EP1649001A2 - Anticorps et utilisations de ceux-ci - Google Patents

Anticorps et utilisations de ceux-ci

Info

Publication number
EP1649001A2
EP1649001A2 EP04777308A EP04777308A EP1649001A2 EP 1649001 A2 EP1649001 A2 EP 1649001A2 EP 04777308 A EP04777308 A EP 04777308A EP 04777308 A EP04777308 A EP 04777308A EP 1649001 A2 EP1649001 A2 EP 1649001A2
Authority
EP
European Patent Office
Prior art keywords
antibody
fragment
cells
seq
patient
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP04777308A
Other languages
German (de)
English (en)
Inventor
Daniel Plaksin
Avigdor Levanon
Esther Szanton
Yocheved Hagay
Rachel Ben-Levy
Yael Nisgav
Yariv Kanfi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bio Technology General Israel Ltd
Bio Tech General (Israel) Ltd
Original Assignee
Bio Technology General Israel Ltd
Bio Tech General (Israel) Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Bio Technology General Israel Ltd, Bio Tech General (Israel) Ltd filed Critical Bio Technology General Israel Ltd
Publication of EP1649001A2 publication Critical patent/EP1649001A2/fr
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • 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
    • 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, HIV 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.
  • Tissue-selective targeting of therapeutic agents is an emerging discipline in the pharmaceutical industry. New cancer treatments based on targeting have been designed to increase the specificity and potency of the treatment while reducing toxicity, thereby enhancing overall efficacy.
  • Mouse monoclonal antibodies (MAbs) to tumor-associated antigens have been employed in an attempt to target toxin, radionucleotide, and chemotherapeutic conjugates to tumors.
  • differentiation antigens such as CD19, CD20, CD22, and CD25, have been exploited as cancer specific targets in treating hematopoietic malignancies.
  • this approach has several limitations. One limitation is the difficulty of isolating appropriate MAbs that display selective binding.
  • a second limitation is the need for high antibody immunogenicity as a prerequisite for successful antibody isolation.
  • a third limitation is that the final product has non-human sequences, which induce immune responses; e.g., when a mouse MAb is given to a human, a human anti-mouse antibody (HAMA) response will be generated.
  • the HAMA response often results in a shorter serum half-life and prevents repetitive treatments, thus diminishing the therapeutic value of the antibody.
  • This latter limitation has stimulated interest both in engineering chimeric or humanized monoclonal antibodies of murine origin and in discovering human antibodies.
  • Another limitation of this approach is that it enables the isolation of only a single antibody species directed against only known and purified antigens. Moreover, this method is not selective insofar as it allows for the isolation of antibodies against cell surface markers that are present on normal, as well as malignant, cells.
  • MAbs have been generally more responsive to treatment with antibodies than solid tumors, such as carcinomas.
  • MAbs rapidly bind to leukemia and lymphoma cells in the bloodstream and easily penetrate to malignant cells in lymphatic tissue, thus making lymphoid tumors excellent candidates for MAb-based therapy.
  • An ideal system entails identifying a MAb that recognizes a marker on the cell surface of stem cells that are producing malignant progeny cells.
  • Phage libraries are used to select random single chain variant fragments (scFvs) that bind to isolated, pre-determined target proteins such as antibodies, hormones, and receptors.
  • scFvs random single chain variant fragments
  • target proteins such as antibodies, hormones, and receptors.
  • antibody display libraries in general, and phage scFv libraries in particular, facilitates an alternative means of discovering unique molecules for targeting specific, yet unrecognized and undetermined, cell surface moieties.
  • 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 immuno logical characteristics.
  • B-ALL B cell ALL
  • CLL Chronic lymphocytic leukemia
  • AML Acute myelogenous leukemia
  • AML is a heterogeneous group of neoplasms with 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.
  • AML primarily affects adults, peaking 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.
  • MAbs have been developed that induce cytolytic activity against tumor cells.
  • the murine MAb muMab4D5 produced against the extracellular domain of HER2 (PI 85) and found to markedly inhibit the proliferation of human tumor cells over-expressing HER2 was humanized to produce the drug HERCEPTIN® (trastuzumab), which was approved by the FDA and is being used to treat human breast cancer (US Patent Nos.
  • the antibody is capable of inhibiting tumor cell growth that is dependent on the HER2 growth factor receptor.
  • a chimeric antibody against CD20, Rituxan® (rituximab) which causes rapid depletion of peripheral B cells, including those associated with lymphoma, was recently approved by the FDA (US Patent No. 5,843,439).
  • the binding of this antibody to target cells results in complement-dependent lysis.
  • This product has recently been approved and is currently being used in the clinic to treat low-grade B cell non- Hodgkin's lymphoma.
  • an additional anti-CD33 antibody (HumM195), currently in clinical trials, was conjugated to several cytotoxic agents, including the gelonin toxin (McGraw et al., Cancer Immunol. Immunother. 39: 367-74 (1994)) and radioisotopes 131 I (Caron et al., Blood 83: 1760-68 (1994)), 90 Y (Jurcic et al., Blood Supp. 92: 613a (1998)) and 213 Bi (Humm et al., Blood Supplement 38: 23 IP (1997)).
  • Gelonin toxin McGraw et al., Cancer Immunol. Immunother. 39: 367-74 (1994)
  • radioisotopes 131 I Caron et al., Blood 83: 1760-68 (1994)
  • 90 Y Jurcic et al., Blood Supp. 92: 613a (1998)
  • 213 Bi Human et al., Blood Supplement 38: 23 IP (1997)
  • a chimeric antibody against the leukocyte antigen CD45 (cHuLym3) is in clinical studies for treatment of human leukemia and lymphoma (Sun et al., Cancer Immunol. Immunother. 48: 595-602 (2000)). In in vitro assays, specific cell lysis was observed in ADCC (Antibody Dependent Cell-mediated Cytotoxicity) assays (Henkart, Immunity 1 : 343-46 (1994); Squier and Cohen, Current Opin. Immunol. 6: 447-52 (1994)). [10] Therapeutic antibodies have also been specifically engineered to have higher affinity to their target, to be more stable, and for optimal biodistribution. See, e.g., Presta, Current Pharma. Biotechnol., 3: 237-56 (2002); Presta et al., Biochem. Society Transactions, 30(4): 487-90 (2002).
  • Phage display technology is more specifically described in the following publications: Smith, Science 228: 1315 (1985); Scott et al., Science 249: 386-90 (1990); Cwirla et al., PNAS 87: 6378-82 (1990); Devlin et al., Science 249: 404-06 (1990); Griffiths et al., EMBOJ. 13(14): 3245-60 (1994); Bass et al., Proteins 8: 309-14 (1990); McCafferty et al., Nature 348: 552-54 (1990); Nissim et al., EMBOJ. 13: 692-98 (1994); U.S. Patent Nos. 5,427,908, 5,432,018, 5,223,409 and 5,403,484.
  • Platelets, fibrinogen, GPIb, selectins, and PSGL-1 (P-Selectin Glycoprotein Ligand- 1) each play an important 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 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 vessels 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 the 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 thus play an important role in hemostasis.
  • platelets Upon vascular injury, platelets adhere to damaged tissue surfaces and attach 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. Importance of Platelets in Metastasis
  • 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, Thrombosis Res. 79: 261-74 (1995)).
  • a single polypeptide chain (60kd) was found to be expressed on surface membrane of HEL cells which 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)).
  • Platelets are also involved in the process of metastasis. 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 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-IX 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 Willebrand 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.
  • vWF von Willebrand factor
  • the GPIb-IX complex is one of the major components of the outer surface of the platelet plasma membrane.
  • the GPIb-IX complex comprises three membrane-spanning polypeptides - a disulfide-linked 130 kDa ⁇ -chain and 25 kDa ⁇ -chain of GPIb and a noncovalently associated GPIX (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 ⁇ transmembrane and cytoplasmic sequences.
  • vWF and thrombin-binding domain of the GPIb-IX complex reside in a globular region that encompasses approximately 300 amino acids at the amino terminus of GPIb ⁇ .
  • the human platelet GPIb-IX 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. Further, 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. Moreover, GPIb ⁇ contains a high-affinity binding site for ⁇ -thrombin, which facilitates platelet activation by an as-yet poorly defined mechanism.
  • 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 in 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 CD 162) is a mucin glycoprotein ligand for P-Selectin, E- Selectin, and L-Selectin that shares structural similarity with GPIb (Afshar-Kharghan et al. (2001), supra).
  • PSGL-1 is a disulfide-linked homodimer that has a PACE (Paired Basic Amino Acid Converting Enzymes) cleavage site.
  • PACE Panaired Basic Amino Acid Converting Enzymes
  • the extracellular portion of PSGL- 1 contains three N-linked glycosylation sites and has numerous sialyated, fucosylated O- linked oligosaccharide branches (Moore et al., J. Biol. Chem. 118: 445-56 (1992)).
  • O-glycan sites Most of the N-glycan sites and many of the O-glycan sites are occupied.
  • the structures of the O-glycans of PSGL-1 from human HL-60 cells have been determined. Subsets of these O- glycans are core-2, sialylated and fucosylated structures that are required for binding to selectins.
  • 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. PSGL-1 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 is also expressed on most blood leukocytes, such as neutrophils, monocytes, leukocytes, subset of B cells, and all T cells (Kishimoto et al. (1997), supra).
  • 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)).
  • Leukocyte rolling is important in inflammation, and interaction between P-Selectin (expressed by activated endothelium and on platelets, which may be immobilized at sites of injury) and PSGL-1 is instrumental for tethering and rolling of leukocytes on vessel walls (Ramachandran et al., PNAS 98(18): 10166-71 (2001); Afshar-Kharghan et al. (2001), supra).
  • Cell rolling is also important in metastasis, and P- and E-Selectin on endothelial cells is believed to bind metastatic cells, thereby facilitating extravasation from the blood stream into the surrounding tissues.
  • 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 in leukocytes. Frenette et al., J. Exp. Med. 191(8): 1413-22 (2000)).
  • KPL1 A commercially available monoclonal antibody to human PSGL- 1 , KPL1 , was generated and shown to inhibit the interactions between PSGL-1 and P-selectin and between PSGL-1 and L-selectin.
  • the KPL1 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)).
  • Fibrinogen There are two forms of normal human fibrinogen - normal ( ⁇ ) and ⁇ ', each of which is found in normal individuals.
  • 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 ⁇ ' 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.
  • 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.
  • 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 the negatively-charged N-terminal globular domain of GPIb contains three tyrosine residues known to undergo sulfation.
  • GPIb ⁇ (CD42), which is expressed by platelets and megakaryocytes and mediates platelet attachment to and rolling on subendothelium via binding with vWF, also contains a cluster of negatively charged amino acids between Asp-269 and Asp-287.
  • tyrosylprotein sulfotransferase specifically recognizes and sulfates tyrosines adjacent to acidic amino residues (Bundgaard et al., J. Biol.
  • PSGL-1 this protein has three potential sulfation sites (on each of the three tyrosine residues at the N-terminal domain of the molecule) followed by 10-16 decamer repeats that are high in proline, serine, and threonine.
  • Sulfation of PSGL-1 is known to be relevant for the binding of P-selectin, and stereo-specific contributions of individual tyrosine sulfates on PSGL-1 are important for the binding of P-selectin to PSGL-1. There are some indications, however, that sulfation of only one tyrosine residue is sufficient for P-selectin binding. (J. Biol. Chem., Vol. 273, 12, 7078-87).
  • 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)).
  • sulfated N-terminal tyrosines influence the role of CC- chemokine receptors, such as CCR5, which serve as co-receptors with related seven transmembrane segment (7TMS) receptor for entry of human and simian immunodeficiency viruses (HIN-1, HIN-2, and SIN) into target cells.
  • sulfated ⁇ -terminal tyrosines contribute to the binding of CCR5 to MlP-l ⁇ , MlP-l ⁇ , and HIV-1 gpl20/CD4 complexes and to the ability of HIV- 1 to enter cells expressing CCR5 and CD4.
  • 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 in CXCR4-dependent HIN-1 entry than CCR5-dependent entry; thus demonstrating a possible role for tyrosine sulfation in the CXC-chemokine family and underscores a general difference in HIV-1 utilization of CCR5 and CXCR4 (Farzan et al., J. Biol. Chem., 277(33): 29,484-89 (2002)).
  • Antibodies that bind to PSGL-1 and/or GPIb were identified using a phage library and disclosed in U.S. Application ⁇ os. 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 ⁇ os. PCT/USOl/49,442 and PCT/US01/49,440. Specific examples of antibodies disclosed in these applications include the Yl, Y17, and L32 antibodies.
  • the sulfated epitopes previously identified as 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.
  • Such epitopes are also found on diseased cells, such as T-ALL cells, B-CLL cells, AML cells, multiple myeloma cells, and metastatic cells.
