EP1333859A2 - Compositions therapeutiques destinees a des etats immunitaires et procede associes - Google Patents

Compositions therapeutiques destinees a des etats immunitaires et procede associes

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Publication number
EP1333859A2
EP1333859A2 EP00993870A EP00993870A EP1333859A2 EP 1333859 A2 EP1333859 A2 EP 1333859A2 EP 00993870 A EP00993870 A EP 00993870A EP 00993870 A EP00993870 A EP 00993870A EP 1333859 A2 EP1333859 A2 EP 1333859A2
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EP
European Patent Office
Prior art keywords
animal
binding agent
antibody
composition
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00993870A
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German (de)
English (en)
Inventor
Ragupathy Madiyalakan
Antoine A. Noujaim
Beatrice Leveugle
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Altarex Medical Corp
Original Assignee
ALTAREX CORP
Altarex Corp
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Publication date
Application filed by ALTAREX CORP, Altarex Corp filed Critical ALTAREX CORP
Publication of EP1333859A2 publication Critical patent/EP1333859A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to compositions for the treatment and/or prevention of immune conditions, particularly inflammation and cancer, and methods for making and using such compositions.
  • Diaz-Gonz ⁇ lez and Sanchez-Madrid teach that the process of leukocyte migration requires an interaction between leukocytes and the endothelial cells lining the blood vessel.
  • Leukocyte-endothelial cell interaction is a multi- step process, which involves a cascade of sequential cellular adhesive events that can be divided into four successive steps as illustrated in Figure 1.
  • the initial contact between leukocytes and the blood vessel wall is a reversible process that involves rolling of leukocytes on the activated endothelium (step one).
  • This rolling of the leukocytes on endothelial cells is facilitated by the adhesion of a selectin molecule (e.g., L-selectin) on the leukocyte to its ligand (e.g. , sialyl Lewis X or Sialyl Lewis A) on the endothelial cell.
  • a selectin molecule e.g., L-selectin
  • its ligand e.g. , sialyl Lewis X or Sialyl Lewis A
  • autoimmune diseases are a group of chronic diseases, in which the immune system not only fail to perform its regular function in protecting the host from invading agents, but also reacts particularly against some of the host autoantigens. This autoimmune response is initiated by cellular, humoral, or both mechanisms, and leads to disease with local inflammation and tissue damage.
  • MS multiple sclerosis
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • selectins and their carbohydrate ligands have been considered very important targets in inflammatory conditions because of their important role in the initial contact between leukocytes and the vascular endothelium at sites of inflammation.
  • McMurray, R.W. (Seminars in Arthritis & Rheumatism 25: 215-234, 1996) teaches that selectins are carbohydrate binding adhesion molecules that share a common structural component consisting of a Ca (2+) dependent N-terminal lectin binding domain.
  • SLe(x) [NeuAca2-3Galbl-4(Fucal-3) GlcNAc-] is expressed on the cell surface of monocytes, a polymorpho nuclear leukocyte (PMNL), and on approximately 10% or peripheral blood T lymphocytes.
  • PMNL polymorpho nuclear leukocyte
  • SLe(x) is recognized by all selectin adhesion molecules.
  • SLe(x) isomeric epitope
  • SLe(a) [NeuAca2-3Galbl-3(Fucal-4) GlcNAc-]
  • E and P- selectins and possibly L-selectin (Paavonen and Renkonen, American Journal of Pathology 141(6): 1259-1261, 1992).
  • Takada et al. (BiochemBiophys Res Commun 179: 713-19, 1991) teach that SLe(a) is absent on most peripheral blood cells. Blocking selectins or their ligands with antibodies or oligosaccharides has been attempted in various animal models of inflammation.
  • results from this study showed an allegedly reduced accumulation of macrophages and lymphocytes by 60% and 90%, respectively, at 48 hours.
  • MEL- 14 mAb allegedly dramatically inhibited granulocyte influx by 80% at 6 hours and by 50% at 24 and 48 hours.
  • the invention provides compositions for reducing an immune condition.
  • the invention provides methods of administration of an agent, preferably a binding agent (such as an antibody) to an adhesion molecule, particularly an adhesion molecule involved in the extravasion of white blood cells or cancer cells from the blood into the surrounding tissue, to an animal suffering from the immune condition (or predisposed to develop the immune condition).
  • an agent preferably a binding agent (such as an antibody) to an adhesion molecule, particularly an adhesion molecule involved in the extravasion of white blood cells or cancer cells from the blood into the surrounding tissue
  • the relatively low dosage of the agent according to the compositions and methods of the invention allow for a reduction in the risk of a treated animal developing antibody-dependent cellular cytotoxicity (ADCC) or antibody-mediated toxicity.
  • ADCC antibody-dependent cellular cytotoxicity
  • the invention provides a method for reducing an immune condition in an animal suffering from the immune condition, comprising administering to the animal a therapeutically effective amount of a binding agent that specifically binds to an adhesion molecule expressed on the surface of or secreted by a blood- borne cell or expressed on the surface of or released by an endothelial cell lining a blood vessel, wherein the binding agent induces an immune response in the animal to the binding agent.
  • a binding agent that specifically binds to an adhesion molecule expressed on the surface of or secreted by a blood- borne cell or expressed on the surface of or released by an endothelial cell lining a blood vessel, wherein the binding agent induces an immune response in the animal to the binding agent.
  • the animal is a domesticated. More preferably, the animal is a mammal, such as a human.
  • the adhesion molecule is a molecule comprising a carbohydrate epitope.
  • the carbohydrate epitope is Sialyl Lewis X or Sialyl Lewis A.
  • the adhesion molecule is a selectin.
  • the selectin is P-selectin or E-selectin.
  • the immune condition is inflammation.
  • the blood-borne cell is a white blood cell.
  • the white blood cell is selected from the group consisting of a lymphocyte, a neutrophil, a polymorphonuclear leukocyte, and a monocyte.
  • the immune condition is cancer.
  • the blood-borne cell is a cancer cell.
  • the cancer cell is selected from the group consisting of a breast cancer cell, a melanoma cell, a liver cancer cell, a lung cancer cell, a leukemic cell, and a lymphoma cell.
  • the binding agent is an antibody, such as a monoclonal antibody.
  • the binding agent is a murine monoclonal antibody.
  • the immune response results in the generation of an antibody that specifically binds to the binding agent.
  • the immune response results in the generation of an antibody that specifically binds to the adhesion molecule.
  • the immune response comprises a humoral and a cellular immune response.
  • the therapeutically effective amount of the binding agent is a dosage of the binding agent that does not induce antibody-dependent cellular cytotoxicity (ADCC) in the animal. In certain embodiments, the therapeutically effective amount of the binding agent is a dosage of the binding agent that does not induce antibody-mediated toxicity in the animal. In certain embodiments, the therapeutically effective amount of the binding agent is a dosage of the binding agent that is less than about 8 mg per 30 kg body weight of the animal.
  • ADCC antibody-dependent cellular cytotoxicity
  • the invention provides a composition for reducing an immune condition in an animal suffering from the immune condition comprising a therapeutically effective amount of a binding agent that specifically binds to an adhesion molecule expressed on the surface of or secreted by a blood-borne cell or expressed on the surface of or released by an endothelial cell lining a blood vessel, wherein the binding agent induces an immune response in the animal to the binding agent.
  • the animal is a domesticated. More preferably, the animal is a mammal, such as a human.
  • the adhesion molecule is a molecule comprising a carbohydrate epitope.
  • the carbohydrate epitope is Sialyl Lewis X or Sialyl Lewis A.
  • the adhesion molecule is a selectin.
  • the selectin is P-selectin or E-selectin.
  • the immune condition is inflammation.
  • the blood-borne cell is a white blood cell.
  • the white blood cell is selected from the group consisting of a lymphocyte, a neutrophil, a polymorphonuclear leukocyte, and a monocyte.
  • the immune condition is cancer.
  • the blood-borne cell is a cancer cell.
  • the cancer cell is selected from the group consisting of a breast cancer cell, a melanoma cell, a liver cancer cell, a lung cancer cell, a leukemic cell, and a lymphoma cell.
  • the binding agent is an antibody, such as a monoclonal antibody.
  • the binding agent is a murine monoclonal antibody.
  • the immune response results in the generation of an antibody that specifically binds to the binding agent. In certain embodiments, the immune response results in the generation of an antibody that specifically binds to the adhesion molecule. In some embodiments, the immune response comprises a humoral and a cellular immune response.
  • the therapeutically effective amount of the binding agent is a dosage of the binding agent that does not induce antibody-dependent cellular cytotoxicity (ADCC) in the animal. In certain embodiments, the therapeutically effective amount of the binding agent is a dosage of the binding agent that does not induce antibody-mediated toxicity in the animal. In certain embodiments, the therapeutically effective amount of the binding agent is a dosage of the binding agent that is less than about 8 mg per 30 kg body weight of the animal. In a certain preferred embodiment of the second aspect of the invention, the composition also comprises a pharmaceutically-acceptable carrier.
  • the invention provides a therapeutic composition
  • a therapeutic composition comprising an agent that is capable of reducing inflammation and reducing cancer in an animal when administered to the animal in a therapeutically effective amount.
  • the agent is a binding agent.
  • the invention provides a method for reducing an immune condition in an animal suffering from cancer or inflammation or predisposed to suffer from cancer and inflammation, comprising administering to the animal a therapeutically effective amount of an agent that is capable of reducing inflammation and reducing cancer in an animal
  • the agent is a binding agent.
  • agents of the invention may be administered in a therapeutically effective amount to an animal suffering from inflammation, cancer, or predisposed to suffer from inflammation or cancer.
  • a therapeutically effective amount of an antibody that specifically binds to Sialyl Lewis X may be administered with a pharmaceutically-acceptable carrier (e.g., physiological sterile saline solution) via any route of administration to a patient suffering from inflammation in an attempt to alleviate any resulting disease symptom (e.g., necrosis).
