of labeled TF-antibody complexes.
  • the source of TF may be tumor cells or other potentially-metastatic tissues, or the source may be normal cells or tissues, which may be processed to achieve various degrees of purity.
  • the present invention contemplates a method for identifying compounds or molecules capable of inhibiting, suppressing or preventing the expression of human tissue factor (huTF) on the surface of tumor cells.
  • a method for identifying compounds or molecules capable of inhibiting, suppressing or preventing the expression of human tissue factor (huTF) on the surface of tumor cells comprises culturing tumor cells; admixing the cells with a compound to be tested — preferably, a compound which does not affect the viability of the cells; incubating the admixture for a predetermined period of time; and assaying for or determining the amount of TF expressed per cell.
  • a therapeutic composition comprises, in unit dose form, molecules capable of preventing or inhibiting the expression of huTF on the surface of tumor cells, in a pharmaceutically acceptable excipient.
  • the molecules are immunoglobulin molecules and are capable of binding to huTF.
  • a therapeutic composition of the present invention comprises, in unit dose form, molecules capable of inhibiting or disrupting huTF function, in a pharmaceutically acceptable excipient.
  • the molecules may comprise immunoglobulin molecules or immunologically active fragments of immunoglobulin molecules.
  • the molecules comprise anti-huTF antibody molecules, such as polyclonal or monoclonal antibodies, including, for example, monoclonal antibodies selected from the group consisting Of TF8-5C4, TF8-5G9, TF8-11D12, TF9-1F1, TF9-1D5, TF9-1E7, TF9-1B8, TF9-1B9, TF9-2C4, TF9-2F6, TF9-4D11, TF9-5G4, TF9-5B.7, TF9-5C7, TF9-6B4, TF9-6G4, TF9-6C9, TF9-7E10, TF9-8E8, TF9-9E1, TF9-9C3, TF9-9B4, and TF9-10C2.
  • monoclonal antibodies TF8-5G9, TF9-6B4, and TF9-6G4 are particularly preferred.
  • the molecules are capable of inhibiting the binding of factor VII or Vila to huTF, or they are capable of disrupting huTF-VIIa complex function.
  • the present invention also contemplates methods of inhibiting huTF synthesis in the vasculature using anti-sense oligonucleotides, as well as methods and compositions for inhibiting the catalytic site of factor Vila in the huTF-VIIa complex. Further contemplated is the use of peptidyl and other pharmaceutical compositions to interfere with huTF ⁇ — ⁇ Vll/VIIa interactions. Disruption or inhibition of the TF pathway using compounds, compositions and methods of the present invention is also contemplated.
  • BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates TF expression by cultured human melanocytes and melanoma cell lines.
  • TF specific activity per cell line is also illustrated.
  • TF functional activity was analyzed in a plasma coagulation assay after octyl- glucopyranoside lysis of cell pellets of normal human epidermal melanocytes (NHEM) and melanoma cell lines WM35, M24met, and C8161.
  • TF specific activity was assessed after blocking TF in cell lysate by a 30 minute preincubation with mAb TF8-5G9 (30 ⁇ g/ml) . Means from four independent experiments are illustrated.
  • Figures 2A and B illustrate the surface expression of TF on metastatic melanoma cell lines. In each instance, the number of cells is plotted on the vertical axis, while fluorescence intensity is shown on the horizontal axes. Indirect immunofluorescence and flow cytometry were used to demonstrate TF antigen on the surface of M24met (Fig. 2A) and C8161 (Fig. 2B) melanoma cell lines. Cells were incubated with 50 ⁇ g/ml isotype matched control Ab (TIB115; solid line) , or anti-TF mAb TF8-5G9 (bold line) and TF9-10H10 (dotted line) , respectively.
  • TIB115 isotype matched control Ab
  • TF8-5G9 bold line
  • TF9-10H10 dotted line
  • FIG. 3A illustrates functional activity of human TF in human and mouse plasma. Clotting time in seconds is plotted on the vertical axis, whereas the amount of human TF (in ng/ml) is shown on the horizontal axis. The ability of phospholipid reconstituted recombinant human TF to initiate coagulation in mouse plasma (open circles) was evaluated in a standard clotting assay in comparison to pooled normal human plasma (closed circles) .
  • Figure 3B shows that, in either case, a preincubation with 30 ⁇ g/ml TF8-5G9 — but not with TF9- 10H10 — inhibited TF-induced coagulation.
  • procoagulant activity mU/ml
  • results of preincubation of human vs. mouse plasma with 30 ⁇ g/ml control (TIB115) , 10H10 (TF9-10H10) or 5G9 (TF8-5G9) mAb is shown.
  • Figure 5 illustrates the ability of anti-TF monoclonal antibodies to inhibit tumor associated TF in vivo and the TF expression of M24met melanoma cells in vivo.
  • Tissue factor procoagulant activity in mU/mg is indicated on the vertical axis
  • TF expression in the presence or absence of mAb TF8-5G9 in cultured cells, subcutaneous tumors, and lymph node metastasis is represented by the dark bars (- TF8-5G9) and light bars (+ TF8-5G9) extending vertically from the horizontal axis.
  • amino acid The amino acid residues described herein are preferred to be in the natural L- configuration. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property of immunoglobulin binding is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • amino acid residue sequences are represented herein by formulae whose left to right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a bond to a further sequence of one or more amino acid residues up to a total of about fifty residues in the polypeptide chain.
  • antibody in its various grammatical forms is used herein to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope.
  • Illustrative antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contain the paratope, including those portions known in the art as Fab, Fab', F(ab') 2 and F(v) .
  • Fab and F(ab') 2 portions of antibody molecules may be prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well known. (See, e.g.. U.S. Pat. No. 4,342,566 to Theofilopoulos, et al., the disclosures of which are hereby incorporated by reference.)
  • Fab' antibody molecule portions are also well known and are produced from F(ab') 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide.
  • An antibody containing intact antibody molecules is preferred, and is utilized as illustrative herein.
  • antibody combining site refers to that structural portion of an antibody molecule comprised of a heavy and light chain variable and hypervariable regions that specifically binds (immunoreacts with) antigen.
  • antigen has been used historically to designate an entity that is bound by an antibody, and also to designate the entity that induces the production of the antibody. More current usage limits the meaning of antigen to that entity bound by an antibody, while the word “immunogen” is used for the entity that induces antibody production.
  • the antigen and immunogen are the same entity as where a synthetic polypeptide is utilized to induce production of antibodies that bind to the polypeptide.
  • the same polypeptides may also be able to induce antibodies that bind to a whole protein, in which case the polypeptide is both immunogen and antigen, while the whole protein is an antigen.
  • an entity discussed herein is both immunogenic and antigenic, it will generally be termed an antigen.
  • antigenic determinant refers to the actual structural portion of the antigen that is immunologically bound by an antibody combining site.
  • epitope refers to the actual structural portion of the antigen that is immunologically bound by an antibody combining site.
