EP1646401A2 - Anticorps humains specifiques - Google Patents

Anticorps humains specifiques

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Publication number
EP1646401A2
EP1646401A2 EP04777349A EP04777349A EP1646401A2 EP 1646401 A2 EP1646401 A2 EP 1646401A2 EP 04777349 A EP04777349 A EP 04777349A EP 04777349 A EP04777349 A EP 04777349A EP 1646401 A2 EP1646401 A2 EP 1646401A2
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European Patent Office
Prior art keywords
antibody
fragment
cells
patient
cell
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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.)
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EP04777349A
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German (de)
English (en)
Other versions
EP1646401A4 (fr
Inventor
Avigdor Levanon
Tikva Vogel
Daniel Plaksin
Tuvia Peretz
Boaz Amit
Lena Cooperman
Yocheved Hagay
Esther Szanton
Yariv Kanfi
Rachel Ben-Levy
Tali Szrajber
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Bio Technology General Israel Ltd
Bio Tech General (Israel) Ltd
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Bio Technology General Israel Ltd
Bio Tech General (Israel) Ltd
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Publication of EP1646401A2 publication Critical patent/EP1646401A2/fr
Publication of EP1646401A4 publication Critical patent/EP1646401A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/65—Tetracyclines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to antibodies that bind to particular epitopes that are present on cells, such as cancer cells, metastatic cells, leukemia cells, leukocytes, and platelets, and that are important in such diverse physiological phenomena as cell rolling, metastasis, inflammation, and auto-immune diseases. More particularly, the antibodies may have anti-cancer activity, anti-metastatic activity, anti-leukemia activity, anti-viral activity, anti-infection activity, and/or activity against other diseases, such as inflammatory diseases, autoimmune diseases, viral infection, cardiovascular diseases such as myocardial infarction, retinopathic diseases, and diseases caused by sulfated tyrosine- dependent protein-protein interactions. In addition, the antibodies of the present invention may be used as a targeting agent to direct a therapeutic to a specific cell or site within the body.
  • Leukemia, lymphoma, and myeloma are cancers that originate in the bone marrow and lymphatic tissues and are involved in uncontrolled growth of cells.
  • Acute lymphoblastic leukemia (ALL) is a heterogeneous disease that is defined by specific clinical and immunological characteristics. Like other forms of ALL, the definitive cause of most cases of B cell ALL (B-ALL) is not known; although, in many cases, the disease results from acquired genetic alterations in the DNA of a single cell, causing abnormalities and continuous multiplication. Prognosis for patients afflicted with B-ALL is significantly worse than for patients with other leukemias, both in children and in adults.
  • Chronic lymphocytic leukemia (CLL), one example of which is B cell CLL (B-CLL) is a slowly progressing form of leukemia, characterized by an increased number of lymphocytes.
  • Acute myelogenous leukemia (AML) is a heterogeneous group of neoplasms having a progenitor cell that, under normal conditions, gives rise to terminally differentiated cells of the myeloid series (erythrocytes, granulocytes, monocytes, and platelets).
  • AML is associated with acquired genetic alterations that result in replacement of normally differentiated myeloid cells with relatively undifferentiated blasts, exhibiting one or more type of early myeloid differentiation.
  • AML generally evolves in the bone marrow and, to a lesser degree, in the secondary hematopoietic organs. Primarily, AML affects adults and peaks in incidence between the ages of 15-40, but it is also known to affect both children and older adults. Nearly all patients with AML require treatment immediately after diagnosis to achieve clinical remission, in which there is no evidence of abnormal levels of circulating undifferentiated blast cells.
  • Platelets, fibrinogen, GPIb, selectins, and PSGL-1 [03] Platelets, fibrinogen, GPIb, selectins, and PSGL-1 (P-Selectin Glycoprotein
  • Ligand- 1) each play an impprtant role in several pathogenic conditions or disease states, such as abnormal or pathogenic inflammation, abnormal or pathogenic immune reactions, autoimmune reactions, metastasis, abnormal or pathogenic adhesion, thrombosis and/or restenosis, and abnormal or pathogenic aggregation.
  • pathogenic conditions or disease states such as abnormal or pathogenic inflammation, abnormal or pathogenic immune reactions, autoimmune reactions, metastasis, abnormal or pathogenic adhesion, thrombosis and/or restenosis, and abnormal or pathogenic aggregation.
  • antibodies that bind to or cross-react with platelets and with these molecules would be useful in the diagnosis and treatment of diseases and disorders involving these and other pathogenic conditions.
  • Platelets are well-characterized components of the blood system and play several important roles in hemostasis, thrombosis and/or restenosis. Damage to blood vessel sets in motion a process known as hemostasis, which is characterized by a series of sequential events.
  • the initial reaction to damaged blood vessels is the adhesion of platelets to the affected region on the inner surface of the vessel.
  • the next step is the aggregation of many layers of platelets onto the previously adhered platelets, forming a hemostatic plug and sealing the vessel wall.
  • the hemostatic plug is further strengthened by the deposition of fibrin polymers. The clot or plug is degraded only when the damage has been repaired.
  • Circulating platelets are cytoplasmic particles released from the periphery of megakaryocytes. Platelets play an important role in hemostasis. Upon vascular injury, platelets adhere to damaged tissue surfaces and attach to one another (cohesion). This sequence of events occurs rapidly, forming a structureless mass (commonly called a platelet plug or thrombus) at the site of vascular injury.
  • the cohesion phenomenon also known as aggregation, may be initiated in vitro by a variety of substances, or agonists, such as collagen, adenosine-diphosphate (ADP), epinephrine, serotonin, and ristocetin. Aggregation is one of the numerous in vitro tests performed as a measure of platelet function.
  • Tumor metastasis is perhaps the most important factor limiting the survival of cancer patients. Accumulated data indicate that the ability of tumor cells to interact with host platelets represents one of the indispensable determinants of metastasis (Oleksowicz, Tlirombosis Res. 79: 261-74 (1995)). When metastatic cancer cells enter the blood stream, multicellular complexes composed of platelets and leukocytes coating the tumor cells are formed. These complexes, which may be referred to as microemboli, aid the tumor cells in evading the immune system. The coating of tumor cells by platelets requires expression of P-selectin by the platelets.
  • GPIb Complex a single polypeptide chain (60kd) was found to be expressed on surface membrane of HEL cells that is closely related to GPIb and corresponds to an incompletely or abnormally O-glycosylated GPIb ⁇ subunit (Kieffer et al., J. Biol. Chem. 261(34): 15854-62 (1986)).
  • Each step in the process of hemostasis requires the presence of receptors on the platelet surface.
  • One receptor that is important in hemostasis is the glycoprotein Ib-LX complex (also known as CD42). This receptor mediates adhesion (initial attachment) of platelets to the blood vessel wall at sites of injury by binding von WiUebrand factor (vWF) in the subendothelium. It also has crucial roles in two other platelet functions important in hemostasis: (a) aggregation of platelets induced by high shear in regions of arterial stenosis and (b) platelet activation induced by low concentrations of thrombin.
  • the GPIb-IX complex is one of the major components of the outer surface of the platelet plasma membrane.
  • This complex comprises three membrane-spanning polypeptides - a disulfide-linked 130 kDa ⁇ -chain and 25 kDa ⁇ -chain of GPIb and a noncovalently associated GPLX (22 kDa). All of the subunits are presented in equimolar amounts on the platelet membrane for efficient cell-surface expression and function of CD42 complex, indicating that proper assembly of the three subunits into a complex is required for full expression on the plasma membrane.
  • the ⁇ -chain of GPIb consists of three distinct structural domains: (1) a globular N-terminal peptide domain containing leucine-rich repeat sequences and Cys-bonded flanking sequences; (2) a highly glycosylated mucin-like macroglycopeptide domain; and (3) a membrane-associated C- terminal region that contains the disulfide bridge to GPIb ⁇ and transmembrane and cytoplasmic sequences.
  • vWF and thrombin-binding domain of the GPIb-IX complex reside in a globular region encompassing approximately 300 amino acids at the amino terminus of GPIb ⁇ .
  • human platelet GPIb-LX complex is a key membrane receptor mediating both platelet function and reactivity
  • recognition of subendothelial-bound vWF by GPIb allows platelets to adhere to damaged blood vessels.
  • binding of vWF to GPIb ⁇ also induces platelet activation, which may involve the interaction of a cytoplasmic domain of the GPIb-LX with cytoskeleton or phospolipase A2.
  • GPIb ⁇ contains a high-affinity binding site for ⁇ -thrombin, which facilitates platelet activation by an as-yet poorly defined mechanism.
  • the N-terminal globular domain of GPIb ⁇ contains a cluster of negatively charged amino acids.
  • Protein sulfation is a widespread post-translational modification that involves enzymatic covalent attachment of sulfate, either to sugar side chains or to the polypeptide backbone. This modification occurs in the trans-Golgi compartment.
  • Sulfated proteins include secretory proteins, proteins targeted for granules, and the extracellular regions of plasma membrane proteins.
  • Tyrosine is an amino acid residue presently known to undergo sulfation. Kehoe et al., Chem. Biol. 7: R57-61 (2000).
  • Other amino acids, e.g., threonine may also undergo sulfation, particularly in diseased cells.
  • GPIb ⁇ (CD42), which is expressed by platelets and megakaryocytes and mediates platelet attachment to and rolling on subendothelium via binding with vWF, also contains numerous negative charges at its N-terminal domain.
  • CC-chemokine receptors such as CCR5, which serve as co-receptors with related seven transmembered segment (7TMS) receptor for entry of human and simian immunodeficiency viruses (HTV-1, HIV-2, and SIV) into target cells.
  • 7TMS seven transmembered segment
  • HTV-1, HIV-2, and SIV human and simian immunodeficiency viruses
  • CXCR4 another important HIV-1 co-receptor, is also sulfated (Farzan et al., Cell 96(5): 667-76 (1999)).
  • Tyrosine sulfation plays a less significant role i CXCR4-dependent HIV-1 entry than CCR5-dependent entry; thus demonstrating a possible role for tyrosine sulfation in the CXC-chemokine family and underscoring a general difference in HIV-1 utilization of CCR5 and CXCR4 (Farzan et al., J. Biol. Chem. 277(33): 29,484-89 (2002)).
  • the P-, E-, and L- Selectins are members of a family of adhesion molecules that, among other functions, mediate rolling of leukocytes on vascular endothelium.
  • P- Selectin is stored as granules in platelets and is transported to the surface after activation by thrombin, histamine, phorbol ester, or other stimulatory molecules.
  • P-Selectin is also expressed on activated endothelial cells.
  • E-Selectin is expressed on endothelial cells
  • L-Selectin is expressed on neutrophils, monocytes, T cells, and B cells.
  • PSGL-1 (also called CD162) is a mucin glycoprotein ligand for P-Selectin,
  • PSGL-1 is a disulfide-linked homodimer that has a PACE (Paired Basic Amino Acid Converting Enzymes) cleavage site. PSGL-1 also has three potential tyrosine sulfation sites followed by 10-16 decamer repeats that are high in proline, serine, and threonine. The extracellular portion of PSGL-1 contains three N-linked glycosylation sites and has numerous sialylated, fucosylated O-linked oligosaccharide branches (Moore et al., J. Biol. Chem.
  • PSGL-1 has 361 residues in HL60 cells, with a 267 residue extracellular region, 25 residue trans-membrane region, and a 69 residue intracellular region, and forms a disulfide-bonded homodimer or heterodimer on the cell surface (Afshar-Kharghan et al., Blood 97: 3306-12 (2001)).
  • the sequence encoding PSGL-1 is in a single exon, so alternative splicing should not be possible.
  • PSGL-1 in HL60 cells has 15 consecutive repeats of a 10 residue consensus sequences present in the extracellular region, although there are 14 and 16 repeats of this sequence in polymorphonuclear leukocytes, monocytes, and several other cell lines, including most native leukocytes.
