Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • 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
  • 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
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • 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
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/10—Cells modified by introduction of foreign genetic material
• • 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
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • 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
  • C07K2317/60—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)

Definitions

• a single polypeptide chain (60kd) was found to be expressed on surface membrane of HEL cells that is closely related to GPIb and corresponds to an incompletely or abnormally O-glycosylated GPIb ⁇ subunit (Kieffer et al., J. Biol. Chem. 261(34): 15854-62 (1986)).
• the N-terminal globular domain of GPIb ⁇ contains a cluster of negatively charged amino acids.
• Several lines of evidence indicate that in transfected CHO cells expressing GPIb-IX complex and in platelet GPIb ⁇ , the three tyrosine residues contained in this domain (Tyr-276, Tyr-278, and Tyr-279) undergo sulfation.
• 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-Go lgi compartment.
• Sulfated proteins include secretory proteins, proteins targeted for granules, and the extracellular regions of plasma membrane proteins.
• Tyrosine is an amino acid residue presently known to undergo sulfation. Kehoe et al, Chem. Biol. 7: R57-61 (2000).
• Other amino acids, e.g., threonine may also undergo sulfation, particularly in diseased cells.
• GPIb ⁇ (CD42), which is expressed by platelets and megakaryocytes and mediates platelet attachment to and rolling on subendothehum via binding with vWF, also contains numerous negative charges at its N-terminal domain.
• CC-chemokine receptors such as CCR5, which serve as co-receptors with related seven transmembered segment (7TMS) receptor for entry of human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV) into target cells.
• 7TMS seven transmembered segment
• HV-1, HIV-2, and SIV human and simian immunodeficiency viruses
• CXCR4 another important HIV-1 co-receptor, is also sulfated (Farzan et al., Cell 96(5): 667-76 (1999)).
• Tyrosine sulfation plays a less significant role in CXCR4-dependent HIV-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)).
• P-, E-, and L- Selectins are members of a family of adhesion molecules that, among other functions, mediate rolling of leukocytes on vascular endothelium.
• P- Selectin is stored as granules in platelets and is transported to the surface after activation by thrombin, histamine, phorbol ester, or other stimulatory molecules.
• P-Selectin is also expressed on activated endothelial cells.
• E-Selectin is expressed on endothelial cells
• L-Selectin is expressed on neutrophils, monocytes, T cells, and B cells.
• PSGL-1 also called CD162
• PSGL-1 is a mucin glycoprotein ligand for P-Selectin
• PSGL-1 is a disulfide-linked homodimer that has a PACE (Paired Basic Amino Acid Converting Enzymes) cleavage site. PSGL-1 also has three potential tyrosine sulfation sites followed by 10-16 decamer repeats that are high in proline, serine, and threonine. The extracellular portion of PSGL-1 contains three N-linked glycosylation sites and has numerous sialylated, fucosylated O-linked oligosaccharide branches (Moore et al., J. Biol. Chem.
• PSGL-1 has 361 residues in HL60 cells, with a 267 residue extracellular region, 25 residue trans-membrane region, and a 69 residue intracellular region, and forms a disulfide-bonded homodimer or heterodimer on the cell surface (Afshar-Kharghan et al., Blood 97: 3306-12 (2001)).
• the sequence encoding PSGL-1 is in a single exon, so alternative splicing should not be possible.
• PSGL-1 in HL60 cells has 15 consecutive repeats of a 10 residue consensus sequences present in the extracellular region, although there are 14 and 16 repeats of this sequence in polymorphonuclear leukocytes, monocytes, and several other cell lines, including most native leukocytes.
• PSGL-1 is expressed on neutrophils as a dimer, with apparent molecular weights of both 250 kDa and 160 kDa, whereas on HL60 the dimeric form is approximately 220 kDa. When analyzed under reducing conditions, each subunit is reduced by half. Differences in molecular mass may be due to polymorphisms in the molecule caused by the presence of different numbers of decamer repeats (Leukocyte Typing VI. Edited by T. Kishimoto et al. (1997)).
• PSGL-1 has been found on all leukocytes: neutrophils, monocytes, lymphocytes, activated peripheral T cells, granulocytes, eosinophils, platelets, and on some CD34 positive stem cells and certain subsets of B cells.
• P-Selectin is selectively expressed on activated platelets and endothelial cells. Interaction between P-Selectin and PSGL-1 promotes rolling of leukocytes on vessel walls, and abnormal accumulation of leukocytes at vascular sites results in various pathological inflammations. Stereo-specific contributions of individual tyrosine sulfates on PSGL-1 are important for the binding of P- Selectin to PSGL-1.
• PSGL-1 tyrosine sulfation supports slower rolling adhesion at all shear rates and supports rolling adhesion at much higher shear rates (Rodgers et al., Biophys. J. 81 : 2001-09 (2001)). Moreover, it has been suggested that PSGL-1 expression on platelets is 25-100 fold lower than that of leukocytes (Frenette et al, J Exp. Med. 191(8): 1413-22 (2000)).