  • the agent of the invention may be delivered subcutaneously, intravenously, intraperitoneally, intra-arterially, intradermally, or intra-muscularly.
  • Pharmaceutically-acceptable carriers and their formulations are well-known and generally described in, for example, Remington's Pharmaceutical Sciences (18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, 1990).
  • Figure 1 is a schematic representation illustrating the different steps in the adhesion cascade between leukocytes and endothelial cells (Dfaz-Gonza ez and Sanchez-Madrid, Immunology Today 19(4): 1-3, 1998).
  • Figure 2 is a schematic representation of the idiotypic cascade, a non-limiting theory of the present invention, showing reactivity with ligand and/or receptor by mirror image.
  • Abl the injected antibody which specifically binds to and blocks the receptor antigen on the surface of a cell (e.g., a tumor cell) is depicted as having a diamond shape.
  • the injected animal then generates an immune response to Abl resulting in the production of Ab2, depicted in this schematic as having a "Y" shape.
  • Ab2 in this schematic specifically binds to the natural ligand (depicted as a solid triangle) of the receptor.
  • the presence of Ab2 results in the production by the immune system of the animal a third antibody, Ab3, which is depicted as having a pentagonal shape that mimicks the natural ligand of the receptor.
  • Figure 3 is a schematic representation illustrating the use of the enzyme linked immunosorbent assay (ELISA) for the detection of Ab2 antibodies. Note that where the Abl antibody is a murine antibody which is injected into a rat, this ELISA will also detect rat anti- mouse antibodies (RAMA).
  • ELISA enzyme linked immunosorbent assay
  • FIG. 4 is a schematic representation illustrating the use of ELISA for the detection of Ab3. Note that the CA19-9 antigen bears the Sialyl Lewis A antigen.
  • FIG. 5 is a schematic representation showing SDS-polyacrylamide Gel Electrophoresis of purified monoclonal antibodies (10% acrylamide Tris-HCl under reducing conditions). Molecular weight (MW) is indicated at the left for the standard loaded into Lane 1.
  • the antibodies are as follows: F5 (IgM) (Lane 2; purified by Mannan binding protein affinity chromatography); 6C2C5C12 (IgM) (Lane 3; purified by the euglobulin precipitation method); ALT-4 (also called HBl; IgM) (Lane 4; purified by Mannan binding protein affinity chromatography); Alt-4 (also called HBl; IgM) (Lane 5; old sample of purified antibody); Flopc-21 (IgG3) (Lane 6; purified by protein A affinity chromatography) ALT-3 (IgG3) (Lane 7; purified by the euglobulin precipitation method); and ALT-3 (IgG3) (Lane 8;
  • Figure 6 is a schematic representation showing the binding of various representative, non-limiting murine monoclonal antibodies (7C2C5C12, F5, ALT-3, and Flopc-21 (at the indicated concentrations) to immobilized tumor antigen CA19-9, a non-limiting representative antigen of the invention.
  • Figure 7 is a schematic representation showing the binding of various representative, non-limiting murine monoclonal antibodies (7C2C5C12, F5, ALT-3, and Flopc-21 (at the indicated concentrations) to immobilized polyacrylamide-sialyl Lewis A oligosaccharide, a non- limiting representative antigen of the invention.
  • Figure 8 is a schematic representation showing the inhibitory effect of sialyl Lewis A antigen, a non- limiting representative antigen of the invention, on the binding of Iodine- 125 radiolabeled HBl (also called ALT-4) antibody, a non-limiting representative therapeutic antibody of the invention, to immobilized C A19-9 antigen, a non-limiting antigen of the invention.
  • Radio-immunoassay plates were coated with CA19-9 at 65 U/mL and incubated with radiolabeled HBl at 0.33 ⁇ g/mL (a non-saturating amount) and increasing concentrations of Sialyl Lewis A antigen (0-100 ⁇ g/ml).
  • Figure 9 is a schematic representation showing Scatchard analysis of the binding of Iodine- 125 radiolabeled HBl mAb (also called ALT-4), a non-limiting representative therapeutic monoclonal antibody of the invention, to CAI 9-9 antigen, a non-limiting representative antigen of the invention.
  • Radioimmunoassay strips were coated with CA19-9 antigen at 65 U/mL, and radiolabeled antibody was added to strips at different dilutions ranging from 0.0005 ⁇ g/mL to 10.5 ⁇ g/mL.
  • the affinity of HBl antibody to CA19-9 was determined by measuring the radioactivity of free and bound fractions in individual wells by Gamma counter.
  • Figures 10 A, 10B and 10C are schematic representations showing the inflammatory response in different rat groups for Example I as measured by the caliper method. Eight measurements were performed, and the results are represented as AUC percent change in effect.
  • Figures 10A, 10B and 10C represent the same data, illustrated as a bar graph (Fig. 10A), a column scatter (Fig. 10B), and a whicker box (Fig. 10C).
  • Figures 11 A, 11B, and 11C are schematic representations showing the inflammatory response in different rat groups for Example I as measured by the caliper method. Eight measurements were performed, and the results are represented as percent change in maximum effect. These figures represent the same data, illustrated as a bar graph (Fig. 11 A), a column scatter (Fig. 11B), and a whicker box (Fig. 11C).
  • Figures 12A, 12B, and 12C are schematic representations showing the inflammatory response in different rat groups for Example I as measured by the water displacement method. Eight measurements were performed, and the results are represented as AUC percent change in effect.
  • Figures 12 A, 12B and 12C represent the same data, illustrated as a bar graph (Fig. 12A), a column scatter (Fig. 12B), and a whicker box (Fig. 12C).
  • Figures 13A, 13B, and 13C are schematic representations showing the inflammatory response in different rat groups for Example I as measured by the water displacement method. Eight measurements were performed, and the results are represented as percent change in maximum effect. These figures represent the same data, illustrated as a bar graph (Fig. 13A), a column scatter (Fig. 13B), and a whicker box (Fig. 13C).
  • Figure 14 is a schematic representation of a column scatter graph showing the RAMA response with the inflammatory response in different rat groups for Example I.
  • Figs. 10B and 12B are reiterated below Fig. 14 to allow comparison the of the extent of the inflammatory response as measured by the caliper method (Fig. 10B) or the water displacement method (Fig. 12B) with the level of immune response (Fig. 14)
  • Figures 15A, 15B and 15C are schematic representations showing the inflammatory response in different rat groups for Example II as measured by caliper method. Eight measurements were performed, and the results are represented as AUC percent change. Figures 15A, 15B and 15C represent the same data, illustrated as a bar graph (Fig. 15A), a column scatter (Fig. 15B), and a whicker box (Fig. 15C).
  • Figures 16A, 16B, and 16C are schematic representations showing the inflammatory response in different rat groups for Example II as measured by caliper method. Eight measurements were performed, and the results are represented as percent change in maximum effect. These figures represent the same data, illustrated as a bar graph (Fig. 16A), a column scatter (Fig. 16B), and a whicker box (Fig. 16C).
  • Figures 17A, 17B, and 17C are schematic representations showing the inflammatory response in different rat groups for Example II as measured by water displacement method. Eight measurements were performed, and the results are represented as AUC percent change. These figures represent the same data, illustrated as a bar graph (Fig. 17A), a column scatter (Fig. 17B), and a whicker box (Fig. 17C).
  • Figures 18 A, 18B, and 18C are schematic representations showing the inflammatory response in different rat groups for Example II as measured by water displacement method. Eight measurements were performed, and the results are represented as percent change in maximum effect. These figures represent the same data, illustrated as a bar graph (Fig. 18A), a column scatter (Fig. 18B), and a whicker box (Fig. 18C).
  • Figure 19 is a schematic representation of a column scatter graph showing the RAMA response with the inflammatory response in different rat groups for Example II.
  • Figs. 15B and 17B are reiterated below Fig. 19 to allow comparison the of the extent of the inflammatory response as measured by the caliper method (Fig. 15B) or the water displacement method (Fig. 17B) with the level of immune response (Fig. 19)
  • Figures 20A-20H are schematic representations of line graphs showing the correlation between the RAMA response and the inflammatory response at different time measurements in the group of rats treated with HB 1 antibody, a non-limiting representative therapeutic antibody of the invention, in Example II.
  • the inflammatory response was measured at 1.5 hours (Fig. 20A), 2.5 hours (Fig. 20B), 3.5 hours (Fig. 20C), 4.5 hours (Fig. 20D), 5.5 hours (Fig. 20E), 6.5 hours (Fig. 20F), or 7.5 hours (Fig. 20G) after injection of carrageenan.
  • Fig. 20H the AUC percent change in effect was plotted against the RAMA (Ab2) response.
  • Figures 21A, 21B and 21C are schematic representations showing the inflammatory response in different rat groups for Example III as measured by the caliper method. Seven measurements were performed, and the results are represented as AUC percent change in effect. Figures 21A, 21B, and 21C represent the same data, illustrated as a bar graph (Fig. 21A), a column scatter (Fig. 21B), and a whicker box (Fig. 21C).
  • Figures 22 A, 22B, and 22C are schematic representations showing the inflammatory response in different rat groups for Example III as measured by the caliper method. Seven measurements were performed, and the results are represented as percent change in maximum effect. These figures represent the same data, illustrated as a bar graph (Fig. 22A), a column scatter (Fig. 22B), and a whicker box (Fig. 22C).
  • Figures 23 A, 23B, and 23C are schematic representations showing the inflammatory response in different rat groups for Example III as measured by the water displacement method. Seven measurements were performed, and the results are represented as AUC percent change in effect. Figures 23 A, 23B, and 23C represent the same data, illustrated as a bar graph (Fig. 23A), a column scatter (Fig. 23B), and a whicker box (Fig. 23C).