  • PSGL-1 is expressed on neutrophils as a dimer, with apparent molecular weights of both 250 kDa and 160 kDa, whereas on HL60 the dimeric form is approximately 220 kDa. When analyzed under reducing conditions, each subunit is reduced by half. Differences in molecular mass may be due to polymorphisms in the molecule caused by the presence of different numbers of decamer repeats (Leukocyte Typing VI. Edited by T. Kishimoto et al. (1997)).
  • PSGL-1 mediates rolling of leukocytes on activated endothelium, on activated platelets, and on other leukocytes and inflammatory sites and mediates rolling of neutrophils on P-Selectin. PSGL-1 may also mediate neutrophil-neutrophil interactions via binding with L-Selectin, thereby mediating inflammation (Snapp et al., Blood 91(1): 154-64 (1998)).
  • PSGL-1 has been found on all leukocytes: neutrophils, monocytes, lymphocytes, activated peripheral T cells, granulocytes, eosinophils, platelets and on some CD34 positive stem cells and certain subsets of B cells.
  • P-Selectin is selectively expressed on activated platelets and endothelial cells. Interaction between P-Selectin and PSGL-1 promotes rolling of leukocytes on vessel walls, and abnormal accumulation of leukocytes at vascular sites results in various pathological inflammations. Stereo-specific contributions of individual tyrosine sulfates on PSGL-1 are important for the binding of P- Selectin to PSGL-1.
  • PSGL-1 tyrosine sulfation supports slower rolling adhesion at all shear rates and supports rolling adhesion at much higher shear rates (Rodgers et al., Biophys. J. 81: 2001-09 (2001)). Moreover, it has been suggested that PSGL-1 expression on platelets is 25-100 fold lower than that of leukocytes (Frenette et al., J. Exp. Med. 191(8): 1413-22 (2000)).
  • KPLl A commercially available monoclonal antibody to human PSGL- 1 , KPLl , has been shown to inhibit the interactions between PSGL-1 and P-selectin and between PSGL-1 and L-selectin.
  • the KPLl epitope was mapped to the tyrosine sulfation region of PSGL-1 (YEYLDYD) (SEQ ID NO:l) (Snapp et al., Blood 91(l):154-64 (1998)).
  • Normal fibrinogen which is the more abundant form (approximately 90% of the total fibrinogen found in the body), is composed of two identical 55 kDa ⁇ chains, two identical 95 kDa ⁇ chains, and two identical 49.5 kDa ⁇ chains.
  • Normal variant fibrinogen which is the less abundant form (approximately 10% of the fibrinogen found in the body), is composed of two identical 55 kDa ⁇ chains, two identical 95 kDa ⁇ chains, one 49.5 kDa ⁇ chain, and one variant 50.5 kDa ⁇ prime chain.
  • the gamma and gamma prime chains are both coded for by the same gene, with alternative splicing occurring at the 3' end.
  • Normal gamma chain is composed of amino acids 1-411 and normal variant gamma prime chain is composed of 427 amino acids, of which amino acids 1-407 are the same as those in the normal gamma chain and amino acids 408-427 are VRPEHPAETEYDSLYPEDDL (SEQ ID NO:2). This region is normally occupied with thrombin molecules.
  • Fibrinogen is converted into fibrin by the action of thrombin in the presence of ionized calcium to produce coagulation of the blood. Fibrin is also a component of thrombi, and acute inflammatory exudates.
  • an object of the invention is to provide various antibodies or polypeptides that bind sulfated PSGL-1 and methods of use thereof.
  • an object of the invention is to provide methods of activating
  • ADCC or stimulating natural killer (NK) or T cells by administering the antibodies of the present invention.
  • Another specific obj ect of the invention is to provide a method of inducing cell death.
  • Yet another specific object of the invention is to provide a method of preventing infection by a virus, such as HIV, comprising administering to a patient in need thereof an antibody as herein.
  • a virus such as HIV
  • Another specific object of the invention is to provide a method of introducing an agent into a cell that expresses sulfated PSGL-1 having the following steps: coupling or complexing the agent to an antibody as described herein and administering the antibody-agent couple or complex to the cell is provided
  • the present invention provides antibodies or polypeptides that bind an epitope of PSGL-1 comprising the motif D-X-Y-D (SEQ ID NO:3), wherein X represents any amino acid or the covalent linkage between D and Y, and Y is sulfated, which antibody can be coupled to or complexed with multiple copies of an agent selected from the group consisting of anti-cancer, anti-leukemic, anti-metastasis, anti-neoplastic, anti- disease, anti-adhesion, anti-thrombosis, anti-restenosis, anti-autoimmune, anti- aggregation, anti-bacterial, anti-viral, and anti-inflammatory agents.
  • an agent selected from the group consisting of anti-cancer, anti-leukemic, anti-metastasis, anti-neoplastic, anti- disease, anti-adhesion, anti-thrombosis, anti-restenosis, anti-autoimmune, anti- aggregation, anti-bacterial, anti-viral,
  • the antibodies of the invention can be used in a method of inducing antibody-dependent cell cytotoxicity and/or stimulating natural killer (NK) cells or T cells.
  • a method of inducing cell death is provided.
  • a method of preventing infection by a virus (e.g., HIV) by administering to a patient in need thereof an antibody of the present invention is also provided.
  • the present invention also provides a method of introducing an agent into a cell that expresses sulfated PSGL-1 by coupling or complexing an agent to an antibody of the present invention and administering the antibody-agent couple or complex to the cell.
  • the present invention further provides a method of identifying, isolating and purifying tumor cell markers.
  • the present invention provides methods of diagnosis, prognosis and staging using the present antibodies.
  • Figure 1 shows a Western blot of partially purified AML-R1 cell lysate before and after passage through Yl-IgG affinity column.
  • Figure 2 shows that, of three tyrosines in the purified protein's sulfated- tyrosine motif, tyrosines 2 and 3 are sulfated.
  • Figure 3 shows Yl-IgG (20 ⁇ g/ml) mediated ADCC (percent cytotoxicity) in primary B-CLL samples.
  • Figure 4 shows Yl -IgG-mediated ADCC (percent cytotoxicity) by PBMC against AML cells.
  • Figure 5 shows increaseed ADCC (percent cytotoxicity) in ML-2 cells as a function of Yl-IgG concentration.
  • Figure 6 shows ADCC (percent cytotoxicity) by PBMC against ML-2 as a function of competition between Yl-IgG and KPL-1.
  • Figure 7 A shows analysis of Yl-IgG-mediated ADCC (percent cytotoxicity) by natural killer cells from normal donors and B-CLL patients against ML2 cells.
  • Figure 7B shows the involvement of CD 14+ cells (monocytes) in ADCC against M12 targets.
  • Figure 8 shows expression of CD69 (an early activation marker) on NK cells mediated by Yl .
  • Figure 9 shows apoptotic effect of Yl-IgG on mononuclaer cells (CD 19+,
  • CD5+ from B-CLL patients by FACS analysis.
  • Figure 10 shows analysis of ADCC activity (percent cytotoxicity) against mononuclear cells from human B-CLL patients, i.e., primary human B-CLL cells (KBC115 and KBC116 cells) mediated by Yl-IgG and Rituximab.
  • Figure 11 shows analysis of CDC activity (percent lysis) against mononuclear cells from human B-CLL patients (KBC156, KBC159, KBC160, KBC166, and RAJI cells) mediated by Yl-IgG, Rituximab, and Campath ® in the presence and absence of patient plasma.
  • Figure 12 shows reaction scheme for preparation of antibody linked to morpholino-doxorubicin.
  • Figure 13 shows cytotoxicity of an antibody-agent complex, namelyYl- morpholinodaunorubicin (Yl-M-DNR) and Yl-morpholinodoxorubicin (Yl-M-Dox) complexes in cord blood and AML cells.
  • Yl-M-DNR Yl-morpholinodaunorubicin
  • Yl-M-Dox Yl-morpholinodoxorubicin
  • Figure 14 shows cytotoxicity of the antibody-agent complex Yl-M-DNR against 2 patient AML samples (M4 and M5 stage) and against CD34+ cells.
  • Figure 15 shows cytotoxicity of various Yl complexes as a percent of control in B-ALL cells.
  • Figure 16A shows binding of Yl scFv to KU812 cells and Figure 16B > shows the surface expression of GPIb on sulfate starved KU812 cells.
  • Figure 17A shows inhibitory effects of sulfated peptides DLYDYYPE on the binding of Yl-scFv to platelets.
  • Figure 17B shows the effects of substitution mutant peptides in Yl-scFv platelet binding assay.
  • Figure 18A shows effects of mutant peptides in the inhibition of Yl-scFv binding to purified glycocalicin.
  • Figure 18B shows the binding of YlscFv to peptides covalently coupled to CovaLinkTM Plates by ELISA.
  • Figure 19 shows binding of Yl to immobilized, sulfated peptides derived from PSGL-1.
  • Figure 20 shows percent activity of Yl binding to non-sulfated PSGL-1 and to PSGL-1 sulfated in the first, second, and third positions.
  • Figure 21 some potential Yl binding motifs that are highly acidic and have sulfated tyrosines.
  • Figure 22 shows recognition of small cell lung carcinoma (SCLC) lysate byYl.
  • SCLC small cell lung carcinoma
  • Figure 23 shows endocytosis of Yl into primary AML cells.
  • Figure 24 shows endocytosis of Yl into primary AML cells.
  • Figure 25 shows analysis of Yl binding to healthy CD34+ stem cells.
  • Figure 26 shows analysis of Yl binding to healthy CD34+ stem cells.
  • Figure 27 shows internalization of Yl into primary AML cells at 37° C.
  • Figure 28 shows visualization of Yl staining in primary AML cells after stripping membrane-bound protein by acid treatment.
  • Figure 29 shows visualization of Yl staining in primary AML cells after stripping membrane-bound protein by pronase treatment.
  • Figure 30 shows visualization of Yl staining in primary AML cells after acid treatment or sucrose pre-incubation at 4° C (Fig. 30A) and at 37° C (Fig. 30B).
  • Figure 31 shows that Yl-scFv effectively inhibits binding of activated human platelets to ML2 cells.
  • Figure 32 shows the effect of Yl-scFv (10 ⁇ g ml) on ML2 cell rolling on immobilized rh-P-Selectin at low density (0.2 ⁇ g/ml).
  • Figure 33 shows the effect of Yl-scFv (10 ⁇ g/ml) on ML2 cell rolling on immobilized rh-P-Selectin at high density (1.0 ⁇ g/ml).
  • Figure 34 shows the effect of Yl-IgG (1 ⁇ g/ml) on ML2 cell rolling on immobilized rh-P-Selectin (1.0 ⁇ g/ml) at various shear stress forces.
  • Figure 35 shows the effect of increasing concentrations of Yl-scFv on human neutrophil rolling on immobilized rh-P-Selectin at high density (1.0 ⁇ g/ml).
  • Figure 36 shows the effect of Y 1 -IgG on human neutrophil rolling on immobilized rh-P-Selectin at high density (1.0 ⁇ g/ml).
  • Antibodies or immunoglobulins (Igs) are protein molecules that bind to antigen. Each functional binding unit of naturally occurring antibodies is composed of units of four polypeptide chains (2 heavy and 2 light) linked together by disulfide bonds. Each of the chains has a constant and variable region. Naturally occurring antibodies can be divided into several classes including IgG, IgM, IgA, IgD, and IgE, based on their heavy chain component. The IgG class encompasses several subclasses including, but not restricted to, IgGi, IgG 2 , IgG 3 , and IgG 4 . Immunoglobulins are produced in vivo by B lymphocytes, and each such molecule recognizes a particular foreign antigenic determinant and facilitates clearing of that antigen.
  • Antibodies may be produced and used in many forms, including antibody complexes.
  • antibody complex or “antibody complexes” is used to mean a complex of one or more antibodies with another antibody or with an antibody fragment or fragments, or a complex of two or more antibody fragments.
  • antibody fragments include Fv, Fab, F(ab') 2 , Fc, and Fd fragments. Therefore, an antibody according to the present invention encompasses a complex of an antibody or fragment thereof.