  • antibodies and polypeptides in methods for diagnosing, prognosing, or staging various disease states of an individual, such as, e.g., AML, T-ALL, B-leukemia, B-CLL, Pre-B-ALL, multiple myeloma, metastasis, HIN 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.
  • these antibodies of the present invention may be used as a targeting agent to direct a therapeutic to a specific cell or site.
  • Another object of the present invention is to provide a method of purging tumor cells.
  • the present invention provides an antibody and polypeptide comprising a consensus sequence: X ⁇ -X 2 -X 3 -Pro-X 5 -X (SEQ ID NO:3), wherein Xi and X 6 are hydrophobic amino acids, and X 2 , X 3 and X 5 are any amino acid, wherein X 2 is preferably a basic amino acid, and wherein the consensus sequence is arranged from N-terminus to C-terminus or from C-terminus to N-terminus.
  • the consensus sequence is preferably within the hypervariable regions of the antibody and more preferably within the CDR3 region.
  • the consensus sequence excludes a CDR3 region comprising the amino acid sequence of SEQ ID NO:4.
  • the present invention moreover provides an antibody and polypeptide having the binding capabilities of an scFv antibody of SEQ ID NO:5 or SEQ ID. NO:6.
  • the present invention additionally provides a process for producing an antibody or polypeptide comprising the steps of providing a phage display library, providing a peptide of SEQ ID NO:7 that binds to an antibody or polypeptide having the binding capabilities of an scFv antibody fragment of SEQ ID NO:4, panning the phage display library for an scFv antibody fragment that binds to the peptide of SEQ ID NO:7, and producing an antibody or polypeptide comprising the scFv antibody fragment that binds to the peptide of SEQ ID NO:7.
  • the present invention also provides a library of immunoglobulin binding domains, specifically scFv molecules, comprising a diverse antigen-binding domain for complementary binding, wherein the library has diversity only in heavy chain CDR3.
  • the present invention also provides pharmaceutical compositions comprising the antibodies and polypeptides of the present invention. These pharmaceutical compositions may be used to treat, diagnose, prognose or stage various conditions including conditions related to or involving cell rolling; inflammation; auto-immune disease; platelet aggregation; restenosis; HIV infection; metastasis; growth and/or replication of tumor cells;; and growth and/or replication of leukemia cells.
  • compositions may be used for inhibiting cell rolling; inhibiting inflammation; inhibiting auto-immune disease; inhibiting platelet aggregation; inhibiting restenosis; inhibiting HIV infection; inhibiting metastasis; inhibiting growth and/or replication of tumor cells, increasing mortality of tumor cells, inhibiting growth and/or replication of leukemia cells, increasing the mortality rate of leukemia cells; altering the susceptibility of diseased cells to damage by anti-disease agents; increasing the susceptibility of tumor cells to damage by anti-cancer agents; increasing the susceptibility of leukemia cells to damage by anti- leukemia agents; inhibiting increase in number of tumor cells in a patient having a tumor; decreasing the number of tumor cells in a patient having cancer; inhibiting increase in number of leukemia cells in a patient having leukemia; and decreasing the number of leukemia cells in a patient having leukemia.
  • 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 cells, Pre-B-ALL, multiple myeloma, metastasis, HIN 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.
  • various disease states such as, e.g., AML, T-ALL, B- leukemia, B-CLL cells, Pre-B-ALL, multiple myeloma, metastasis, HIN 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.
  • the present invention also provides a method of diagnosing, prognosing, or staging a disease in a patient by providing a sample containing a cell from the patient and determining whether the antibodies or polypeptides of the present invention bind to the cell of the patient, thereby indicating that the patient is at risk for or has the disease.
  • the present invention also provides a method of purging tumor cells from a patient by providing a sample containing cells from the patient and incubating the cells from the patient with an antibody or polypeptide of the present invention.
  • 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 sub-classes 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 4 Ser) 3 (SEQ ID NO:8).
  • 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
  • CDR Determining Regions
  • a fragment of a Fv molecule is defined as any molecule smaller than the original
  • 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/IL99/00581) (3) similar bodies having a fragment of the light chain, and (4) similar bodies having a functional unit of a light chain variable region.
  • 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.
  • a 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.
  • Hydrophobic amino acids are generally valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tryptophan (W), cysteine (C), alanine (A), tyrosine
  • Basic amino acids are generally arginine, histidine, and lysine.
  • 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.
  • affinity is a measure of the binding strength (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.
  • 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 in suspension rather than being in solution.
  • the term 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) [76] 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.
  • 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 or larger), each phage particle displaying a peptide sequence.
  • a pharmaceutical composition refers to a formulation which comprises a 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 refers to an agent that 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 warm-blooded animal.
  • the agent is selected from the group of radioisotope, toxin, oligonucleotide, recombinant protein, antibody fragment, pharmaceutical agents, anti-cancer agents, anti-leukemic agents, anti-metastasis agents, anti-neoplastic agents, anti-disease agents, anti-adhesion agents, anti-thrombosis agents, anti-restenosis agents, anti-autoimmune agents, anti- aggregation agents, anti-bacterial agents, anti-viral agents, and anti-inflammatory agents.
  • 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
  • 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.
  • An anti-cancer agent is an agent with anti-cancer activity.
  • anticancer agents include agents that inhibit or halt the growth of cancerous or immature pre- cancerous cells, agents that kill cancerous or pre-cancerous, 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 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 be 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 may be 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 II).
  • 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 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.
  • FIG. 1 depicts the numerical data from phage ELISA of selected clones according to the present invention to analyze binding to PSGL-1
  • FIG. 2 depicts numerical data from scFv ELISA of selected clones according to the present invention to analyze binding to PSGL-1.
  • FIG. 3 depicts numerical data from FACS analysis of scFv from selected clones according to the present invention and L32 to analyze binding to ML-2 cells that express
  • FIG. 4 depicts numerical data from ELISA of scFv from selected clones according to the present invention and L32 to analyze binding to glycocalicin.
  • FIG. 5 depicts numerical data from FACS analyses of scFv from selected clones according to the present invention and L32 to analyze binding to platelets and granulocytes.
  • FIG. 6 shows a comparison of the granulocyte/platelet binding ratio of selected clones according to the present invention and L32.
  • FIG. 7 depicts results analyzing the binding of S15 to ML-2 cells in the presence and absence of KPL-1.
  • FIG. 8 depicts numerical data from FACS analyses providing a comparison of the binding of purified scFvs to ML-2 cells in PBS.
  • FIG. 9 depicts numerical data from FACS analyses providing a comparison of the binding of purified scFvs to ML-2 cells in PBS.
  • FIG. 10 depicts numerical data from FACS analyses providing a comparison of the binding of purified scFvs to ML-2 cells in 50% plasma.
  • FIG. 11 depicts numerical data from FACS analyses providing a comparison of the binding of purified scFvs to ML-2 cells in 50% plasma.
  • FIG. 12 depicts a FACS analysis of the dose response of purified scFvs to ML-2 cells.
  • Fig 13 depicts the binding of selected phage clones to GPIb and PSGL-1 sulfated peptides.
  • Fig 14 shows binding of scFvs to glycocalicin.
  • FIG. 15 is a graph of binding of various scFvs at increasing concentrations to washed platelets using flow cytometry
  • FIG. 16 depicts binding of various scFvs to glycocalicin via an ELISA assay.
  • FIG. 17 depicts the effect of A3R scFv on platelet aggregation induced by ristocetin.
  • FIG. 18 depicts the effect of Yl scFv, A3R scFv and control PBS on platelet adhesion to polystyrene using CPA assay.
  • FIG. 19 depicts the level of A3R scFv bound to guinea pig platelets following bolus injection.
  • FIG. 20 depicts plasma concentration of A3R scFv in guinea pig following bolus injection.
  • FIG. 21 depicts numerical data from direct binding of scFv to peptides based on sulfated regions of PSGL-1, GPIb and CCR5.
  • FIG. 22 depict S 15 IgG induced ADCC in B-CLL patient samples.
  • FIG. 23 depicts involvement of different effector cell populations in S15 IgG induced ADCC in B-CLL patient samples.
  • FIG. 24 depicts the ability of S 15 IgG and rituximab to induce apoptosis in B-CLL patient samples.
  • the present invention relates to an antibody or fragment thereof comprising a consensus sequence: X ! -X 2 -X -Pro-X 5 -X 6 (SEQ ID NO:3), wherein Xi and X 6 are hydrophobic amino acids and X 2> X 3 and X 5 are any amino acid, wherein X is preferably a basic amino acid, and wherein the consensus sequence can be ananged either from the N- terminus to the C-terminus or from the C-terminus to the N-terminus (such antibody generally referred to herein as the consensus antibody).
  • X 2 is selected from the group consisting of arginine and lysine and Xi and X 6 are selected from the group consisting of leucine, valine, methionine, alanine, phenylalanine, and isoleucine.
  • the consensus antibody of this embodiment comprises a consensus sequence selected from the group consisting of SEQ ID NO:9 and SEQ ID NO: 10. More preferably, in this embodiment, the consensus antibody comprises SEQ ID NO: 5 (the CDR3 sequence is SEQ ID NO:9) and such consensus antibody is designated and refened to herein as SI 5.
  • the consensus antibody in this embodiment more preferably is SEQ ID NO:6 (the CDR3 sequence is SEQ ID NO: 10) and such consensus antibody is designated and refened to herein as A3R.
  • X 2 and X 3 are arginine and the hydrophobic amino acid X 6 is preferably isoleucine.
  • the consensus antibody of this another embodiment comprises a consensus sequence selected from the group consisting of SEQ ID NO:9 and SEQ ID NO: 10. More preferably, the consensus antibody of this another embodiment is A3R and or SI 5.
  • Xi is selected from the group consisting of leucine and methionine
  • X 2 and X 3 are arginine
  • X 5 is selected from the group consisting of serine and valine
  • X 6 isoleucine.
  • the consensus antibody of this another embodiment comprises a consensus sequence selected from the group consisting of SEQ ID NO:9 and SEQ ID NO: 10.
  • Xi is leucine
  • X 2 is selected from the group consisting of a basic amino acid
  • X 6 is selected from the group of hydrophobic amino acids.
  • Prefened consensus antibodies of this embodiment comprises D series antibodies of SEQ ID NO:ll to SEQ ID NO:16.
  • the consensus antibody of this embodiment is Dl (the CDR3 region is SEQ ED NO:14 and the full scFv is SEQ ID NO:55) or D3 (the CDR3 region is SEQ ID NO:13 and the full scFv is SEQ ID NO:56).
  • the consensus antibody of the present invention preferably binds preferentially a first epitope over a second epitope, wherein at least one of the first and second epitope is sulfated.
  • the first and second epitope include a PSGL-1 epitope and a GPIb epitope. More preferably, the consensus antibody binds epitopes of both PSGL-1 and GPIb and preferably shows binding with stronger affinity to a PSGL-1 epitope over a GPIb epitope or binding with stronger affinity to a GPIb epitope over a PSGL-1 epitope.
  • the consensus antibody binds the first and second epitopes (e.g., PSGL-1 and GPIb) with a similar affinity.
  • Suitable antibodies that bind to PSGL-1 and GPIb with similar affinity include Dl and D3, either the full scFv or the CDR3 region.
  • the consensus antibody is A3R
  • the consensus antibody binds an epitope of sulfated GPIb with stronger affinity than its binding to a sulfated PSGL-1 epitope.
  • S15 and A3R at similar concentrations it has been found that A3R binds to healthy GPIb platelets with stronger affinity than SI 5.
  • the present invention provides an antibody that binds specifically to sulfated PSGL-1 and/or sulfated GPIb with an affinity substantially similar to that of SI 5 and preferably binds to PSGL-1 with a stronger affinity than to GPIb. More preferably the antibody binds to a sulfated PSGL-1 epitope that is sulfated at the third N-terminal tyrosine residue at position 51.
  • the antibody of the present invention binds specifically to sulfated PSGL-1 and/or sulfated GPIb with an affinity substantially similar to that of A3R and preferably binds to GPIb with a stronger affinity than to PSGL-1. More preferably, the antibody of this embodiment binds to a sulfated GPIb epitope that is sulfated at the first N-terminal tyrosine residue at position 46.
  • the present invention provides an antibody that binds specifically to sulfated PSGL-1 (preferably sulfated at the third N-terminal tyrosine residue at Tyr-51) and to sulfated GPIb (preferably sulfated at the first N-terminal tyrosine at Tyr-276) with an affinity substantially similar to that of Dl and/or D3
  • the Xi and X 5 of the consensus sequence of the consensus antibody of the present invention may contribute to the binding of the consensus antibody to tyrosine sulfation sites.