  • Figures 24 A, 24B, and 24C are schematic representations showing the inflammatory response in different rat groups for Example III as measured by the water displacement method. Seven measurements were performed, and the results are represented as percent change in maximum effect. Figures 24A, 24B and 24C represent the same data, illustrated as a bar graph (Fig. 24A), a column scatter (Fig. 24B), and a whicker box (Fig. 24C).
  • Figure 25 is a schematic representation of a column scatter graph showing the RAMA response with the inflammatory response in different rat groups for Example III.
  • Figures 2 IB and 23B are reiterated below Figure 20 to allow comparison the of the extent of the inflammatory response as measured by the caliper method (Fig. 2 IB) or the water displacement method (Fig. 23B) with the level of immune response (Fig. 25)
  • Figures 26A-26G are schematic representations showing the correlation between the RAMA response and the inflammatory response at different time measurements in the group of rats treated with HBl antibody, a non- limiting representative therapeutic antibody of the invention, in Example III.
  • the inflammatory response was measured at 1.5 hours (Fig. 26A), 2.5 hours (Fig. 26B), 3.5 hours (Fig. 26C), 4.5 hours (Fig. 26D), 5.5 hours (Fig. 26E), 6.5 hours (Fig. 26F), or 7.5 hours (Fig. 26G) after injection of carrageenan.
  • Figures 27A-27G are schematic representations showing the correlation between the
  • the inflammatory response was measured at 1.5 hours (Fig. 27 A), 2.5 hours (Fig. 27B), 3.5 hours (Fig. 27C), 4.5 hours (Fig. 27D), 5.5 hours (Fig. 27E), 6.5 hours (Fig. 27F), or 7.5 hours (Fig. 27G) after injection of carrageenan.
  • Figures 28A, 28B and 28C are schematic representations showing the inflammatory response in different rat groups for Example IV as measured by caliper method. Eight measurements were performed, and the results are represented as AUC percent change. These figures represent the same data, illustrated as a bar graph (Fig. 28A), a column scatter (Fig. 28B), and a whicker box (Fig. 28C).
  • Figures 29 A, 29B, and 29C are schematic representations showing the inflammatory response in different rat groups for Example IV as measured by caliper method. Eight measurements were performed, and the results are represented as percent change in maximum effect. These figures represent the same data, illustrated as a bar graph (Fig. 29 A), a column scatter (Fig. 29B), and a whicker box (Fig. 29C).
  • Figures 30 A, 30B, and 30C are schematic representations showing the inflammatory response in different rat groups for Example IV as measured by water displacement method. Eight measurements were performed, and the results are represented as AUC percent change. These figures represent the same data, illustrated as a bar graph (Fig. 30A), a column scatter (Fig. 30B), and a whicker box (Fig. 30C).
  • Figures 31 A, 3 IB, and 31C are schematic representations showing the inflammatory response in different rat groups for Example IV as measured by water displacement method. Eight measurements were performed, and the results are represented as percent change in maximum effect. These figures represent the same data, illustrated as a bar graph (Fig. 31 A), a column scatter (Fig. 3 IB), and a whicker box (Fig. 31C).
  • Figure 32 is a schematic representation of a column scatter graph showing the RAMA (Ab2) response with the inflammatory response in different rat groups for Example IV.
  • Figs. 28B and 30B are reiterated below Fig. 32 to allow comparison the of the extent of the inflammatory response as measured by the caliper method (Fig. 28B) or the water displacement method (Fig. 30B) with the level of immune response (Fig. 32)
  • Figures 33A, 33B and 33C are schematic representations showing the inflammatory response in different rat groups for Example V as measured by caliper method. Eight measurements were performed, and the results are represented as AUC percent change. These figures represent the same data, illustrated as a bar graph (Fig. 33A), a column scatter (Fig. 33B), and a whicker box (Fig. 33C).
  • Figures 34 A, 34B, and 34C are schematic representations showing the inflammatory response in different rat groups for Example V as measured by caliper method. Eight measurements were performed, and the results are represented as percent change in maximum effect. These figures represent the same data, illustrated as a bar graph (Fig. 34A), a column scatter (Fig. 34B), and a whicker box (Fig. 34C).
  • Figures 35A, 35B, and 35C are schematic representations showing the inflammatory response in different rat groups for Example V as measured by water displacement method. Eight measurements were performed, and the results are represented as AUC percent change. These figures represent the same data, illustrated as a bar graph (Fig. 35A), a column scatter (Fig. 35B), and a whicker box (Fig. 35C).
  • Figures 36A, 36B, and 36C are schematic representations showing the inflammatory response in different rat groups for Example V as measured by water displacement method. Eight measurements were performed, and the results are represented as percent change in maximum effect. These figures represent the same data, illustrated as a bar graph (Fig. 36A), a column scatter (Fig. 36B), and a whicker box (Fig. 36C).
  • Figure 37 is a schematic representation of a column scatter graph showing the RAMA (Ab2) response with the inflammatory response in different rat groups for Example V.
  • Figs. 33B and 35B are reiterated below Figure 37 to allow comparison the of the extent of the inflammatory response as measured by the caliper method (Fig. 33B) or the water displacement method (Fig. 35B) with the level of immune response (Fig. 37)
  • Figures 38A-38H are schematic representations of histograms showing the binding of therapeutic monoclonal antibodies to human neutrophils as determined by single step indirect immunofluorescence flow cytometry (FACS).
  • Figs. 38A and 38B colo 205 cells, which are known to express the Sialyl Lewis A epitope, were used as a positive control.
  • Figs. 38C- 38H are neutrophils stained with control (no primary antibody) (Fig. 38C), ALT-4 IgM antibody (Fig. 38D), ALT-3 IgG3 antibody (Fig. 38E), 7C2C5C12 IgM antibody (Fig. 38F), F5 IgM antibody (Fig. 38G), and Flopc-21 IgG3 antibody (Fig. 38H).
  • Figures 39A and 39B are schematic representations showing the instrumental setup and the schematic diagram of the in vitro inhibition of leukocyte rolling and adhesion in microchips channels coated with selectins.
  • Figure 40 is a schematic representation of a bar graph showing the inhibition of human polymorphonuclear leukocyte (PMNL) rolling in microchip channels coated with recombinant E-selectin, a non- limiting representative adhesion molecule of the invention, by the various indicated agents.
  • the graph bars show the percentage of human PMNL rolling.
  • Figure 41 is a schematic representation showing the inhibition of human PMNL rolling in microchip channels coated with recombinant P-selectin, a non-limiting representative adhesion molecule of the invention, by the various indicated agents.
  • the graph bars show the percentage of human PMNL rolling.
  • the present inventors have developed methods and therapeutic compositions for reducing inflammation.
  • the methods and therapeutic compositions of the invention are useful as analytical tools and as therapeutic tools.
  • the invention also provides methods and therapeutic compositions which may be manipulated and fine-tuned to fit the condition(s) to be treated while producing fewer side effects.
  • Standard reference works setting forth the general principles of the technology described herein include Ausubel et al, Current Protocols in Molecular Biology. John Wiley & Sons, New York, 1994; Sambrook et al, Molecular Cloning: A Laboratory Manual. 2d Ed., Cold Spring Harbor Laboratory Press, Plainview, New York, 1989; Kaufman et al. (Eds.), Handbook of Molecular and Cellular Methods in Biology and Medicine.
  • the invention provides a method for reducing an immune condition in an animal suffering from the immune condition or predisposed to suffer from the immune condition, comprising administering to the animal a therapeutically effective amount of a binding agent that specifically binds to an adhesion molecule expressed on the surface of or secreted by a blood-borne cell or expressed on the surface of or released by an endothelial cell lining a blood vessel, wherein the binding agent induces an immune response in the animal to the binding agent.
  • the binding agent is administered with a pharmaceutically-acceptable carrier.
  • the invention provides a therapeutic composition for reducing an immune condition in an animal suffering from the immune condition or predisposed to suffer from the immune condition, comprising a therapeutically effective amount of a binding agent that specifically binds to an adhesion molecule expressed on the surface of or secreted by a blood-borne cell or expressed on the surface of or released by an endothelial cell lining a blood vessel, wherein the binding agent induces an immune response in the animal to the binding agent.
  • the composition further comprises a pharmaceutically-acceptable carrier.
  • the invention provides a therapeutic composition comprising an agent that is capable of reducing inflammation and reducing cancer in an animal when administered to the animal in a therapeutically effective amount.
  • the composition further comprises a pharmaceutically-acceptable carrier.
  • the invention provides a method for reducing an immune condition in an animal suffering from cancer or inflammation or predisposed to suffer from cancer and inflammation, comprising administering to the animal a therapeutically effective amount of an agent that is capable of reducing inflammation and reducing cancer in an animal.
  • agent is meant a molecule of any size which may be naturally occurring or synthetic.
  • an agent includes, without limitation, a chemical, a protein, a carbohydrate, a lipid, a nucleic acid, an acid, a base, a synthetic polymer, and a resin.
  • immune condition is meant cancer and/or inflammation.
  • predisposed to suffer from an immune condition is meant an animal that has a genetically or environmentally caused predisposition to suffer from an immune condition, but is not yet suffering from the immune condition.
  • the animal may have a certain type of MHC haplotype rendering it more susceptible to develop an immune condition, such as rheumatoid arthritis, but has not yet developed the condition.
  • Another example is an animal which has been exposed to intense sunlight, but has not yet developed a sunburn.
  • Another example is an animal which has been exposed to a carcinogen, but has not yet developed cancer.
  • the immune condition is cancer.
  • cancer is meant the excessive growth of an abnormal cell in an animal resulting in the migration of metastatic cells via the blood stream from the original anatomical site of the original abnormal cell to other parts in the body.