• KPLl A commercially available monoclonal antibody to human PSGL-1, KPLl, has been shown to inhibit the interactions between PSGL-1 and P-selectin and between PSGL-1 and L-selectin.
• the KPLl epitope was mapped to the tyrosine sulfation region of PSGL-1 (YEYLDYD) (Snapp et al., Blood 91(l):154-64 (1998)).
• Normal fibrinogen which is the more abundant form (approximately 90% of the total fibrinogen found in the body), is composed of two identical 55 kDa ⁇ chains, two identical 95 kDa ⁇ chains, and two identical 49.5 kDa ⁇ chains.
• Normal variant fibrinogen which is the less abundant form (approximately 10% of the fibrinogen found in the body), is composed of two identical 55 kDa ⁇ chains, two identical 95 kDa ⁇ chains, one 49.5 kDa ⁇ chain, and one variant 50.5 kDa ⁇ prime chain.
• the gamma and gamma prime chains are both coded for by the same gene, with alternative splicing occurring at the 3' end.
• Normal gamma chain is composed of amino acids 1-411 and normal variant gamma prime chain is composed of 427 amino acids, of which amino acids 1-407 are the same as those in the normal gamma chain and amino acids 408-427 are VRPEHPAETEYDSLYPEDDL. 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.
• diseased cells such as AML cells, T-ALL cells, Pre-B-ALL cells, B-leukemia, B-CLL cells, multiple myeloma cells, and metastatic cells.
• Another objective of the present invention includes the use of such antibodies in the development and provision of medicaments for the inhibition of cell rolling, inflammation, immune reactions, infection, autoimmune reactions, and metastasis, yet are not involved in adhesion, thrombosis and/or restenosis and aggregation, and for the treatment of diseases, such as AML, T-ALL, B-leukemia, B-CLL, Pre-B-ALL, multiple myeloma, metastasis, cardiovascular diseases such as myocardial infarction, retinopathic diseases, diseases caused by sulfated tyrosine-dependent protein-protein interactions, or other diseases in which such cellular functions or actions play a significant role.
• diseases such as AML, T-ALL, B-leukemia, B-CLL, Pre-B-ALL, multiple myeloma, metastasis, cardiovascular diseases such as myocardial infarction, retinopathic diseases, diseases caused by sulfated tyrosine-dependent protein-protein interactions, or other diseases
• Another object of the present invention is to provide a method of purging tumor cells.
• Yet another object of the invention is to provide methods of activating
• the present invention provides antibodies or fragments thereof having the binding capabilities of an scFv antibody fragment of SEQ ID NO:l.
• the present invention also provides antibodies or fragments thereof, wherein at least one antibody, or binding fragment thereof, has a first hypervariable region of SEQ ID NO:2, a second hypervariable region of SEQ ID NO:3, and/or a third hypervariable region of SEQ ID NO:4.
• the antibodies or fragments thereof, of the present invention preferably bind to, or crossreact with, an epitope of PSGL-1.
• the antibodies or fragments thereof of the present invention bind to an epitope on at least one cell type selected from the group consisting of T-ALL, AML, B-leukemia, B-CLL and multiple myeloma leukemia cells.
• the present invention also provides isolated epitopes having an amino acid sequence that binds to the antibodies or binding fragments thereof of the present invention.
• the isolated epitope is located between amino acids 1 and 17 of the mature PSGL-1, which is within a cluster of negatively charged amino acids.
• compositions and processes for production of such antibodies or fragments thereof are provided, including conditions related to inhibiting or treating cell rolling; inhibiting or treating inflammation; inhibiting or treating an auto-immune disease; inhibiting or treating an infection (e.g., a viral infection such as HIV); inhibiting or treating metastasis; inhibiting or treating 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; alters 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
• 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. Accordingly, the term "antibody or fragment thereof as used herein includes an antibody complex or antibody complexes.
• 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 .
• the Fv molecule itself, is comprised of a first chain and a second chain, each chain having a first, second and third hypervariable region.
• the hypervariable loops within the variable domains of the light and heavy chains are termed Complementary Determining Regions (CDRs).
• CDRs Complementary Determining Regions
• the most variable of these regions in nature is the CDR3 region of the heavy chain.
• the CDR3 region is understood to be the most exposed region of the Ig molecule and, as shown and provided herein, is the site primarily responsible for the selective and/or specific binding characteristics observed.
• a fragment of an Fv molecule is defined as any molecule smaller than the original Fv that still retains the selective and/or specific binding characteristics of the original Fv.
• fragments include but are limited to (1) a minibody, which comprises a fragment of the heavy chain only of the Fv, (2) a microbody, which comprises a small fractional unit of antibody heavy chain variable region (International Application No. PCT/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. It should be appreciated that a fragment of an Fv molecule can be a substantially circular or looped polypeptide.
• Fab fragment is a monovalent antigen-binding fragment of an immunoglobulin.
• a Fab fragment is composed of the light chain and part of the heavy chain.
• An F(ab') 2 fragment is a bivalent antigen binding fragment of an immunoglobulin obtained by pepsin digestion. It contains both light chains and part of both heavy chains.