  • epitopope refers to the actual structural portion of the antigen that is immunologically bound by an antibody combining site.
  • epitope refers to the actual structural portion of the antigen that is immunologically bound by an antibody combining site.
  • immunosorbent in its various forms is used herein to refer to binding between an antigenic determinant-containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or a portion thereof.
  • the term “specifically bound” refers to a non-random binding reaction between a cell surface receptor and a ligand molecule.
  • the phrase "monoclonal antibody” in its various grammatical forms refers to an antibody containing or having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen, e.g., a bispecific (chimeric) monoclonal antibody.
  • the phrase "monoclonal antibody composition" in its various grammatical forms refers to a population of antibody molecules that contain only one species of antibody combining site capable of immunoreacting with a particular antigen.
  • a monoclonal antibody composition thus typically displays a single binding affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody composition may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen, e.g., a bispecific monoclonal antibody.
  • a monoclonal antibody composition is typically composed of antibodies produced by clones of a single cell called a hybridoma that secretes (produces) but one kind of antibody molecule.
  • the hybridoma cell is formed by fusing an antibody-producing cell and a myeloma or other self-perpetuating cell line.
  • Such antibodies were first described by Kohler and Milstein, Nature 256: 495-497 (1975) , which description is incorporated by reference herein.
  • a monoclonal antibody composition can also be produced by methods well known to those skilled in the art of producing chimeric antibodies. Those methods include isolating, manipulating, and expressing the nucleic acid that codes for all or part of an immunoglobulin variable region including both the portion of the variable region comprised by the variable region of immunoglobulin light chain and the portion of the variable region comprised by the variable region of immunoglobulin heavy chain. Methods for isolating, manipulating, and expressing the variable region coding nucleic acid in procaryotic and eucaryotic hosts are disclosed in Robinson et al., PCT Publication No. WO 89/0099; Winter et al., European Patent Publication No. 0239400; Reading, U.S. Patent No.
  • the nucleic acid codes for all or part of an immunoglobulin variable region that binds a preselected antigen (ligand) .
  • sources of such nucleic acids are well known to one skilled in the art and, for example, may be obtained from a hybridoma producing a monoclonal antibody that binds the preselected antigen, or the preselected antigen may be used to screen an expression library coding for a plurality of immunoglobulin variable regions, thus isolating the nucleic acid.
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a mammal, particularly a human, without the production of allergic or untoward physiological effects such as nausea, dizziness, gastric upset and the like.
  • substantially simultaneously or “substantially concurrently” is used herein to mean within a time period sufficient to produce concurrent results, e.g., prevention or inhibition of the spread of malignant cells — i.e., metastasis — that may occur as a result of an invasive surgical procedure by administration of an anti-huTF antibody, anti-huTF antibody-containing composition, or a subcombination or combination thereof, as described herein.
  • pg means picogram
  • ng means nanogram
  • ⁇ g means microgram
  • g means milligram
  • kg means kilogram
  • ⁇ l means microliter
  • ml means milliliter
  • 1 means liter
  • the present invention contemplates methods of treating and/or preventing metastasis, and compositions useful therefor.
  • Patients in need of such treatment include those at risk for or suffering from metastatic or potentially-metastatic tumors, malignancies, or neoplasias, such as melanoma and the like.
  • Patients particularly able to benefit from the present invention are those with diagnosed tumors, neoplasias, or other malignancies, particularly those with a tendency to metastasize.
  • the present invention contemplates a method of preventing or inhibiting metastasis by administering to a patient in need of such treatment a therapeutically effective amount of a molecule that interferes with cell-surface expression of TF or with TF function.
  • Methods of interfering with the cell-surface expression of TF may preferentially utilize molecules or compounds capable of interfering with or modulating the expression of TF-related genes, for example.
  • molecules capable of disrupting cell-surface expression of TF must be of sufficiently small size to facilitate the entry of such molecules into the cell.
  • such molecules are preferably non-toxic to the cells.
  • Molecules capable of disrupting or modulating cell-surface TF expression may include, for example, nucleotide sequences encoding regulatory molecules, or the regulatory molecules themselves.
  • Other examples of molecules which may suppress cell-surface expression of TF include molecules which specifically modulate cellular processing and degradation of TF.
  • Methods of interfering with TF function may preferentially employ immunoglobulins, such as anti-TF antibodies.
  • a more preferred method utilizes an anti- huTF antibody.
  • An even more preferred method utilizes an inhibitory anti-huTF antibody specific for locus I or II epitopes — or both — as described in Ruf, et al., Biochem. J. 278: 729-733 (1991), which is incorporated herein by reference.
  • antibodies to the locus I epitope include inhibitory (e.g., TF8-5G9) and non-inhibitory (e.g., TF9-10H10) antibodies.
  • Antibodies to the locus II epitope include inhibitory (e.g.
  • TF9-6B4, TF9-6G4 and non-inhibitory antibodies as well.
  • the locus I epitope appears to be encoded by exons 4 and 5 of huTF, whereas the locus II epitope appears to be encoded by exon 3 of huTF. Applicants do not intend, however, for the present invention to be limited by a particular hypothesis or theory of operation.
  • Exemplary antibody molecules include intact immunoglobulin molecules, substantially intact immunoglobulin molecules, and those portions of an immunoglobulin molecule that contain the paratope, including those portions known in the art as Fab, Fab', F(ab) 2 and F(v) .
  • therapeutically effective amount is used herein to mean an amount sufficient to inhibit, and preferably inhibit by at least about 10 percent, more preferably by about 20-30 percent, even more preferably by at least about 50 percent, or about 70 percent, and most preferably by at least 90 percent, a clinically significant increase in the surface expression of TF on potentially metastatic cells, e.g., tumor cells.
  • Preferred therapeutically effective amounts for the agents used herein as active ingredients are provided below.
  • a clinically significant increase in the cell surface expression of TF on tumor cells is an increase of about 10 times the normal level to about 50,000 times the normal level. Often, a clinically significant increase is an increase of about 100 to 1,000 times the normal level of surface-expressed huTF.
  • a therapeutically effective amount of the agents used herein may also comprise an amount sufficient to inhibit about 1-20 percent, and preferably inhibit by about 20-50 percent, more preferably by about 40-70 percent, even more preferably by about 80-100 percent, the functional activity of huTF.
  • Methods of assessing the functional activity of TF are known in the art; a preferred method is that described hereinbelow in Section G.
  • the method comprises administering to a patient in need of treatment (i.e., with metastatic tumors or at risk for metastasis) a therapeutically effective amount of an anti-human TF (huTF) antibody, preferably an amount sufficient to inhibit huTF functional activity in vivo.
  • the method comprises administering to a patient in need of treatment a therapeutically effective amount of a molecule capable of inhibiting or preventing the cell-surface expression of huTF.
  • a therapeutic method of the present invention comprises administering a therapeutically effective amount of an anti-human TF antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb.
  • an anti-human TF antibody preferably an affinity-purified polyclonal antibody, and more preferably a mAb.