  • detection of a secondary site of cancer caused by the migration of a metastatic cell is not required for an animal to be diagnosed as suffering from cancer. Rather, an animal (e.g., a human) may have a cell growth biopsied. Upon a physician's inspection of the biopsied tissue and an assessment that the biopsied cells are likely malignant (i.e., capable of metastasis), the animal may be treated in accordance with the invention.
  • One non-limiting cancer of the invention is melanoma.
  • Other non-limiting cancers of the invention include breast cancer, colorectal cancer, bladder cancer, leukemia, lymphoma, cervix cancer, prostate cancer, testicular cancer, Uver cancer, lung cancer, ovarian cancer, and pancreatic cancer.
  • the immune condition is inflammation.
  • inflammation is meant a local response to cellular injury that is marked by any or all of capillary dilatation, leukocyte infiltration, redness, heat, and pain and that serves as a mechanism initiating the elimination of the inflammation- inducing antigen and of damaged tissue.
  • the term includes acute inflammation, chronic inflammation, and excessive inflammation, such as that leading to inflammatory disease.
  • Acute inflammation exhibits a rapid onset and is of short duration.
  • the characteristic signs of an acute localized inflammatory response generally include swelling, redness, heat, pain and loss of function (Kuby, J., Immunology, 3 rd ed., W.H. Freeman, New York, pages 361-377, 1997).
  • Infiltration of leukocytes into the tissue peaks within the first 6 hours of an acute inflammatory response, and most of them disappear from the inflamed area within 24- 48 hours (Kuby, J., supra).
  • Chronic inflammation develops during persistence of an antigen due to infection or various pathologic conditions.
  • the characteristic hallmark of chronic inflammation is the formation of granuloma.
  • an immune condition in an animal suffering from the immune condition (or predisposed to suffer from the immune condition (e.g., an animal exposed to the sun which has not yet developed a sunburn; an animal who has been exposed to a carcinogen which has not yet developed cancer; or an animal genetically predisposed to develop an immune condition which has not yet developed the immune condition)) when administered to that animal.
  • the alleviation of symptoms and/or prevention of onset is reduced in a treated animal as compared to the immune condition in an animal to which an agent of the invention has not been administered.
  • the reduction in the immune condition in an animal to which has been administered a binding agent of the invention is at least 10% lower as compared to an untreated animal; more preferably, the reduction is at least 25% lower; still more preferably, the reduction is at least 50% lower; even more preferably, the reduction is at least 75% lower; and most preferably, the reduction in the immune condition in an animal to which has been administered a binding agent of the invention is at least 100% lower as compared to an untreated animal (i.e., an animal to which a binding agent of the invention has not been administered).
  • Methods for determining the amount of an immune condition in an animal, where the condition is inflammation include, without limitation, visual detection of redness and measurement of swelling by the caliper method or by the water displacement method described below.
  • Methods for determining the amount of an immune condition in an animal, where the condition is cancer include, without limitation, detection of the presence of a metastatic cancer cell and detection of cancer cell-induced angiogenesis.
  • the animal suffering from an immune condition is preferably a domesticated animal including, without limitation, domesticated fowl (e.g., ducks, geese, chickens, turkeys, Cornish hens, and ostriches), domesticated livestock (e.g., cattle, llamas, elephants, camels, pigs, and sheep), domesticated pets (e.g., horses, cats, dogs, ferrets, hamsters, and guinea pigs); and laboratory animals (e.g., primates (e.g., baboons, Rhesus monkeys) and rodents (e.g., mice, rats)).
  • domesticated fowl e.g., ducks, geese, chickens, turkeys, Cornish hens, and ostriches
  • domesticated livestock e.g., cattle, llamas, elephants, camels, pigs, and sheep
  • domesticated pets e.g., horses, cats, dogs,
  • the animal suffering from the immune condition is a mammal. In a preferred embodiment of the first and second aspects of the invention, the animal suffering from the immune condition is a human.
  • inces an immune response is meant that a cellular and/or humoral immune response is induced or increased in an animal to which has been administered a binding agent of the invention, such that the immune response is directed toward the administered binding agent.
  • the immune response comprises both a humoral and a cellular immune response.
  • the presence of such an immune response can be detected by any of the well known immune response detection methods including, without limitation, detection of the presence of a T cell (e.g., a. cytotoxic T cell or helper T cell) whose T cell receptor specifically binds to the binding agent and/or to the adhesion molecule (in the context of the major histocompatibility complex), detection of the presence of a B cell whose B cell receptor (i. e. , surface-expressed IgM) specifically binds to the binding agent and/or to the adhesion molecule, and detection of the presence of an antibody (of any serotype) which specifically binds to the binding agent and/or to the adhesion molecule.
  • a T cell e.g., a. cytotoxic T cell or helper T cell
  • B cell receptor i. e. , surface-expressed IgM
  • an antibody of any serotype
  • the immune response results in the generation of an antibody that specifically binds to the binding agent.
  • an antibody that specifically binds to the binding agent is generated as a result of an immune response in the treated animal to the administered binding agent.
  • One non- limiting theory explaining such an immune response is the idiotypic cascade first described by Jerne (Jerne, NK., Annals of Immunology 125C: 373-389, 1974). This theory, as illustrated for an idiiotypic cascade of antibodies, where the first antibody specifically binds to a tumor associated antigen, is illustrated on Figure 2 (Kingsbury et al, Leukemia 12: 982-991, 1998).
  • an administered binding agent e.g., an antibody
  • Abl reacts with epitopic determinants on the adhesion molecule.
  • the animal to which has been administered a binding agent of the invention generates an immune response to that binding agent.
  • the immune response comprises antibodies that bind to the binding agent, these antibodies are designated Ab2.
  • Some Ab2 antibodies namely the Ab2 ⁇ , recognize the adhesion molecule- binding site of Abl and resemble the original adhesion molecule epitope recognized by Abl.
  • Other Ab2 antibodies recognize sites present elsewhere on the variable region, and may (Ab2 ⁇ ) or may not (Ab2 ) interfere with the binding site (Steinitz et al, Journal of Immunology 141: 3516-3522, 1988).
  • each Ab2 might serve as the antigen for an additional antibody (Ab3).
  • This Ab3 antibody like the original binding agent (Abl) specifically binds to the adhesion molecule to which Abl specifically bound.
  • the immune response results in the generation of an antibody that specifically binds to the adhesion molecule.
  • a selectin e.g., E-selectin
  • Absl specific binding agent
  • the first mechanism of this non-Umiting example is dependent on the direct effect of Abl that will act to block the selectin on either the white blood ceU surface or endotheUal ceU membrane, and make it unavailable for binding by a selectin ligand (e.g., sialyl Lewis A)- bearing cell.
  • a selectin ligand e.g., sialyl Lewis A
  • Ab2 anti-idiotypic
  • Ab3 anti-anti-idiotypic
  • a carbohydrate selectin ligand e.g., sialyl Lewis A
  • specific binding agent e.g., melanoma cell
  • rolUng metastatic cancer cell
  • the first mechanism is dependent on the direct effect of Abl that wiU act to block selectin ligand on either white blood ceU surface or endotheUal ceU membrane, and make it unavailable for binding by a selectin-bearing cell.
  • the direct effect of Abl would be direct but short- term.
  • the second mechanism is dependent on the induction of anti-idiotypic (Ab2) antibodies which mimic the selectin Ugand and anti-anti-idiotypic (Ab3) antibodies which mimic the Abl antibody that specificaUy bound to the selectin Ugand.
  • Ab2 would mimic a selectin Ugand, thus inhibiting cancer ceU rolling on endotheUal cells and extravasation through its binding to selectin molecules.
  • Ab3 will inhibit cancer cell rolling and extravasaion by competing with the naturaUy existing selectin molecule in binding to carbohydrate selectin Ugand.
  • Ab2 and Ab3 i.e. , the indirect effect of Abl
  • the effect of Ab2 and Ab3 would likely to be more efficient than the direct effect of Abl for the inhibition of leukocyte or cancer ceU migration, since Ab2 and Ab3 antibodies are produced by the treated animal and remain in the circulation for a considerable period of time (usuaUy several months).
  • the term "therapeutically effective amount” is used to denote a dosage of binding agent effective to reduce an immune condition in the treated animal and/or a dosage of binding agent effective to induce an immune response to the binding agent in the treated animal.
  • such administration should be intravenous, intra-arterial, subcutaneous, parenteral, or transdermal.
  • a binding agent of the invention is administered at a dosage of less than about 8 mg per 30 kg body weight of the animal to be treated; preferably less than about 5 mg per 30 kg body weight; preferably less than about 3 mg per 30 kg body weight; still more preferably from about 0.5 mg to about 2 mg per 30 kg body weight; still more preferably from about 0.5 mg to about 1.5 mg per 30 kg body weight; and most preferably, a binding agent of the invention is administered at a dosage of about 1 mg per 30 kg body weight of the animal to be treated.
  • the therapeutically effective amount of a binding agent is a dosage of the binding agent that, when administered to an animal, does not induce antibody-dependent cellular cytotoxicity (ADCC) in the treated animal.
  • the therapeutically effective amount of a binding agent is a dosage of the binding agent that is the maximum amount of binding agent that, when administered to an animal, does not induce antibody-dependent cellular cytotoxicity (ADCC) in the treated animal.
  • ADCC may be assessed by incubating 51 Cr-labeled tumor ceUs with a binding agent according to the invention and adding fresh human peripheral blood mononuclear ceUs (PBMCs), foUowed by incubation for four hours and then measurement of specific lysis (as determined by 51 Cr release). ADCC is absent if specific lysis is less than 15%.
  • PBMCs peripheral blood mononuclear ceUs
  • the therapeutically effective amount of a binding agent is a dosage of the binding agent that, when administered to an animal, does not induce antibody-mediated toxicity in the treated animal.