  • the amino acids in the Xi and X 5 positions of the consensus sequence may be specifically selected depending on the particular sulfated epitope is targeting for binding. In other words, positions Xj and X 5 may be altered to tailor an antibody to specifically bind to a particular sulfated epitope.
  • S15 preferably binds to a PSGL-1 epitope comprising sulfate modification at the third N-terminal tyrosine and A3R preferably binds to a GPIb epitope having a sulfate modification at the first N-terminal tyrosine (while Dl and D3 bind both PSGL-1 and A3R equally), Xi and X 5 of the consensus sequence may be relevant to binding of SI 5 and A3R to the third and first sulfated tyrosine of PSGL-1 and GPIb, respectively.
  • Antibodies that bind to PSGL-1 and/or GPIb were identified using a phage 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 USOl/49,442 and PCT/US01/49,440. Specific examples of antibodies disclosed in these applications include the Yl, Y17, and L32 antibodies.
  • the sulfated epitopes previously identified as 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.
  • Such epitopes are also found on diseased cells, such as T-ALL cells, B-CLL cells, AML cells, multiple myeloma cells, and metastatic cells.
  • the consensus antibodies of the present invention which were isolated from the DP32 family, bind to proteins having sulfated tyrosine epitopes.
  • proteins include, but are not limited to, PSGL-1, GPIb, ⁇ -2-antiplasmin; aminopeptidase B; CC chemokine receptors such as CCR2, CCR5, CCR3, CXCR3, CXCR4, CCR8, and CCR2b; seven- transmembrane-segment (7TMS) receptors; coagulation factors such as factor V, VIII, and IX; fibrinogen gamma chain; heparin cofactor II; secretogranins such as secretogranin I and II; vitronectin, amyloid precursor, ⁇ -2-antiplasmin; cholecystokinin; ⁇ - choriogonadotropin; complement C4; dermatan sulfateproteoglycan; fibronectin; and castrin.
  • the consensus antibody of the present invention binds to sulfated CC chemokine receptors such as CCR5, CXCR4, 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.
  • the binding of the antibodies of the present invention may be dependent on the stage of development of the cell (AML subtype is classified based on the French- American-British system using the morphology observed under routine processing and cytochemical staining).
  • the antibodies may 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 show low binding to normal, healthy bone manow (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. [123] Therefore, the consensus antibody may not bind undifferentiated cells in the bone manow such as M 0 , Mi, M 2 , and M 3 cells.
  • PSGL-1 to which the consensus antibody binds, is not expressed in significant levels or is not sulfated on these undifferentiated cells.
  • the consensus antibody of the present invention may also not bind to healthy bone manow cells (such as CD34+ cells).
  • the consensus antibody of the present invention binds to sulfated PSGL-1.
  • the S15 antibody in particular, exhibits enhanced selectivity for 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, NLA-4, and NCAM-1 in the inflammatory processes of diseases such as atherosclerosis (Huo and Ley, Acta 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 interference of the consensus antibody, and particularly SI 5, with any of these central molecules suggests a potential role for the consensus 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 enhanced scFv selectivity of the consensus antibody and SI 5 for sulfated PSGL-1 may make them a superior molecule for treating a variety of malignant and inflammatory diseases.
  • the consensus antibody of the present invention particularly in embodiments where X 2 and X 3 are arginine, and X is isoleucine, and preferably A3R, also exhibits enhanced selectivity for sulfated GPIb.
  • GPIb is involved in aggregation of platelets involved by high shear in regions of arterial stenosis and platelet activation induced by low concentrations of thrombin. Based on this understanding of GPIb, it is apparent that the enhanced scFv selectivity of the consensus antibody and A3R for sulfated GPIb may make it a superior molecule for treating a variety of cardiovascular and inflammatory diseases.
  • the consensus antibody of the present invention binds to an epitope present on at least one cell type involved in inflammation or tumorogenesis, including T- ALL cells, AML cells, Pre-B-ALL cells, B-leukemia cells, B-CLL cells, multiple myeloma cells, and metastatic cells.
  • the consensus 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 consensus antibody of the present invention cross-reacts 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 or fragments thereof of the present invention may be internalized into the AML cells, for example, following binding to PSGL-1. Such internalization may occur via endocytosis and an active process that is process, time and temperature dependent.
  • CDRs complementarity-determining regions
  • DP32 which is one of the 49 germ lines present in the phage display library, is the specific germ line of the phage library from which the consensus antibody of the present invention was isolated. Therefore, DP32 provides the antibodies of the present invention with at least the heavy and light chain framework variable regions, light chain CDR1, CDR2, and CDR3 regions, and/or heavy chain CDR1 and CDR2. DP32 also provides a three-dimensional structure on which the hypervariable regions were conformed. It is well known that the specificity of an antibody is determined by its three-dimensional conformation. Thus, the limitations imposed by DP32 may have a significant role in determining the specificity of the antibodies of the present invention.
  • DP32 has various charged amino acids, which may have a structural role in the antibodies' antigen recognition.
  • CDRs may also be inserted into cassettes to produce antibodies.
  • a cassette as applied to polypeptides and as defined in the present invention, 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.
  • the amino acid sequence of the cassette may ostensibly be fixed, whereas the replaced, inserted, or attached sequence can be highly variable.
  • the cassette can be comprised of several domains, each of which encompasses a function crucial to the final construct.
  • the cassette of a particular embodiment of the present invention comprises, from the N-terminus, framework region 1 (FR1), CDRl, framework region 2 (FR2), CDR2, framework region 3 (FR3), and framework region 4 (FR4).
  • the CDR2 and CDRl hypervariable regions of the cassette may be replaced or modified by non-conservative or, preferably, conservative amino acid substitutions.
  • the consensus antibody and the antibodies that bind with substantially the same affinity as A3R and SI 5 have a heavy and a light chain, and each chain has a first, second, and third hypervariable region, which are the CDR3, CDR2, and CDRl regions, respectively.
  • the binding selectivity and specificity are determined particularly by the CDR3 region of a chain, possibly by the CDR3 region of the light chain and, preferably, by the CDR3 region of the heavy chain, and secondarily by the CDR2 and CDRl regions of the light chain and, preferably, of the heavy chain.
  • the binding selectivity and specificity may also be secondarily influenced by the upstream or downstream regions flanking the first, second, and/or third hypervariable regions.
  • the consensus sequence of the consensus antibody is within the hypervariable regions of the consensus antibody.
  • the consensus sequence may be in the CDR3 region, the CDR2 region, or the CDRl region of the consensus antibody. All of or only a portion of the consensus sequence may be in the CDR3 region, the CDR2 region, or the CDRl region.
  • the consensus sequence may overlap two hypervariable regions or may be partially within one or more hypervariable regions and partially within another part of the variable region of the consensus antibody.
  • the consensus sequence is in the CDR3 region.
  • the consensus sequence excludes a CDR3 region comprising the amino acid sequence of SEQ ID NO:4.
  • the consensus antibody preferably includes one or more amino acid sequences of SEQ ID NO:9, SEQ ID NO:17, and SEQ ID NO:18.
  • the consensus sequence preferably includes one or more amino acid sequences of SEQ ID NO:10, SEQ ID NO:17, and SEQ ID NO:18.
  • the amino acid sequences are preferably within the hypervariable regions of the consensus antibody.
  • the amino acid sequences may be in the CDR3 region, the CDR2 region, or the CDRl region of the consensus antibody. All or only a portion of the amino acid sequences may be in the CDR3 region, the CDR2 region, or the CDRl region.
  • the amino acid sequence of SEQ ID NO:9 or SEQ ID NO: 10 is in the CDR3 region
  • the amino acid sequence of SEQ ID NO: 17 is in the CDR2 region
  • the amino acid sequence of SEQ ID NO: 18 is in the CDRl region of the consensus antibody.
  • the present invention also provides antibodies that bind to sulfated PSGL-1 and/or sulfated GPIb with substantially the same affinity as S15, A3R, SI, SI 1, Dl and/or D3.
  • the antibodies bind to sulfated PSGL-1 and/or sulfated GPIb with substantially the same affinity as S15 and in this embodiment the antibody preferably comprise one or more of amino acid sequence of SEQ ID NO:9, SEQ ID NO: 17, and SEQ ID NO:18.
  • these amino acid sequences are in the hypervariable region of the antibody.
  • the amino acid sequences may be in the CDR3 region, the CDR2 region, or the CDRl region of the antibody. All or only a portion of the amino acid sequences may be in the CDR3 region, the CDR2 region, or the CDRl region.
  • amino acid sequence of SEQ ID NO:9 is in the CDR3 region
  • amino acid sequence of SEQ ID NO: 17 is in the CDR2 region
  • amino acid sequence of SEQ ID NO: 18 is in the CDRl region of the antibody.
  • the antibodies of the present invention bind to sulfated PSGL-1 and/or sulfated GPIb with substantially the same affinity as A3R and in this embodiment the antibody preferably comprise one or more of amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 17, and SEQ ID NO: 18.
  • these amino acid sequences are in the hypervariable region of the antibody.
  • the amino acid sequences may be in the CDR3 region, the CDR2 region, or the CDRl region of the antibody. All or only a portion of the amino acid sequences may be in the CDR3 region, the CDR2 region, or the CDRl region.
  • amino acid sequence of SEQ ID NO: 10 is in the CDR3 region
  • amino acid sequence of SEQ ID NO: 17 is in the CDR2 region
  • amino acid sequence of SEQ ED NO: 18 is in the CDRl region of the antibody.
  • the antibodies of the present invention bind to sulfated PSGL-1 and/or sulfated GPIb with substantially the same affinity as SI and in this embodiment the antibody preferably comprise one or more of amino acid sequence of SEQ ID NO:28, SEQ ID NO: 17, and SEQ ID NO: 18.
  • amino acid sequences are in the hypervariable region of the antibody.
  • the amino acid sequences may be in the CDR3 region, the CDR2 region, or the CDRl region of the antibody. All or only a portion of the amino acid sequences may be in the CDR3 region, the CDR2 region, or the CDRl region.
  • amino acid sequence of SEQ ID NO:28 is in the CDR3 region
  • amino acid sequence of SEQ ED NO: 17 is in the CDR2 region
  • amino acid sequence of SEQ ED NO: 18 is in the CDRl region of the antibody.
  • the antibodies of the present invention bind to sulfated PSGL-1 and/or sulfated GPIb with substantially the same affinity as SI 1 and in this embodiment the antibody preferably comprise one or more of amino acid sequence of SEQ ID NO:31, SEQ ID NO: 17, and SEQ ID NO: 18.
  • amino acid sequences are in the hypervariable region of the antibody.
  • the amino acid sequences may be in the CDR3 region, the CDR2 region, or the CDRl region of the antibody. All or only a portion of the amino acid sequences may be in the CDR3 region, the CDR2 region, or the CDRl region.
  • amino acid sequence of SEQ ID NO:31 is in the CDR3 region
  • amino acid sequence of SEQ ID NO: 17 is in the CDR2 region
  • amino acid sequence of SEQ ID NO: 18 is in the CDRl region of the antibody.
  • the antibodies of the present invention bind to sulfated PSGL-1 and/or sulfated GPIb with substantially the same affinity as Dl and in this embodiment the antibody preferably comprise one or more of amino acid sequence of SEQ ID NO:14, SEQ ID NO:17, and SEQ ID NO:18.
  • amino acid sequences are in the hypervariable region of the antibody.
  • the amino acid sequences may be in the CDR3 region, the CDR2 region, or the CDRl region of the antibody. All or only a portion of the amino acid sequences may be in the CDR3 region, the CDR2 region, or the CDRl region.
  • amino acid sequence of SEQ ID NO: 14 is in the CDR3 region
  • amino acid sequence of SEQ ID NO: 17 is in the CDR2 region
  • amino acid sequence of SEQ ID NO: 18 is in the CDRl region of the antibody.
  • the antibodies of the present invention bind to sulfated PSGL-1 and/or sulfated GPIb with substantially the same affinity as D3 and in this embodiment the antibody preferably comprise one or more of amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 17, and SEQ ID NO: 18.
  • amino acid sequences are in the hypervariable region of the antibody.
  • the amino acid sequences may be in the CDR3 region, the CDR2 region, or the CDRl region of the antibody. All or only a portion of the amino acid sequences may be in the CDR3 region, the CDR2 region, or the CDRl region.
  • amino acid sequence of SEQ ID NO: 13 is in the CDR3 region
  • amino acid sequence of SEQ ID NO: 17 is in the CDR2 region
  • amino acid sequence of SEQ ID NO: 18 is in the CDRl region of the antibody.
  • amino acid sequences of ⁇ 25 amino acid residues described and detailed herein include within their scope one or two amino acid substitution(s) and that preferably the substitutions are conservative amino acid substitutions.
  • amino acid sequences of >25 amino acid residues described and detailed herein it is to be understood and considered as an embodiment of the invention that these amino acid sequences include within their scope an amino acid sequence with > 90% sequence similarity to the original sequence (Altschul et al., Nucleic Acids Res. 25: 3389-402 (1997)).