  • the anti-huTF antibody molecules used herein may be in the form of Fab, Fab 1 , F(ab') 2 or F(v) portions of whole antibody molecules.
  • the amount of anti- huTF antibody administered is sufficient to reduce by at least about 20 percent, preferably by at least 40 percent, and more preferably by at least 60 or 80 percent, a clinically significant increase in the levels of tumor cell surface-expressed, functional huTF in a patient diagnosed as having tumors, malignancies, or neoplasia, or displaying at least one of the symptoms associated therewith.
  • patients capable of benefiting from this method include those suffering from malignancies in which metastasis is not uncommon.
  • Methods for isolating TF and for inducing anti- huTF antibodies are well known in the art. (See, for example, U.S. Patent No.
  • compositions disclosed herein include, as an active ingredient, molecules capable of inhibiting or preventing the cell-surface expression of huTF.
  • Molecules capable of disrupting or modulating cell- surface TF expression may include, for example, nucleotide sequences encoding regulatory molecules, or the regulatory molecules themselves.
  • Other examples of molecules which may suppress cell-surface expression of TF include molecules which specifically modulate cellular processing and degradation of TF.
  • compositions of the present invention also include those in which the active ingredient is capable of inhibiting TF function.
  • active ingredients according to the present invention include, for example, immunoglobulins and fragments thereof, including antibodies and antibody fragments.
  • an anti-huTF antibody used in a therapeutic method of this invention is an affinity purified polyclonal antibody. More preferably, the antibody is a monoclonal antibody (mAb) .
  • preferred anti-huTF antibody molecules used herein may be in the form of Fab, Fab 1 , F(ab') 2 or F(v) portions of whole antibody molecules.
  • Preferred monoclonal antibodies useful in practicing the present invention are those capable of being produced by a hybridoma such as hybridoma TF8-5G9 or TF9-6B4, described in U.S. Pat. No. 5,110,730 to Edgington, et al.
  • mAbs such as TF8-5G9 and TF9- 6B4 can be produced by hybridoma culture, the invention is not so limited. Also contemplated is the use of mAbs produced by an anti-TF immunoglobulin expressing nucleic acid cloned from a hybridoma such as TF8-5G9, TF9-6B4, or TF9-6G4. That is, the nucleic acid expressing the anti-TF antibody molecules secreted by hybridomas TF8-5G9, TF9-6B4, or TF9-6G4 and the like can be transferred into another cell line to produce a transformant.
  • the transformant is genotypically distinct from the original hybridoma but is also capable of producing anti-huTF antibody molecules, including immunologically active fragments of whole antibody molecules, corresponding to those secreted by the hybridoma. See, for example, United States Patent No. 4,642,334 to Reading; PCT Publication No. WO 890099 to Robinson et al.; European Patent Publications No.
  • immunoreactivity in its various grammatical forms refers to the concentration of antigen required to achieve a 50% inhibition of the immunoreaction between a given amount of the antibody and a given amount of huTF antigen. That is, immunoreactivity is the concentration of antigen required to achieve a B/B 0 value of 0.5, where B 0 is the maximum amount of antibody bound in the absence of competing antigen and B is the amount of antibody bound in the presence of competing antigen, and both B 0 and B have been adjusted for background. (See, e.g., Robard, Clin. Chem. 20: 1255-1270 (1974).)
  • Preferred anti-huTF antibodies according to the present invention also include antibodies that inhibit or interfere with huTF function.
  • Such preferred antibodies include antibodies to huTF which are capable of inhibiting the binding of factor VII or Vila. More preferred are huTF antibodies which are specific for primates, and more preferably humans (i.e., such antibodies do not cross-react with TF from non- primates, and more preferably, do not cross-react with TF from non-human primates) .
  • Exemplary preferred monoclonal antibodies are listed in Table 1 below.
  • TF peptide analogs are a polypeptide capable of competitively inhibiting the binding of factor VII or Vila to TF expressed on the surface of tumor cells.
  • Preferred TF peptide analogs include the epitopes identified as locus I or locus II, or both.
  • Other preferred anti-huTF antibodies useful in practicing the present invention immunologically cross-react with recombinant TF or with TF mutants which are functionally similar to, or include the same functional epitope as, native huTF.
  • mutant TFs include, without limitation, TF A165A166 (Ruf, et al., J. Biol. Chem. 267: 6375-6381 (1992)).
  • a monoclonal antibody composition contains, within detectable limits, only one species of antibody combining site capable of effectively binding huTF.
  • a monoclonal antibody composition of the present invention typically displays a single binding affinity for huTF, even though it may contain antibodies capable of binding proteins other than huTF.
  • a monoclonal antibody composition contains antibodies capable of inhibiting the function of huTF.
  • a monoclonal antibody composition contains antibody molecules that immunoreact with huTF and a polypeptide analog of the tissue factor epitope located near the carboxy terminus of huTF, preferably pl06-219, and more preferably, the locus I epitope.
  • the antibody molecules immunoreact with huTF and a polypeptide analog of the tissue factor epitope identified as the locus II epitope.
  • the antibody molecules contained in these compositions immunoreact with huTF at or near its locus I or II epitopes and thereby neutralize its functionality and/or its ability to bind factor VII or Vila.
  • a monoclonal antibody composition of the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate polypeptide specificity.
  • the culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium.
  • the antibody- containing medium is then collected.
  • the antibody molecules can then be further isolated by well-known techniques.
  • An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco, et al., Virol. 8: 396 (1959)) supplemented with 4.5g/l glucose, 20mM glutamine, and 20% fetal calf serum.
  • DMEM Dulbecco's minimal essential medium
  • An exemplary inbred mouse strain is the BALB/c strain.
  • the monoclonal antibody compositions produced by the above methods can be used, for example, in diagnostic and therapeutic modalities wherein formation of an huTF- containing immunoreaction product is desired.
  • compositions which contain polypeptides, antibody molecules, or portions thereof as active ingredients are well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions; however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • the active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, or pH buffering agents which enhance the effectiveness of the active ingredient.
  • a therapeutic composition of the present invention may also take the form of tablets, pills, capsules, sustained release formulations, or powders.
  • the compositions preferably contain from about 10%-95% active ingredient, and more preferably, about 25%-70% active ingredient.
  • a polypeptide or antibody can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids,or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids,or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides
  • the therapeutic polypeptide- or antibody- containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e.., carrier, or vehicle.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • the quantity of the effective ingredient to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of inhibition or neutralization of TF-binding capacity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual.
  • suitable dosage ranges are of the order of one to several milligrams of active ingredient per individual per day, preferably one to several milligrams of active ingredient per kilogram bodyweight of individual per day, more preferably about 0.05mg/kg/day to about 20mg/kg/day, and even more preferably about lmg/kg/day to about 5mg/kg/day, and depend on the route of administration.
  • Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. For example, a single bolus of about 500 ⁇ g/kg may be administered (e.g., intravenously) for therapeutic benefit.