  • the therapeutically effective amount of a binding agent is a dosage of the binding agent that is the maximum amount of binding agent that, when administered to an animal, does not induce antibody-mediated toxicity in the treated animal.
  • antibody-mediated toxicity is meant cUnical toxicity, such as abnormal serum chemistries, impaired renal function, signs and symptoms of serum sickness, or anaphylaxis.
  • Antibody-mediated toxicity is well-known to immunologist and described, for example, in Abbas et al, Cellular and Molecular Immunology, 3 rd ed., W. B. Saunders Co., Philadelphia, PA 1997 (see particularly pages 425- 434).
  • a single such dosage will therapeuticaUy treat the animal.
  • the treatment may be ongoing, e.g. , administration of a dosage of a binding agent four times a year for three or more years.
  • the administration of a dosage of a binding agent will be performed one injection per month for about three months to years.
  • blood-borne ceU means any cell that can be carried by the blood stream.
  • the definition includes bone marrow derived cells such as red blood cells and white blood ceUs which include, without Umitation, lymphocytes (both T and B), natural killer ceUs, neutrophils (also caUed polymorphonuclear leukocytes or PMNL), eosinophils, basophils, monocytes, megakaryocytes, and platelets.
  • lymphocytes both T and B
  • neutrophils also caUed polymorphonuclear leukocytes or PMNL
  • eosinophils basophils
  • monocytes monocytes
  • megakaryocytes megakaryocytes
  • platelets also included as blood-borne ceUs are metastatic cancer cells which have migrated (or are attempting to migrate) from the site of the original abnormal cell.
  • the blood-borne ceU is a white blood ceU.
  • the white blood ceU is, without Umitation, a lymphocyte, a platelet, a granulocyte (i.e., a a neutrophil, eosinophil, or a basophil), or a monocyte.
  • the blood-borne cell is a cancer cell. Any cancer ceU that is carried by the blood stream is included in the invention.
  • the cancer ceU is, without Umitation, a breast cancer ceU, a melanoma cell, a Uver cancer cell, a lung cancer ceU, a leukemic cell, and a lymphoma cell.
  • adheresion molecule is meant a molecule which is expressed on the surface of or released by either a blood-borne ceU or a ceU (e.g., an endotheUal cell) that lines a blood vessel and which can form an association with another adhesion molecule expressed on the surface of either a blood-borne cell or a ceU Uning a blood vessel.
  • released by means an adhesion molecule that is either cleaved from the cell surface of the indicated ceU, secreted by the indicated ceU, or released from the indicated ceU upon lysis of that ceU.
  • an adhesion molecule of the invention is a molecule expressed on the surface of or released by either on a blood-borne ceU or an endothelial ceU lining a blood vessel that forms an association with a second adhesion molecule on a blood-borne ceU or an endotheUal cell lining a blood vessel, such that association formed is between a blood-borne ceU-expressed or released adhesion molecule and an endotheUal cell-expressed or released adhesion molecule.
  • an adhesion molecule of the invention is one that is involved in the extravasion of white blood ceUs or cancer cells from the blood into the surrounding tissue.
  • adhesion molecule excludes the B ceU receptor, the T ceU receptor, and/or the signaUing molecules comprising the B cell receptor complex (e.g. , Ig- ⁇ ) and the T ceU receptor complex (e.g., CD3).
  • the site on an adhesion molecule of the invention which is specifically bound by the binding agent is called an "epitope.”
  • Non-Umiting examples of adhesion molecules include the molecules which are members of the integrin family of proteins including, without Umitation, LFA-1 (CDlla/CD18), VLA-4 (CD49d/CD29), and Mac-1 (CDllb/CD18).
  • Adhesion molecules also include those molecules which are members of the immunoglobulin gene family of proteins including, without Umitation, ICAM-1, B7, CD4, and CD28. Adhesion molecules also include those molecules which are members of the selectin family of molecules including, without Umitation, P-selectin ELAM-1, CD62E), E-selectin (ELAM-1, CD62E) and L-selectin (CD62L) (Bevilacqua and Nelson, Journal of Clinical Investigation 91: 370- 387, 1993. Thus, in certain embodiments of the first and second aspects of the invention, the adhesion molecule is a selectin, such as P-selectin, E-selectin, or L-selectin.
  • a selectin such as P-selectin, E-selectin, or L-selectin.
  • Adhesion molecules of the invention also include glycoproteins, such as PSGL-1 (P- selectin glycoprotein Ugand 1) and molecules (e.g., mucins or mucoproteins) comprising carbohydrate epitopes, including the Sialyl Lewis A (SLe(a)) carbohydrate epitope and the Sialyl Lewis X (SLe(x)) carbohydrate epitope.
  • the adhesion molecule of the invention is a molecule comprising a carbohydrate epitope, such as a molecule comprising a Sialyl Lewis A epitope.
  • a molecule comprising a carbohydrate epitope is specifically bound by a selectin molecule.
  • the agent is a binding agent that specifically binds to an adhesion molecule.
  • binding agent is meant a molecule or macromolecule that specifically binds to an adhesion molecule either in water, under physiological conditions (e.g., in an animal's body), or under conditions which approximate physiological conditions with respect to ionic strength, e.g., 140 mM NaCl, 5 mM MgCl 2 .
  • specifically binds is meant a binding agent of the invention recognizes and forms a covalent association or, preferably, a non-covalent association with an adhesion molecule of the invention, but does not substantially recognize and form a covalent or non-covalent association with other molecules in a sample, e.g., proteins that are not adhesion molecules.
  • an adhesion molecule of the invention bound by a binding agent of the invention that specifically binds to that adhesion molecule is said to be "specifically bound” by that binding agent.
  • a binding agent of the invention that specifically binds to an adhesion molecule of the invention forms an association with that adhesion molecule with an affinity of at least 10 6 M "1 , more preferably, at least 10 7 M " ⁇ even more preferably, at least 10 8 M _1 , and most preferably, at least 10 9 NT l .
  • a binding agent of the invention is a peptide, a pe tido mimetic, or a carbohydrate.
  • peptide is meant a molecule comprised of a Unear array of two or more amino acid residues connected to each other in the Unear array by peptide bonds.
  • peptidomimetic is meant a non-peptide molecule that mimics the structure and/or function of a peptide.
  • carbohydrate or oligosaccharide is meant a molecule comprising sugar residues.
  • Non-Umiting binding agents that are carbohydrates include SLe(x) oUgosaccharide (Buerke et al, Journal of Clinical Investigation 93: 1140- 1148, 1994; Lefer et al, Circulation 90: 2390-2401, 1994), and SLe(a) oUgosaccharide.
  • Non-Umiting binding agents of the invention include antibodies.
  • the binding agent is an antibody, such as a monoclonal antibody.
  • the binding agent is a murine monoclonal antibody.
  • the monoclonal antibody of the invention HBl (also caUed ALT-4), is a murine IgM monoclonal antibody (mAb) secreted by the HBl hybridoma cell line.
  • HBl specificaUy binds to the tumor marker, CA19-9, which comprises the carbohydrate epitope Sialyl Lewis A (Mukae et al, Am. Rev. Respir. Dis.
  • HBl hybridoma ceUs were maintained in standard media (RPMI-1640 supplemented with 2 mM L-glutamine, 50 U/ml penicillin and 50 U/ml streptomycin, 10% v/v fetal bovine serum (FBS)).
  • HB2 also caUed ALT-3
  • IgG3 murine monoclonal antibody secreted by the HB2 hybridoma cell line HB2 specifically binds to the tumor marker CA19-9, and has also been shown to specificaUy bind to sialyl Lewis A.
  • HB2 hybridoma ceUs were maintained in standard media.
  • binding agents that are antibodies include PB1.3, an anti- P selectin monoclonal antibody (Weiser et al, Shock 5(6): 402-407, 1996; Yamada et al, European Journal of Pharmacology 346: 222-224, 1998); ARP2-41 , an anti-rat-P-selectin mAb (Tojo et al, Glycobiology 6(4): 463-9, 1996; Ohnishi et al.
  • P91090M and clone no. 5D8.8.12 (PL2) (Catalog No. P91209M) and the various anti-SLe(a) mAb commerciaUy available from Biodesign Intl., Saco, ME (e.g.. clone no. 241 (Catalog No. M37241B); clone no. 59 (95-330-07) (Catalog No. M37059M); clone no. 62 (Catalog No. M37062M); clone no. 67 (Catalog No. M37067M); clone no. 192 (Catalog No. M37192M); and clone no. 239 (Catalog No. M37239M)).
  • clone no. 241 Catalog No. M37241B
  • clone no. 59 95-330-07
  • clone no. 62 Catalog No. M37062M
  • binding agents of the invention include the anti-human E- selectin and anti-human P-selectin antibodies commerciaUy available from R & D Systems (MinneapoUs, MN, USA).
  • the binding agents of the invention comprises a complementarity determining region (CDR) of an antibody or a T ceU receptor that specifically binds to an adhesion molecule of the invention.
  • CDR complementarity determining region
  • CDR complementarity determining region
  • CDR complementarity determining region of a T ceU receptor
  • a portion of a T ceU receptor that specifically binds to an epitope e.g., in context of the major histocompatibUity complex
  • an epitope e.g., in context of the major histocompatibUity complex
  • the CDR of the invention is derived from a human or a murine antibody.
  • peptidomimetics having binding characteristics similar or superior to such complementarity determining regions, wherein such peptidomimetics are included as binding agents of the invention.
  • Peptidomimetics are well known and described, for example, in HorweU, D.C., Bioorg. Med. Chem. 4: 1573-1576, 1996; Liskamp et al, Reel Trav. Chim. Pays-Bas. 1: 113, 1994; Gante et al., Angew. Chem. Int. Ed. Engl 33: 1699, 1994; and Seebach et al, Helv. Chim. Acta 79: 913, 1996.