  • Similar or homologous amino acids are defined as non-identical amino acids which display similar properties, e.g., acidic, basic, aromatic, size, positively or negatively charged, polarity, non-polarity.
  • Percent amino acid similarity or homology or sequence similarity is determined by comparing the amino acid sequences of two different peptides or polypeptides. Antibody sequences were determined by DNA sequencing. The two sequences are aligned, usually by use of one of a variety of computer programs designed for the purpose, and amino acid residues at each position are compared. Amino acid identity or homology is then determined. An algorithm is then applied to determine the percentage amino acid similarity.
  • Protein comparison can take into account the presence of conservative amino acid substitutions, whereby a mismatch may yet yield a positive score if the non-identical amino acid has similar physical and/or chemical properties (Altschul et al. (1997), supra).
  • the three hypervariable regions of each of the light and heavy chains can be interchanged between the two chains and among the three- hypervariable sites within and/or between chains.
  • the consensus antibodies and the antibodies that bind with substantially the same affinity as S15, A3R and/or D1/D3 include IgG, IgA, IgD, IgE, or IgM antibodies.
  • the IgG class encompasses several sub-classes including IgG,, IgG 2 , IgG 3 , and IgG 4 .
  • Antibodies may be provided in many forms, such as fragments, complexes, and multimers.
  • antibody fragments include Fv, scFv, dsFv, Fab, Fab 2 , and Fd molecules.
  • Smaller antibody fragments such as fragments of Fvs and fragments of Fabs, are also included in the term "fragments", as long as they retain the binding characteristics of the original antibody or larger fragment. Examples of such fragments would be (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/IL99/00581) (3) similar bodies having a fragment of the light chain, and (4) similar bodies having a functional unit of a light chain variable region.
  • Constructs include, for example, multimers such as diabodies, triabodies, and tetrabodies.
  • the term "antibody” is intended to encompass all of these molecules, as well as derivatives, combinations, modifications, homologs, mimetics, and variants thereof, unless it is specified otherwise or indicated otherwise based on context and/or knowledge in the art.
  • scFv monomers are designed with the C-terminal end of the V H domain tethered by a polypeptide linker to the N-terminal residue of the V L .
  • an inverse orientation is employed: the C-terminal end of the V L domain is tethered to the N- terminal residue of V H through a polypeptide linker (Power et al., J. Immun. Meth. 242: 193-204 (2000)).
  • the polypeptide linker is typically around fifteen amino acids in length. When the linker is reduced to about three to seven amino acids, the scFvs can not fold into a functional Fv domain and instead associate with a second scFv to form a diabody. Further reducing the length of the linker to less than three amino acids forces the scFv association into trimers or tetramers, depending on the linker length, composition and Fv domain orientations. (Powers (2000), supra).
  • a scFv may be employed as a blocking agent to bind a target receptor and thus block the binding of the "natural" ligand.
  • this higher affinity may be useful when the target receptors are involved in adhesion and rolling or when the target receptors are on cells present in areas of high sheer flow, such as platelets.
  • peptoid modification For example, peptoid modification, semipeptoid modification, cyclic peptide modification, N terminus modification, C terminus modification, peptide bond modification, backbone modification, and residue modification may be performed. It is also within the ability of the skilled worker following the guidance of the present specification to test the modified antibodies or fragments to assess whether their binding characteristics have been changed. [154] Likewise, it is within the ability of the skilled worker using the guidance provided herein to alter the binding characteristics of an antibody to obtain a molecule with more desirable characteristics. For example, once an antibody having desirable properties is identified, random or directed mutagenesis may be used to generate variants of the antibody, and those variants may be screened for desirable characteristics.
  • additional antibodies that have the binding capabilities of the consensus antibody and/or that bind specifically to sulfated PSGL-1 or sulfated GPIb and bind with affinity substantially similar to that of the S15 and A3R.
  • additional antibodies such as Dl and D3, can be isolated using the biopanning methods described herein, wherein the molecule or cell that the consensus antibody binds is used to screen a particular phage display library, particularly a library prepared from a leukemia, lymphoma, and myeloma patient.
  • Antibodies according to the present invention may also have a tag that may be inserted or attached thereto to aid in the preparation and identification thereof, and in diagnostics.
  • the tag can later be removed from the molecule.
  • useful tags include: AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSN, HTTPHH, IRS, KT3, Protein C, S-TAG ® , T7, N5, and VSV-G (Jarvik and Telmer, Ann. Rev. Gen., 32, 601-18 (1998)).
  • the tag is preferably c-myc or KAK.
  • scFv is defined as a molecule which 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.
  • a scFv construct may be a multimer (e.g., dimer, trimer, tetramer, and the like) of scFv molecules that incorporate one or more of the hypervariable domains of the antibody.
  • All scFv derived constructs and fragments retain enhanced binding characteristics so as to bind selectively and/or specifically to a target cell in favor of other cells.
  • the binding selectivity and/or specificity is primarily determined by hypervariable regions.
  • the antibodies of the subject invention can be constructed to fold into multivalent Fv forms, which may improve binding affinity and specificity and increased half-life in blood.
  • Mulitvalent forms of scFv have been designed and produced by others. One approach has been to link two scFvs with linkers. Another approach involves using disulfide bonds between two scFvs for the linkage.
  • Another method was designed to make tetramers by adding a sfreptavidin coding sequence at the c- terminus of the scFv.
  • Sfreptavidin is composed of 4 subunits, so when the scFv- streptavidin is folded, 4 subunits accommodate themselves to form a tetramer (Kipriyanov et al., Hum Antibodies Hybridomas 6(3): 93-101 (1995)).
  • a free cysteine is introduced in the protein of interest.
  • a peptide-based cross linker with variable numbers (2 to 4) of maleimide groups was used to cross link the protein of interest to the free cysteines (Cochran et al., Immunity 12(3): 241- 50 (2000)).
  • the phage library (as described herein above) can be designed to display scFvs, which can fold into the monovalent form of the Fv region of an antibody.
  • the construct is suitable for bacterial expression.
  • the genetically engineered scFvs comprise heavy chain and light chain variable regions joined by a contiguously encoded 15 amino acid flexible peptide spacer.
  • the prefened spacer is (Gly 4 Ser) 3 (SEQ ID NO: 8).
  • the length of this spacer, along with its amino acid, constituents provides for a nonbulky spacer, which allows the V H and the V L regions to fold into a functional Fv domain that provides effective binding to its target.
  • Varying the length of the spacers is yet another prefened method of forming dimers, trimers, and triamers (often refened to in the art as diabodies, triabodies, and tetrabodies, respectively). Dimers are formed under conditions where the spacer joining the two variable chains of a scFv is shortened to generally 5-12 amino acid residues. This shortened spacer prevents the two variable chains from the same molecule from folding into a functional Fv domain. Instead, the domains are forced to pair with complimentary domains of another molecule to create two binding domains. In a prefened method, a spacer of only 5 amino acids (Gly 4 Ser) (SEQ ID NO:19) was used for diabody construction.
  • This dimer can be formed from two identical scFvs, or from two different populations of scFvs and retain the selective and/or specific enhanced binding activity of the parent scFv(s), and/or show increased binding strength or affinity.
  • triabodies are formed under conditions where the spacer joining the two variable chains of a scFv is shortened to generally less than 5 amino acid residues, preventing the two variable chains from the same molecule from folding into a functional Fv domain. Instead, three separate scFv molecules associate to form a trimer. In a prefened method, triabodies were obtained by completely removing this flexible spacer.
  • the triabody can be formed from three identical scFvs, or from two or three different populations of scFvs, and retain the selective and/or specific enhanced binding activity of the parent scFv(s), and/or show increased binding strength or affinity.
  • Tefrabodies are similarly formed under conditions where the spacer joining the two variable chains of a scFv is shortened to generally less than 5 amino acid residues, preventing the two variable chains from the same molecule from folding into a functional Fv domain. Instead, four separate scFv molecules associate to form a tetramer.
  • the tetrabody can be formed from four identical scFvs, or from 1 - 4 individual units from different populations of scFvs and should retain the selective and/or specific enhanced binding activity of the parent scFv(s), and/or show increased binding strength or affinity. Whether triabodies or tefrabodies form, under conditions where the spacer is generally less than 5 amino acid residues long, depends on the amino acid sequence of the particular scFv(s) in the mixture and the reaction conditions.
  • the present invention also provides polypeptides comprising a consensus sequence: X ⁇ -X 2 -X 3 -Pro-Xs-X 6 (SEQ ED NO:3), wherein X] and X 6 are hydrophobic amino acids and X 2 , X , and X 5 are any amino acid.
  • X 2 is selected from the group consisting of arginine, lysine, and Xi and X 6 are selected from the group consisting of leucine, valine, methionine, alanine, phenylalanine, and isoleucine.
  • the polypeptide may preferably comprise SEQ ID. NO:5.
  • the polypeptide may comprise SEQ ID NO:6.
  • X and X 3 are arginine and the hydrophobic amino acid X 6 is preferably isoleucine.
  • Xi is selected from the group consisting of leucine and methionine
  • X 2 and X 3 are arginine
  • X 5 is selected from the group consisting of serine and valine
  • X 6 is isoleucine.
  • the polypeptides of the present invention may be substantially circular or looped.
  • the present invention also provides polypeptides that bind specifically to PSGL-1, wherein the polypeptide binds with an affinity substantially similar to SI 5.
  • the polypeptides can bind specifically to GPIb with an affinity substantially similar to A3R
  • the present invention further provides isolated or purified polypeptides, such as recombinant nucleic acids, that encode the antibodies and polypeptides of the present invention.
  • isolated or purified polypeptides can be produced in prokaryotic or eukaryotic expression systems.
  • Such expression systems include expression vectors and host cells transfected with such expression vectors.
  • Methods for producing antibodies and polypeptides in prokaryotic and eukaryotic systems including culturing recombinant host cells under conditions permitting expression of such antibodies and isolating or purifying such antibodies from the recombinant host cells or from culture medium are well-known in the art.
  • An eukaryotic cell system refers to an expression system for producing peptides or polypeptides by genetic engineering methods, wherein the host cell is an eukaryote.
  • An 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 prefened 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 o ⁇ Aeromonas aminopeptidase under suitable conditions (U.S.
  • the present invention also provides a process for selecting entities, e.g., antibodies or fragments thereof or alternatively small inorganic chemical entities, that bind sulfated epitopes. These methods involve panning a library (e.g., a phage display library to identify antibodies or fragments thereof and combinatorial libraries to identify small inorganic chemical entities) against a peptide having a sulfated epitope.
  • entities e.g., antibodies or fragments thereof or alternatively small inorganic chemical entities, that bind sulfated epitopes.
  • Suitable sulfated epitopes for panning may be based on or derived from, for example, PSGL-1, GPIb, ⁇ -2- antiplasmin; aminopeptidase B; CC chemokine receptors such as CCR2, CCR5, CCR3, CXCR3, CXCR4, CCR8, and CCR2b; seven-transmembrane-segment (7TMS) receptors; coagulation factors such as factor V, VIII, and IX; fibrinogen gamma chain; heparin cofactor II; secretogranins such as secretogranin I and II; vitronectin, amyloid precursor, ⁇ -2-antiplasmin; cholecystokinin; ⁇ -choriogonadotropin; complement C4; dermatan sulfateproteoglycan; fibronectin; or castrin.
  • CC chemokine receptors such as CCR2, CCR5, CCR3, CXCR3, CXCR4, CCR8, and C
  • the peptide can be sulfated at any position.
  • the peptide comprises the sulfated epitope is derived from or based on a region of PSGL-1 (especially when sulfated at the tyrosine residue at position 51 from the N- terminus), GPIb (especially when sulfated at the tyrosine residue at position 276 and to a lesser extend the tyrosine residue at position 279), or CCR5 (especially when sulfated at the tyrosine residue at position 10).
  • the method comprises immobilizing the peptide on a solid support.
  • the method comprises competitive panning using a non-sulfated, soluble peptide or a soluble peptide sulfated at an alternate tyrosine position.
  • Panning an appropriate combinatorial library to identify a small inorganic chemical entity can, of course, also be used to carry out these methods.