  • an Fv is defined as a molecule that is made up of a variable region of a heavy chain of a human antibody and a variable region of a light chain of a human antibody, which may be the same or different, and in which the variable region of the heavy chain is connected, linked, fused, or covalently attached to, or associated with, the variable region of the light chain.
  • the Fv can be a single chain Fv (scFv) or a disulfide stabilized Fv (dsFv).
  • An scFv is comprised of the variable domains of each of the heavy and light chains of an antibody, linked by a flexible amino-acid polypeptide spacer, or linker.
  • the linker may be branched or unbranched.
  • the linker is 0-15 amino acid residues, and most preferably the linker is (Gly Ser) 3 .
  • the Fv molecule itself, is comprised of a first chain and a second chain, each chain having a first, second and third hypervariable region.
  • the hypervariable loops within the variable domains of the light and heavy chains are termed Complementary Determining Regions (CDRs).
  • CDRs Complementary Determining Regions
  • the most variable of these regions in nature is the CDR3 region of the heavy chain.
  • the CDR3 region is understood to be the most exposed region of the Ig molecule and, as shown and provided herein, is the site primarily responsible for the selective and/or specific binding characteristics observed.
  • a fragment of an Fv molecule is defined as any molecule smaller than the original Fv that still retains the selective and/or specific binding characteristics of the original Fv.
  • fragments include but are limited to (1) a minibody, which comprises a fragment of the heavy chain only of the Fv, (2) a microbody, which comprises a small fractional unit of antibody heavy chain variable region (International Application No. PCT/LL99/00581), (3) similar bodies having a fragment of the light chain, and (4) similar bodies having a functional unit of a light chain variable region.
  • a fragment of an Fv molecule can be a substantially circular or looped polypeptide.
  • Fab fragment is a monovalent antigen-binding fragment of an immunoglobulin. A Fab fragment is composed of the light chain and part of the heavy chain.
  • An F(ab') 2 fragment is a bivalent antigen binding fragment of an immunoglobulin obtained by pepsin digestion. It contains both light chains and part of both heavy chains.
  • An Fc fragment is a non-antigen-binding portion of an immunoglobulin. It contains the carboxy-terminal portion of heavy chains and the binding sites for the Fc receptor.
  • An Fd fragment is the variable region and first constant region of the heavy chain of an immunoglobulin.
  • Polyclonal antibodies are the product of an immune response and are formed by a number of different B lymphocytes. Monoclonal antibodies are derived from one clonal B cell.
  • a cassette refers to a given sequence of consecutive amino acids that serves as a framework and is considered a single unit and is manipulated as such. Amino acids can be replaced, inserted into, removed, or attached at one or both ends. Likewise, stretches of amino acids can be replaced, inserted into, removed, or attached at one or both ends.
  • epitope is used herein to mean the antigenic determinant or recognition site or antigen site that interacts with an antibody, antibody fragment, antibody complex or a complex having a binding fragment thereof or T cell receptor.
  • epitope is used interchangeably herein with the terms ligand, domain, and binding region.
  • Selectivity is herein defined as the ability of a targeting molecule to choose and bind one entity or cell state from a mixture of entities or entity states, all entities or entity states of which may be specific for the targeting molecule.
  • association constant between a binding molecule (e.g., one binding site on an antibody) and a ligand (e.g., antigenic determinant).
  • the strength of the sum total of noncovalent interactions between a single antigen-binding site on an antibody and a single epitope is the affinity of the antibody for that epitope.
  • Low affinity antibodies bind antigen weakly and tend to dissociate readily, whereas high-affinity antibodies bind antigen more tightly and remain bound longer.
  • the term "avidity" differs from affinity, because the former reflects the valence of the antigen-antibody interaction.
  • antigen-antibody reaction is specific, in some cases antibodies elicited by one antigen can cross-react with another unrelated antigen. Such cross-reactions occur if two different antigens share a homologous or similar structure, epitope, or an anchor region thereof, or if antibodies specific for one epitope bind to an unrelated epitope possessing similar structure conformation or chemical properties.
  • a platelet is a disc-like cytoplasmic fragment of a megakaryocyte that is shed in the marrow sinus and subsequently circulates in the peripheral blood stream. Platelets have several physiological functions including a major role in clotting. A platelet contains centrally located granules and peripheral clear protoplasm, but has no definite nucleus.
  • Agglutination as used herein means the process by which suspended bacteria, cells, discs, or other particles of similar size are caused to adhere and form into clumps. The process is similar to precipitation but the particles are larger and are hi suspension rather than being in solution.
  • aggregation means a clumping of platelets induced in vitro, and thrombin and collagen, as part of a sequential mechanism leading to the formation of a thrombus or hemostatic plug.
  • Conservative amino acid substitution is defined as a change in the amino acid composition by way of changing one or two amino acids of a peptide, polypeptide or protein, or fragment thereof.
  • the substitution is of amino acids with generally similar properties (e.g., acidic, basic, aromatic, size, positively or negatively charged, polarity, non-polarity) such that the substitutions do not substantially alter peptide, polypeptide or protein characteristics (e.g., charge, isoelectric point, affinity, avidity, conformation, solubility) or activity.
  • Typical substitutions that may be performed for such conservative amino acid substitution may be among the groups of amino acids as follows: glycine (G), alanine (A), valine (V), leucine (L) and isoleucine (I) aspartic acid (D) and glutamic acid (E) alanine (A), serine (S) and threonine (T) histidine (H), lysine (K) and arginine (R) asparagine (N) and glutamine (Q) phenylalanine (F), tyrosine (Y) and tryptophan (W)
  • Conservative amino acid substitutions can be made in, e.g., regions flanking the hypervariable regions primarily responsible for the selective and/or specific binding characteristics of the molecule, as well as other parts of the molecule, e.g., variable heavy chain cassette. Additionally or alternatively, modification can be accomplished by reconstructing the molecules to form full-size antibodies, diabodies (dimers), triabodies (timers), and/or tetrabodies (tetramers) or to form minibodies or microbodies.
  • a phagemid is defined as a phage particle that carries plasmid DNA.
  • Phagemids are plasmid vectors designed to contain an origin of replication from a filamentous phage, such as ml 3 of fd. Since it carries plasmid DNA, the phagemid particle does not have sufficient space to contain the full complement of the phage genome.
  • the component that is missing from the phage genome is information essential for packaging the phage particle. In order to propagate the phage, therefore, it is necessary to culture the desired phage particles together with a helper phage strain that complements the missing packaging information.
  • a promoter is a region on DNA at which RNA polymerase binds and initiates transcription.
  • a phage display library (also termed phage peptide/antibody library, phage library, or peptide/antibody library) comprises a large population of phages (10 8 or larger), each phage particle displaying a different peptide or polypeptide sequence. These peptide or polypeptide fragments may constructed to be of variable length.
  • the displayed peptide or polypeptide can be derived from, but need not be limited to, human antibody heavy or light chains.
  • a pharmaceutical composition refers to a formulation which comprises an antibody or peptide or polypeptide of the invention and a pharmaceutically acceptable carrier, excipient or diluent thereof, or an antibody-pharmaceutical agent (antibody-agent) complex and a pharmaceutically acceptable carrier, excipient or diluent thereof.
  • An agent in the context of the present invention is useful in the treatment of active disease, prophylactic treatment, or diagnosis of a mammal including, but not restricted to, a human, bovine, equine, porcine, murine, canine, feline, or any other warmblooded animal.
  • the agent is selected from the group of radioisotope, toxin, oligonucleotide, recombinant protein, antibody fragment, anti-cancer agents, anti- leukemic, anti-metastasis, anti-neoplastic, anti-disease, anti-adhesion, anti-thrombosis, anti-restenosis, anti-autoimmune, anti-aggregation, anti-bacterial, anti-viral, and anti- inflammatory agents.
  • antiviral agents including acyclovir, ganciclovir, and zidovudine
  • anti-thrombosis/restenosis agents including cilostazol, dalteparin sodium, reviparin sodium, and aspirin
  • anti-inflammatory agents including zaltoprofen, pranoprofen, droxicam, acetyl salicylic 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, amtolmetin, celecoxib, indomethacin, rofecoxib, and nimesulid
  • anti-autoimmune agents including leflunomide, denileukin diftitox, subreum, WinRho SDF, defibrotide, and cyclophosphamide
  • anti- adhesion/anti-aggregation agents including limaprost, clorcromene, and
  • An anti-leukemia agent is an agent with anti-leukemia activity.
  • anti-leukemia agents include agents that inhibit or halt the growth of leukemic or immature pre-leukemic cells, agents that kill leukemic or pre-leukemic cells, agents that increase the susceptibility of leukemic or pre-leukemic cells to other anti-leukemia agents, and agents that inhibit metastasis of leukemic cells.
  • an anti- leukemia agent may also be an agent with anti-angiogenic activity that prevents, inhibits, retards or halts vascularization of tumors.
  • the expression pattern of a gene can be studied by analyzing the amount of gene product produced under various conditions, at specific times, in various tissues, etc.
  • a gene is considered to be "over-expressed" when the amount of gene product is higher than that found in a normal control, e.g., non-diseased control.
  • a given cell may express on its surface a protein having a binding site (or epitope) for a given antibody, but that binding site may exist in a cryptic form (e.g., be sterically hindered or blocked, or lack features needed for binding by the antibody) in the cell in a state, which may be called a first stage (stage I).
  • Stage I may be, e.g., a normal, healthy, non-diseased status.
  • the epitope maybe exposed by, e.g., undergoing modifications itself, or being unblocked because nearby or associated molecules are modified or because a region undergoes a conformational change.
  • modifications include changes in folding, changes in post-translational modifications, changes in phospholipidation, changes in sulfation, changes in glycosylation, and the like.
  • Such modifications may occur when the cell enters a different state, which may be called a second stage (stage H).
  • second states, or stages include activation, proliferation, transformation, or in a malignant status.
  • the epitope may then be exposed, and the antibody may bind.
  • Peptido-mimetics are molecules (e.g., antibodies) that no longer contain any peptide bonds, i.e., amide bonds, between amino acids; however, in the context of the present invention, the term peptide mimetic is intended to include molecules that are no longer completely peptidic in nature, such as pseudo-peptides, semi- peptides and peptoids. Whether completely or partially non-peptide, peptidomimetics according to this invention provide a spatial arrangement of reactive chemical moieties that closely resembles the three-dimensional arrangement of active groups in the peptide on which the peptidomimetic is based. These molecules include small molecules, lipids, polysaccharides, or conjugates thereof.
  • Phagemids are plasmid vectors designed to contain an origin of replication from a filamentous phage, such as Ml 3 or fd.
  • the subject invention provides for peptides or polypeptides that comprise an Fv molecule, a construct thereof, a fragment thereof, a construct of a fragment thereof, or a fragment of a construct, all of which have enhanced binding characteristics.
  • the Fv can be a scFv or a dsFv.
  • the Fv molecule described above can be used to target a diseased cell.
  • the diseased cell can be, for example, a cancer cell.
  • types of cancer that are amenable to diagnosis and/or treatment by specific targeting include, but are not limited to, carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma.
  • Leukemia, lymphoma, and myeloma are cancers that originate in the bone marrow and lymphatic tissues and are involved in uncontrolled growth of cells.
  • Antibodies that bind to PSGL-1 and or GPIb were identified using a phage display library and disclosed in U.S. Application Nos. 10/032,423; 10/032,037; 10/029,988; 10/029,926; 09/751,181; 10,189,032; and 60/258,948 and International Application Nos. PCT/US01/49442 and PCT/US01/49440. Specific examples of antibodies disclosed in these applications include the Yl, Y17, and L32 antibodies.
  • the sulfated epitopes binding to Y1/Y17/L32 are characterized by the presence of sulfated moieties, such as sulfated tyrosine residues or sulfated carbohydrate or lipid moieties, preferably within a cluster of two or more acidic amino acids, which are found on ligands and receptors that play important roles in such diverse processes as inflammation, immune reactions, infection, autoimmune reactions, metastasis, adhesion, thrombosis and/or restenosis, cell rolling, and aggregation.