  • the binding agents of the present invention are preferably purified binding agents.
  • purified is meant that a binding agent is at least 60%, preferably at least 75%, even more preferably at least 90%, and most preferably at least 95% free from contaminants, such as endotoxins.
  • the binding agents of the invention are monoclonal antibodies
  • the antibodies were purified from ascites.
  • ascites preparation adult mice (age 6 weeks) were primed by injecting 0.5 ml of pristane (2,6,10,14-tetramethyldecanoic acid) into the peritoneum (i.p.). Two weeks later the mice were injected i.p., with 5 x 10 6 hybridoma ceUs (e.g., HBl or HB2) resuspended in 0.5 ml of PBS.
  • Ascetic fluid was tapped from mice 2 to 3 times in due time, and the coUected fluid was incubated for 1 hour at room temperature and transferred to 4°C overnight. The fluid was centrifuged at 2000 rpm for 15 minutes and the supernatant was stored at -20 °C. The monoclonal antibodies were then purified and concentrated.
  • the foUowing purification methods are non- limiting examples of methods that can be used for purifying antibodies from ascites. It should be noted that these methods can also be used to purify and concentrate antibodies from the supernatant of hybridoma ceUs grown in vitro under standard tissue culture conditions.
  • the ascetic fluid containing the IgM was dialyzed against binding buffer (10 Mm Tris, 1.25 M NaCl, 20 mM CaCl 2 , and 0.02 % Sodium azide (NaN 3 ) at a pH of 7.4), after which the ascetic sample was diluted 1: 1 v/v with binding buffer, and filtered with 0.22 mm filter.
  • binding buffer 10 Mm Tris, 1.25 M NaCl, 20 mM CaCl 2 , and 0.02 % Sodium azide (NaN 3 ) at a pH of 7.4
  • the MBP column (commercially available from Pierce, Rockford, IL, USA) was prewashed with 2 column volume (20 ml) of elution buffer (10 mM Tris, 1.25 M NaCl, 2mM EDTA and 0.02% NaN 3 ), and then equilibrated with 4 column volume (40 ml) of binding buffer.
  • the ascetic fluid (maximum 1.5 ml/ 5 ml of gel) was appUed to the column, allowed to completely enter the gel, and incubated at 4 °C for 30 minutes.
  • the column was washed with 9 column volumes of the binding buffer to remove the unbound proteins. The wash was monitored for the presence of proteins by measuring the absorbance at 280 nm, using the binding buffer as a reference.
  • the column was removed from the cold and incubated with the elution buffer at room temperature for 1 hour, after which the column was washed with the elution buffer, and minimum of 14 fractions were coUected each with a volume of 3 ml.
  • the elution of IgM was monitored for the presence of proteins by spectrophotometer at 280 nm using the elution buffer as a reference.
  • the column was washed with 2 column volume of deionized water and then by 2 column volumes of 4 °C binding buffer and stored at 4 °C.
  • the euglobulin precipitation method is a non-chromatographic method used for the purification of both murine IgG 3 and IgM mAbs, which takes advantage of their euglobulin properties. This method was performed as essentially described by (Garcia-Gonzalez et al., Journal of Immunological Methods 111: 17-23, 1988). Briefly, CaCl 2 was added to the ascetic fluid (final concentration, 25 mM) to generate fibrin formation. When the clot was formed, it was removed by paper filtration.
  • the filtered ascetic fluid was dialyzed for 2 hours at 20 °C (IgG3), or for 15 hours at 4 °C (IgM) against lOOx volume of demineralized water (pH 5.5).
  • the ascetic fluid was centrifuged in a BECKMAN L8-55 ultracentrifuge at 22,000 x g for 30 minutes and the precipitate was recovered and suspended in IM NaCl/0.1 M Tris-HCl pH 8. Dialysis and precipitation were repeated twice. Purified peUet with a high lipid content was mixed with 1.7 M NaCl and centrifuged for 3 hours at 27,000 x g.
  • Protein A affinity chromatography purification a 5 ml Protein A column (commerciaUy available from Pierce, Rockford, IL, USA) was washed with 2 column volumes of elution buffer (0.1 mM Glycine, ImM NaCl, 0.001 % tween 20, pH 4). Then the column was equilibrated with 5 column volumes of binding buffer (50 mM Tris-HCl, ImM NaCl, 0.001 % tween 20, pH 8).
  • the ascetic sample containing IgG 3 antibody was diluted 1:1 v/v with binding buffer and loaded to the column at a rate of 0.75 ml/ minute, then the column was washed with 50 ml of binding buffer, at a rate of 1.5 ml / minute.
  • the collected fractions containing unbound proteins were checked by spectrophotometer at 280 nm, using the binding buffer as a reference.
  • the column was washed with 15 ml of elution buffer. Then the eluted fractions were collected at a rate of 1 ml /minute and adjusted to a pH of 7 using 50 mM Tris-HCl.
  • Elution of bound proteins was monitored by spectrophotometer at 280 nm, using the elution buffer as a reference.
  • a 10 ml Detoxi-Gel column (commercially available from Pierce, Rockford, IL, USA) was regenerated by washing the gel with 5 column volumes of 1% sodium deoxycholate. Then the column was washed with 3 column volumes of pyrogen-free water. The purified and concentrated immunoglobuUn was loaded to the column, and then the sample was coUected by gravity flow using PBS as elution buffer.
  • the ultrafilter YM-30 membrane (MW cutoff 30,000 Dalton) (commercially available from Fisher Scientific, Nepean, ON, Canada) was floated with distilled water for one hour, changing the water three times. Then the membrane was mounted in an ultrafiltration cell and rinsed with distilled water at 20 psi (3.7 atm) for at least 5 minutes. The samples of purified Igs were loaded in the cell, and the sample flow through the ultrafilter membrane was operated under a maximum pressure of 40 psi (4.7 atm). The antibody solution was concentrated approximately lOx and then washed at least three time using PBS.
  • MW cutoff 30,000 Dalton commercially available from Fisher Scientific, Nepean, ON, Canada
  • control F5 antibody is murine IgM monoclonal antibody secreted by the F5 hybridoma cell line (commercially available from the American Type Culture Collection, Maryland, USA).
  • Control 7C2C5C12 antibody is a murine IgM monoclonal antibody which specifically binds to Trichinella spiralis and which is secreted by the 7C2C5C12 hybridoma cell line (commerciaUy available from the American Type Culture CoUection, Maryland, USA).
  • F5 hybridoma cells and 7C2C5C12 hybridoma cells were maintained in standard media (RPMI-1640 supplemented with 2 mM L-glutamine, 50 U/ml penicillin and 50 U/ml streptomycin, 10% v/v fetal bovine serum (FBS)).
  • standard media RPMI-1640 supplemented with 2 mM L-glutamine, 50 U/ml penicillin and 50 U/ml streptomycin, 10% v/v fetal bovine serum (FBS)).
  • microtiter strips were coated with 100 ml of PBS containing either 2.5 ⁇ g/ml of goat anti-mouse Ig (whole molecule), 75 U/ml of CA19-9 antigen (commercially available from Altarex Corp., Edmonton, Alberta, Canada), or 2.5 ⁇ g ml of polyacrylamide-SLe(a) (commerciaUy available from Altarex Corp., Edmonton, Alberta, Canada) and incubated overnight at 4 °C.
  • the coating solution was discarded and 150 ⁇ l of blocking buffer (2% sucrose, 2% BSA, 0.06% thimerosal in PBS) was added to each well in order to block the non-specific binding sites on the weUs. After one hour of incubation at room temperature the plates were washed three times with PBST. 100 ⁇ l of binding buffer containing mAbs was added in different dilutions. AU wells were incubated for 1 hour at room temperature, followed by three washings with PBST (PBS containing 0.1% Tween-20, pH 7.1)).
  • blocking buffer 2% sucrose, 2% BSA, 0.06% thimerosal in PBS
  • binding buffer containing goat anti- mo use Ig (H+L) -HRP labeled (commerciaUy available from Southern Biotechnology Associates Inc., Birmingham, AL, USA) was added to each well at a dilution of 1/5000 v/v, incubated for 1 hour, and foUowed by three washings with PBST.
  • the activity of bound mAbs was determined by adding a 100 ⁇ l of ABTS (Peroxidase solution B and ABTS peroxidase substrate, 1:1 v/v dilution) to each well (ABTS Peroxidase solution and ABTS peroxidase substrate commercially available from LifeTechnologies GIBCO BRL (BurUngton, Ontario).
  • ABTS Peroxidase solution and ABTS peroxidase substrate commercially available from LifeTechnologies GIBCO BRL (BurUngton, Ontario).
  • the optical density was measured at dual wavelength, 405 and 492 nm using an ELISA reader (T
  • SDS-polyacrylamide gel electrophoresis was used to check the purity of each of the purified mAbs.
  • Ab samples were dUuted 1:4 v/v with SDS-PAGE sample buffer (0.5 M Tris-HCl at pH 6.8, 20% glycerol, 10% w/v SDS, 10% b- mercaptoethanol and 0.5 % bromphenol blue) and heated for 4 minutes at 95 °C.
  • the Tris-HCL ready gel sandwich (10% acrylamide) (commerciaUy available from BioRad, Hercules, CA, USA) was inserted into the Mini-PROTEAN II ceU clamp assembly and aUgned properly.
  • the gel sandwich was attached to the inner core of the Mini-PROTEAN II ceU (commercially available from BioRad).
  • the upper and the lower buffer chambers were filled with approximately 200 ml of lx running buffer (0.3% Tris base, 1.45% glycine, 0.1% SDS, pH 8.3).
  • 6 ⁇ l of molecular weight markers in sample buffer was applied to one of the wells, while the antibody samples, each with a volume of 30 ⁇ l (1.5-5 ⁇ g of antibody) were applied to the rest of the weUs in the gel.