• An Fc fragment is a non-antigen-binding portion of an immunoglobulin. It contains the carboxy-terminal portion of heavy chains and the binding sites for the Fc receptor.
• An Fd fragment is the variable region and first constant region of the heavy chain of an immunoglobulin.
• Polyclonal antibodies are the product of an immune response and are formed by a number of different B lymphocytes. Monoclonal antibodies are derived from one clonal B cell.
• a cassette refers to a given sequence of consecutive amino acids that serves as a framework and is considered a single unit and is manipulated as such. Amino acids can be replaced, inserted into, removed, or attached at one or both ends. Likewise, stretches of amino acids can be replaced, inserted into, removed, or attached at one or both ends.
• epitope is used interchangeably herein with the terms ligand, domain, and binding region.
• Selectivity is herein defined as the ability of a targeting molecule to choose and bind one entity or cell state from a mixture of entities or entity states, all entities or entity states of which may be specific for the targeting molecule.
• association constant between a binding molecule (e.g., one binding site on an antibody) and a ligand (e.g., antigenic determinant).
• the strength of the sum total of noncovalent interactions between a single antigen-binding site on an antibody and a single epitope is the affinity of the antibody for that epitope.
• Low affinity antibodies bind antigen weakly and tend to dissociate readily, whereas high-affinity antibodies bind antigen more tightly and remain bound longer.
• the term "avidity" differs from affinity, because the former reflects the valence of the antigen-antibody interaction.
• antigen-antibody reaction is specific, in some cases antibodies elicited by one antigen can cross-react with another unrelated antigen. Such cross-reactions occur if two different antigens share a homologous or similar structure, epitope, or an anchor region thereof, or if antibodies specific for one epitope bind to an unrelated epitope possessing similar structure conformation or chemical properties.
• a platelet is a disc-like cytoplasmic fragment of a megakaryocyte that is shed in the marrow sinus and subsequently circulates in the peripheral blood stream. Platelets have several physiological functions including a major role in clotting. A platelet contains centrally located granules and peripheral clear protoplasm, but has no definite nucleus.
• Agglutination as used herein means the process by which suspended bacteria, cells, discs, or other particles of similar size are caused to adhere and form into clumps. The process is similar to precipitation but the particles are larger and are in suspension rather than being in solution.
• aggregation means a clumping of platelets induced in vitro, and thrombin and collagen, as part of a sequential mechanism leading to the formation of a thrombus or hemostatic plug.
• Conservative amino acid substitution is defined as a change in the amino acid composition by way of changing one or two amino acids of a peptide, polypeptide or protein, or fragment thereof.
• the substitution is of amino acids with generally similar properties (e.g., acidic, basic, aromatic, size, positively or negatively charged, polarity, non-polarity) such that the substitutions do not substantially alter peptide, polypeptide or protein characteristics (e.g., charge, isoelectric point, affinity, avidity, conformation, solubility) or activity.
• Typical substitutions that may be performed for such conservative amino acid substitution may be among the groups of amino acids as follows:
• G glycine
• A alanine
• V valine
• L leucine
• I isoleucine
• A alanine
• S serine
• T threonine
• H histidine
• K lysine
• R arginine
• a phagemid is defined as a phage particle that carries plasmid DNA.
• a phage display library (also termed phage peptide/antibody library, phage library, or peptide/antibody library) comprises a large population of phages (10 8 or larger), each phage particle displaying a different peptide or polypeptide sequence. These peptide or polypeptide fragments may constructed to be of variable length.
• the displayed peptide or polypeptide can be derived from, but need not be limited to, human antibody heavy or light chains.
• a 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, pharmaceutical agent, oligonucleotide, recombinant protein, antibody fragment, anti-cancer 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-vir 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.
• a given cell may express on its surface a protein having a binding site (or epitope) for a given antibody, but that binding site may exist in a cryptic form (e.g., be sterically hindered or blocked, or lack features needed for binding by the antibody) in the cell in a state, which may be called a first stage (stage I).
• Stage I may be, e.g., a normal, healthy, non-diseased status.
• the epitope 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 (e.g., antibodies) that no longer contain any peptide bonds, i.e., amide bonds, between amino acids; however, in the context of the present invention, the term peptide mimetic is intended to include molecules that are no longer completely peptidic in nature, such as pseudo-peptides, semi- peptides and peptoids. Whether completely or partially non-peptide, peptidomimetics according to this invention provide a spatial arrangement of reactive chemical moieties that closely resembles the three-dimensional arrangement of active groups in the peptide on which the peptidomimetic is based. These molecules include small molecules, lipids, polysaccharides, or conjugates thereof.
• FIG. 1 depicts FACS analysis following TM3.13 scFv staining of T-ALL cells.
• FIG. 2 depicts numerical data from Lumiaggregometer analysis of platelet aggregation in the presence of scFv antibodies as a percent of control aggregation of either washed platelets or PRP.
• FIG. 3 depicts FACS analysis comparing binding of various scFv antibodies to platelets:
• Fig. 3A is AN51-PE (FSC)
• Fig. 3B is AN51-PE (FL2-H)
• Fig. 3C is a negative control
• Fig. 3D is Yl-myc+
• Fig. 3E is Yl
• Fig. 3F is L32
• Fig. 3G is TM1.1.