  • the process for producing an entity that binds sulfated epitopes comprises the steps of: (a) providing a library (e.g., phage display library); (b) providing a peptide of PSGL-1 of SEQ ID NO:7; (c) panning the library to select for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ID NO:7; and (d) producing the selected entity (e.g., antibody or polypeptide comprising the scFv antibody) that binds to the peptide of SEQ ID NO:7.
  • a library e.g., phage display library
  • a peptide of PSGL-1 of SEQ ID NO:7 e.g., phage display library
  • panning the library to select for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ID NO:7
  • producing the selected entity e.g., antibody or poly
  • the process for producing an entity comprises the steps of: (a) providing a library (e.g., phage display library); (b) providing an immobilized peptide of PSGL-1 (SEQ ID NO:7); (c) panning the library to select for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ID NO:7 in the presence of a soluble unsulfated PSGL-1 peptide (SEQ ID NO:26); and (d) producing the entity (e.g., antibody or polypeptide comprising the scFv antibody) that binds to the peptide of SEQ ID NO:7.
  • a library e.g., phage display library
  • an immobilized peptide of PSGL-1 SEQ ID NO:7
  • panning the library to select for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ID NO:7 in the presence of a soluble unsulfated
  • the process for producing an entity comprises the steps of: (a) providing a library (e.g., phage display library); (b) providing an immobilized peptide of PSGL-1 (SEQ ID NO:7); (c) panning the library for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ID NO:7 in the presence of a soluble sulfated GPIb peptide (SEQ ID NO:44 and/or 50); and (d) producing the entity (e.g., antibody or polypeptide comprising the scFv antibody) that binds to the peptide of SEQ ID NO:7.
  • a library e.g., phage display library
  • an immobilized peptide of PSGL-1 SEQ ID NO:7
  • panning the library for an entity e.g., phage particle
  • SEQ ID NO:44 and/or 50 a soluble sulfated GPIb peptide
  • the process for producing an entity comprises the steps of: (a) providing a library (e.g., phage display library); (b) providing an immobilized peptide of PSGL-1 (SEQ ID NO:7); (c) panning the library for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ID NO:7 in the presence of a soluble sulfated GPIb peptide (SEQ ID NO:44 and/or 50) or unsulfated PSGL-1 peptide (SEQ ID NO:26); and (d) producing the entity (e.g., antibody or polypeptide comprising the scFv antibody) that binds to the peptide of SEQ ID NO:7.
  • a library e.g., phage display library
  • an immobilized peptide of PSGL-1 SEQ ID NO:7
  • panning the library for an entity e.g., phage particle
  • SEQ ID NO:44 and/or 50
  • the process for producing an entity comprises the steps of: (a) providing a library (e.g., phage display library); (b) providing an immobilized peptide of PSGL-1 (SEQ ID NO:7); (c) panning the library for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ID NO:7 in the presence of a soluble sulfated GPIb peptide (SEQ ID NO:44 and/or 50) and soluble unsulfated GPIb peptide (SEQ ID NO:43); and (d) producing the entity (e.g., antibody or polypeptide comprising the scFv antibody) that binds to the peptide of SEQ ID NO:7.
  • a library e.g., phage display library
  • an immobilized peptide of PSGL-1 SEQ ID NO:7
  • panning the library for an entity e.g., phage particle
  • the process for producing an entity comprises the steps of: (a) providing a library (e.g., phage display library); (b) providing an immobilized peptide of PSGL-1 (SEQ ID NO:7); (c) panning the library for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ID NO:7 in the presence of a soluble sulfated GPIb peptide (selected from SEQ ID NO:44, 50, 57 and 58 or combination thereof) and/or unsulfated PSGL-1 peptide (SEQ ID NO:43); and (d) producing the entity (e.g., antibody or polypeptide comprising the scFv antibody) that binds to the peptide of SEQ ID NO:7.
  • a library e.g., phage display library
  • an immobilized peptide of PSGL-1 SEQ ID NO:7
  • panning the library for an entity e.g., phage particle
  • the process for producing an entity comprises the steps of: (a) providing a library (e.g., phage display library); (b) providing an immobilized peptide of GPIb peptide (SEQ ID NO:44); (c) panning the library for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ID NO:44 and (d) producing the entity (e.g., antibody or polypeptide comprising the scFv antibody) that binds to the peptide of SEQ ID NO:44.
  • a library e.g., phage display library
  • SEQ ID NO:44 an immobilized peptide of GPIb peptide
  • producing the entity e.g., antibody or polypeptide comprising the scFv antibody
  • the process for producing an entity comprises the steps of: (a) providing a library (e.g., phage display library); (b) providing an immobilized peptide of GPIb peptide (SEQ ID NO:44); (c) panning the library for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ID NO:44 in the presence of a soluble unsulfated GPIb peptide (SEQ ID NO:43); and (d) producing the entity (e.g., antibody or polypeptide comprising the scFv antibody) that binds to the peptide of SEQ ID NO:44.
  • a library e.g., phage display library
  • SEQ ID NO:44 an immobilized peptide of GPIb peptide
  • the process for producing an entity comprises the steps of: (a) providing a library (e.g., phage display library); (b) providing an immobilized peptide of GPIb peptide (SEQ ID NO:44); (c) panning the library for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ED NO:44 in the presence of a one or more of the soluble sulfated PSGL-1 peptides of SEQ ED NO:7, 48, 49 or 59; and (d) producing the entity (e.g., antibody or polypeptide comprising the scFv antibody) that binds to the peptide of SEQ ID NO:44.
  • a library e.g., phage display library
  • an immobilized peptide of GPIb peptide SEQ ID NO:44
  • the process for producing an entity comprises the steps of: (a) providing a library (e.g., phage display library); (b) providing an immobilized peptide of GPIb peptide (SEQ ED NO:44); (c) panning the library for an entity (e.g., phage particle) that binds to the immoblized peptide of of SEQ ED NO:44 in the presence of a one or more of the soluble sulfated PSGL-1 peptides of SEQ ED NO:7, 48, 49 or 59 and soluble unsulfated PSGL-1 peptide (SEQ ED NO:26); and (d) producing the entity (e.g., antibody or polypeptide comprising the scFv antibody) that binds to the peptide of SEQ ID NO:44.
  • a library e.g., phage display library
  • SEQ ED NO:44 an immobilized peptide of GPIb peptide
  • the process for producing an entity comprises the steps of: (a) providing a library (e.g., phage display library); (b) providing an immobilized peptide of GPIb peptide (SEQ ID NO:44); (c) panning the library for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ID NO:44 in the presence of a one or more of the soluble sulfated PSGL-1 peptides of SEQ ID NO:7, 48, 49 or 59 and soluble unsulfated GPIb peptide (SEQ DD NO:43); and (d) producing the entity (e.g., antibody or polypeptide comprising the scFv antibody) that binds to the peptide of SEQ ID NO:44.
  • a library e.g., phage display library
  • SEQ ID NO:44 an immobilized peptide of GPIb peptide
  • the process for producing an entity comprises the steps of: (a) providing a library (e.g., phage display library); (b) providing an immobilized peptide of GPIb peptide (SEQ ID NO:44); (c) panning the library for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ID NO:44 in the presence of a one or more of the soluble sulfated PSGL-1 peptides of SEQ ID NO:7, 48, 49 or 59 and one or more of the soluble sulfated GPIb peptides (SEQ ID NO:44 and/or 50); and (d) producing the entity (e.g., antibody or polypeptide comprising the scFv antibody) that binds to the peptide of SEQ ID NO:44.
  • a library e.g., phage display library
  • an immobilized peptide of GPIb peptide SEQ ID NO:44
  • the process for producing an entity comprises the steps of: (a) providing a library (e.g., phage display library); (b) providing an immobilized peptide of CCR5 (SEQ ID NO:53); (c) panning the library for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ID NO:53 and (d) producing the entity (e.g., antibody or polypeptide comprising the scFv antibody) that binds to the peptide of SEQ ID NO:53.
  • a library e.g., phage display library
  • an immobilized peptide of CCR5 SEQ ID NO:53
  • panning the library for an entity e.g., phage particle
  • the entity e.g., antibody or polypeptide comprising the scFv antibody
  • the process for producing an entity comprises the steps of: (a) providing a library (e.g., phage display library); (b) providing an immobilized peptide of CCR5 (SEQ ID NO:53); (c) panning the library for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ID NO: 53 in the presence of a soluble unsulfated GPIb peptide (SEQ ED NO:43) and/or unsulfated soluble PSGL-1 peptide (SEQ ED NO:26) (d) producing the entity (e.g., antibody or polypeptide comprising the scFv antibody) that binds to the peptide of SEQ ED NO:53.
  • a library e.g., phage display library
  • an immobilized peptide of CCR5 SEQ ID NO:53
  • the process for producing an entity comprises the steps of: (a) providing a library (e.g., phage display library); (b) providing an immobilized peptide of CCR5 (SEQ ID NO:53); (c) panning the library for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ID NO: 53 in the presence of a soluble sulfated GPIb peptide (SEQ ED NO:44 and/or 50) and/or soluble sulfated PSGL-1 peptides (SEQ ID NO:7, 48, 49 and/or 59) and (d) producing the entity (e.g., antibody or polypeptide comprising the scFv antibody) that binds to the peptide of SEQ ID NO:53.
  • a library e.g., phage display library
  • an immobilized peptide of CCR5 SEQ ID NO:53
  • the process for producing an entity comprises the steps of: (a) providing a library (e.g., phage display library); (b) providing an immobilized peptide of CCR5 (SEQ ED NO:53); (c) panning the library for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ED NO: 53 in the presence of a soluble sulfated GPIb peptide (SEQ ED NO:44 and/or 50) and/or unsulfated soluble PSGL-1 peptide (SEQ ID NO:26); and (d) producing the entity (e.g., antibody or polypeptide comprising the scFv antibody) that binds to the peptide of SEQ ED NO:53.
  • a library e.g., phage display library
  • an immobilized peptide of CCR5 SEQ ED NO:53
  • the process for producing an entity comprises the steps of: (a) providing a library (e.g., phage display library); (b) providing an immobilized peptide of CCR5 (SEQ ID NO:53); (c) panning the library for an entity (e.g., phage particle) that binds to the immobilized peptide of SEQ ED NO: 53 in the presence of a soluble sulfated GPIb peptide (SEQ ID NO:44 and/or 50) and or unsulfated soluble GPIb peptide (SEQ ID NO:43); and (d) producing the entity (e.g., antibody or polypeptide comprising the scFv antibody) that binds to the peptide of SEQ ID NO:53.
  • a library e.g., phage display library
  • an immobilized peptide of CCR5 SEQ ID NO:53
  • 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 nanow 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 may be 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.
  • the present invention also provides a library for identifying human antibodies that bind to sulfated epitopes.
  • the library is of immunoglobulin binding domains comprising a diverse antigen-binding domain for complementary binding, wherein the library has diversity only in heavy chain CDR3.
  • the immunoglobulin binding domains are scFv molecules.
  • the immunoglobulin binding domains have heavy chain complementarity determining regions (CDRs) 1 and 2 derived from DP32 and, more preferably, also have light chain variable regions derived from DP32.
  • the immunoglobulin binding domains of the present library can be displayed on the surface of any suitable vector, such as filamentous bacteriophage particles, for example.
  • the libraries of the present invention can be used to select for sulfated motifs or epitopes.
  • the antibodies and binding fragments thereof of the subj ect invention can be associated with, combined, fused, or linked to various agents, such as drugs, toxins, pharmaceuticals, and radioactive isotopes with, optionally, a pharmaceutically effective carrier, to form drug-peptide compositions, fusions or conjugates having anti-disease and/or anti-cancer activity.
  • agents such as drugs, toxins, pharmaceuticals, and radioactive isotopes
  • a pharmaceutically effective carrier to form drug-peptide compositions, fusions or conjugates having anti-disease and/or anti-cancer activity.
  • conjugates and fusions may also be used for diagnostic, prognostic, or staging purposes.
  • Examples of carriers useful in the invention include dexfran, HPMA (a hydrophilic polymer), or any other polymer, such as a hydrophilic polymer, as well as derivatives, combinations and modifications thereof.
  • decorated liposomes 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); and (d) carboxylic acid hydrazides selected from 2-5 carbon atoms, and derivatives, combinations, modifications, and analogues thereof.
  • dicarboxylic acids such as succinic acid, glutaric acid, and adipic acid
  • maleimido hydrazides such as N-[maleimidocaproic acid] hydrazide, 4-[N- maleimidomethyl]cyclohexan
  • 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, prognostic, or staging and imaging purposes. Kits having such radioisotope-antibody (or fragment) conjugates are provided.
  • radioisotopes useful for diagnostics, prognostics, staging and imaging include u indium, 113 indium, 99m rhenium, 105 rhenium, 101 rhenium, 99m technetium, 121m tellurium, I22m tellurium, 125m telluriunm 165 thulium, 167 thulium 168 thulium 123 iodine, iodine, iodine, iodine, krypton, xenon, yttrium, bismuth, bromine, 18 fluorine, 95 ruthenium, 97 ruthenium, 103 ruthenium, 105 ruthenium, 107 mercury, 203 mercury, 67 gallium, and 68 gallium.
  • Prefened 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 include fluorescein, phycoerythrin, or rhodamine, or modifications or conjugates thereof.
  • Antibodies and polypeptides conjugated to indicative markers may be used to diagnose, prognose, or stage disease states.
  • the present invention also provides a method of purging tumor cells from a patient by providing a sample containing cells from the patient and incubating the cells from the patient with an antibody of the present invention. Such activities 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.