  • sulfated moieties such as sulfated tyrosine residues or sulfated carbohydrate or lipid moieties, preferably within a cluster of two or more acidic amino acids, which are found on ligands and receptors that play important roles in such diverse processes as inflammation, immune reactions, infection, autoimmune reactions, metastasis, adhesion, thrombosis and/or restenosis, cell rolling, and
  • Such epitopes are also found on diseased cells, such as T-ALL cells, B-leukemia cells, B-CLL cells, AML cells, multiple myeloma cells, and metastatic cells. These epitopes are useful targets for the therapeutic mediation of these processes (as well as targeting agents) and for diagnostic procedures.
  • the antibodies of the present invention bind to AML cells that are of subtype M3 or above, but not M0 or Ml subtype cells.
  • the antibodies may or may not bind M2 subtype cells.
  • the antibodies of the present invention do not bind normal, healthy bone marrow (e.g., CD34+ cells). It is thought that such differences are based on alterations in PSGL-1 expression and/or sulfation, as well as possible conformational changes in PSGL-1 that expose a slightly different epitope.
  • KU812 cells a human chronic myeloid leukemia cell line that expresses low levels of GPIb, binds the Yl antibody. Following growth of KU812 cells in sodium chlorate, which inhibits sulfation but not expression of the GPIb protein, binding of Yl to the cells was reduced by 50%.
  • tyrosine-sulfated peptides based on amino acids 273 to 285 of GPIb competitively inhibit binding of the Yl antibody to platelets, while non-sulfated peptides do not inhibit binding of the Yl antibody to platelets.
  • the invention comprises or employs an antibody or fragment thereof that recognizes and binds to an epitope comprising a sulfated tyrosine motif.
  • a motif comprises a peptide sequence that is rich in acidic residues (aspartate and glutamate) and contains at least one tyrosine. Recognition and binding depend at least in part on at least one of the tyrosines being sulfated.
  • One such antibody is Yl or a fragment thereof. Although Yl is the antibody referred to in the embodiments described herein, this should not be understood as limiting the invention to embodiments that employ Yl.
  • the invention includes embodiments that use other antibodies that bind to an epitope comprising a sulfated tyrosine motif, including but not limited to antibodies related to Yl and fragments thereof that retain binding specificity.
  • an antibody or fragment thereof that binds to an epitope comprising a sulfated tyrosine motif, wherein the binding is dependent on at least one tyrosine of the motif being sulfated.
  • the antibody mediates antibody-dependent cell cytotoxicity.
  • the antibody is Yl or a related antibody, or a fragment thereof.
  • the invention further provides an agent complexed with (e.g., associated, combined, fused, or linked to) such an antibody or fragment thereof. Between 1 and 16 agent molecules, or more, can be bound to each antibody.
  • the antibody has four disulfide bonds at the hinge region that can be selectively reduced to eight thiol groups. By using a linker that can covalently bond to thiol functions and which carries one agent molecule, up to eight agent molecules can be attached to the antibody. By using a linker that similarly reacts with thiol functions but carries n agent molecules, up to 8n agent molecules can be attached to the antibody.
  • each heavy chain is complexed with about 2 copies of the agent and each light chain is complexed with about 2 copies of the agent.
  • agent molecules can be linked to the antibody by using intermediate drug carriers such as natural (e.g. dextran) and synthetic (e.g. HPMA) polymers as well as liposomes (e.g., antibody — linker — carrier — agent).
  • Agents can also be linked directly or indirectly to free amino groups of the antibody.
  • agents can be linked to free ⁇ - or ⁇ -amino groups via a linker.
  • an agent is joined to a linker directly, or first to a carrier, which is then joined to a linker.
  • the linker-agent or linker-carrier-agent complex is then joined to the antibody.
  • the antibody-agent complex can be internalized by a tumor cell, wherein the agent brings about the cell's death.
  • the antibody-agent linkage can be broken inside the cell by, for example, acid cleavage or enyzme cleavage.
  • the antibody is Yl or a related antibody, or a fragment thereof.
  • the invention further provides a composition for treating a disease comprising Yl or a Yl-agent complex.
  • the present invention provides methods of inducing or activating ADCC by administering the antibodies of the present invention. Accordingly, these antibodies may activate ADCC and or stimulate natural killer (NK) cells (e.g. CD56+), ⁇ T-cells, and/or monocytes, which may result in cell lysis.
  • NK natural killer
  • an antibody comprising an Fc region or portion of the antibody
  • said antibody binds to an Fc receptor (FcR) on effector cells, for example, NK cells, triggering the release of perform and granzyme B and/or induction of Fas B expression, which then leads to apoptosis.
  • FcR Fc receptor
  • Binding of FasL expressed on effector cells to the Fas receptor on the target cell surface may induce target cell apoptosis via activation of the Fas receptor signal transduction pathway.
  • the antibody of the invention induces FasL expression on effector cells.
  • cytokines IL-2 and G-CSF, for example
  • incubation time the number of receptors present on the surface of the cells, and antibody affinity.
  • a method of inducing cell death by administering to a patient in need thereof an antibody of the present invention coupled or complexed to an agent, wherein the antibody-agent couple or complex enters the cell by internalization and the antibody-agent conjugate or complex is cleaved, releasing the agent is provided.
  • Internalization can take place by any suitable means, for example, by endocytosis or by phagocytosis.
  • the invention thus provides a means of treating a disease (e.g., treating can include ameliorating the effects of a disease, preventing a disease, or inhibiting the progress of a disease) in a patient.
  • an antibody is used to introduce an agent into a cell.
  • the antibody binds to proteins preferentially expressed on the surface of diseased cells, such as proteins with sulfated tyrosine residues.
  • the agent is a toxin such as doxorubicin, morpholino-doxorubicin, or mo holino-daunorubicin.
  • the toxin is linked to the antibody via an adipic acid linker or an [N- ⁇ -Maleimidocaproic acid hydrazide linker.
  • the adipic acid linker has been used to bind to the ⁇ amino groups, whereas the N-[maleimidocaproic acid] hydrazide linker has been used to bind to both the ⁇ amino groups and also to the SH groups of the reduced disulphide linkages (via the maleimido group to form a C-S bond).
  • a hydrazone bond is formed between the drug and the N-[maleimidocaproic acid] hydrazide linker.
  • the invention provides a composition for treating a disease comprising such an antibody-toxin conjugate.
  • Another embodiment provides an analogous method for introducing a non- toxic agent into a cell.
  • the non-toxic agent can be used to change the behavior or activity of the cell, for example by directly or indirectly activating or repressing the activity of a specific gene.
  • the present invention provides a method of preventing infection by a virus comprising administering to a patient in need thereof an antibody as herein.
  • a means of treating a disease is accomplished by administering an antibody that blocks infection.
  • the cell expresses on its surface a protein containing a sulfated-tyrosine motif-containing epitope that is recognized by the antibody and that is also necessary for infection by the infectious agent.
  • the antibody binds to the protein, thereby blocking infection. Proteins that the preferred antibody is known to bind via a sulfated tyrosine motif-containing epitope include fibrinogen ⁇ chain, GPlb- ⁇ chain, complement C4, and PSGL-1.
  • Proteins that the preferred antibody is believed to bind via a sulfated tyrosine motif-containing epitope include CCR5 and CXCR4. Either of CCR5 and CXCR4 can function as a coreceptor necessary for HIV infection.
  • the antibody could be used to block infection by an HIV strain.
  • the antibody is Yl.
  • a method for introducing an agent into a cell that expresses sulfated PSGL-1 having the following steps: coupling or complexing the agent to an antibody as described herein and administering the antibody-agent couple or complex to the cell.
  • the antibody of the present invention binds to sulfated PSGL-1.
  • White cells involved in inflammation such as monocytes, neutrophils, and lymphocytes, are primarily recruited by the four adhesion molecules, PSGL-1, P-selectin, VLA-4, and VCAM-1 in the inflammatory processes of diseases such as atherosclerosis (Huo and Ley, Ada Physiol. Scand., 173: 35-43 (2001); Libby, Sci. Am. May: 48-55 (2002); Wang et al., J. Am. Coll. Cardiol. 38: 577-582 (2001)).
  • the antibody's interference with any of these central molecules may suggest a potential role for the antibody in abrogating related diseases.
  • P-selectin controls cell attachment and rolling. Additionally, P- selectin - PSGL-1 interactions activate a number of other molecules on cells which are integrally connected with tumorigenesis (when concerned with malignant cells) and inflammatory responses (when concerned with white blood cells) (Shebuski and Kilgore, J. Pharmacol. Exp. Ther. 300: 729-735 (2002)). Based on this understanding of P- selectin's ability to regulate cellular processes, it is apparent that the antibody's enhanced scFv selectivity for sulfated PSGL-1 may make it a superior molecule for treating a variety of malignant and inflammatory diseases.
  • the antibody of the present invention binds to an epitope present on at least one cell type involved in inflammation or tumorigenesis, including T-ALL cells, AML cells, Pre-B-ALL cells, B-leukemia cells, B-CLL cells, multiple myeloma cells, and metastatic cells.
  • the antibody of the present invention may bind to epitopes on a lipid, carbohydrate, peptide, glycolipid, glycoprotein, lipoprotein, and/or lipopolysaccharide molecule.
  • Such epitopes preferably have at least one sulfated moiety.
  • the antibody of the present invention crossreacts with two or more epitopes, each epitope having one or more sulfated tyrosine residues, and at least one cluster of two or more acidic amino acids, an example of which is PSGL-1.
  • These antibodies, antigen-binding fragment or complex thereof, of the present invention may be internalized into a cell following binding to PSGL-1 on the surface of the cell. Such internalization may occur via endocytosis as an active process, which is manner, time and temperature dependent. For example, Yl is specifically internalized into cells from AML patients via PSGL-1.
  • the antibody of the present invention binds to proteins having tyrosine sulfation sites.
  • Such proteins include PSGL-1, GPIb, ⁇ -2antiplasmin; aminopeptidase B; CC chemokine receptors such as CCR2, CCR5, CCR3, CXCR3, CXCR4, CCR8, CCR2b, and CXCI; seven-transmembrane-segment (7TMS) receptors; coagulation factors such as factor V, VIII, and LX; fibrinogen gamma chain; heparin cofactor II; secretogranins such as secretogranin I and II; vitronectin, amyloid precursor, ⁇ -2-antiplasmin; cholecystokinin; ⁇ -choriogonadotropin; complement C4; dermatan sufaieprobizlycan; fibrinectin; and castrin.
  • TMS seven-transmembrane-segment
  • the antibody of the present invention binds to sulfated CC chemokine receptors such as CCR5, CXCR4, CXCI, and CCR2b.
  • sulfated tyrosines may contribute to the binding of CCR5 to MlP-l ⁇ , MlP ⁇ , and HIV-1 gpl20/CD4 and to the ability of HIV-1 to enter cells expressing CCR5 and CD4.
  • Antibodies, peptides, polypeptides, proteins, and fragments and constructs thereof can be produced in either prokaryotic or eukaryotic expression systems. Methods for producing antibodies and fragments in prokaryotic and eukaryotic systems are well- known in the art.
  • a eukaryotic cell system refers to an expression system for producing peptides or polypeptides by genetic engineering methods, wherein the host cell is a eukaryote.
  • a eukaryotic expression system may be a mammalian system, and the peptide or polypeptide produced in the mammalian expression system, after purification, is preferably substantially free of mammalian contaminants.
  • Other examples of a useful eukaryotic expression system include yeast expression systems.
  • a preferred prokaryotic system for production of the peptide or polypeptide of the invention uses E. coli as the host for the expression vector.
  • the peptide or polypeptide produced in the E. coli system, after purification, is substantially free of E. coli contaminating proteins.
  • Use of a prokaryotic expression system may result in the addition of a methionine residue to the N-terminus of some or all of the sequences provided for in the present invention. Removal of the N-terminal methionine residue, after peptide or polypeptide production to allow for full expression of the peptide or polypeptide, can be performed as is known in the art, one example being with the use of Aeromonas aminopeptidase under suitable conditions (U.S. Patent No. 5,763,215).