  • a therapeutically effective amount for intravenous administration is an amount sufficient to produce plasma levels of the effective ingredient (e.g., compounds which bind to TF, such as monoclonal antibodies) of about O.l ⁇ g/ml to about lO ⁇ g/ml are contemplated.
  • administration of an amount sufficient to produce plasma levels of said molecules of about l ⁇ g/ml to about 5 ⁇ g/ml are contemplated.
  • the primary mode of communication and delivery of substances is via the circulatory system.
  • the circulatory system comprises the blood vascular system and the lymphatic system. Of particular interest are the capillaries.
  • Capillaries are typically comprised of simple endothelial cells that connect the arterial and venous sides of the circulatory system.
  • Meshes of the capillary network are present throughout the body, varying in size and in shape in different tissues and organs. The intensity of metabolism in a region generally determines the closeness of the mesh. Therefore, there is a close network in the lungs, liver, kidneys, mucous membranes, glands, and skeletal muscle, as well as in the grey matter of the brain.
  • the network has a large mesh and is sparse in tissues such as tendons, nerves, smooth muscle, and serous membranes.
  • the ability to transfer substances through the wall of capillaries is referred to as permeability. Permeability varies regionally and, under changed conditions, locally.
  • tumor vessels are anatomically heterogeneous structures. Often, they consist of relatively undifferentiated channels, lined by a simple endothelium and with fewer pericytes and smooth muscle cells than would be expected of comparably sized vessels in normal tissues.
  • One property of tumor vessels on which most investigators agree is that, relative to normal vessels, tumor vessels are hyperpermeable to circulating macromolecules. This observation demands explanation because of its obvious relevance to an understanding of the localization of monoclonal antibodies and tumoricidal drugs in solid tumors. (See, e.g., Dvorak, et al., Am. J. Pathol.
  • vasoactive agent may under some circumstances increase the perfusion of tumor relative to other tissues and increase tumor uptake and concentration of pharmaceuticals.
  • the immunoglobulins of the present invention may also be appropriate to enhance delivery of the immunoglobulins of the present invention by administering them in conjunction with, or by conjugating them to, other therapeutically useful agents.
  • the anti-huTF mAbs of the present invention may be administered in conjunction with one or more vasoactive agents.
  • immunoglobulin-agent conjugates would retain the ability to bind with antigen; more preferably, they would also fail to bind with any component of blood or normal, intact, non-inflamed endothelium. It is also preferable that such conjugates would show little or no tendency to pass across the endothelium of normal blood vessels from blood into tissues. It is also conceivable that anti- huTF mAbs of the present invention may be conjugated to other antibodies or fragments thereof, such as mAbs that are specifically targeted to an antigen present on a specific type of tumor. Methods for making and using such conjugates are known in the art and will thus not be discussed at length herein.
  • Vasoactive agents which improve the extravascular penetration and binding of monoclonal antibodies (as well as other drugs or molecules) may also be useful and may be administered in conjunction with, or conjugated to, the immunoglobulins of the present invention.
  • conjugates other than vasoactive agent-immunoglobulin conjugates are also contemplated by the present invention, immunoglobulin-vasoactive agent conjugates (or "immunoconjugates") shall be discussed herein as exemplary. It is expressly to be understood that the present invention is not limited to such conjugates.
  • Immunoconjugates useful as disclosed herein may be prepared by various methods including genetic approaches, or by covalently or otherwise linking a selected clinically useful immunoglobulin to a selected biologically active agent which is inflammation provoking, and preferably vasoactive.
  • the linking agent and the chemical procedure of assembling the immunoconjugate should be selected and carried out so as not to compromise the effectiveness of the antibody in binding to its target or the effectiveness of the vasoactive agent in stimulating natural defense mechanisms.
  • Vasoactive agents as described herein are typically used to increase the flow of blood and/or the vessel permeability in the tumor so as to improve the extravascular penetration and binding of monoclonal antibodies and other drugs or molecules in vivo. They may act directly by increasing the volume of tumor blood flow or the degree of tumor blood vessel "leakiness,” or indirectly by inducing an inflammatory immune response at the tumor site.
  • Inflammation can be induced by chemotactic factors which attract polymorphonuclear leukocytes, macrophages, eosinophils, basophils, mast cells, T-cells and other cells associated with inflammation. These cells, when stimulated, secrete immunomodulatory factors which then act on the tumor blood flow and blood vessel permeability to increase the percent of the injected dose penetrating and binding to the tumor.
  • Vasoactive agents having the described reactivity at the tumor site and suitable for linking to monoclonal antibodies in an immunoconjugate are found in several biochemical classes, including peptides, carbohydrates, and lipids, and their derivatives.
  • vasoactive agents examples include tachykinins, leukotrienes, anaphylatoxins, lymphokines, inflammagens, human eosinophil acidic tetrapeptides, proteases, platelet-activating factor (PAF) , prostaglandins, prostaglandin derivatives, histamine, and vasoactive carbohydrate compounds such as glucan.
  • vasoactive agents are selected according to availability and applicability to the stated goals of therapy or study.
  • the structural link between the immunoglobulin and the agent to which it is conjugated, and the chemical method by which they are joined, should be chosen so that the binding ability of the immunoglobulin and the biological activity of the agent, when joined in the conjugate, are minimally compromised.
  • ECDI l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • ECDI l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • Conjugates are derived by condensation of the antibody and conjugant under acidic conditions with ECDI. This method provides a rapid and simple means of conjugation.
  • SPDP N-succinimidyl 3-(2-pyridyldithio) propionate
  • SPDP is a heterobifunctional reagent which introduces thiol groups to the terminal amino of proteins, and has been used in a number of immunoconjugates.
  • Periodate oxidation can be used to oxidize and cleave carbon-carbon bonds in a sugar ring.
  • the exposed terminal groups can then link to NH 2 groups on proteins in a Schiff base linkage which is reduced with NaBH 4 .
  • N-hydroxysuccinimide (NHS) activates a terminal COOH group, for example, of a peptide, to form an active ester derivative that can be covalently coupled to the protein of the monoclonal antibody. This method has been used to attach 30 molecules of chlorambucil/antibody with little loss of binding activity. (Smyth, et al., J. Natl. Cancer Inst. 76(3): 503-510 (1986).)
  • the genetic sequence of the agent can be engineered into the genetic sequence of the immunoglobulin.
  • the genetic sequence of a vasoactive peptide may be engineered into that of one of the antibodies of the present invention.
  • the dose of vasoactive agent or immunoconjugate to be given is based on criteria of medical judgment and experience, both objective and subjective. However, an adequate measure of an effective dose is that amount required to increase localization of a subsequently- or concurrently-administered diagnostic or therapeutic agent (such as the immunoglobulins of the present invention) to an extent which improves the clinical efficacy of therapy, or accuracy of diagnosis, to a statistically significant degree. Comparison is typically made between treated and untreated tumor host animals to whom equivalent doses of the diagnostic or therapeutic agent are administered.