  • the present invention thus 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 FITC, PE, and the like, and 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.
  • the present invention also provides a method of diagnosing, prognosing, or staging a disease in a patient by providing a sample containing a cell from the patient and determining whether the antibodies of the present invention bind to the cell of the patient, thereby indicating that the patient is at risk for or has the disease.
  • Such activities 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 imacceptable 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 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: TI, NO, MO which mean:Tl - Tumor is 2 cm or less in diameter, NO - No regional lymph node metastasis, MO - No distant metastasis.
  • TI TMN
  • NO TMN
  • MO which mean:Tl - Tumor is 2 cm or less in diameter
  • NO - No regional lymph node metastasis MO - 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 conelate with specific cytogenetic findings and AML associated with myelodysplasia. Others have also proposed pathologic classifications.
  • AML World Health Organization
  • Another proposal specific for AML includes disease types that conelate 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)).
  • Antibodies and polypeptides of the present invention may be bound to, conjugated to, or otherwise associated with anti-cancer agents, anti-neoplastic agents, anti-viral 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 agents.
  • 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
  • Exemplary pharmaceutical agents include anthracyclines such as doxorubicin (adriamycin), daunorubicin, idarubicin, detorubicin, carminomycin, epirubicin, esorubicin,morpholinodoxorubicin, morpholinodaunorubicin, methoxymorpholinyldoxorubicin,methoxymo holinodaunorubicin and methoxymorpholinyldoxorubicin and substituted derivatives, combinations and modifications thereof.
  • anthracyclines such as doxorubicin (adriamycin), daunorubicin, idarubicin, detorubicin, carminomycin, epirubicin, esorubicin,morpholinodoxorubicin, morpholinodaunorubicin, methoxymorpholinyldoxorubicin,methoxymo holinodaunorubicin and meth
  • 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.
  • 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.
  • 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.
  • the present invention provides methods of inducing or activating ADCC by administering the present antibodies.
  • the consensus antibody and the antibodies that bind with substantially the same affinity as S15 and A3R of the present invention 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
  • the 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 FasL 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.
  • an IgG antibody comprising the consensus sequence of the invention induces FasL expression on effector cells.
  • Various factors can affect ADCC, including the type of effector cells involved, cytokines (IL-2 and G-CSF, for example), incubation time, the number of receptors present on the surface of the cells, and antibody affinity.
  • Examples of anti-disease, anti-cancer, and anti-leukemic agents to which antibodies and polypeptides of the present invention may usefully be linked include toxins, radioisotopes, and pharmaceuticals.
  • Examples of toxins include gelonin, Pseudomonas exotoxin (PE), PE40, PE38, diphtheria toxin, ricin, or derivatives, combinations and modifications thereof.
  • Examples of radioisotopes 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, morpholmodoxorubicin, morpholinodaunorubicin, methoxymo ⁇ holinyldoxorubicin, methoxymo ⁇ holinodaunorubicin and methoxymo ⁇ holinyldoxorubicin and substituted derivatives, combinations and modifications thereof.
  • anthracyclines such as doxorubicin (adriamycin), daunorubicin, idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, morpholmodoxorubicin, morpholinodaunorubicin, 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 comprising any of the consensus sequences of the present invention and a pharmaceutically acceptable canier.
  • 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 in which case the consensus antibody preferably comprises A3R. The antibody, or polypeptide can also be present in an amount effective to inhibit HIV entry and/or treat HIV infection. 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 with antibody or fragment thereof and of the pharmaceutical composition, mode of administration of the pharmaceutical 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 phannaceutical 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 and polypeptides 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.
  • injectable 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 freating a disease (e.g., freating can include ameliorating the effects of a disease, preventing a disease, or inhibiting the progress of a disease) in patients in need thereof.
  • freating can include ameliorating the effects of a disease, preventing a disease, or inhibiting the progress of a disease
  • 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 HIN entry in cells and also, as a result of such inhibition, blocking replication of HIN and thereby treating HIN infection.
  • Such methods further include preventing or inhibiting cardiovascular diseases such as restenosis.
  • the present invention moreover provides antibodies and polypeptides for use in 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, HEN 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 present example demonstrates selection, production, and initial characterization of S15 scFv antibody fragments, including the binding capabilities of SI 5 antibody fragments.
  • a phage display library based on a specific scaffold of VH- NL with a random CDR3-VH of six amino acids was utilized to identify a scFv antibody that binds sulfated PSGl-1.
  • the scFv antibody was obtained by panning against a synthetic sulfated peptide having the sequence of amino acids 1-17 of the mature PSGL-1 molecule from ⁇ -terminus to C-terminus or from C-terminus to ⁇ -terminus (conesponding to amino acids 42-58 of the immature PSGL-1 molecule i.e. including the signal sequence).
  • Flow cytometry particularly fluorescence-activated cell sorting (FACS) and ELISA was used for identifying and characterizing specific phage clones that bind to the synthetic sulfated peptide or to whole cells expressing PSGL-1.
  • the phage display library was constructed from a scaffold of a clone isolated from a combinatorial phage antibody library (CAT) in a pHE ⁇ vector.
  • the scaffold contained a VH3 (1-3, 3-20) and a VL (11-7).
  • the library was constructed by randomization of the CDR3 hypervariable loop of six amino acids in length.
  • the Eagl restriction site at the 3' end of the VL was mutated by excising an Xmal - ⁇ otl fragment from the pHE ⁇ -Yl and inserting a new fragment (by annealing an oligonucleotide of SEQ ED ⁇ O:20 and an oligonucleotide of SEQ ID NO:21) with a mutation of the ligation part of the Notl site.
  • the mutation was required in order to generate two unique sites (Eagl and Xmal) and to introduce a PCR product of the same size but with randomized CDR3. After ligation, the plasmid was sequenced at the insertion region and the mutation was confirmed. The new pHEN-Yl-mut was restricted with Eagl and Xmal and the large fragment was the vector for subsequent ligation.
  • the template for the preparation of the variable CDR3 was made by PCR with an oligonucleotide of SEQ ID NO:22 and an oligonucleotide of SEQ ID NO:23.
  • the PCR product (template A) was isolated from a SDS-polyacrylamide gel and purified for further amplifications.
  • Template A was used for amplification with oligonucleotides of SEQ ED NO:24 and SEQ ED NO:23, the product was purified and designated template B.
  • Template B was amplified with oligonucleotides of SEQ ED NO:25 and SEQ ED NO:23, purified, and restricted with Eagl and Xmal restriction enzymes.
  • the restricted vector pHEN-Yl-mut with Eagl and Xmal was purified and ligated to the last PCR product restricted with the same enzymes.
  • the product of the ligation was used to transform TGI cells.
  • the yield of transformation from 3 ligations yielded 2.4 x 10 6 independent colony-forming units (CFUs).
  • the library was amplified by plating in 10 (13 cm) SOBAG (20g Bacto-tryptone, 5g Bacto-yeast, 8.5mM NaCl, 10 mM MgCl 2 , 0.1M glucose, O.lmg/ml ampicillin per liter; for plates 15g of Bacto-agar) plates.
  • the bacteria was resuspended in SOBAG medium from plates and kept at -70°C in 20% glycerol.
  • the titer of the amplified library was 1.5 x 10 CFU/ml.
  • the eluted phage clones were optionally amplified before additional cycles of binding and optional amplification, enriching the pool of specific sequences in favor of those phage clones bearing antibody fragments which best bind to the peptides. After several cycles of panning, individual phage clones were characterized, and the sequences of the clones were determined.
  • the S 15 antibody clone was identified by panning a phage display library in solution with sfreptavidin-magnetic beads bound to a biotinylated sulfated peptide of SEQ ED NO:7.
  • This peptide was chemically synthesized and is based on the highly acidic sequence found at amino acids 42 to 58 within PSGL-1, including sulfation of the 3rd tyrosine residue.
  • the N-terminus of the synthetic peptide was extended with aminocaproic acid and biotinylated at the amino group of the caproic acid to avoid steric hindrance of the sulfated epitope.
  • the synthetic sulfated peptide of SEQ ED NO:7 was immobilized on sfreptavidin-magnetic beads (Dynal) by incubation of the peptide in excess followed by washing with PBST (PBS + 0.05% Tween-20) and blocking by PBST-M (PBST supplemented with 5% low fat milk). For panning, the peptide-bound beads were incubated with 2 x 10 n phages. Peptide of the same linear sequence (SEQ.ED. No:45) that was neither biotinylated nor sulfated was added to the panning solution in order to avoid isolating scFv clones that bind to non-sulfated peptides.
  • FIG. 1 shows that all of the panned and selected phage clones tested specifically bound to the synthetic sulfated peptide.
  • FIG. 2 shows that all of the scFv expressed on clones selected after 3 cycles of panning specifically bound to the synthetic sulfated peptide.
  • Table 2 provides a summary of all the clones used in the study of Example 1 and the amino acid sequences of their CDR3 regions.
  • scFv from selected clones were analyzed by FACS to evaluate binding to PSGL-1 on ML-2, an AML M4 cell line which expresses PSGL-1. As shown in FIG. 3, the scFv from all of the selected clones, as well as L32 bound to PSGL-1 on ML-2 cells at different sensitivities.
  • FIGS. 3 and 4 show that S15 displays high affinity for ML-2 cells that express PSGL-1 and low affinity for platelets, based on binding to glycocalicin. This conclusion was confirmed by FACS analysis of scFv binding to granulocytes (which are PSGL-1 expressing cells) and to platelets, the results of which are shown in FIG. 5.
  • FIG. 5 clearly shows that SI 5 binds strongly to granulocytes, but only weakly to intact platelets.
  • FIG. 6 shows a comparison of the granulocyte/platelet binding ratio of scFv.
  • S15 has a granulocyte/platelet binding ratio significantly higher than that of
  • FIG. 7 shows the results of analysis of the binding of S 15 to PSGL- 1 on ML-2 cells in the presence and absence of KPL-1, a murine antibody directed against PSGL-1.
  • SI 5 was purified on a Protein A affinity column for further experimentation described in Example 2.
  • Example 2 [246] The present example describes further characterization of the purified S15 scFv antibody, including its binding capabilities, compared to similarly purified L32 and Yl scFv.
  • Example 3 This example describes the identification of two additional antibodies comprising the consensus sequence which were obtained by panning the library described in Example 1 against a synthetic peptide conesponding to residues 268-285 of GPIb including sulfation at the first tyrosine position.
  • the present example demonstrates selection, production, and initial characterization of Dl and D3 scFv antibody fragments, including the binding capabilities of Dl and D3 antibody fragments.
  • a phage display library based on a specific scaffold of VH-VL with a random CDR3-VH of six amino acids was utilized to identify an scFv antibody that binds sulfated GPIb.
  • the scFv antibody was obtained by panning against a synthetic sulfated peptide having the sequence of amino acids 268-285 (GDEGDTDLY(SO 4 )DYYPEEDTE) (SEQ ID NO:44) of the mature GPIb molecule from N-terminus to C-terminus (conesponding to amino acids 284-301 of the immature GPIb molecule, i.e., including the signal sequence).
  • Flow cytometry, particularly fluorescence- activated cell sorting (FACS) and ELISA was used for identifying and characterizing specific phage clones that bind to the synthetic sulfated peptide or to platelets expressing GPIb.
  • the phage display library used is described in Example 1.
  • Biopanning was carried out by incubating immobilized peptide of SEQ ID NO:44 (GDEGDTDLY S DYYPEEDTE) with the phage display library, removing unbound phage by washing, and specifically eluting the bound phage.
  • the eluted phage clones were optionally amplified before additional cycles of binding and optional amplification, enriching the pool of specific sequences in favor of those phage clones bearing antibody fragments which best bind to the peptides. After several cycles of panning, individual phage clones were characterized, and the sequences of the clones were determined.
  • the Dl and D3 antibody clones were identified by panning a phage display library in solution with a chemically synthesized sulfated peptide of SEQ ID NO:44 covalently bound to magnetic beads.
  • the sulfated peptide of SEQ ID NO:44 is based on the highly acidic sequence found at amino acids 268-285 within mature GPIb, including sulfation of the first tyrosine residue.
  • the synthetic sulfated peptide of SEQ ID NO:44 was covalently bound to amine-magnetic beads (Dynal) by a reaction involving EDC/NHS, according to the manufacturer instructions.
  • the beads were washed with PBST (PBS + 0.05% Tween-20) and blocked by PBST-M (PBST supplemented with 5% low fat milk).