  • the antibodies and polypeptides of the subject invention can be complexed with e.g. associated with, combined, fused, or linked to various pharmaceutical agents, such as drugs, toxins, and radioactive isotopes and optionally, with a pharmaceutically effective carrier, to form peptide-drug compositions comprising an antibody/polypeptide and a pharmaceutical agent having anti-disease and/or anti-cancer activity. Such compositions may also be used for diagnostic purposes.
  • conjugation or complexing of antlracyclines to antibodies is generally known in the art (Dubowchik & Walker, Pharmacol. & Tliera. 83: 67-123 (1999); Trail et al., Cancer Immunol. Immunother. 52: 328-337 (2003)).
  • Such conjugation can be by direct conjugation or via linkers, such as acid cleavable linkers or enzyme cleavable linkers and may involve the use of intermediate carriers such as dextran and synthetic polymers.
  • Antliracyclines have been complexed to the antibodies of the present invention via (1) ⁇ amino groups (about pH 8) to produce a drug:antibody ratio of 4:1 (in which case two drug molecules are attached to the heavy chain and two to the light chain); and (2) disulfide linkages to produce a drug antibody ratio of between 4:1 and 8:1 depending or the method used.
  • the drug antibody ratio can, for example, be doubled, tripled or quadrupled, etc, by using a two, three, four, etc., branched linker.
  • One skilled in the art may make chemical modifications to the antibody, linker, carrier and/or drug in order to make reactions more convenient for the purposes of preparing a conjugate.
  • the two disulfide linkages in the Fc region were reduced with mercaptoethylamine and was then reacted with the drug linker at about pH 7, which leads to a drug antibody ratio of 4:1 (in which case all the four drugs are attached in the heavy chains).
  • the four disulfide bonds in the hinge region were reduced with DTT (about pH 7) and was then reacted with the drug linker, which leads to a drug antibody ratio of about 7:1 to 8:1 (in which case 5 or 6 of the drug molecules are attached to the heavy chains and one or two of the drug molecules are attached to the light chains).
  • Examples of carriers useful in the invention include dextran, HPMA (a hydrophilic polymer), or any other polymer, such as a hydrophilic polymer, as well as derivatives, combinations and modifications thereof.
  • decorated liposomes also known as immunoliposomes, can be used, such as liposomes decorated with scFv Yl molecules, such as Doxil, a commercially available liposome containing large amounts of doxorubicin.
  • Such liposomes can be prepared to contain one or more desired agents and be admixed with the antibodies of the present invention to provide a high drug to antibody ratio.
  • the link between the antibody or polypeptide and the agent may be a direct link.
  • a direct link between two or more neighboring molecules may be produced via a chemical bond between elements or groups of elements in the molecules.
  • the chemical bond can be, for example, an ionic bond, a covalent bond, a hydrophobic bond, a hydrophilic bond, an electrostatic bond, or a hydrogen bond.
  • the bonds can be, for example, amide, carbon-sulfide, peptide, and/or disulfide bonds.
  • amine, carboxy, hydroxyl, thiol and ester functional groups may be used, as is known in the art to form covalent bonds.
  • linker compound is defined as a compound that joins two or more moieties.
  • the linker can be straight-chained or branched.
  • a branched linker compound may be composed of a double-branch, triple branch, or quadruple or more branched compound.
  • Linker compounds useful in the present invention include those selected from the group having dicarboxylic acids, malemido hydrazides, PDPH, carboxylic acid hydrazides, and small peptides.
  • linker compounds useful, according to the present invention include: (a) dicarboxylic acids such as succinic acid, glutaric acid, and adipic acid; (b) maleimido hydrazides such as N-[maleimidocaproic acid] hydrazide, 4-[N- maleimidomethyl]cyclohexan-l-carboxylhydrazide, and N-[maleimidoundecanoic acid] hydrazide; (c) (3-[2-pyridyldithio]propionyl hydrazide) derivatives, combinations, modifications and analogs thereof; and (d) carboxylic acid hydrazides selected from 2-5 carbon atoms.
  • dicarboxylic acids such as succinic acid, glutaric acid, and adipic acid
  • maleimido hydrazides such as N-[maleimidocaproic acid] hydrazide, 4-[N- maleimidomethyl]cyclohexan-l-car
  • Linking via direct coupling using small peptide linkers is also useful.
  • direct coupling between the free sugar of, for example, the anti-cancer drug doxorubicin and a scFv may be accomplished using small peptides.
  • small peptides include AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, Protein C, S-TAG ® , T7, V5, VSV-G, and KAK.
  • Antibodies and polypeptides of the present invention may be bound to, conjugated to, complexed with, or otherwise associated with imaging agents (also called indicative markers), such as radioisotopes, and these conjugates can be used for diagnostic and imaging purposes. Kits having such radioisotope-antibody (or fragment) complexes are provided.
  • radioisotopes useful for diagnostics include ⁇ indium,
  • radioactive isotopes are opaque to X-rays or any suitable paramagnetic ions.
  • the indicative marker molecule may also be a fluorescent marker molecule.
  • fluorescent marker molecules examples include fluorescein, phycoerythrin, or rhodamine, or modifications or conjugates thereof.
  • Antibodies and polypeptides conjugated to indicative markers may be used to diagnose, prognose, or monitor disease states.
  • such methods include providing a sample of at least one cell from a patient and determining whether the antibody or fragment thereof of the present invention binds to the cell of the patient, thereby indicating that the patient is at risk for or has the disease.
  • Such monitoring may be carried out in vivo, in vitro, or ex vivo.
  • the imaging agent is preferably physiologically acceptable in that it does not harm the patient to an unacceptable level. Acceptable levels of harm may be determined by clinicians using such criteria as the severity of the disease and the availability of other options.
  • staging a disease in a patient generally involves determining the classification of the disease based on the size, type, location, and invasiveness of the tumor.
  • One classification system to classify cancer by tumor characteristics is the "TNM Classification of Malignant Tumours" (6th Edition) (L.H. Sobin, Ed.), which is incorporated by reference herein and which classifies stages of cancer into T, N, and M categories with T describing the primary tumor according to its size and location, N describing the regional lymph nodes, and M describing distant metastases.
  • TNM Classification of Malignant Tumours (6th Edition) (L.H. Sobin, Ed.)
  • N describing the primary tumor according to its size and location
  • N describing the regional lymph nodes
  • M describing distant metastases the numbers I, II, III and IV are used to denote the stages and each number refers to a possible combination of TNM factors.
  • a Stage I breast cancer is defined by the TMN group: Tl, NO, M0 which mean:Tl - Tumor is 2 cm or less in diameter, NO - No regional lymph node metastasis, M0 - No distant metastasis.
  • Tl Tumor
  • M0 No distant metastasis.
  • Another system is used to stage AML, with subtypes of classified based on the French- American-British system using the morphology observed under routine processing and cytochemical staining.
  • a recently proposed World Health Organization (WHO) staging or classification of neoplastic diseases of the hematopoietic and lymphoid tissues includes (specifically for AMLs) traditional FAB-type categories of disease, as well as additional disease types that correlate with specific cytogenetic findings and AML associated with myelodysplasia.
  • Others have also proposed pathologic classifications.
  • one proposal specific for AML includes disease types that correlate with specific cytogenetic translocations and can be recognized reliably by morphologic evaluation and immunophenotyping and that incorporate the importance of associated myelodysplastic changes. This system would be supported by cytogenetic or molecular genetic studies and could be expanded as new recognizable clinicopathologic entities are described (Arber, Am. J. Clin. Pathol. 115(4): 552-60 (2001)).
  • the present invention provides for a diagnostic kit for in vitro analysis of treatment efficacy before, during, or after treatment, having an imaging agent having a peptide of the invention linked to an indicative marker molecule, or imaging agent.
  • the invention further provides for a method of using the imaging agent for diagnostic localization and imaging of a cancer, more specifically a tumor, having the following steps: (a) contacting the cells with the composition; (b) measuring the radioactivity bound to the cells; and hence (c) visualizing the tumor.
  • suitable imaging agents include fluorescent dyes, such as
  • fluorescent proteins such as green fluorescent proteins.
  • Other examples include radioactive molecules and enzymes that react with a substrate to produce a recognizable change, such as a color change.
  • the imaging agent of the kit is a fluorescent dye, such as
  • FITC and the kit provides for analysis of treatment efficacy of cancers, more specifically blood-related cancers, e.g., leukemia, lymphoma, and myeloma.
  • FACS analysis is used to determine the percentage of cells stained by the imaging agent and the intensity of staining at each stage of the disease, e.g., upon diagnosis, during treatment, during remission and during relapse.
  • Antibodies and polypeptides of the present invention may be bound to, conjugated to, or otherwise associated with anti-cancer agents, anti-neoplastic agents, antiviral agents, anti-metastatic agents, anti-inflammatory agents, anti-thrombosis agents, anti- restenosis agents, anti-aggregation agents, anti-autoimmune agents, anti-adhesion agents, anti-cardiovascular disease agents, pharmaceutical agents, or other anti-disease.
  • An agent refers to an agent that is useful in the prophylactic treatment or diagnosis of a mammal including, but not restricted to, a human, bovine, equine, porcine, murine, canine, feline, or any other warm-blooded animal.
  • anti- viral agents including acyclovir, ganciclovir and zidovudine
  • anti-thrombosis/restenosis agents including cilostazol, dalteparin sodium, reviparin sodium, and aspirin
  • anti-inflammatory agents including zaltoprofen, pranoprofen, droxicam, acetyl salicylic 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, amtolmetin, celecoxib, indomethacin, rofecoxib, and nimesulid
  • anti-autoimmune agents including leflunomide, denileukin diftitox, subreum, WinRho SDF, defibrotide, and cyclophosphamide
  • anti- adhesion/anti-aggregation agents including limaprost, clorcromene, and
  • Exemplary pharmaceutical agents include anthracyclines such as doxorubicin (adriamycin), daunorubicin, idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, mo ⁇ holinodoxorabicin, morpholinodaunorubicin, methoxymo ⁇ holinyldoxorubicin, methoxymorpholinodaunorubicin and methoxymo ⁇ holinyldoxorubicin and substituted derivatives, combinations and modifications thereof.
  • anthracyclines such as doxorubicin (adriamycin), daunorubicin, idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, mo ⁇ holinodoxorabicin, morpholinodaunorubicin, methoxymo ⁇ holinyldoxorubicin, methoxymorpholinodaun
  • exemplary pharmaceutical agents include cis-platinum, taxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, fludarabine, idarubicin, chlorambucil, interferon alpha, hydroxyurea, temozolomide, thalidomide and bleomycin, and derivatives, combinations and modifications thereof.
  • An anti-cancer agent is an agent with anti-cancer activity.
  • anti-cancer agents include agents that inhibit or halt the growth of cancerous or immature pre-cancerous cells, agents that kill cancerous or pre-cancerous cells, agents that increase the susceptibility of cancerous or pre-cancerous cells to other anti-cancer agents, and agents that inhibit metastasis of cancerous cells.
  • an anti-cancer agent may also be an agent with anti-angiogenic activity that prevents, inhibits, retards, or halts vascularization of tumors.
  • Inhibition of growth of a cancer cell includes, for example, the (i) prevention of cancerous or metastatic growth, (ii) slowing down of the cancerous or metastatic growth, (iii) the total prevention of the growth process of the cancer cell or the metastatic process, while leaving the cell intact and alive, (iv) interfering contact of cancer cells with the microenvironment, or (v) killing the cancer cell.
  • an antibody could effect the killing of a cancer cell by binding to the cancer cell and thereby stimulating T cells or natural killer cells to kill the bound cell by antibody-dependent cell cytotoxicity.
  • An anti-leukemia agent is an agent with anti-leukemia activity.
  • anti-leukemia agents include agents that inhibit or halt the growth of leukemic or immature pre-leukemic cells, agents that kill leukemic or pre-leukemic, agents that increase the susceptibility of leukemic or pre-leukemic cells to other anti-leukemia agents, and agents that inhibit metastasis of leukemic cells.
  • an anti- leukemia agent may also be agent with anti-angiogenic activity that prevents, inhibits, retards or halts vascularization of tumors.