  • the immunodiagnostic dose may comprise an immunoglobulin having a specificity for huTF and having a label which is detectable in vivo. In a preferred embodiment, this label comprises a radioactive isotope.
  • the immunotherapeutic dose may similarly comprise a clinically useful immunoglobulin such as those described herein, administered in conjunction with a vasoactive agent, or alone.
  • This immunoglobulin may further be attached to :a tu oricidal agent, for example, a radioisotope, a chemotherapeutic drug or a toxin.
  • a myeloma or other self- perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with huTF or a receptor-binding portion thereof.
  • Methods for producing hybridomas capable of producing (secreting) antibody molecules having a desired immunospecificity — i.e., having the ability to immunoreact with a particular protein and/or polypeptide — are well known in the art. Particularly applicable is the hybridoma technology described by Niman, et al., PNAS USA 80: 4949-4953 (1983).
  • Preferred hybridomas are those which secrete antibodies capable of immunoreacting with huTF. Even more preferred are those which secrete antibodies capable of immunoreacting with an huTF epitope, and more particularly, with the epitope located at or near the carboxy terminus of native huTF and defined or encompassed by amino acid residue nos. 106-219.
  • the hybridomas of the present invention are characterized as producing antibody molecules that immunoreact with huTF. Such antibodies may further be capable of immunoreacting with polypeptide analogs TF epitopes, e.g. p204-226.
  • Preferred methods of producing and screening antibodies useful according to the present invention are set forth in U.S. Patent No. 5,110,730 to Edgington, et al. and is incorporated herein by reference. (Also see U.S. Patent No. 5,223,427 to Edgington, et al., which is also incorporated herein by reference.)
  • Exemplary hybridomas include those described in U.S. Pat. App. No. 5,110,730. Especially preferred hybridomas are those identified by ATCC accession numbers HB9382 and HB9381 (ATCC, Bethesda, MD) . Other preferred hybridomas are those identified in Table 1 of Morrissey, et al., Thromb. Res. 52: 247-261 (1988) . The monoclonal antibodies listed in Table 1 of that reference were prepared essentially as follows. The immunogen, human brain TF isolated by the factor VH-affinity method, was lyophilized and extracted twice with 80% acetone to remove detergent.
  • TF was dissolved in normal saline and emulsified with adjuvant as required.
  • Denatured TF was prepared by boiling for 5 minutes in TBS containing 0.5% SDS and 1% 2- mercaptoethanol.
  • Carrier mouse serum albumin was added followed by acetone extraction (as described in Morrissey, et al., supra) .
  • Female BALB/c mice from The Scripps Research
  • mice were injected subcutaneously with 5 ⁇ g of huTF in Ribi adjuvant (monophosphoryl lipid A, trehalose dimycolate, squalene and Tween-80 emulsion; from Ribi Immunochem Research, Inc., Hamilton, MT) .
  • Ribi adjuvant monophosphoryl lipid A, trehalose dimycolate, squalene and Tween-80 emulsion; from Ribi Immunochem Research, Inc., Hamilton, MT
  • Mouse TF8 received native TF
  • mouse TF9 received denatured TF.
  • each mouse was again injected subcutaneously with 5 ⁇ g of denatured huTF in Ribi adjuvant, and again on day 28 intraperitoneally with 33 ⁇ g of native huTF in complete Freund's adjuvant (Difco, Detroit, MI).
  • mouse TF8 received 15 ⁇ g of native TF in saline intravenously, followed on day 58 by harvest of the spleen for hybridoma production.
  • mouse TF9 received lO ⁇ g of native TF intravenously prior to spleen harvest on day 156. Fusion of spleen cells to the P3Ag8.653.1 myeloma cell line and isolation of hybridomas were performed as described in Soule, et al., PNAS USA 80: 1332-1336 (1983) and Kohler and Milstein, Nature 256: 495-497 (1975) .
  • Hybridomas from fusion TF8 were screened against native TF on dot- blots and also against native 125 I-TF in the radioimmunoassay.
  • the hybridomas from fusion TF9 were screened against reduced, denatured TF on western blots and also against native 125 ⁇ -TF in the radioimmunoassay. Each hybridoma was recloned at least twice.
  • Isotypes were determined using an ELISA test (Hyclone Laboratories, Logan, UT) . Hybridoma culture supernatants were used for most experiments with these antibodies. When required, IgG was purified from ascites fluid using affinity chromatography on immobilized S. aureus protein A (MAPS II System; BioRad Laboratories, Richmond, CA) .
  • MAMS II System BioRad Laboratories, Richmond, CA
  • Hybridomas TF8-5G9 and TF9-6B4 were deposited pursuant to Budapest Treaty requirements with the ATCC on March 27, 1987, and were assigned accession numbers HB9382 and HB9381, respectively. (See also Ruf, et al., Thromb. Haemost. 66: 529-533 (1991) and Morrissey, et al., Thromb. Res. 52: 247-261 (1988).)
  • Monoclonal antibody TF9-10H10 which does not exhibit the presently-preferred immunospecificity, is useful as disclosed herein.
  • Antibody TF9-10H10 is secreted by hybridoma TF9-10H10, which was deposited with the ATCC on March 27, 1987, pursuant to Budapest Treaty requirements, and was assigned accession number HB9383.
  • Metastasis is a multi-step process that has specific requirements which must be met by both tumor cells and host factors, in order for the process to be completed successfully.
  • Monoclonal antibody TF9-10H10 is further described and characterized in U.S. Patent No. 5,110,730 to Edgington, et al. (which has been incorporated herein by reference) . Distribution and fate of radiolabeled tumor cells after I.V. injection indicated at least two phases of melanoma cell arrest in the microvasculature of the lungs. Thus, initial adherence is not influenced by specific inhibitors of tumor cell TF and is apparently not sufficient for prolonged tumor cell survival.
  • NHEM normal human epidermal melanocytes
  • MGM melanocyte growth medium
  • WM35 melanocyte growth medium
  • WM35 was maintained in MCDB153/L15 medium supplemented with insulin (5 ⁇ g/ml) and two percent fetal bovine serum (FBS) .
  • FBS fetal bovine serum
  • the human melanoma cell line M24met has been described in detail (Mueller, et al. Cancer Res. 51; 2193-2198 (1991)).
  • the M24met line was derived from M24, which was established from a lymph node metastasis (Guiliano, et al. Cancer Immunol. Immunother. 10: 243- 249 (1981); Tsuchida, et al., J. Natl. Cancer Inst. 78: 55-59 (1987)).
  • the human melanoma cell line C8161 was also derived from a metastatic melanoma (Welch, et al.. Int. J. Cancer 47: 227-237 (1991)).
  • M24met and C8161 were maintained in RPMI 1640 tissue culture medium supplemented with 10% FBS (Sigma, St. Louis, MO) .
  • WM35 and M24met were cultured on tissue culture plastic that was pretreated with 1% gelatin to enhance cellular attachment. All cells were routinely tested from mycoplasma contamination.