  • PBST-M PBST supplemented with 5% low fat milk.
  • Peptide of the same linear sequence SEQ.ID. NO:43 but lacking sulfation was added to the panning solution in order to avoid isolating scFv clones that bind to non-sulfated peptides.
  • GDEGDTDLYDYYPEEDTE in "background" wells as well as sulfated and non- sulfated peptides derived from PSGL1-1 respectively SEQ ED NO:7 (QATEYEYLDY S DFLPETE) and SEQ ID NO:26 (QATEYEYLDYDFLPETE).
  • Peptides were coated on NH-CovaLinkTM plates (NUNC) as recommended by the manufacturer. Binding data obtained from the background wells were subtracted from the binding data from the conesponding systems in the test wells, to generate the results shown in FIG. 13. A randomly chosen phage clone was used as the negative confrol (NC) for binding to the synthetic sulfated peptide.
  • FIG. 13 shows that each of the selected phage clones specifically bound to both of the synthetic sulfated peptides i.e. GPIb and PSGL-1. Wlender the clones exhibited a range of binding strength to the synthetic sulfated peptides, each clone displayed about the same behavior with respect to both GPIb and PSGL-1.
  • scFv from selected clones were analyzed by ELISA to evaluate binding to glycocalicin (the outer membrane portion derived from GPIb expressed in platelets). As shown in FIG. 14, scFvs Dl and D3 exhibited significant binding, while D2 and D16 exhibited weak binding and D5 and D9 were similar to the negative control.
  • scFvs (1 ⁇ g per experiment) from selected clones were analyzed by FACS to evaluate binding to ML-2, an AML M4 cell line expressing PSGL-1. As shown in Table 4, all of the scFvs bound to ML-2 cells, at different sensitivities. Dl and D3 exhibited the strongest binding to ML-2, presumably via interaction with an epitope of PSGL-1.
  • the present example demonstrates binding of several scFv antibodies having CDR3 sequences falling within the consensus sequence of the present invention. Briefly, using the CDR3 sequence of the Yl-scFv as a scaffold, mutant scFv's were produced and the effect of these mutations on the binding to platelets and granulocytes was assessed. [262] Using site directed mutagenesis in the CDR3 of the heavy chain (CDR3H) of Yl- scFv, additional CDR3 regions falling within the consensus sequence were generated. These additional scFvs were then tested to determine the relative binding to platelets and granulocytes.
  • CDR3H heavy chain
  • Yl-scFv is known to bind the negatively charged GPIb ⁇ epitope found on platelets.
  • an arginine residue i.e. a positively charged amino acid, in the second position.
  • scFvs were constructed. Mutant R2A has the argimne residue replaced by alanine; mutant A3R has an additional arginine at position 3, replacing alanine; mutant V5R has an additional arginine at position 5, replacing valine.
  • the fourth scFv was a scrambled mutant.
  • the scFv mutants are summarized in Table 5.
  • Anti-scFv antibody was generated by immunization of a rabbit with 400 ⁇ g of a mixture of scFvs, and labeled using the PhycolinkTM. R-phycoerythrin conjugation kit (ProZyme, San Leandro, CA) according to the manufacturer's instructions. Aliquots (10 ⁇ g/mL) of scFvs were incubated with 10 7 washed platelets for 1 hour at room temperature. The platelets were then washed in PBS containing 1% BSA and incubated with R- phycoerythrin -anti-scFv antibody for 1 hour at room temperature. Platelets were then washed, resuspended in PBS and samples (10 4 platelets) were analyzed by FACS (VACScan, Becton-Dickinson, CA).
  • FIG. 15 shows the average binding +SEM from 3 experiment using platelets from different donors. Mutant R2A does not exhibit binding to platelets, compared to the Yl parent scFv, suggesting that the arginine in the second position of CDR3H may play a role in the platelet binding function. Mutant N5R bound to platelets in a manner similar to wild type Yl-scFv, in the concentration range between 1 to 10 ⁇ g/ml. At a concentration of 20 ⁇ g/ml however, binding of V5R was two fold higher as compared toYl scFv (FIG. 15).
  • mutant A3R scFv exhibited nine-fold higher binding to platelets compared to Yl scFv at all the concenfrations tested, suggesting that an additional arginine residue adjacent to the arginine residue at position 2 can augment the binding to platelets.
  • the scrambled scFv failed to bind to platelets (FIG. 15), as would be expected of a mutant lacking arginine at the second position.
  • FIG. 16 shows these results as the average binding of 5 ⁇ g/ml scFv + STDV from 2 experiments. Mutant R2A scFv and the scrambled scFv did not bind to purified GPIb ⁇ nor to any of the GPIb ⁇ -derived peptides. V5R bound to purified glycocalicin and to GPIb ⁇ -derived peptides in a fashion similar to Yl. Mutants A3R exhibited enhanced binding to glycocalicin compared to Yl.
  • FIG. 17 shows that this mutant exhibits more effective inhibition of ristocetin-induced, vWF-dependent platelet aggregation in washed platelets, as compared to Yl scFv (IC 50 of 0. 2 ⁇ M and 0.8 ⁇ M respectively).
  • IC 50 0. 2 ⁇ M and 0.8 ⁇ M respectively.
  • Example 5 The biological activity of A3R and of Yl scFvs was further assessed in a Cone and Plate(let) Analyzer (CPA) assay, which is a new method for clinically evaluating whole blood platelet adhesion and aggregation on a polystyrene surface under high shear rates, thus mimicking physiological conditions (Varon et al., (1997) Thromb. Res. 85(4): 283- 294; Shenkman et al., (2000) Thromb. Res. 99(4): 353-361).
  • CPA Cone and Plate(let) Analyzer
  • Example 6 The present example describes a comparison of the binding characteristics of S 15 , A3R, and Yl to healthy platelet rich plasma (PRP).
  • i Neg stands for negative and is defined as when Geo Mean is below 10.
  • the present example describes a comparison of the binding characteristics of S 15, A3R, and Yl to healthy whole blood cells containing granulocytes (G), lymphocytes (L), and monocytes (M).
  • This example describes the development of a non-human in vivo animal model system to further study the properties of the antibodies comprising the consensus sequence.
  • An initial goal was to identify a non-human mammalian species in which one (or more) of the antibodies both (a) binds to platelets and (b) is capable of inhibiting
  • scFv Yl showed the weakest relative binding among the antibodies comprising the consensus sequence. This observation supports the conclusion that the mutant (A3R) and library selected (S 15, Sl l, SI and Dl) scFvs all have enhanced binding capacity for sulfated epitopes, as occur on platelet GPIb.
  • scFvs A3R and S15 exhibit 5-9 fold greater (relative to Yl) binding capacity on human and guinea pig platelets, while binding capacity of SI 1, SI and Dl to those species is about 25-fold greater than Yl.
  • SI 1 With respect to the species anay of non-human platelets bound by the panel of scFvs, SI 1, SI and Dl each bind platelets derived from dog, guinea pig and rabbit.
  • SI 5 binds platelets from dog (highest affimty), guinea pig and pig, but not from rabbit, mice or monkey (baboon and cynomalogus monkey).
  • guinea pig is the single non- human species identified in which all the antibodies comprising the consensus sequence are capable of binding platelets. Effect on platelet aggregation
  • the inhibitory effect of scFv S 15 on guinea pig platelet aggregation (IC 50 80- 160 ug/ml) was found to be about 8-10 times lower relative to its inhibitory effect on human derived platelets. This may be due to the relative lower binding of scFv SI 5 antibody to guinea pig derived-platelets.
  • Plasma was obtained by centrifuging blood samples at 3000 x g for 15 minutes and stored at -20°C and PRP was obtained by centrifuging blood samples at 150 x g for 10 minutes.
  • Platelet bound- A3R scFv was tested in guinea pig PRP by FACS analysis using anti-scFv PE- labeled antibodies. Plasma scFv A3R antibody concentrations were be assayed by a specific ELISA.
  • Plasma level of A3R-scFv was measured by ELISA using plates coated with GPIb peptide having sulfated tyrosine at Tyr-276 (1 ⁇ M/ well). Bound scFv was detected by addition of rabbit anti-VL (variable light) antibody, followed by anti-rabbit HRP (Sigma) and 3,3', 5,5'-tetramethyl-benzidine (TMB) (Sigma, St Louis, MO) substrate. The intensity of the color produced was read by an ELISA plate reader (Anthos, Salzburg) at
  • the plasma concentration of the scFv in each sample was calculated from standard curves constructed by adding known amount of these antibodies to guinea pig plasma or to PBS containing 0.05% Tween 20 and 2% skim milk.
  • Results show that the plasma concentration of scFv A3R peaked 3 minutes after bolus injection and gradually declined over 6 hours (FIG. 20). The half-life of A3R in guinea pig was 139 ⁇ 9 minutes.
  • Example 9 The BIAcore biosensor uses surface plasmon resonance detection and permits realtime kinetic analysis of two interacting species. This system was used to measure the binding kinetics of the scFvs Yl, A3R and S15 to glycocalicin, a polypeptide derived from platelet GPIb.
  • Binding affinities (K D ) of the antibodies for glycocalicin were determined using BIAevaluation 3.1 software (BIAcore), from average Ka (association rate) and K d (dissociation rate) kinetics.
  • A3R-scFv binds to platelet glycocalicin with higher affinity than the Yl-scFv ( ⁇ 7 fold), as indicated by its more rapid association rate (Table 11). These results are in accordance with results obtained by FACS analysis on intact platelet. S15-scFv also binds to glycocalicin with higher affinity than the Yl-scFv but lower than A3R.
  • This example describes direct binding of antibodies comprising the consensus sequence to synthetic peptides based on those regions of PSGL-1, GPIb and CCR5 which undergo sulfation at tyrosine residues.
  • PSGL-1 (residues 42-58) derived
  • GPIb (residues 268-285) derived
  • GDEGDTDLY S DYYPEEDTE (sulfated at 1 st tyrosine position) SEQ ID NO:44
  • MDYQVSSPIYDINYYTSE non-sulfated
  • SEQ ED NO:51 MDY S QVSSPIYDINYYTSE (sulfated at 1 st tyrosine position)
  • SEQ ED NO:52 MDYQVSSPEY 5 DENYYTSE (sulfated at 2 nd tyrosine position)
  • SEQ ED NO:53 MDYQVSSPIYDINY 5 YTSE (sulfated at 3 rd tyrosine position)
  • the scFvs used were N06 (negative control, Yl (PO3), S15, Sl l, SI, S9, s.c.3.1 and Sl l.
  • protein-A purified scFv antibodies (0.5 ⁇ g / well) were
  • anti-scFv rabbit anti- human V L polyclonal antibody
  • PBS buffer containing 0.05% Tween PBS buffer containing 0.05% Tween.
  • Goat anti-rabbit HRP-labeled antibody in blocking buffer was added for incubation for 1 hour at room temperature, and the excess was removed by washing 10 times.
  • TMB Developer was added for 5 minutes and
  • Table 12 and FIG. 21 indicate that all scFv antibodies tested comprising the consensus sequence bound significantly to PSGL-1 -derived peptide sulfated at the third tyrosine position, but not to that sulfated at the first or second tyrosine position.
  • Single chain antibodies SI, S9 and 3.1 showed the strongest binding to the PSGL-1 -derived peptide sulfated at the third tyrosine position.
  • Example 11 Studies were carried out to demonstrate that an IgG antibody comprising the consensus sequence of the invention is capable of mediating antibody dependent cell cytotoxicity (ADCC) of target cells, particularly B-CLL cells derived from patient samples.
  • ADCC antibody dependent cell cytotoxicity
  • hyper cross-linking of S15-IgG with secondary anti-human Fc antibodies demonstrated that an apoptotic mechanism also contributes to cell killing.
  • ADCC Antibody-Dependent Cell Cytotoxicity
  • FIG. 22B shows that SI 5 IgG mediated effector cell cytotoxicity in all three samples, with 30-50% ADCC as compared to control.
  • SI 5-IgG ADCC is mediated by natural killer and monocytic cells. Effector cells were analyzed for their capability of effecting SI 5 IgG-mediated ADCC of B-CLL cells. Natural killer (CD56+) and monocytic (CD 14+) cells from both normal donors and B- CLL patients were isolated using commercially available magnetic beads.
  • NK cells from a normal donor and B-CLL patients were capable of effecting ADCC, resulting in about 50% and 35% killing respectively.
  • Monocytes from both a normal donor and from B-CLL patients were also capable of effecting ADCC (about 5-13%).
  • CD56+ NK cells constituted the more significant effector cell population for SI 5 IgG-mediated ADCC of B-CLL cells.
  • Apoptosis experiments were as follows. Mononuclear cells from B-CLL patients were separated on FICOLL and the cells were incubated in the presence or in the absence of S15-IgG or control antibodies for 10 minutes at 37°C. Anti-human Fc antibodies were then added and incubated for 4-24 hours at 37°C.
  • Example 8 Screening of Inorganic Compound Library.
  • 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-sfretavidin 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Oncology (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
  • Hematology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Cardiology (AREA)
  • Urology & Nephrology (AREA)
  • Transplantation (AREA)
  • AIDS & HIV (AREA)
  • Diabetes (AREA)
  • Vascular Medicine (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des anticorps ou des fragments de ceux-ci se liant à des cellules cancéreuses et jouant un rôle important dans les phénomènes physiologiques, tels que le roulement de cellules et la métastase. L'invention concerne également des procédés thérapeutiques et diagnostiques, ainsi que des compositions utilisant de tels fragments d'anticorps. Les procédés et compositions selon l'invention peuvent être utilisés dans le ciblage d'agents thérapeutiques et dans le diagnostic, le pronostic et l'évaluation du stade de maladies et la thérapie destinée à celles-ci, telles que le cancer, notamment la croissance tumorale et la métastase, la leucémie, une maladie auto-immune et une maladie inflammatoire. L'invention concerne enfin une bibliothèque de domaines de liaison à l'immunoglobuline comprenant un domaine de liaison aux antigènes différent destiné à une liaison complémentaire, la bibliothèque présentant une diversité uniquement dans une chaîne lourde CDR3.
EP04777308A 2003-06-30 2004-06-30 Anticorps et utilisations de ceux-ci Ceased EP1649001A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US61123803A 2003-06-30 2003-06-30
PCT/US2004/021002 WO2005010153A2 (fr) 2003-06-30 2004-06-30 Anticorps et utilisations de ceux-ci