• FIG. 4 depicts FACS analyses that compare the ability of scFv antibodies
• Fig. 4 A is 0 ng
• Fig. 4B is 100 ng
• Fig. 4C is 250 ng
• Fig. 4D is 500 ng
• Fig. 4E is 1000 ng
• Fig. 4F is 2500 ng
• Fig. 4G is 5000 ng.
• FIG. 5 depicts numerical data from FACS analyses that compare the ability of scFv antibodies (NOl, Yl-myc+, and L32) to compete and interfere with the binding of labeled Yl antibody to KG-1 cells.
• FIG. 6 depicts numerical data from ELISA analyses providing comparisons of the binding of different concentrations of scFv antibodies (TM1.1, Yl-myc+, Yl, and L32) to glycocalicin.
• FIG. 7 depicts Western analysis of L32 and Yl scFv antibody binding to
• FIG. 8 depicts numerical data from ELISA analyses providing comparisons ofthe binding of scFv antibodies (Yl-myc+, TMl.l, and L32) to fibrinogen, PSGL-1, and GPIb ⁇ -related peptides.
• FIG. 9 depicts numerical data from FACS analyses following staining of platelets with Y17 scFv antibody, in the presence of varying concentrations of GPIb- derived peptides. The results are presented as percent reduction in the geo mean ofthe response obtained with the scFv antibody alone.
• FIG. 10 depicts numerical data from ELISA analyses following binding of
• the present invention relates to an antibody or fragment thereof having binding capabilities of an scFv antibody fragment of SEQ ID NO:l, that binds PSGL-1.
• these antibodies ofthe present invention have similar binding affinity as SEQ ID NO:l.
• the scFv fragment of SEQ ID NO:l has been designated L32.
• an antibody ofthe present invention is L32. This antibody was identified by screening a phage library, which has diversity only in the heavy chain CDR3 regions, against a leukemia cell to select specific antibodies that recognize leukemia cell surface determinants, wherein the specific receptor was not previously known or characterized. Using this same method, another antibody, L31, was identified. Athough the present invention encompasses many antibodies, L32 will be used hereafter exemplarily. [86] Previously, other antibodies that bind to leukemic cells were identified in
• L32 antibody and the antibodies disclosed in the Yl/Yl 7 applications bind leukemic cells, although L32 binds to leukemic cells with approximately five times greater affinity than Yl. Based on this fact, as well as the fact that the antibodies were isolated from a common germ line (DP32), comparison studies were done to determine the correlation between their respective binding epitopes. It was subsequently determined that L32 appears to bind the same sulfated epitope as Yl/Yl 7. As the Y1/Y17 epitope is specifically present on platelets, although it has been suggested that the levels of expression are 25-100 fold lower than that of leukocytes (Frenette et al., J. Exp. Med.
• the sulfated epitopes previously identified as binding to Yl/Yl 7 are characterized by the presence of sulfated moieties, such as sulfated tyrosine residues or sulfated carbohydrate or lipid moieties, preferably within a cluster of two or more acidic amino acids, which are found on ligands and receptors that play important roles in such diverse processes as inflammation, immune reactions, infection, autoimmune reactions, metastasis, adhesion, thrombosis and/or restenosis, cell rolling, and aggregation.
• sulfated moieties such as sulfated tyrosine residues or sulfated carbohydrate or lipid moieties, preferably within a cluster of two or more acidic amino acids, which are found on ligands and receptors that play important roles in such diverse processes as inflammation, immune reactions, infection, autoimmune reactions, metastasis, adhesion, thrombosis and/or restenosis, cell rolling,
• Such epitopes are also found on diseased cells, such as B-leukemia cells, B-CLL cells, AML cells, multiple myeloma cells, and metastatic cells. These epitopes are useful targets for the therapeutic mediation of these processes and for diagnostic or prognostic procedures, including staging.
• the L32 scFv has 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, VLA-4, and VCAM-1 in the inflammatory processes of diseases such as atherosclerosis (Huo and Ley, Ada Physiol. Scand., 173: 35-43 (2001); Libby, Sci. Am. May: 48-55 (2002); Wang et al., J. Am. Coll. Cardiol. 38: 577-582 (2001)). L32 interference with any of these central molecules may suggest a potential role for L32 in abrogating related diseases.
• the antigen-binding site is complementary to the structure ofthe epitopes to which the antibodies bind, therefore these binding sites are referred to as complementarity-determining regions (CDRs).
• CDRs complementarity-determining regions
• the most variable of these regions is the CDR3 region ofthe heavy chain.
• the CDR3 region is understood to be the most exposed region ofthe Ig molecule and, as provided herein, has a central role in determining the selective and/or specific binding characteristics observed.
• CDRs may be inserted into cassettes to produce antibodies.
• 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.
• the amino acid sequence ofthe 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 ofthe 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 ofthe cassette may be replaced or modified by non-conservative or, preferably, conservative amino acid substitutions.