  • the murine anti-human TF monoclonal antibodies TF8-5G9 and TF9-10H10 have been previously characterized (Ruf, et al., Thromb. Haemost. 66: 529- 533 (1991); Ruf, et al., Biochem. J. 278; 729-733 (1991)). Both mAbs are of IgGl isotype.
  • the IgGl mAb to the SV40 large T viral antigen (TIB115, ATCC, Rockville, MD) was used as isotype matched irrelevant control.
  • the mAbs were purified on protein A, and Fab fragments of mAb TF8-5G9 were produced by papain digestion (Ruf, et al., Thromb. Haemost.
  • HBS HEPES-buffered saline
  • Clotting times were determined for the cell lysates in a one-stage clotting assay (equal volumes of sample, plasma, and 20mM CaCl 2 ) and converted to units based on a calibration curve using purified and phospholipid-reconstituted TF.
  • the clotting times obtained in the one-stage clotting assay were converted into units of TF activity using a calibration curve based on serial dilutions of a cell line expressing wild-type TF, as previously described in detail
  • TF was quantified by converting clotting times into procoagulant activity units based on a calibration curve using purified and phospholipid reconstituted recombinant TF.
  • the activity which yields a 50s clotting time was defined as the unit lU/ml.
  • the procoagulant activity was demonstrated to be TF- specific by blocking TF function in the cell lysates by a 30-minute preincubation -with mAb TF8-5G9 (30 ⁇ g/ml) .
  • the functional activity of human recombinant TF was compared in mouse plasma (Sigma, St. Louis, MO) and pooled normal human plasma using a standard coagulation assay.
  • TF The cell surface expression of TF was determined via flow cytometry, as described herein.
  • Melanoma cells (10° cells/tube) were suspended in phosphate buffered saline (PBS) with 50 ⁇ g/ml mAb for 45 minutes on ice. Cells were washed, and bound primary mAb was detected by incubation with goat anti-mouse IgG antibody labeled with fluorescein for 45 minutes on ice. After washing to remove unbound secondary antibody, cells were analyzed with a FACS 440 flow cytometer. Cell surface TF was quantified on M24met melanoma cells by radioligand analysis.
  • PBS phosphate buffered saline
  • the mAb TF8-5G9 was labeled to a specific activity of 5 nCi (185 Bq)/ng with Na 125 I (Amersham, Arlington Heights, IL) with Enzymobeads (BioRad, Richmond, CA) . Radioligand binding analysis with the radiolabeled mAb was performed on melanoma cell monolayers as described (Mueller, et al., J. Immunol. 144: 1382-1386 (1990)). The number of TF molecules per cell and the dissociation constant of the ligand were determined by Scatchard plot analysis. I. TF Expression
  • an assay for identifying such compounds may comprise the steps of: (a) culturing tumor cells; (b) admixing said tumor cells with a test compound, preferably one that is not cytotoxic; (c) incubating the resulting admixture for a predetermined period of time; and (d) determining the amount of TF expressed (e.g. , per cell) .
  • test compound may comprise a nucleotide sequence such as an antisense nucleotide sequence.
  • it may comprise a molecule which binds to at least a portion of the TF-encoding sequence or a nucleotide sequence which affects the rate or frequency of transcription of a TF-encoding sequence, such as the TF promoter or regulatory enhancer elements.
  • Such molecules would thus include transcription repressors.
  • TF genes include molecules that mimic growth factors or otherwise compete with same for binding sites on corresponding cognate cellular receptors, to inhibit the initiation of transcription of the TF gene.
  • molecules that mimic growth factors or otherwise compete with same for binding sites on corresponding cognate cellular receptors to inhibit the initiation of transcription of the TF gene.
  • Molecules which alter the post-translational regulation of TF expression are also potentially useful as therapeutic agents.
  • potentially-useful molecules would likely include those that impact sequestration, for example.
  • Other potentially-useful candidates include molecules which alter TF degradation mechanisms.
  • Additional molecules or compounds potentially useful as modifiers of expression levels of TF include such substances as interleukins (e.g., IL-1) and other cytokines, as well as growth factors and substances such as tumor necrosis factor (TNF) , which exhibit cytokine-like effects.
  • interleukins e.g., IL-1
  • TNF tumor necrosis factor
  • LPS LPS
  • antigen-antibody complexes may induce expression of TF and may thus be useful as disclosed herein.
  • VPF vascular permeability factor
  • Example I Experimental Metastasis Assay Human melanoma cells (0.5 to 1.0 X 10 6 cells in
  • mice 200 ⁇ l PBS were injected into the lateral tail vein of 6-8 week old female C.B-17 scid/scid mice (from The Scripps Research Institute Rodent Breeding Facility, La Jolla, CA) . Prior to injection, cells were incubated for 45 minutes at room temperature. Buffer controls were incubated in PBS and a total of lmg mAb or Fab fragment was present in the experimental groups. When a 2mg total dose was administered, mice received a second intravenous injection of lmg mAb after 24 hours. Twenty-one days later, these animals were sacrificed and necropsy was performed. All major organs were systematically examined for the presence of tumors. The lungs were removed and their weight determined prior to fixation in Bouin's solution. Tumor foci on the lung surface were counted under a low magnification microscope. Data were analyzed for statistical significance by the non-parametric Wilcoxon rank sum test.
  • Example II Determination of Distribution of Radiolabeled Melanoma Cells After Intravenous Injection
  • the initial localization of radiolabeled tumor cells to organs was analyzed using the methodology described by Esumi, et al. (Cancer Res. 51: 4549-4556 (1991)). Cells were cultured for 24 hours in the presence of 0.1 ⁇ Ci (3.7 kBq) /ml 5-( 123 I)-iodo-2 • - deoxyuridine (Amersham, Arlington Heights, IL) resulting in the incorporation of 50,000 cpm per 10 6 M24met cells. This had no effect on the viability of the cells.
  • mice After incubation with mAb or saline for 45 minutes, 10 6 cells in 200 ⁇ l were injected into the lateral tail vein of female scid mice. Mice were bled from the orbital sinuses (50 ⁇ l/mouse) and sacrificed at 10 min. , 90 min. , 240 min. and 24 hours post-injection. Lungs, livers, spleens, and kidneys were collected and washed in 70% ethanol for 3 days prior to gamma- counting.
  • Example III Assay for Expression of TF in Metastatic Melanomas
  • the expression of TF on cultured melanocytes and melanoma cell lines was analyzed with a sensitive coagulation assay (see Fig. 1) .
  • the various cell lines used are shown on the vertical axis; procoagulant activity expressed in mU/10 6 cells is shown on the horizontal axis.
  • TF functional activity was analyzed in a plasma coagulation assay after octyl- glucopyranoside lysis of cell pellets of normal human epidermal melanocytes (NHEM) and melanoma cell lines WM35, M24met, and C8161.