  • the core assembly was inserted into the lower buffer tank and the gel was run at 200 V constant voltage for approximately 45 minutes.
  • the gel was then removed from the gel sandwich, immersed in staining solution (10% acetic acid, 0.025% Coomassie BlueG-250, methanol 40%) for 1.5 hour, and destained in destaining solution (10% acetic acid, 40% methanol) untU the desired destaining was reached.
  • staining solution (10% acetic acid, 0.025% Coomassie BlueG-250, methanol 40%) for 1.5 hour, and destained in destaining solution (10% acetic acid, 40% methanol) untU the desired destaining was reached.
  • Antibodies of the invention were purified and then resolved by SDS-polyacrylamide gel electrophoresis. As shown in Figure 5, antibodies purified by the mannan binding protein affinity chromatograph (lanes 2 and 4), by the euglobin precipitation method (lanes 3 and 7), and by protein A affinity chromatography (lane 6) are "purified" for the purposes of the present invention (compare to lanes 5 and 8 for old samples of purified monoclonal antibodies.
  • the binding agent of the invention is a monoclonal antibody
  • the binding of the various murine monoclonal antibodies to tumor antigen CA19-9 and to sialyl Lewis A was determined.
  • the various purified monoclonal antibodies were tested for binding to purified CA19-9.
  • a range of mAb concentration from 0 to 0.64 mg/ml was used.
  • a second ELISA assay (as described above) was performed which used the carbohydrate antigen SLe(a) conjugated to polyacrylamide in order to carry a direct binding assay.
  • both HBl (ALT-4) and HB2 (ALT-3) mAbs recognized the carbohydrate epitope SLe(a).
  • Figure 7 demonstrated that Flopc-21 mAb is directed against a different CA19-9 epitope determinant than the SLe(a) epitope recognized by HB2 and HBl and, thus, does not interact with SLe(a) carbohydrate oUgosaccharide.
  • HBl was first radiolabeled with 12D I (iodinated). 50 ⁇ g of HBl mAb was mixed with approximately 600 ⁇ Ci of Na 125 I. 10 ml of 2.5 mg/ml of chloramine-T (CT) (commercially available from Sigma Chemical Co.) was added to the reaction vial and gently shaken for 30 seconds. Then, 10 ⁇ l of 5 mg/ml sodium metabisulfate (Na-Met) was added to the mixture and shaken gently for 15 seconds. Finally, 20 ⁇ l of IM sodium iodide (Nal) was added and the whole mixture was gently shaken for 5 seconds.
  • CT chloramine-T
  • the reaction mixture was loaded to the Desalting Econo- pac 10 DG chromatographic column (commercially available from BioRad, Hercules, CA, USA) pre-washed three times with 0.5% BSA in PBS.
  • the vial was rinsed with 100 ⁇ l of PBS and transferred to the column.
  • the column was then washed with 0.5 ml PBS fractions 12 to 14 times. Each elute was collected separately in numbered tubes. The radioactivity of elutes was monitored by a Geiger counter.
  • the fractions containing the mAbs were pooled, labeled, placed in lead containers, and stored at 4 °C.
  • a nonsaturating concentration (0.33 ⁇ g/ml) of iodinated HBl (ALT-4) mAb was chosen in order to obtain a final count per minute (c.p.m.) concentration of approximately 40,000 c.p.m./well. 100 ⁇ l of each dilution was added to different weUs and aUowed to incubate for 1.5 hours. After that the supernatant was discarded, the strips were washed with PBST three times and broken down to individual wells and the radioactivity was measured (bound fraction).
  • HB 1 (ALT-4) mAb to Cal9-9 antibody was next determined by scatchard analysis using iodinated HBl mAb. To do this, radio-immunoassay (RIA) strips were coated with 100 ⁇ l of PBS containing 65 U/ml of CA19-9 antigen overnight at 4°C. After discarding the coating solution, the nonspecific binding sites were blocked with 150 ⁇ l/well of blocking buffer (2% sucrose, 2% BSA, 0.06% thimerosal in PBS).
  • blocking buffer 2% sucrose, 2% BSA, 0.06% thimerosal in PBS.
  • the weUs were incubated for 1 hour at room temperature with gentle shaking, and then washed three times with PBST (PBS, 0.1% Tween-20, pH 7.1). Each strip was then incubated with a range of concentration from 0.005 to 10.5 ⁇ g/ml of I 125 labeled HBl antibody in 100 ⁇ l of binding buffer (1% BSA, 0.02% thimerosal in PBS) at room temperature for 1 hour. Then, 50 ⁇ l of the HB 1 supernatant was taken from each well and the radioactivity was measured (free fraction) gamma counter. The strips were again washed three times with PBST, broken into individual wells and the radioactivity of each well was measured (bound fraction) by Gamma counter.
  • PBST PBS, 0.1% Tween-20, pH 7.1
  • the affinity of iodinated HBl antibody to CA19-9 antigen was determined by measuring the radioactivity of free and bound fractions in individual wells by Gamma counter. As shown in Figure 9, the Kd value from this experiment was calculated to be equal to 0.94 nM.
  • KLH keyhole Umpet hemocyanin
  • the mAbs were conjugated to KLH in order to enhance their immunogenicity. Chemical conjugation is usually very efficient but is likely to decrease the binding activity of the mAbs. For each conjugation, we therefore determined the remaining binding activity of the KLH-mAbs. This was done by direct ELISA as described above and schematically depicted in Figure 4 using CA19-9 coated plates.
  • the rats were then subjected to acute inflammation in the right hind paw by injection of carrageenan (carrageenan lambda type IV (gelatin, vegetable, Irish moss); commercially available from Sigma Chemical Co.) five days after the last injection of antibody.
  • carrageenan carrageenan lambda type IV (gelatin, vegetable, Irish moss); commercially available from Sigma Chemical Co.
  • 0.05 ml of 1% carrageenan in 0.9 % NaCl as injected into the rats five days after the last immunization with antibody (e.g., KLH conjugated HBl, F5, or PBS only).
  • the animals were Ughtly anaesthetized with ethyl ether for the injections and for inflammation measurements.
  • Paw edema was used as a characteristic sign of inflammation, and was quantitated at regular interval of time after carrageenan injection, with a total of 8 measurements. Paw edema was measured by two different methods. The caUper method was used to measure paw thickness, and water displacement method to measure paw volume. The thickness of rat paw was measured in inches by a caliper device before and after carrageenan injection at desired time intervals. The paw volume was measured by immersing the paw up to the tibio- tarsae articulation in a cylinder filled with water, and the fluid volume replaced by immersed paw was measured before and after carrageenan injection at desired time intervals.
  • AUC percent change in effect means the percent change in inflammatory response in each rat from time zero to each consecutive time of inflammation measurement.
  • AUC percent change in effect (area under the curve) means the sum of the percent changes in inflammatory response from time zero to each consecutive time of inflammation measurement for each rat. The average of AUC percent change in effect was calculated per rat group.
  • Percent change in maximum effect means the percent change in inflammatory response from time zero to the time where maximum inflammation has occurred in each rat.
  • Paw thickness measurement results are illustrated as AUC percent change in effect in Figures 10A, 10B, and IOC; and as percent change in maximum effect in Figures 11 A, 11B, and llC.
  • Paw volume measurement results are Ulustrated as AUC percent change in effect in figure's 12A, 12B, and 12C; and as percent change in maximum effect in Figures 13A, 13B, and 13C.
  • the HBl therapeutic mAb-injected group showed absorbance values above background level that corresponds to either anti-isotypic, anti-idiotypic or anti-allotypic immune response or a combination of them, among which only the idiotypic response wnT have the therapeutic effect.
  • mice Male Sprague Dawley rats (age six weeks) were injected four times with 500 ⁇ g/rat of either KLH-conjugated HB 1 , control KLH-conjugated F5, control KLH-conjugated 7C2C5C12 mAbs, or have simply received PBS injection via i.p. route.
  • RAMA and Ab3 responses were measured between each injection. Induction and measurement of inflammation were done by the same methods as described in Example I. Paw edema was observed and measured at intervals of time. Paw thickness and paw volume measurement results were analyzed and plotted as described in Example I. Paw thickness measurement results are illustrated as AUC percent change in effect in Figure 15A, 15B, and 15C; and as percent change in maximum effect in Figures 16 A, 16B, and 16C. Paw volume measurement results are illustrated as AUC percent change in effect in Figures 17A, 17B, and 17C; and percent change in maximum effect in Figures 18A, 18B, and 18C.
  • Example III Male Sprague Dawley rats (age six weeks) were injected four times with 500 mg/rat of either HBl, control F5 mAbs mixed with QUIL A adjuvant (commercially avaUable from Calbiochem, San Deigo, USA), or have simply received PBS injection via subcutaneous (s.e.) route. RAMA and Ab3 responses were measured between each injection at regular intervals. Induction and measurement of inflammation was done by the same methods as described in Example I. Paw edema was observed and measured at regular intervals of time.
  • QUIL A adjuvant commercially avaUable from Calbiochem, San Deigo, USA
  • Paw thickness and paw volume measurement results were analyzed and plotted as in Example I. Paw thickness measurement results are Ulustrated as AUC percent change in effect in Figures 21A, 21B, and 21C; and as percent change in maximum effect in Figures 22 A, 22B, and 22C. Paw volume measurement results are Ulustrated as AUC percent change in effect in Figures 23A, 23B, and 23C; and as percent change in maximum effect in Figures 24A, 24B, and 24C.
  • Paw thickness and paw volume measurement results were analyzed and plotted as in Example I. Paw thickness measurement results are illustrated as AUC percent change in effect in Figures 28A, 28B, and 28C; and as percent change in maximum effect in Figures 29 A, 29B, and 29C. Paw volume measurement results are Ulustrated as AUC percent change in effect in Figures 30A, 30B, and 30C; and as percent change in maximum effect in Figures 31 A, 3 IB, and 31C.