• the antibody or fragment thereof has 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.
• a CDR3 region of one chain either the CDR3 region ofthe light chain or, preferably, the CDR3 region ofthe heavy chain and, more preferably, both heavy and light chain CDR3 regions determine binding selectivity and specificity.
• the binding selectivity and specificity are determined by the CDR2 and CDRl regions ofthe light chain and, preferably, ofthe heavy chain.
• the upstream or downstream regions flanking the first, second, and/or third hypervariable regions may also secondarily influence the binding selectivity and specificity.
• At least one antibody or fragment thereof has a first hypervariable region (CDR3) of SEQ ID NO:2.
• at least one antibody or fragment thereof has a second hypervariable region (CDR2) of SEQ ID NO:3.
• at least one antibody pr fragment thereof has a third hypervariable region (CDRl) of SEQ ID NO:4. More preferably, at least one antibody or fragment thereof has a first hypervariable region (CDR3) of SEQ ID NO:2 and a second hypervariable region (CDR2) of SEQ ID NO:3 and a third hypervariable region (CDRl) of SEQ ID NO:4.
• At least one antibody, or binding fragment thereof, ofthe antibody or fragment thereof is an scFv having SEQ ID NO:l.
• 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 ofthe 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. It is generally preferable to compare amino acid sequences, due to the greatly increased sensitivity to detection of subtle relationships between the peptide, polypeptide or protein molecules. 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 ofthe light and heavy chains can be interchanged between the two chains and among the three-hypervariable sites within and/or between chains.
• the sequences ofthe hypervariable regions can be altered to span two or more ofthe CDRs. Also in framework variable regions - also so that may only partially be ine 1 CDR
• the present invention provides for a peptide or polypeptide having an antibody or antigen binding fragment thereof, a construct thereof, or a construct of a fragment.
• antibodies include IgG, IgA, IgD, IgE, or IgM antibodies.
• the IgG class encompasses several sub-classes including IgG], IgG , 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 , and Fd molecules.
• fragments such as fragments of Fvs and fragments of Fabs
• fragments are also included in the term "fragments", as long as they retain the binding characteristics ofthe original antibody or larger fragment.
• Constructs include, for example, multimers such as diabodies, triabodies, and tetrabodies.
• the phrases "antibody, binding fragment thereof, or complex having an antibody or binding fragment thereof and “antibody or fragment” are 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.
• Varying the length ofthe spacers is yet another preferred method of forming dimers, trimers, and triamers (often referred 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, hi a preferred method, a spacer of only 5 amino acids (Gly 4 Ser) 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.
• additional antibodies can be isolated using the biopanning methods described herein, wherein the molecule or cell that L32 binds is used to screen a particular phage display library, particularly a library prepared from a leukemia, lymphoma, and myeloma patient.
• Antibodies and fragments, 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 or prognostics, including staging.
• the tag can later be removed from the molecule.
• useful tags include: AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, Protein C, S-TAG®, T7, V5, and VSV-G (Jarvik and Telmer, Ann. Rev. Gen., 32, 601-18 (1998)).
• the tag is preferably c-myc or KAK.
• a eukaryotic cell system refers to an expression system for producing peptides or polypeptides by genetic engineering methods, wherein the host cell is a eukaryote.
• a eukaryotic expression system may be a mammalian system, and the peptide or polypeptide produced in the mammalian expression system, after purification, is preferably substantially free of mammalian contaminants.
• Other examples of a useful eukaryotic expression system include yeast expression systems.
• the process for producing an antibody or fragment thereof has the steps of: (a) providing a phage display library; (b) providing a molecule or cell that an antibody or fragment thereof having the binding capabilities of an scFv antibody fragment of SEQ ID NO:l may bind to; (c) panning the phage display library for a phage particle displaying an oligopeptide or polypeptide that binds to the molecule or cell; and (d) producing an antibody or fragment thereof having at least one antibody or binding fragment thereof having an antibody or binding fragment thereof, having the peptide or polypeptide that binds to the molecule or cell.
• 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.
• 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.
• radioisotopes useful for diagnostics, prognostics, or staging include ⁇ indium, 113 indium, 99m rhenium, 105 rhenium, 101 rhenium, 99m technetium, 121m tellurium, 122m 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, 7 gallium, and 8 gallium.
• Preferred radioactive isotopes are opaque to X-rays or any suitable paramagnetic ions.
• the indicative marker molecule may also be a fluorescent marker molecule.
• fluorescent marker molecules examples include fluorescein, phycoerythrin, or rhodamine, or modifications or conjugates thereof.
• Antibodies or fragments conjugated to indicative markers may be used to diagnose, prognose or stage disease states by providing a sample containing a cell from the patient and determimng whether the antibodies ofthe 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 ofthe present invention. Such activities may be carried out in vivo, in vitro, or ex vivo. Where carried out in vivo ox 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 ofthe disease and the availability of other options.
• staging a disease in a patient generally involves determining the classification ofthe disease based on the size, type, location, and invasiveness ofthe 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 inco ⁇ orated by reference herein and which classifies stages of cancer into T, N, and M categories with T describing the primary tumor according to its size and location, N describing the regional lymph nodes, and M describing distant metastases.