Publications (1)

Publication Number Publication Date
EP1649001A2 true EP1649001A2 (fr) 2006-04-26

Family

ID=34103137

Family Applications (1)

Application Number Title Priority Date Filing Date
EP04777308A Ceased EP1649001A2 (fr) 2003-06-30 2004-06-30 Anticorps et utilisations de ceux-ci

Country Status (11)

Country Link
EP (1) EP1649001A2 (fr)
JP (1) JP2007528711A (fr)
KR (1) KR20060106631A (fr)
CN (1) CN101300021A (fr)
AU (1) AU2004259406A1 (fr)
BR (1) BRPI0411945A (fr)
CA (1) CA2536644A1 (fr)
IL (1) IL172510A0 (fr)
MX (1) MXPA06000252A (fr)
RU (1) RU2006102570A (fr)
WO (1) WO2005010153A2 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1714978A1 (fr) * 2005-04-19 2006-10-25 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Peptides servant au diagnostic et au traitement des tumeurs
US20070298034A9 (en) * 2005-12-09 2007-12-27 Angela Widom Sulfotyrosine specific antibodies and uses therefor
EP2161072A1 (fr) * 2008-09-03 2010-03-10 Gambro Lundia AB Membranes d'hémodialyse avec un seuil de coupure pour le traitement de patients nécessitant des hémodialyses régulières
EP2246364A1 (fr) * 2009-04-29 2010-11-03 Pierre Fabre Médicament Anticorps anti CXCR4 pour le traitement du VIH
EP2371863A1 (fr) * 2010-03-30 2011-10-05 Pierre Fabre Médicament Anticorps humanisé anti CXCR4 pour le traitement du cancer
CN104507967B (zh) * 2012-01-20 2018-10-12 建新公司 抗cxcr3抗体
ES2877171T3 (es) * 2014-07-08 2021-11-16 Sanford Burnham Med Res Inst Moduladores de PSGL-1 y usos de los mismos
RU2766000C2 (ru) * 2016-01-08 2022-02-07 АльтруБио Инк. Четырехвалентные антитела к psgl-1 и их применения

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL137121A0 (en) * 1998-10-30 2001-06-14 Miller Jonathan L Variable heavy chain and variable light chain regions of antibodies to human platelet glycoprotein ib alpha
CZ20031982A3 (cs) * 2000-12-29 2004-09-15 Bio-Technology General Corp. Izolované molekuly zahrnující epitopy obsahující sulfatované sloučeniny, protilátky proti takovým epitopům a jejich použití

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2005010153A2 *

Also Published As

Publication number Publication date
MXPA06000252A (es) 2006-12-15
KR20060106631A (ko) 2006-10-12
BRPI0411945A (pt) 2006-10-24
CN101300021A (zh) 2008-11-05
RU2006102570A (ru) 2007-08-20
WO2005010153A2 (fr) 2005-02-03
IL172510A0 (en) 2006-04-10
AU2004259406A1 (en) 2005-02-03
JP2007528711A (ja) 2007-10-18
CA2536644A1 (fr) 2005-02-03
WO2005010153A3 (fr) 2008-03-13

Similar Documents

Publication Publication Date Title
US20090220486A1 (en) Antibodies and uses thereof
US20040002450A1 (en) Y17 - isolated molecules comprising epitopes containing sulfated moieties, antibodies to such epitopes, and uses thereof
US20040001822A1 (en) Y1-isolated molecules comprising epitopes containing sulfated moieties, antibodies to such epitopes, and uses thereof
US20040001839A1 (en) Multimers - isolated molecules comprising epitopes containing sulfated moieties, antibodies to such epitopes, and uses thereof
JP2005503756A5 (fr)
JP2005503756A (ja) 硫酸化部分を含むエピトープを含む単離分子、当該エピトープに対する抗体、及びその使用
US20040202665A1 (en) Compositions and methods for therapeutic treatment
JP2004524023A (ja) 選択的癌療法のための特異的ヒト抗体
US20080274100A1 (en) Antibodies and uses thereof
EP1649001A2 (fr) Anticorps et utilisations de ceux-ci
WO2004003166A2 (fr) Anticorps et leurs applications
US20050152906A1 (en) Specific human antibodies
WO2004002528A9 (fr) Compositions et procedes pour traitement therapeutique
WO2005005455A2 (fr) Anticorps humains specifiques
US20040208877A1 (en) Antibodies and uses thereof
AU2002246738A1 (en) Isolated molecules comprising epitopes containing sulfated moieties, antibodies to such epitopes, and uses thereof

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20060130

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL HR LT LV MK

DAX Request for extension of the european patent (deleted)
PUAK Availability of information related to the publication of the international search report

Free format text: ORIGINAL CODE: 0009015

RIC1 Information provided on ipc code assigned before grant

Ipc: C12P 21/08 20060101ALI20080409BHEP

Ipc: C07K 16/00 20060101ALI20080409BHEP

Ipc: C07K 1/00 20060101ALI20080409BHEP

Ipc: A61K 38/04 20060101ALI20080409BHEP

Ipc: A61K 38/12 20060101ALI20080409BHEP

Ipc: A61K 38/00 20060101AFI20080409BHEP

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED

18R Application refused

Effective date: 20081120