  • the RAMA immune response was detected (using the method depicted in Figure 3) in one rat that belonged to the therapeutic HBl group.
  • AUC percent change in effect from the measurements of caliper and water displacement methods showed that one rat in the therapeutic HB 1 group had responded with minimal inflammatory response as shown in Figures 28B and 30B, respectively. Therefore, the extent of the inflammatory response as shown in Figures 28B and 30B was compared to the level of RAMA immune response in each rat in Figure 32.
  • the same rat that showed the highest RAMA immune response as shown in Figure 32 also showed the least inflammatory response measured by both caUper and water displacement methods as shown in Figures 28B and 30B, respectively.
  • Rats were injected four times with 500 mg/rat of either KLH-conjugated HB2, control KLH-conjugated Flopc-21 mAbs, or have simply received PBS injection via i.p. route.
  • RAMA and Ab3 immune responses were measured between each injection. Induction and measurement of inflammation were done by the same methods as described in Example I. Paw edema was observed and measured at regular intervals of time.
  • Paw thickness and paw volume measurement results were analyzed and plotted as described in Example I. Paw thickness measurement results are illustrated as AUC percent change in effect in Figures 33A, 33B, and 33C; and as percent change in maximum effect in Figures 34A, 34B, and 34C. Paw volume measurement results are Ulustrated as AUC percent change in effect in Figures 35A, 35B, and 35C; and as percent change in maximum effect in Figures 36A, 36B, and 36C.
  • HBl ALT-4
  • HB2 HB2
  • control monoclonal antibodies F5 IgM
  • 7C2C5C12 IgM
  • Flopc-21 IgG3
  • Human umbilical vein endotheUal cells is an endotheUal cell line derived from the vein of a normal, human umbilical cord, obtained from American Type Culture CoUection (Maryland, USA). HUV-EC-C ceUs were maintained in MCDB 131 media supplemented with 10 ng/ ml of HEGF, 12 mg/ ml of BBE, 1 mg/ ml of hydrocortisone, 5 % v/v FBS, 0.05 mg/ ml of Gentamycin suUate, and 0.05 mg/ ml of amphotericin B.
  • the tubes were centrifuged at 1500 rpm (400 g) for 15 minutes at 21 °C.
  • the peUet was retrieved and washed three times with 10 ml of 3 mM EDTA, 1% FBS in PBS solution without magnesium and without calcium and centrifuged at 800 rpm (250 x g) for 10 minutes.
  • the pellet was suspended in 2 ml of RPMI + 5% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • the purified ceU pellet (mononuclear or PMNL ceUs) was suspended at lx 10 7 ceUs/ml in RPMI media, 5% FBS and kept at 37 °C until used. CeU viability was determined by exclusion of 0.4% trypan blue.
  • FACS analysis 1x10° white blood cells or HUV-EC-C washed in PBS, 1% bovine serum albumin (BSA), were incubated with primary antibody (i.e., HBl, HB2, F5, 7C2C5C12, or Floppc-21) at a concentration of 5 ⁇ g/ ml in PBS, 1% BSA.
  • primary antibody i.e., HBl, HB2, F5, 7C2C5C12, or Floppc-21
  • Colo 205 ceUs which were used as a positive control (Figs. 38A and 38B), are known to express the Sialyl Lewis A epitope on their surface.
  • Colo 205 ceUs are a human adenocarcinoma cell Une derived from metastatic colon cancer that produces CA19-9 antigen.
  • Colo 205 ceUs were obtained from the American Type Culture CoUection (Maryland, USA), and were maintained in standard media (RPMI-1640 supplemented with 2 mM L-glutamine, 50 U/ml penicillin and 50 U/ml streptomycin, 10% v/v fetal bovine serum (FBS)).
  • microchip glass wafer device that consists of channels in which in vitro ceU roUing and adhesion can be observed under flow conditions, the process that mimics the in vivo cell roUing and adhesion events.
  • a 10 cm x 10 cm glass wafer with a series of simple "Y" mixer flow manifolds was used in this assay.
  • the "Y” mixer flow manifold consisted of 50 microns in depth and 300 microns in width channels with a flow region downstream. These channels are structured in similar way to capUlaries and therefore, the use of such channels aUows the observation of cell roUing and adhesion in vitro that mimics the in vivo cell rolling and adhesion events.
  • the microchip was secured into an aluminum microchip holder, and the microchip reservoirs were glued onto the microchip with 5-minute epoxy resin.
  • the channels were conditioned with concentrated nitric acid (HNO 3 ), 2 M of sulfuric acid (H 2 SO 4 ) and 1 M of sodium hydroxide (NaOH) (10 minutes each) before and after each experiment.
  • the channels were then coated with 20 ⁇ g/ml of recombinant human E-selectin or P-selectin (commercially avaUable from R & D Systems, MinneapoUs, MN, USA) for 2 hours at 37 °C to mimic the surface of a vessel wall endotheUal Uning.
  • a microsyringe pump that operated as a negative pressure source was used to drive the ceUs through the channels at a flow rate of 2.5 ml/min for the 50 micron deep device.
  • Example VI suspended in RPMI, 5% FBS was pumped to the channel through an injection route and allowed to run at an optimum flow rate for the purpose of achieving the rolling step. Since each channel had two sides, one side of each channel was used to test the agent under study and the other side of the channel was used as a control for ceU roUing only without the addition of any agents (see Figure 39B).
  • the cell suspensions added to different sides of the channel were regulated by a luminal fluidic flow that allowed these ceU suspensions to continue running at different sides of the channel and prevented ceU diffusion from one side of the channel to the other.
  • the foUowing agents were tested at a volume of 10 ⁇ l and at a dUution of 1:10 v/v in RPMI and 5% FBS: fresh human serum (negative control), fresh rat serum, HBl rat serum; and F5 rat serum.
  • 10 ⁇ l of 100 ⁇ g/ml of anti-human E-selectin antibody (commercially available from R & D Systems, MinneapoUs, MN, USA) was used as positive control for E- selectin coated channels.
  • Serum of rats immunized with HB 1 monoclonal antibody from Example III was obtained from rats who showed a high RAMA response (rat # 2, 4, 5, 6, 7, and 8, collectively).
  • Serum of rats immunized with F5 monoclonal antibody from Example III was obtained from rats who showed a high RAMA response (rat # 4, 6, and 7, coUectively).
  • 10 ⁇ l of each of the above mentioned agent was added to different channels.
  • the pump sucked the added sample into the channel to start the inhibition step.
  • the images of cell roUing and inhibition steps were captured at different times and the number of cells was counted in both sides of the channel. Non-adherent cells appeared as streaks on the video image.
  • the cell counts were conducted on a PC computer instaUed with a perception video board and image Pro Plus software.
  • P-selectin coated channels were also tested on P-selectin coated channels under the same conditions as with E-selectin coated channels.
  • anti-human P- selectin antibody commercially available from R & D Systems, MinneapoUs, MN, USA was used as a positive control.
  • Example VIII A rodent model of melanoma is used to determine the effect of the binding agents of the invention in reducing melanoma.
  • a murine melanoma cell line such as clone B16 (commercially available as Catalog No. CRL-6322 from the American Type Culture CoUection, Manassas, VA) is injected subcutaneously or intra-peritoneaUy into syngeneic mice (e.g., C57BL/6J mice) or immunodeficient nude mice (both commercially avaUable from The Jackson Laboratory, Bar Harbor, ME) at an estabUshed primary site in an amount sufficient for the melanoma cells to estabUsh tumor growth at the primary tumor site in the mice.
  • a first set of mice is injected, and the time needed for metastasis of the melanoma cells to occur is recorded.
  • the experimental set of rats is injected with the melanoma cells.
  • the experimental set is divided into different groups of animals, each of which is administered a therapeutically effective amount of the various binding agents of the invention including, without Umitation, HBl, HB2, ARP2-41, DREG-200, MEL-14, BBIG-E5, and anti-sialyl Lewis X antibodies via a subcutaneous, intraperitoneal, or intravenous injection.
  • Control antibodies include 7C2C5C12 and F5.
  • mice injected with binding agents of the invention that specifically bind to adhesion molecules expressed either on the cell surface of the melanoma cells or on the cell surface of endotheUal cells Uning blood vessel walls are found to have a reduced number of tumor formation at secondary sites (i.e., sites distinct from the primary tumor site) than those mice injected with the control antibodies.

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Abstract

L'invention concerne des compositions permettant de réduire un état immunitaire, tel qu'une inflammation ou un cancer, chez un animal souffrant d'un état immunitaire ou étant prédisposé à souffrir d'un état immunitaire. Les compositions renferment un agent liant se liant de manière spécifique à une molécule d'adhésion exprimée sur la surface d'une cellule à diffusion hématogène ou d'une cellule endothéliale recouvrant un vaisseau sanguin ou libérée par celle-ci, l'agent liant induisant une réponse immunitaire chez l'animal au niveau de l'agent liant. L'invention concerne des compositions supplémentaires renfermant un agent qui est capable de réduire une inflammation et un cancer chez un animal lorsqu'elles sont administrées à l'animal dans une quantité efficace sur le plan thérapeutique. L'invention concerne également des méthodes d'administration de telles compositions à un animal souffrant de l'état immunitaire ou prédisposé à celui-ci.
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EP0508282A2 (fr) * 1991-04-01 1992-10-14 Kyowa Hakko Kogyo Co., Ltd. Anticorps monocloneaux anti-idiotypiques

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RYE ET AL., TUMOR BIOLOGY, vol. 19, 1998, pages 390 - 420 *
SHITARA K ET AL., ANTICANCER RESEARCH, vol. 11, 1991, pages 2003 - 2014, XP008073114 *

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