• TNM Classification of Malignant Tumours (6th Edition) (L.H. Sobin, Ed.)
• N describing the primary tumor according to its size and location
• N describing the regional lymph nodes
• M describing distant metastases.
• the numbers I, II, III and IV are used to denote the stages and each number refers to a possible combination of TNM factors.
• imaging agents include fluorescent dyes, such as
• fluorescent proteins such as green fluorescent proteins.
• Other examples include radioactive molecules and enzymes that react with a substrate to produce a recognizable change, such as a color change.
• the imaging agent ofthe 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 ofthe disease, e.g., upon diagnosis, during treatment, during remission and during relapse.
• Antibodies, and fragments thereof, ofthe 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, and
• Exemplary pharmaceutical agents include the anthracyclines, such as doxorubicin (adriamycin), daunorubicin (daunomycin), idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, as well as mo ⁇ holino and substituted derivates and combinations thereof.
• anthracyclines such as doxorubicin (adriamycin), daunorubicin (daunomycin), idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, as well as mo ⁇ holino and substituted derivates and combinations thereof.
• Inhibition of growth of a cancer cell includes, for example, (i) prevention of cancerous or metastatic growth, (ii) slowing down ofthe cancerous or metastatic growth, (iii) total prevention ofthe growth process ofthe 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 ofthe leukemic or metastatic growth, (iii) the total prevention ofthe growth process ofthe 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.
• anti-disease, anti-cancer, and anti-leukemic agents examples include toxins, radioisotopes, and pharmaceuticals.
• toxins examples include gelonin, Pseudomonas exotoxin (PE), PE40,
• PE38 diphtheria toxin, ricin, or derivatives, combinations and modifications thereof.
• radioisotopes examples include gamma-emitters, positron-emitters, and x-ray emitters that may be used for localization and/or therapy, and beta-emitters and alpha-emitters that may be used for therapy.
• the radioisotopes described previously as useful for diagnostics, prognostics and staging are also useful for therapeutics.
• Non-limiting examples of anti-cancer or anti-leukemia agents include anthracyclines such as doxorubicin (adriamycin), daunorubicin (daunomycin), idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, and mo ⁇ holino and substituted derivatives, combinations and modifications thereof.
• anthracyclines such as doxorubicin (adriamycin), daunorubicin (daunomycin), idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, and mo ⁇ holino and substituted derivatives, combinations and modifications thereof.
• 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 anti-cancer or anti- leukemia is doxorubicin, mo ⁇ holinodoxorubicin, or mo ⁇ holinodaunorubicin.
• the antibody or fragment thereof can be present in an amount effective to increase mortality of tumor cells or leukemia cells. Also alternatively, the antibody or fragment thereof 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 fragment thereof 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.
• Antibodies, constructs, conjugates, and fragments ofthe subject invention may be admimstered 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.
• 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 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 ofthe present invention. In one embodiment, the purging occurs ex vivo.
• the scFv display phage library (Nissim et al., EMBO J, 13: 692-98 (1994)) was used with the agreement of the MRC.
• the library was originally constructed as a phagemid library displaying scFv fragments in which the V H and the V L domains were linked by a flexible polypeptide.
• the scFvs displayed in the phagemid library were fused to the N-terminus ofthe minor coat protein pill ofthe phage, which was then subcloned into the pHENl vector.
• Fresh bacterial cultures ofthe bacterial strains TG-1 and HB2151 were prepared for infection (amplification) by growing the cells to A 600 of 0.5-0.9 (exponentially growing cells). E. coli TG-1 cells were used for phage propagation and E. coli HB2151 cells were used for scFv protein production. Colonies from the large plates were scraped and pooled. An aliquot ( ⁇ 10 7 ) of ampicillin resistant E. coli TG-1 cells was grown in liquid culture to A 60 o of ⁇ 0.5, then infected with helper phage (VSC-M13, Stratagene) to produce a large amplified phagemid stock. Phagemids were recovered by a PEG precipitation procedure (Harrison et al., Methods in Enzymology (1996) 267: 83-
• Clone NOl (reactive to recombinant hepatitis B virus [HBV] particles) belongs to the V H 3-DP35 family, and the sequence ofthe 9-mer V H -CDR3 encoded by this clone, designated NOl CDR3, is SEQ ID NO:7.
• TMl.l-myc+/TMl.l The first clone of the amplified stock ofthe third panning procedure, on immobilized T cell membranes, was designated TMl.l-myc+/TMl.l.
• scFv antibodies were prepared from this clone, in particular TMl.l and a variant thereof with a myc tag (TMl.l-myc+).
• the amplified stock designated TM2
• the amplified stock was used for an additional round of panning on T- lymphoma cells, as above.
• the final stock was designated TM3.
• the sequences of scFv isolated as a result of following the TM protocol are presented in Table 4. Binding activity following FITC labeling ofthe scFv was also assessed to verify retention ofscFv specificity (see Example 7). For example, the specificity of TM3.13 binding to T-ALL cells was verified by its binding according to FACS analysis (see FIG. 1).