  • Inhibition of growth of a leukemia cell includes, for example, the (i) prevention of leukemic or metastatic growth, (ii) slowing down of the leukemic or metastatic growth, (iii) the total prevention of the growth process of the leukemia cell or the metastatic process, while leaving the cell intact and alive, (iv) interfering contact of cancer cells with the microenvironment, or (v) killing the leukemia cell.
  • Examples of anti-disease, anti-cancer, and anti-leukemic agents to which antibodies and fragments of the present invention may usefully be linked include toxins, radioisotopes, and pharmaceuticals.
  • toxins examples include gelonin, Pseudomonas exotoxin (PE), PE40,
  • PE38 diphtheria toxin, ricin, or derivatives, combinations and modifications thereof.
  • radioisotopes examples include gamma-emitters, positron-emitters, and x-ray emitters that may be used for localization and/or therapy, and beta-emitters and alpha-emitters that may be used for therapy.
  • the radioisotopes described previously as useful for diagnostics are also useful for therapeutics.
  • Non-limiting examples of anti-cancer or anti-leukemia agents include anthracyclines such as doxorubicin (adriamycin), daunorubicin, idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, mo ⁇ holinodoxorubicin, mo ⁇ holinodaunorubicin,methoxymo ⁇ holinyldoxorubicin,methoxymo ⁇ holinodaunorubic in and methoxymo ⁇ holinyldoxorubicin and substituted derivatives, combinations and modifications thereof.
  • anthracyclines such as doxorubicin (adriamycin), daunorubicin, idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, mo ⁇ holinodoxorubicin, mo ⁇ holinodaunorubicin,methoxymo ⁇ holin
  • Exemplary pharmaceutical agents include cis-platinum, taxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, idarubicin, fludarabine, chlorambucil, interferon alpha, hydroxyurea, temozolomide, thalidomide, and bleomycin, and derivatives, combinations and modifications thereof.
  • the pharmaceutical compositions of the present invention have an antibody or polypeptide of the present invention and a pharmaceutically acceptable carrier.
  • the antibody or polypeptide can be present in an amount effective to inhibit cell rolling, inflammation, auto-immune disease, metastasis, growth and/or replication of tumor cells or leukemia cells, or increase in number of tumor cells in a patient having a tumor or leukemia cells in a patient having leukemia.
  • the antibody or polypeptide can be present in an amount effective to increase mortality of tumor cells or leukemia cells.
  • the antibody or polypeptide can be present in an amount effective to alter the susceptibility of diseased cells to damage by anti-disease agents, tumor cells to damage by anti-cancer agents, or leukemia cells to damage by anti-leukemia agents. Further alternatively, the antibody or polypeptide can be present in an amount effective to decrease number of tumor cells in a patient having a tumor or leukemia cells in a patient having leukemia. Yet further alternatively, the antibody or polypeptide can be present in an amount effective to inhibit restenosis. The antibody, or polypeptide can also be present in an amount effective to inhibit HIV entry. Alternatively, the antibody or polypeptide, can be used as a targeting agent to direct a therapeutic to a specific cell or site.
  • Antibodies and polypeptides of the present invention may be administered to patients in need thereof via any suitable method.
  • Exemplary methods include intravenous, intramuscular, subcutaneous, topical, intratracheal, intrathecal, intraperitoneal, intralymphatic, nasal, sublingual, oral, rectal, vaginal, respiratory, buccal, intradermal, transdermal, or intrapleural administration.
  • the formulation preferably will be prepared so that the amount administered to the patient will be an effective amount from about 0.1 mg to about 1000 mg of the desired composition. More preferably, the amount administered will be in the range of about 1 mg to about 500 mg of the desired composition.
  • the compositions of the invention are effective over a wide dosage range and depend on factors such as the particulars of the disease to be treated, the half-life of the peptide, or polypeptide-based pharmaceutical composition in the body of the patient, physical and chemical characteristics of any agent complexed to antibody or fragment thereof and of the pharmaceutical composition, mode of administration of the phamiaceutical composition, particulars of the patient to be treated or diagnosed, as well as other parameters deemed important by the treating physician.
  • composition for oral administration may be in any suitable form. Examples include tablets, liquids, emulsions, suspensions, syrups, pills, caplets, and capsules. Methods of making pharmaceutical compositions are well known in the art (See, e.g., Remington, The Science and Practice of Pharmacy, Alfonso R. Gennaro (Ed.) Lippincott, Williams & Wilkins (pub)).
  • the pharmaceutical composition may also be formulated so as to facilitate timed, sustained, pulsed, or continuous release.
  • the pharmaceutical composition may also be administered in a device, such as a timed, sustained, pulsed, or continuous release device.
  • the pharmaceutical composition for topical administration can be in any suitable form, such as creams, ointments, lotions, patches, solutions, suspensions, lyophilizates, and gels.
  • compositions having antibodies, constructs, conjugates, and fragments of the subject invention may comprise conventional pharmaceutically acceptable diluents, excipients, carriers, and the like.
  • Tablets, pills, caplets, and capsules may include conventional excipients such as lactose, starch, and magnesium stearate.
  • Suppositories may include excipients such as waxes and glycerol.
  • hijectable solutions comprise sterile pyrogen-free media such as saline, and may include buffering agents, stabilizing agents or preservatives. Conventional enteric coatings may also be used.
  • the antibodies and polypeptides of the present invention and pharmaceutical compositions thereof can be used in methods of ameliorating the effects of a disease, preventing a disease, treating a disease, or inhibiting the progress of a disease in patients in need thereof.
  • Such methods include inhibiting cell rolling, inflammation, autoimmune disease, metastasis, growth and/or replication of tumor cells or leukemia cells, or increase in number of tumor cells in a patient having a tumor or leukemia cells in a patient having leukemia.
  • such methods include increasing the mortality rate of tumor cells or leukemia cells, alter the susceptibility of diseased cells to damage by anti-disease agents, tumor cells to damage by anti-cancer agents, or leukemia cells to damage by anti-cancer agents.
  • Such methods also include decreasing number of tumor cells in a patient having tumor or leukemia cells in a patient having leukemia.
  • Such methods also include inhibiting or decreasing HIV entry in cells.
  • Such methods further include preventing or inhibiting cardiovascular diseases such as restenosis.
  • the present invention moreover provides a method of manufacturing a medicament for the treatment of various disease states such as, e.g., AML, T-ALL, B- leukemia, B-CLL, Pre-B-ALL, multiple myeloma, metastasis, HIV infection, cardiovascular diseases, or other diseases in which such cellular functions or actions as cell rolling, inflammation, immune reactions, infection, autoimmune reactions, metastasis, play a significant role.
  • Such medicament comprises the antibodies and the polypeptides of the present invention.
  • the invention provides a method of diagnosing cancer in a person by assaying the ability of Yl to bind specifically to a tissue sample and comparing Yl binding to binding by a control antibody such as KPL-1.
  • the method comprises isolating cell samples from blood or solid tissue from the person, incubating the cells with an antibody or a fragment thereof that recognizes a sulfated tyrosine motif-containing epitope ("the experimental antibody"), washing away the non-specifically bound antibody, and comparing the results to those of a corresponding staining procedure performed with a reference standard such as a control antibody with known binding activity.
  • a control antibody is one that recognizes an epitope containing the unsulfated form of the tyrosine motif or antigens that contain such.
  • the presence of tumor cells is indicated when the experimental antibody binding is substantially greater than binding by the control, as determined by the strength of the staining.
  • the staining procedure can be performed by standard methods.
  • the first antibody can be visualized by using secondary antibodies that recognize the first antibody and that are conjugated to an enzyme substrate which produces a color reaction when acted on by the enzyme.
  • the presence of tumor cells is indicated when both the experimental antibody and the control antibody bind to the cells, but the cells internalize Yl and do not internalize the control antibody.
  • the cancer is a solid tumor.
  • the cancer is a blood-borne tumor.
  • the experimental antibody is Yl or a fragment thereof, or a related antibody or a fragment thereof.
  • the control antibody is KPLl.
  • the invention provides a method of diagnosing a cancer comprising screening cell samples from blood or solid tissue for the presence of tumor cells.
  • Western blots are performed on cell sample lysates using Yl or a fragment thereof, or a related antibody or a fragment thereof.
  • Yl binding can be observed by tagging Yl itself with a detectable label, or by using standard methods that employ a detectable anti-human antibody.
  • the presence of tumor cells is indicated when Yl binding is substantially greater than binding by the control, where the control is defined as above.
  • the presence of tumor cells is indicated when Yl binding is substantially greater than binding by the control.
  • the invention provides a method of identifying protein markers of blood-borne or solid tumors by preparing a cell lysate and purifying the lysate by passing it through an affinity column.
  • the affinity column inco ⁇ orates Yl or a fragment thereof, or a related antibody or fragment thereof, hi one embodiment, the cell lysate is derived from a primary tissue sample collected from a human being.
  • the cell lysate is derived from a tumor cell line.
  • the tumor cell line can be an immortalized cell line.
  • the invention provides a method of monitoring the stage of a blood-borne cancer comprising isolating white blood cells from a patient with a blood-borne cancer, incubating the cells with Yl, determining the extent of Yl binding relative to reference standard.
  • a small inorganic chemical entity may be identified by screening of an appropriate combinatorial library.
  • Such a chemical entity may have a number of advantages over a scFv or IgG-based therapeutic agent.
  • an inorganic chemical entity may be administered orally and have an enhanced biosafety profile, including reduced immuno-crossreactivity. It may provide enhanced selectivity towards the target, particularly following rational drug design to optimize an initially selected lead compound.
  • Other advantages include lower production costs, longer shelf-life and a less complicated regulatory approval process.
  • a ligand-driven approach may be taken to identify inorganic chemical entities, which have very narrow specificity, or alternately, target more than one sulfated tyrosine epitope for disease states such as re-perfusion injury which involves more than one distinct target each bearing such an epitope.
  • the ligand-driven approach significantly shortens the screening process for identifying targets for therapeutic intervention, and enables simultaneous target validation with lead optimization, which maybe carried out with a series of focused libraries.
  • a library of inorganic chemical entities specialized for targeting sulfated tyrosine epitopes may be designed and developed first by analyzing the three dimensional interaction between an antibody such as Yl and its known targets such as residues sulfated Tyr-276 and Asp-277 of GPIb.
  • Chemical libraries composed of entities that mimic the Yl binding site and which provide increased affinity to the target may be developed by computer assisted combinatorial library design.
  • EXAMPLE 1 Identification of Yl ligand from primary AML cells (R1198-3)
  • stage M4 Primary AML cells (stage M4) were collected from a patient and lysed. The lysate was subjected to purification comprising affinity chromatography on a Yl-IgG column (see Fig. 1). The isolated protein was digested with endoproteinase Asp- N, and the resulting peptide sequence was determined using mass spectrometry. The sequence was identical to the published human PSGL-1 N-terminal amino acid sequence. These results indicate that primary AML cells at stage 4 express PSGL-1 that can be bound by Yl-IgG. It was further determined that the purified protein was sulfated at tyrosines 2 and 3 of the Yl recognition motif (see Fig. 2). Internal controls were used to verify the specificity of the immunomodulatory effects of Yl e.g. no induction of mouse interleukin-6 secretion was detected.
  • Yl-IgG is capable of mediating antibody dependent cell cytotoxicity (ADCC) have shown this antibody mediates effector cell cytotoxicity of various target cells, including ML2 (an AML-derived cell line which served as a target in our model system) and B-CLL cells from patient clinical samples.
  • ML2 an AML-derived cell line which served as a target in our model system
  • B-CLL cells from patient clinical samples.
  • Yl-IgG binds these cell types via CD 162 (PSGL-1), a molecule which is substantially absent on healthy B- cells and early stage AML.
  • NK natural killer
  • CD56+ ⁇ T cells
  • monocytes CD14+
  • T-helper cells CD4+
  • cytotoxic T cells CD8+
  • Yl-IgG mediates activation of different types of effector cells, as measured by the appearance of an early activation marker (CD69+), secretion of cytokines, such as TNF ⁇ and IFN ⁇ and induction of FasL.