  • TF specific activity was assessed after blocking TF in cell lysate by a 30 minute preincubation with mAb TF8-5G9 (30 ⁇ g/ml) . Means from four independent experiments are illustrated in Figure l.
  • the procoagulant activity of the cell lysates was shown to be TF-specific by inhibition with anti-TF monoclonal antibodies (see Fig. 1) .
  • Both cultured human melanocytes and the cell line WM35 which was derived from a primary non-metastatic melanoma (Kath, et al.. Cancer Therapy Control 1: 179-191 (1990)), expressed TF at very low levels.
  • the melanoma lines M24met Moeller, et al.. Cancer Res. 51; 2193-2198 (1991)
  • C8161 Welch, et al.. Int. J.
  • Figures 2A and B illustrate the surface expression of TF on metastatic melanoma cell lines. In each instance, the number of cells is plotted on the vertical axis, while fluorescence intensity is shown on the horizontal axes. Indirect immunofluorescence and flow cytometry were used to demonstrate TF antigen on the surface of M24met (Fig. 2A) and C8161 (Fig. 2B) melanoma cell lines. Cells were incubated with 50 ⁇ g/ml isotype matched control Ab (TIB115; solid line) , or anti-TF mAb TF8-5G9 (bold line) and TF9-10H10 (dotted line) , respectively.
  • TIB115 isotype matched control Ab
  • TF8-5G9 bold line
  • TF9-10H10 dotted line
  • Bound mAb was visualized by a secondary goat anti-mouse IgG conjugated to fluorescein isothiocyanate. Results of representative experiments are shown in each of 2A and 2B. Additional anti-TF mAbs to non-overlapping epitopes provided similar results (data not shown) .
  • the cell surface expression of TF on M24met cells was approximately 3.5- to 4-fold lower than on C8161 cells, consistent with the relative functional TF activities of these cell lines determined in the coagulation assay. Cell surface expression on M24met cells was also quantified by radioligand binding analysis with radiolabeled mAb TF8-5G9.
  • Example IV Specific Inhibition of Human TF in Murine Blood
  • a xenograft model in which human melanoma cells are injected intravenously into mice with severe combined immune deficiency (scid) was chosen.
  • Species compatibility was assessed with recombinant human TF reconstituted into phospholipid vesicles.
  • Figure 3A illustrates functional activity of human TF in human and mouse plasma. Clotting time in seconds is plotted on the vertical axis, whereas the amount of human TF (in ng/ml) is shown on the horizontal axis. The ability of phospholipid reconstituted recombinant human TF to initiate coagulation in mouse plasma (open circles) was evaluated in a standard clotting assay in comparison to pooled normal human plasma (closed circles) .
  • Figure 3B shows that, in either case, a preincubation with 30 ⁇ g/ml TF8-5G9 inhibited TF-induced coagulation, whereas preincubation with TF9-10H10 did not.
  • procoagulant activity (mU/ml) is shown on the vertical axis.
  • results of preincubation of human vs. mouse plasma with 30 ⁇ g/ml control (TIB115) , 10H10 (TF9-10H10) or 5G9 (TF8-5G9) mAb is shown.
  • mAb TF9-10H10 does not inhibit binding of factor Vila and function of the TF-VIIa complex
  • mAb TF8-5G9 is a potent and rapid inhibitor of human TF in human plasma due to its ability to compete with substrate access to the TF-VIIa complex.
  • this mAb does not inhibit TF from various non-human primates (except chimpanzee) or any lower mammal species tested to date (Morrissey, et al., Thromb. Res. 52: 247-261 (1988)), thus providing a highly-specific tool to selectively block human TF function in the xenograft model.
  • Both the inhibitory mAb TF8-5G9 and the non-inhibitory mAb TF9-10H10 had identical functional characteristics whether in human or mouse plasma (Fig. 3) .
  • the use of the human TF-specific mAb TF8-5G9 thus allows selective inhibition of melanoma cell TF function without interfering with murine TF expressed on host cells.
  • Example V Melanoma Cell TF Function is Required for Hematogenous Metastasis Intravenous injection of M24met metastatic melanoma cells into scid mice reliably produces a large number of metastases in the lungs.
  • Table 2 illustrates the effect of functional inhibition of TF, in an experimental pulmonary metastasis model. In that table, the number of metastases per mouse in each experiment is plotted against the total dose of monoclonal antibody (or buffer solution only) administered to each subject.
  • mice received a second intravenous injection of lmg mAb after 24 hours. After 21 days, all animals were sacrificed, their lungs fixed, and the number of visible tumor nodules on the lungs was counted. The probability of no difference (p) between buffer control and mAb-treated groups is indicated based on the non-parametric Wilcoxon rank sum test.
  • TF8-5G9 (lmg) 14 16 27 42 94 153 ⁇ 0.0022 TF9-10H10 (lmg) 279 293 346 363 375 377 0.309 IgG, (lmg) 281 291 292 327 368 424 0.24
  • TF9-10H10 (2mg) 2 3 15 21 56 63 0.09
  • the number of experimental pulmonary metastases was significantly reduced when mice were infused with M24met cells in the presence of lmg TF8-5G9, in comparison to a control group injected with M24met cells in buffer (see Table 2).
  • Mice receiving TF8-5G9 also had a significant reduction in metastases when compared to animals treated with an irrelevant isotype matched control mAb (TIB115) or the anti-TF mAb TF9- 10H10, which binds TF with high affinity without blocking its function.
  • TIB115 irrelevant isotype matched control mAb
  • TF9- 10H10 which binds TF with high affinity without blocking its function.
  • Fab fragments of TF8-5G9 effectively inhibited M24met metastasis. This finding further excludes F c -mediated effects, though considered unlikely because of the lack of anti-metastatic effects of the anti-TF mAb TF9-10H10 which is of the same isotype as TF8-5G9.
  • the inhibition by Fab fragments was slightly less effective compared to the intact mAb; this may be a result of an observed 2- to 3-fold poorer affinity of Fab fragments of TF8-5G9 (Ruf, et al.,
  • the role of TF in metastasis was further evaluated with another metastatic melanoma cell line which exhibits high levels of TF expression.
  • mAb TF8-5G9 significantly inhibited experimental lung metastasis of C8161 melanoma cells.
  • FIG. 4 Prolonged Adherence of Melanoma Cells
  • the distribution of intravenously injected radiolabeled tumor cells was studied in order to assess whether initial arrest in various organs and persistence of tumor cells is influenced by blocking of TF function (Fig. 4) .
  • Radiolabeled M24met cells were injected into the tail vein of scid mice. Mice were bled and sacrificed at different time points, as shown. Means and standard deviations are based on four animals per group at each time interval. The probability of no difference occurring between groups at a given time was assessed with the Wilcoxon rank sum test.
  • Example VII Discussion of the Results of Examples I-VI
  • the malignant phenotype of cells typically incorporates aberrations in growth control, as well as their capacity to invade and metastasize.
  • Critical events in the metastatic process include (1) the initial arrest and (2) stable implantation of tumor cells in the microvasculature of target organs.