• pHENl a vector used to construct the original phagemid library, was designed with an amber stop codon encoded at the junction ofthe scFv gene and the pIH gene. Therefore, when the vectors of selected clones are introduced by phagemid infection into E. coli HB2151, which is a non-suppressor strain, this system enables production and secretion of soluble scFv into the bacterial periplasm (Harrison et al., Methods in Enzymol. 267: 83-109 (1996)). The scFv is then readily retrievable from the culture broth.
• VH3 family allowing purification on a Protein A affinity column.
• Periplasmic fractions 100-250 ml, from induced cultures of each clone, were prepared and incubated with Protein A Sepharose beads.
• the bound scFvs were recovered from the column by acid elution (0.1M glycine, pH 3.0), followed by eluate neutralization with Tris, pH.8.0.
• the concentration ofthe recovered protein was determined by A 280 measurement, followed by PBS buffer exchange by dialysis or on a G-25 Sepharose column.
• the scFv of a clone L32 derived from the LI protocol also belongs to the
• VH3 gene family (DP-32).
• 5 mM DTT was required and added prior to loading the sample onto the Protein-A affinity column, after which purification on and recovery from Protein-A Sepharose beads and PBS buffer exchange was performed as described above.
• Proteins were fractionated, and fractions containing the N14 scFv (as detected by SDS- PAGE and Western analysis) were pooled, lyophilized, and suspended in 1/10 volume H 2 O. The N14 scFv (unlabeled and FITC-labeled) was then used as a negative control in FACS analysis experiments.
• scFvs were then labelled with FITC. Approximately 1 mg of purified scFv from each preparation was suspended in PBS and coupled to FITC using a Fluoro-Tag FITC conjugation commercial kit (Sigma-Aldrich Co ⁇ ., St. Louis, MO), according to the manufacturer's instructions. Following purification and FITC labeling, the profile of each preparation (labeled and unlabeled) was analyzed by SDS-PAGE, Western blotting, HPLC using a Superdex-75 column (A 280 and A 495 ), and fluorometry. The analyses indicated 80% purity ofthe N14 scFv, and 90% purity for the VH3 clones, with approximately 2 molecules of FITC conjugated to each scFv molecule (F/P ratio of 2:1).
• the present example demonstrates the binding of L32 scFv to washed platelets and platelet-rich-plasma (PRP) and the effects of L32 scFv on platelet aggregation.
• PRP platelet-rich-plasma
• the reaction mixture contains PRP
• L32 scFv staining (binding) of platelets was also assessed by FACS. This method is useful for measurements based on the intensity of staining by fluorescent markers. As indicated in FIG. 3, staining with Yl and Yl-myc+ scFv (a glycocalicin- reactive Yl scFv antibody with a c-myc tag) resulted in stained platelets from PRP. In contrast, the fluorescent signal following staining of these platelets with L32 scFv was relatively unchanged, as compared with the staining with control antibodies (compare the histograms in FIG. 3, wherein TM1. l-myc+ is a scFv that does not bind to any platelet- associated epitopes).
• FACS FACS. Analysis was carried out following three-step staining with (i) L32; (ii) anti-single chain antibody; and (iii) anti-Rabbit FITC-labeled antibody.
• the different cell lines were classified according to the ratio ofthe geo means ofthe cell population following L32 binding over that the negative control, as shown in Table 5. Low binding was assigned to cells with a ratio of 1, medium binding was assigned to cells with a ratio within the range of 1-4, and high binding was assigned to cells with a ratio greater than 4.
• a crude membrane fraction and extraction of membrane proteins were also prepared. Twenty volumes of homogenization buffer was added to one volume of packed cells.
• the homogenization buffer contained 2% (w/v) Tween 20, 1 mM MgSO 4 , 2 mM CaCl 2 , 150 mM NaCl, and 25 mM Tris-HCI, pH 7.4.
• the following protease inhibitors were also added: lmM PMSF, 5 ⁇ g/ml Leupeptin, and 5 ⁇ g/ml Aprotonin.
• the cells were homogenized using a Potter-Elvehjem homogenizer with a rotating Teflon pestle (Ultra-Torex) at a rate of three to five strokes.
• Plasma protein samples were prepared after diluting pooled plasma from a blood bank of healthy individuals 1:10 (v/v) with PBS. The diluted solution was filtered through 0.45 ⁇ m membranes, and aliquots were stored frozen (-20° C) until they were analyzed. Samples were then run on 10% SDS-PAGE at 140-160 Volts, for 3.5 hours at Sigma Z37, 503-9 appliance. The electrophoresed samples were transferred in Tris Glycine buffer (20% MeOH, 192 mM glycine, 25 mM TRIS, pH 8.3) onto a nitrocellulose membrane overnight at 20 Volts at RT.
• Tris Glycine buffer (20% MeOH, 192 mM glycine, 25 mM TRIS, pH 8.3
• the present example demonstrates comparative binding to sulfated and non-sulfated peptides to L32 and Yl by ELISA.