  • cytokines such as TNF ⁇ and IFN ⁇
  • FasL Hyper cross-linking (XL) of Yl-IgG with secondary anti-human Fc antibodies demonstrated that an apoptotic mechanism also contributes to cell killing.
  • Yl-IgG activity towards primary B-CLL cells in vitro was compared to that of two commercially available humanized antibodies currently used extensively for treatment of various lymphoid malignancies: Rituximab (which binds CD20) and Campath (which binds CD52). While the mechanism of action of Rituximab against B-CLL is not clear, its cytotoxic effects against CD20-positive malignant B cells may involve one or more of complement-dependent cytotoxicity (CDC), ADCC and induction of apoptosis. The cytotoxic effects of Campath against CD52-positive malignant B cells, as well as normal B and T cells, involves CDC, ADCC and induction of apoptosis. Campath administration is associated with complete ablation of all mature normal B and T cells, leading to severe hematological toxicity.
  • CDC complement-dependent cytotoxicity
  • FICOLL ® mononuclear cells from healthy donors were separated on FICOLL ® . Cells were incubated with or without Yl-IgG or control antibodies for 24 hours. FACS analysis with ⁇ CD69 (an early activation marker) antibody was performed for different types of effector cells. Secretion of cytokines such as TNF ⁇ and IFN ⁇ were measured by ELISA.
  • B-CLL cells from different patients were co-incubated for 24 hours with PBMC effector cells at different effector/target cell ratios.
  • Analysis of thirteen different B-CLL clinical samples indicated that Yl-IgG mediated effector cell cytotoxicity in all cases (Fig. 3) with the average extent of cell lysis about 21.4%.
  • Four of thirteen samples (30%) exhibited more than 30% lysis, while only two of thirteen samples (15%) exhibited less than 10% lysis. In some cases, a high degree of lysis was seen even at a low E:T ratio, e.g.
  • PBMC also effected ADCC against primary AML cells from a patient using varying ratios of PBMC:AML (10, 20, or 40: in the presence of 10 or 20 ⁇ g/ml Yl- IgG.
  • ML-2 cells provide a good model for ADCC since Yl-IgG binds without undergoing detectable internalization.
  • ML2 ADCC increases with Yl-IgG concentration: After 24 hours of incubation, cytotoxicity was higher in the presence of Yl-IgG than in its absence at four different effector (PBMC) to target ratios (5:1, 10:1, 20:1, 40:1) (Fig. 5). This effect was diminished or absent when mouse anti-PSGL-1 antibody KPLl was substituted for Yl- IgG and diminished even further when human IgG (which binds to the Fc receptor on effector cells) was used instead of Yl-IgG. A Yl-IgG concentration as low as 5 ⁇ g/ml could induce ADCC when the effecto ⁇ target ratio was 40:1.
  • Yl-IgG-induced ADCC of ML2 involves binding of PSGL-1 by Yl-IgG.
  • NK cells Natural killer (NK) cells (CD56+), ⁇ T cells and cytotoxic T-cells (CD8+) from normal donors and B-CLL patients were isolated using commercially available magnetic beads.
  • NK cells from both normal donors and from B-CLL patients KCS samples in Fig. 7A
  • KCS samples in Fig. 7A are capable of effecting ADCC on ML2 and B-CLL targets (KCS samples in Fig. 7A), resulting in 13 to 68% lysis over control.
  • ⁇ T cells were shown to mediate ADCC of ML2 cells.
  • cytotoxic T-cells do not appear to be involved in Yl- IgG mediated cytotoxicity.
  • CD 14+ cells are also involved in ADCC against ML2 targets, in addition to NK cells (CD56+) and ⁇ + T cells (Fig. 7B). All of the effector cells which are involved in Yl-IgG mediated toxicity express the Fc receptor, CD 16.
  • Yl mediates ADCC by natural killer cells, as measured by expression of CD69. Effector cells from six healthy donors were incubated for 24 hours at 37° C in the presence of Yl-IgG or human IgG or a murine anti- CD62 antibody (KPLl, PL1 or PL2) or in the absence of any antibody (control). FACS analysis was then performed and expression of the early activation marker CD69 on natural killer (NK) cells (CD56+) was determined. As shown in Fig.
  • Rituximab or Campath ® in the presence and absence of 25% of the patients' plasma. As shown in Figure 11, only Campath ® mediated cytotoxicity of primary B-CLL cells via CDC. Neither Rituximab nor Yl-IgG induced cytotoxicity via complement fixation.
  • Yl-IgG-M-Daunorubicin Derivative Antibody- toxin conjugates such as mo ⁇ holino-doxorubicin-Yl-IgG (Fig. 13) and antibody-M- daunorubicin conjugates (see below) were prepared. Daunorubicin was modified, joined to one of two different linkers, and then joined to the antibody via the antibody's free amino groups or via the antibody's reduced disulfide bonds.
  • M-DNR-LINKER refers to both (6-Maleimidoca ⁇ royl)hydrazone of Mo ⁇ hlinyldaunorubicin acetate and to M-DNR-AES.
  • M-DNR-Ac is stable for at least 12 months under argon at -20° C.
  • 6-maleimidocaproylhydrazide was added to a solution of M-DNR-Ac in dry methanol, under argon, followed by trifluoroacetic acid. The clear solution was protected from light and stirred for 24 hours at room temperature.
  • M-DNR-LLNKER in dry DMF was added to the MAb solution at a molar ratio M- DNR-LINKER/Yl-IgG of 23.
  • the mixture was gently shaken overnight at room temperature under argon, then centrifuged.
  • the supernatant was filtered through SPDM-X tubes (Costar) and shaken with Bio-Beads SM-2 (Biorad) for 1 hour at room temperature.
  • the mixture was allowed to stand for 10 minutes.
  • the supernatant was passed through PD-10 columns (Pharmacia) that had been equilibrated with PBS.
  • the conjugate was eluted with PBS and the protein-containing fractions combined.
  • the purified conjugate was sterilized by SPIN-X filtration.
  • the conjugate solutions were frozen and stored at - 70°C.
  • the product conjugate was obtained in 45-50% yield and had the following characteristics: 5% aggregates; 2%-5% absorbed, non-covalently linked M-DNR derivatives; average molecular ratio of drug to antibody is 4.
  • Yl-IgG in a buffer composed of NaCl, MES and EDTA was added to cysteamine hydrochloride solution (Merck) in the same buffer. The mixture was incubated at 37° C for 1.5 hours under argon. The reaction mixture was loaded onto a PD-10 column (Sephadex G-25, Pharmacia) that had been equilibrated with PBS/EDTA. The reduced protein was eluted with PBS EDTA. The fractions with the highest protein concentrations were combined and stored at 4° C. The molecular ratio of free sulfhydryl groups to antibody was at least 3.5.
  • IgG-Yl was reduced by passing it through a PD-10 column (Sephadex -25M, Pharmacia) in EDTA and eluting in PBS/EDTA. The protein-containing fractions were pooled. The molecular ratio of free sulfhydryl groups to antibody was at least 6. (6- Maleimidocaproyl)hydrazone of mo ⁇ hlinyldaunorubicin acetate in dry dimethylformamide was diluted in DMF and added to the reduced protein. The solution was incubated for 30 minutes at 4°C. The protein conjugate was purified on a PD-10 column in PBS and the protein fractions were sterilized by SPIN-X filtration (Corning Life Sciences). The conjugate was frozen and stored at -70°C. Yield was about 50% relative to original antibody. The final product contained less than 5% aggregates, free M-DNR was between 0-2%, and the average molecular ratio of drug to antibody was 7.
  • non-target cells health CD34+ stem cells
  • 1 ⁇ M Yl-IgG-M-DNR survived at least as well as control cells and cells incubated in the presence of 1 ⁇ M b-IgG-M-DNR or 0.1 ⁇ M M-DNR (i.e. a non-lethal dose of free drug).
  • a non-lethal dose of free drug i.e. a non-lethal dose of free drug
  • DNR was three times more inhibitory in inhibiting colony growth, relative to the nonspecific b-IgG-M-DNR conjugate at the same concentration (Fig. 13). That is, 1 ⁇ M Yl- IgG-M-DNR reduced viability of primary AML cells to 40% relative to the control (target cells incubated alone), while 0.1 ⁇ M M-DNR and 1 ⁇ M b-IgG-M-DNR each reduced viability of primary AML cells to 80% relative to the control.
  • B-ALL cells which do not express a Yl epitope (as assessed by failure to bind Yl), are not sensitive to Yl-IgG-M-DNR and thus gave rise to colonies following incubation with 1 ⁇ M Yl-IgG-M-DNR at the same rate as the control (Fig. 15).
  • Synthetic peptides were purified using a Lichrosorb RP-18 column.
  • Yl-scFv platelet binding assay a mixture of synthetic peptide (2.5, 25 or 200 ⁇ M) and Yl-scFv (10 ⁇ g) was incubated with washed. Following washing, platelets were incubated with R-phycoerythrin labeled-anti scFv, washed and analyzed by FACS.
  • each of peptides DLY S DYYPE (SEQ ID NO:6), DLY S DY S YPE (SEQ ID NO:7) and DLY S DYY S PE (SEQ ID NO:8) (sulfated at the first, the first and second, and the first and third tyrosines, respectively) at 25 ⁇ M effectively inhibited Yl-scFv binding.
  • mutant peptides DLY S EYYPE (SEQ ID NO: 12), DLY S NYYPE (SEQ ID NO: 13) and DLY S AYYPE(SEQ ID NO.T4), having Asp-277 replaced by Glu, Asn, and Ala respectively were substantially incapable of inhibiting Yl-scFv binding to platelets.
  • the mutant peptides were also tested for inhibition of Yl-scFv binding to purified glycocalicin (Fig. 18A). Briefly, glycocalicin immobilized on microtiter plates was incubated with Yl-scFv (5 ⁇ g/ml) and peptide (25 ⁇ M). Following washing, bound Yl-scFv was detected using polyclonal anti- scFv (generated by immunization of a rabbit with an scFv mixture) and anti-rabbit IgG antibody conjugated to horseradish peroxidase, and reading the absorbance at 450 nm in an ELISA reader.
  • PSGL-1 is highly expressed on AML patients' blood cells. The results below indicate that Yl specifically recognizes and is internalized by tumor cells expressing PSGL-1.
  • KPL-1 anti-PSGL-1
  • Figures 23 and 24 show live AML patient's cells visualized by confocal microscopy following incubation of the cells at 37°C for 2 hours with Yl-PE (left), KPL1- PE (middle) and Yl-IgG-FITC (right). Cells were scanned in three-dimensional manner (X, Y and Z plans) and the pictures presented here were taken from the center of the sphere in respect to the Z plan. As shown, following incubation Yl-IgG was present in the interior part of the cells (but not in the nucleus), while KPLl was present on the cell membranes and did not internalize.
  • Figures 25 and 26 show live CD34+ cells (from healthy bone marrow and from healthy cord blood, respectively) visualized by Confocal Microscopy following incubation of the cells at 37°C for 2 hours with Yl-IgG-FITC (left) and KPL1-PE (right) and with anti-CD34-PE or FITC. As shown, Yl-IgG did not bind normal CD34+ stem cells. In contrast, KPL-l-PE labeled normal cells including CD34+ cells, as evidenced by double staining of some cells in the lower right panel.
  • Figure 27 shows four individual cells from an AML patient sample incubated with non-labeled Yl-IgG for different time periods, at 37°C or at 4°C. As shown, cell surface binding of Yl-IgG was detected after incubation of the cells in the presence of the antibody, at 4°C with 0.1% NaN3. Incubation of Yl-IgG at 37°C for 10 minutes - 2hours (without NaN 3 ) resulted in capping and patching and in internalized staining. Internalization increased with time. No internalization was observed in cells kept at 4°C. Yl-IgG was detected with rhodamine-labeled anti-human (Fc) antibodies. Visualizing the cells was performed by fluorescence microscopy.
  • Figure 28 shows AML patient's cells that were incubated with non-labeled
  • Yl-IgG for 1 hour, at 37°C.