  • TF the major initiating molecule of the coagulation cascades. Indeed, the cellular expression of this molecule was observed to parallel the progression of melanoma cells to a metastatic phenotype. Whereas normal cultured human melanocytes or a primary melanoma derived cell line had only trace levels of functional TF, at least a 1000-fold higher cell surface expression was detected on metastasizing human melanoma cells, since all of the cell lines were proliferating, expression of TF did not reflect cell cycle difference, but appeared, rather, to be the correlate of the metastatic potential.
  • the functional role of TF expressed on melanoma cells was explored in an experimental metastasis model in scid mice.
  • the mAb TF8-5G9 which selectively binds and functionally neutralizes human TF in the xenograft model, markedly reduced the number of pulmonary metastases of two metastatic melanoma cell lines.
  • Thrombin may trigger critical events in the early phase of tumor cell implantation. Stimulation of endothelial cells by thrombin results in rapid expression of P-selectin (Pober, et al..
  • Melanoma-endothelial cell interaction in metastasis may recapitulate the principles of leukocyte-endothelial cell interactions in inflammation (Butcher, Cell 67: 1033-1036 (1991)). This process is characterized by an initial, transient and reversible adhesion mediated by selectins, followed by more stable adherence resulting from the up-regulation and functional participation of leukocyte integrins and their counter-receptors on the stimulated endothelial cell.
  • Fibrin generation may protect arrested cells from host defensive mechanisms (Gunji, et al., Cancer Res. 48: 5216-5221 (1988)). Fibrin which is deposited locally serves as a matrix for the interaction of tumor cells through their adhesive receptors which may modify or induce the expression of genes relevant for invasion and tumor growth (Hynes, Cell 69: 11-25 (1992)). Thrombin may support invasion of tumor cells by degrading the subendothelial matrix either directly (Liotta, et al., Cancer Res.
  • ligand-induced di erization of TF may provide adaptive or proliferative signals that support, or may even be required for, the metastatic process.
  • the findings disclosed herein establish a requirement for ligand binding and function of TF expressed on the tumor cell surface. Specific functional inhibition of this receptor molecule markedly reduces pulmonary metastasis, providing evidence that locally generated proteolytic activity at the site of tumor cell arrest is a key event in metastatic implantation.
  • Example VIII Functional Inhibition of TF and Metastasis Dosages and Inhibitory Mechanisms
  • the protocol described in Example V above was followed. As noted above, intravenous injection of M24met metastatic melanoma cells into scid mice reliably produces a large number of metastases in the lungs.
  • Tables 3A and 3B illustrate the effect of functional inhibition of TF, in an experimental pulmonary metastasis model.
  • the number of metastases per mouse in each experiment is plotted against the total dose of monoclonal antibody (or buffer solution only) administered to each subject.
  • a dose titration of TF8-5G9 demonstrates efficient inhibition of metastasis at lower doses, which are all capable inhibiting tissue factor function by more than 98%. This suggests that antibodies capable of inhibiting tissue factor function are useful inhibitors of metastasis over a rather wide dosage range.
  • Table 3B depicts data from an experiment in which antibody TF8-5G9 is compared with antibody TF9-6B4, which inhibits tissue factor function via a different means than TF8-5G9, through binding to a distinct epitope in the amino-terminal module of tissue factor.
  • TF9-6B4 inhibits metastasis, though perhaps somewhat less potently than TF8-5G9. This difference seems to be is consistent with the reduced inhibitory potency of TF9-6B4 versus TF8-5G9 observed in plasma.
  • TF8-5G9 Inhibition of Tumor-Associated TF In Vivo lmg of mAb TF8-5G9 was injected into the tail veins of scid mice and plasma was obtained from cardiac puncture after four hours. The inhibition of TF function by the mAb in the mouse plasma was determined by preincubation of relipidated human TF with the plasma for 30 minutes followed by determination of residual TF function. A 1:2500 dilution of the mouse plasma inhibited TF function by more than 90%. Thus, specific functional inhibition by TF8-5G9 is not lost in vivo.
  • TF procoagulant activity was normalized on the basis of the wet weight of tumor tissue or the M24met cell pellet from culture in vitro and the quantitative differences may relate to the relative amount of tumor cells compared to host tissue.
  • FIG. 5 illustrates the ability of anti-TF monoclonal antibodies to inhibit tumor associated TF in vivo and the TF expression of M24met melanoma cells in vivo.
  • Tissue factor procoagulant activity in mU/mg is indicated on the vertical axis
  • TF expression in the presence or absence of mAb TF8-5G9 in cultured cells, subcutaneous tumors, and lymph node metastases is represented by the dark bars (- TF8-5G9) and light bars (+ TF8-5G9) extending vertically from the horizontal axis.
  • Monoclonal antibody TF8-5G9 efficiently inhibited TF in both the subcutaneous tumor and the lymph node metastases. Residual procoagulant activity in the metastasis originates most likely from host-derived activated macrophages which express TF during the immune response. Host TF is not, however, neutralized by TF8-5G9.

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Abstract

L'invention concerne des procédés et des compositions de prévention, d'inhibition ou de traitement des métastases ainsi que des procédés de production et d'utilisation desdites compositions. L'invention se rapporte plus précisément à des molécules qui lient la thromboplastine tissulaire et interrompent le complexe thromboplastine tissulaire.facteur VIIa et inhibent ainsi la naissance et la progression des métastases. De plus, l'invention se rapporte à des molécules capables d'interrompre la fonction de la thromboplastine tissulaire, ou celle des complexes thromboplastine tissulaire.facteur VII. L'invention porte également sur des procédés et des compositions utiles pour interrompre ou moduler l'expression de la thromboplastine tissulaire à la surface de cellules dont, par exemple, des molécules capables de modifier ou d'interrompre la transcription et la traduction des séquences codant la thromboplastine tissulaire, ou des molécules qui modulent spécifiquement la maturation cellulaire ou la dégradation de la thromboplastine tissulaire. L'invention porte également sur des compositions thérapeutiques et des procédés d'inhibition ou d'interruption des interactions hôtes-tumeurs nécessaires au démarrage et au maintien du processus métastatique.
PCT/US1993/008047 1992-08-28 1993-08-27 Inhibitions des metastases tumorales par neutralisation de la fonction de la thromboplastine tissulaire Ceased WO1994005328A1 (fr)

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US7824677B2 (en) 1997-03-10 2010-11-02 Genentech, Inc. Method for using antibodies for inhibiting blood coagulation
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WO1998051321A1 (fr) * 1997-05-09 1998-11-19 Merckle Gmbh Thromboplastine tissulaire destinee a influencer la formation vasculaire
US6268163B1 (en) 1997-07-18 2001-07-31 Novo Nordisk A/S Identification of drug candidates modulating factor VII-mediated intracellular signaling
US6461610B1 (en) 1997-07-18 2002-10-08 Novo Nordisk A/S Methods for modifying cell motility using factor VIIa or inactivated factor VIIa
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