• amino acids 268 to 285 of GPIb and 1-17 of mature PSGL-1) were prepared and used to assess the binding specificity of L32 scFv antibody (ELISA).
• Sulfated and nonsulfated peptides (1 ⁇ M) were attached to microtiter plates suitable for ELISA analysis, and unattached peptides were thoroughly washed before nonspecific binding sites were blocked.
• the plates were incubated with scFv antibodies in the indicated concentrations (see FIG. 8) for one hour at RT.
• the attached scFvs were detected using polyclonal rabbit anti-V L antibodies, followed by horse raddish peroxidase (HRP) anti-rabbit antiserum.
• HRP horse raddish peroxidase
• Y* designates sulfated Tyrosines.
• Yl nor L32 were found to significantly bind the sulfated PSGL-1-related peptides G and D (lacking sulfation at the third residue). Furthermore, neither of Yl and L32 bound sulfated (or non-sulfated) peptides related to the Fibrinogen ⁇ -chain. Neither scFv antibody binds to the peptides in which the tyrosines are not sulfated, indicating that sulfation is required for binding.
• Y17 scFv antibody binding to washed platelets was examined by FACS as described in Example 4.
• the effects ofthe various peptides on Y17 binding to platelets were evaluated by first incubating Y17 together with the indicated concentration of peptide (see FIG. 9), before addition to the platelet preparation.
• GPIb-derived peptide containing sulfated tyrosine at position 276 caused the greatest inhibition of Y17 binding to washed platelets.
• GPIb-derived peptides, containing amino acid changes were prepared and tested to confirm the consensus sequence requirements for Y17 recognition. These results suggest that the first sulfated tyrosine is important for Y17 binding to wash platelets. However, sulfation ofthe second Tyrosine apparently does not have a role in Y17 recognition.
• the negative charge amino acid residues of Aspartate (D) at positions 277 and 275 are also important for Y17 binding. The results were similar to those observed with Yl.
• the present example demonstrates binding of L32 to primary cells from animals of various species.
• the vector pHEN-L32 encoding the original L32 is amplified using PCR for both the V and the V H regions, individually.
• the sense oligonucleotide and the anti- sense oligonucleotide are used for the V L PCR reaction.
• the cDNA product with the expected size is purified, sequenced, and digested with restriction enzymes. The same procedure is employed to amplify the V H region.
• the V H PCR product is digested with restriction enzymes.
• a triple ligation procedure into the pHEN vector, pre-digested, is employed.
• the final vector is designated pTria-L32. Following E.
• the re-folding solution containing the protein was dialyzed in a buffer containing 25 mM Phosphate buffer pH 6, 100 mM Urea, and concentrated to 500 ml.
• the concentrated/dialyzed solution was bound to an SP-sepharose column, and the protein was eluted by a gradient of NaCl (up to IM).
• CHO cells are cultivated in F-12 medium supplemented with 10% fetal calf serum and 40 ⁇ g/ml gentamicin at 37° C in 5% CO 2 atmosphere. One day before transfection 0.8-lxl0 6 cells are seeded on 90mm dishes. The cultures are transfected with 10 ⁇ g of DNA encoding for the light and heavy chains ofthe L32 antibody cloned under the CMV (cytomegalo virus) promoter and the dhfr gene under the sv-40 promoter. Transfection is carried out using the FuGene (Roche) transfection reagent technique.
• CMV cytomegalo virus
• the cells After 2 days of growth in nonselective growth media, the cells are cultured in a media containing lOOnM-5 ⁇ M of methofrexate (MTX) and dialyzed fetal calf serum in order to select for clones (after limiting dilution) that express increased levels ofthe full L32 antibody.
• MTX methofrexate
• Table 16 summarizes the affinity data for L32 and Yl affinity in AML and B-CLL samples.
• the cells are CD19+/CD5-

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• 2003
  • 2003-06-30 BR BR0312483-5A patent/BR0312483A/pt not_active IP Right Cessation
  • 2003-06-30 WO PCT/US2003/020602 patent/WO2004003166A2/en not_active Application Discontinuation
  • 2003-06-30 RU RU2005101621/13A patent/RU2005101621A/ru not_active Application Discontinuation
  • 2003-06-30 PL PL03374439A patent/PL374439A1/xx not_active Application Discontinuation
  • 2003-06-30 JP JP2004518132A patent/JP2005536199A/ja active Pending
  • 2003-06-30 CN CNA038204029A patent/CN1678347A/zh active Pending
  • 2003-06-30 MX MXPA05000272A patent/MXPA05000272A/es unknown
  • 2003-06-30 EP EP03762258A patent/EP1534332A4/de not_active Withdrawn
  • 2003-06-30 KR KR1020047021603A patent/KR20060011925A/ko not_active Application Discontinuation
  • 2003-06-30 AU AU2003263764A patent/AU2003263764A1/en not_active Abandoned
  • 2003-06-30 CA CA002491363A patent/CA2491363A1/en not_active Abandoned
• 2004
  • 2004-12-30 IL IL16606304A patent/IL166063A0/xx unknown

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