  • Cells shown in the lower row were then incubated at room temperature for 5 minutes with 50mM glycine, pH 2.5 to remove surface bound Yl-IgG.
  • the upper panel represents cell surface capping and patching and internalized Yl-IgG. hi the lower panel, only internalized Yl-IgG was detected.
  • Pronase Removing of cell surface proteins by the proteolytic enzyme, pronase, also resulted in removing of cell surface binding of Yl-IgG and enabled detection of internalized Yl-IgG.
  • Figure 29 shows AML patient's cells that were incubated with non-labeled
  • Yl-IgG for 1 hour at 37°C.
  • Cells shown in the lower row were then incubated at room temperature for 60 minutes with lmg/ml pronase to remove surface bound Yl-IgG.
  • the upper panel represents cell surface capping and patching and internalized Yl- IgG.
  • the lower panel only internalized Yl-IgG is detected. Note that the uropods were removed by pronase, which implies that the uropods formed by Yl-IgG cross-linking of CD 162 are formed on the outer surface of the cell surface.
  • Coated-pits mediated endocytosis can occur via coated-pits. Coated-pits-mediated endocytosis can be blocked by incubation of the cells with 0.45M sucrose for 15 minutes at 37°C prior to incubation with Yl-IgG. This method inhibited endocytosis of Yl-IgG without affecting binding of Yl-IgG to the cell surface.
  • Figure 30 shows AML patient's cells that were incubated with non-labeled
  • Yl-IgG for lhr at 4° C (Fig. 30A) or at 37° C (Fig. 30B).
  • Cells shown in the middle row were then incubated at room temperature for 5 minutes with 50 mM Glycine pH 2.5 to remove surface bound Yl-IgG.
  • the upper panel represents cell surface staining of Yl-IgG.
  • the surface bound Yl-IgG was removed by the acid wash (middle panel).
  • capping and patching and internalization of Yl-IgG was observed (upper panel). Acid wash removed cell surface bound Yl-IgG and only the internalized antibody could be detected (middle panel).
  • EXAMPLE 6 Yl-IgG-Inhibition of Leukocyte-Platelet
  • Adherence of leukocytes to vascular surfaces results in organ injury in various disorders, including reperfusion injury, stroke, mesentaric and peripheral vascular disease; organ transplantation and circulatory shock.
  • Reperfusion injury is associated with adherence of leukocytes to vascular endothelium in the ischemic zone, presumably in part due to activation of platelets and endothelium by thrombin and cytokines, which renders their surfaces adhesive for leukocytes.
  • the main initiator of reperfusion injury is the interaction between von Willbrand factor (vWF) and platelet GPIb receptor.
  • vWF von Willbrand factor
  • thrombolytic agents such as tissue plasminogen activator and streptokinase to relieve coronary artery obstruction
  • myocardial necrosis due to reperfusion injury.
  • drugs which are capable of reducing leukocyte adherence to vascular surfaces and which may be administered in conjunction with thrombolytic agents to improve outcome of cardiovascular disorders.
  • Yl both scFv and full IgG binds to distinct sulfated molecules on platelets (i.e. GPIb) and leukocytes (i.e. PSGL-1), this antibody has potential as a therapeutic agent for inhibiting various cell-cell interactions.
  • Figure 31 shows that Yl-scFv effectively inhibits the binding of activated human platelets to ML2 cells (a human AML derived cell line expressing PSGL-1). Optimal inhibition was obtained when the antibody was incubated simultaneously with both platelets and ML2 cells, while partial inhibition was obtained when the antibody was initially incubated with either platelets or ML2 cells, followed by removal of non-bound antibody and subsequent addition of the remaining cell type (Fig. 31).
  • FIG 31 also shows that the murine antibody KPLl (directed against human PSGL-1 N-terminal domain, but not tyrosine sulfation dependent) was also effective in inhibiting binding of activated platelets to ML2, but the inhibition was less than that exerted by Yl-scFv. This might be due to the fact that KPLl does not recognize an epitope present on both cell types, as does Yl-scFv. No inhibition was observed with the murine antibody, PL2 antibody which is also directed against human PSGL-1 (not shown).
  • EXAMPLE 7 Yl-IgG-Inhibition of Cell Rolling on Immobilized
  • H/H medium Human neutrophils (isolated from anti-coagulated blood by dextran sedimentation and density separation over FICOLL) or ML-2 cells were washed in H/H medium (Hanks' balanced salt solution, 10 mM HEPES , resuspended in cell binding medium (H/H medium supplemented with 2 mM CaCl 2 ) at 2 x 10 cells/ml, and perfused at room temperature through the flow chamber at a rate generating wall shear stress at the desired flow rate, generated with an automated syringe pump (Harvard Apparatus, Natick, MA).
  • H/H medium Human neutrophils (isolated from anti-coagulated blood by dextran sedimentation and density separation over FICOLL) or ML-2 cells were washed in H/H medium (Hanks' balanced salt solution, 10 mM HEPES , resuspended in cell binding medium (H/H medium supplemented with 2 mM CaCl 2 ) at 2 x 10
  • the flow rate was elevated to generate a shear stress of 1 dyn/cm 2 , and all cellular interactions were visualized at two different fields of view (each 0.17 mm 2 in area) using a 10x objective of an inverted phase contrast microscope (Diaphot 300, Nikon Inc., Tokyo, Japan).
  • An imaging system was used for analysis of instantaneous velocities of leukocytes, WSCAN-Array-3 (Galai, Migdal-Ha'emek, Israel) as described previously (Dwir et al., J. Biol. Chem. 275, 18682-18691 (2000)).
  • Video frame images consisting of 768 x 574 pixels (with a pixel size of 1.15 ⁇ m using a 10x objective), were digitized using a Matrox Pulsar frame grabber (Matrox Graphics Inc., Dorval, Quebec, Canada), and images were scanned and processed by the WSCAN-Array- 3 imaging software (Galai, Migdal-Ha'emek, Israel), running on an Atlas pentium MMX- 200 work station. Cell motions were identified from images tracked at 0.02-s intervals. The program output provided the co-ordinates of the center point of each cell in successive interlaced fields at 0.02 s apart.
  • Figure 32 shows the effect of Yl -scFv (1 O ⁇ g/ml) on ML2 cell rolling on immobilized rh- P-Selectin at low density (0.2 ⁇ g/ml).
  • the analysis showed that at shear force of 1 dyn/cm 2 , the number of rolling cells per field was totally eradicated in the presence of Yl-scFv. No such effect was obtained when equal amounts of the scFv-N06 (negative control) was used.
  • Figure 33 shows the effect of Yl-scFv (10 ⁇ g/ml) on ML2 cell rolling on immobilized rh-P-Selectin at high density (1.0 ⁇ g/ml) at various shear forces.
  • the analysis showed that at shear forces of 1, 5 and 10 dyn/cm , the number of rolling cells per field was inhibited by 83%, 98% and 100%, respectively in the presence of Yl-scFv. No such effect was obtained with the negative control, N06, or when cells were washed following incubation with Yl-scFv and then tested (Yl wash).
  • Figure 34 shows the effect of Yl-IgG (1 ⁇ g/ml) on ML2 cell rolling on immobilized rh-P-Selectin (1 ⁇ g/ml) at various shear stress forces.
  • the analysis showed that at shear force of ldyn/cm 2 , cell rolling was inhibited by 89%, and that at shear forces of 5 and 10 dyn/cm 2 cell rolling was inhibited by 100%.
  • Yl-IgG wash When cells were washed following incubation with Yl-IgG and then tested (Yl-IgG wash), cell rolling was inhibited by 46%, 48% and 54% at shear forces 1, 5 and 10 dyn/cm 2 , respectively.
  • the murine anti-PSGL-1 antibody KPLl was also capable of 100% inhibition of cell rolling at all shear forces.
  • Figure 35 shows the effect of increasing concentrations of Yl-scFv on human neutrophil rolling on immobilized rh-P-Selectin at high density (1.0 ⁇ g/ml). The analysis showed that at a shear force 1 dyn/cm 2 , Yl-scFv at 1, 5 and 10 ⁇ g/ml inhibited the number of rolling neutrophils by 20%, 81% and 100%, respectively.
  • Figure 36 shows the effect of Yl-IgG on human neutrophil rolling on immobilized rh-P-Selectin at high density (1.0 ⁇ g/ml). The analysis showed that at a shear force ldyn/cm 2 , the number of rolling neutrophils per field was totally eradicated (100% inhibition) in the presence of Yl-IgG (1 ⁇ g/ml). Similar results were obtained with KPL- 1.
  • Synthetic sulfated peptide (sulfated on a given specific tyrosine residue within the known amino acid sequence of the peptide) derived from a specific receptor (protein) can be prepared with a biotin tag (biotinylated) coupled to the synthetic peptide via a short linker such as caproic acid.
  • Control peptides using the same synthetic peptide can be prepared without sulfation and without the biotin tag ("B").
  • synthetic sulfated peptides derived from other, non-related proteins can be prepared without having the biotin tag ("C”) as additional controls.
  • the biotinylated peptide above (“A") can be coupled to strepavidin-coated magnetic beads and excess unbound biotinylated peptide then washed away.
  • the biotin- stretavidin peptide conjugate (“D") can be screened against a small chemical entity library in the presence of large excess of non-sulfated control peptide ("B") under physiological conditions (37° C, pH 7.0-7.4, salts concentration, conductivity etc.) for molecules that bind to "A”.
  • the coupled-magnetic beads are then washed twice with buffer, each time centrifuged to remove excess unbound molecules.
  • Molecules bound to the magnetic beads (“E”) can be eluted, chemically identified and prepared in larger quantity for further screening.
  • Biotin-streptavidin peptide conjugate (“D") can be re-screened with the selected compounds "E” in the presence of large excess of unrelated biotinylated sulfated peptides, "C”. The tube is then centrifuged, the biotin-stretavidin peptide conjugate coupled magnetic beads washed twice with buffer and centrifuged each time to remove excess unbound molecules. Compounds that bound to the magnetic beads can be eluted for chemical identification. Larger quantities of the chemical compound can be prepared for further studies, such as validation of selective binding to "A", and efficacy testing in vitro and in vivo.

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Abstract

Cette invention concerne des anticorps qui se lient à un épitope de PSGL-1 comprenant le motif structural D-X-Y-D dans lequel X désigne un acide aminé quelconque ou la liaison covalente entre D et Y, et Y est sulfaté. Cet anticorps peut être complexé à une ou plusieurs copies d'un agent. Les anticorps de cette invention peuvent être utilisés dans une méthode visant à induire une cytotoxicité cellulaire dépendant des anticorps et/ou à stimuler des cellules tueuses naturelles (NK) ou des lymphocytes T tueurs naturels. Cette invention concerne également une méthode d'induction de mort cellulaire consistant à administrer ces anticorps à un patient ayant besoin d'un tel traitement. Cette invention concerne en outre une méthode de prévention d'une infection par un virus (tel que le VIH) consistant à administrer un anticorps de la présente invention à un patient ayant besoin d'un tel traitement. Cette invention concerne également une méthode d'introduction d'un agent dans une cellule exprimant le PSGL-1 sulfaté consistant à coupler ou à complexer un agent à un anticorps de la présente invention et à administrer ce complexe à la cellule. Cette invention concerne enfin des méthodes de diagnostic, de pronostic et de détermination du stade utilisant les anticorps de la présente invention.
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WO2007067984A2 (fr) * 2005-12-09 2007-06-14 Wyeth Anticorps neutralisants diriges contre le ligand psgl-1 de primate et utilisations a cet effet
FR2894983B1 (fr) * 2005-12-16 2012-08-17 Lab Francais Du Fractionnement Test de caracterisation des anticorps.
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EP2240495B1 (fr) 2008-02-01 2015-07-15 Genentech, Inc. Métabolite de némorubicine et réactifs analogues, conjugués anticorps-médicament et procédés
US8900589B2 (en) * 2008-07-15 2014-12-02 Genetech, Inc. Anthracycline derivative conjugates, process for their preparation and their use as antitumor compounds
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